Registration Dossier

Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-amine
EC Number:
700-140-9
Cas Number:
752984-24-0
Molecular formula:
C11H15N
IUPAC Name:
(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-amine
Specific details on test material used for the study:
Name of test substance: 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)-
Test substance No.: 19/0201-1
Batch identification: B6943 v. 10.04.2019
CAS No.: 752984-24-0
Content: 99.9 area-% (96.6 area-% trans isomer, 3.3 area-% cis diastereomer)
Identity: Confirmed (for details see Final Report, Study code: 19L00134)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: Expiry date: 09 Apr 2021
The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Method

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:
The S9 fraction was prepared according to Ames et al.. 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.

The S9 mix was prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM

The phosphate buffer is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1. To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
sterility control: additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance alone vehicle control: only contains the vehicle used for the test substance at the same concentration and volume for all tester strains
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: with S9 mix: 2-aminoanthracene; without S8 mix: 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 109 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay. The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions (5). The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Checking the tester strains
The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA was checked for UV sensitivity. Histidine and tryptophan auxotrophy was checked in each experiment via the spontaneous rate.
Rationale for test conditions:
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 given purity at the beginning of the study 5.2 mg/plate was used as top dose in the 1st Experiment.
Evaluation criteria:
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 dimethyl sulfoxide (DMSO). 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see also attached table
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see also attached table
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see also attached table
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see also attached table
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see also attached table
Additional information on results:
The test substance 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA.

TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate. In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 33 μg/plate.

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

Any other information on results incl. tables

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E.coli

1st-SPT

Without

2600 – 5200

5200

5200

5200

1000 – 5200

With

5200

5200

1000 – 5200

5200

5200

2nd-PIT

Without

100;

1000 – 2600

1000 – 2600

33;

333 – 2600

2600

2600

With

2600

2600

1000 – 2600

2600

1000 – 2600

Reduced background growth was observed at following concentrations (μg/plate):

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E.coli

1st-SPT

Without

5200

With

5200

2nd-PIT

Without

2600

With

2600

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, it is concluded that 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). 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. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data.