GAMMA-CYCLODEXTRINE, 6A,6B,6C,6D,6E,6F,6G,6H-OCTABROMO-6A,6B,6C,6D,6E,6F,6G,6H-OCTADEOXY-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 - March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ICH. S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline (Into Operation June 2012)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R,3R,5S,6R,8S,10S,11S,13R,15S,16R,18R,20S,21R,23S,25S,26S,28R,30S,31R,33S,35S,36S,38R,40S,41R,42R,43R,44R,45R,46R,47R,48R,49R,50R,51R,52R,53R,54R,55R,56R)-5,10,15,20,25,30,35,40-octakis(bromomethyl)-2,4,7,9,12,14,17,19,22,24,27,29,32,34,37,39-hexadecaoxanonacyclo[36.2.2.2³,⁶.2⁸,¹¹.2¹³,¹⁶.2¹⁸,²¹.2²³,²⁶.2²⁸,³¹.2³³,³⁶]hexapentacontane-41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56-hexadecol

EC Number:

611-044-0

Cas Number:

53784-84-2

Molecular formula:

C48H72Br8O32

IUPAC Name:

(1R,3R,5S,6R,8S,10S,11S,13R,15S,16R,18R,20S,21R,23S,25S,26S,28R,30S,31R,33S,35S,36S,38R,40S,41R,42R,43R,44R,45R,46R,47R,48R,49R,50R,51R,52R,53R,54R,55R,56R)-5,10,15,20,25,30,35,40-octakis(bromomethyl)-2,4,7,9,12,14,17,19,22,24,27,29,32,34,37,39-hexadecaoxanonacyclo[36.2.2.2³,⁶.2⁸,¹¹.2¹³,¹⁶.2¹⁸,²¹.2²³,²⁶.2²⁸,³¹.2³³,³⁶]hexapentacontane-41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56-hexadecol

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: off-white powder
- Storage condition of test material: At room temperature

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate

Experiment 2:
Pre-incubation assay without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A homogeneous suspension could be prepared in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 5 µg/plate in saline for TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9 2.5 µg/plate in DMSO for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene

Remarks:

without S9 10 µg/plate in DMSO for TA98; 15 µg/plate in DMSO for TA1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 650 µg/plate in DMSO for TA100

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 10 µg/plate in DMSO for WP2uvrA

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 in DMSO 1 µg/plate TA98,TA100; 2.5 µg/plate TA1535,TA1537; 5 µg/plate TA100(pre-incubation); 15 µg/plate WP2uvrA

Details on test system and experimental conditions:

METHOD:
Experiment 1: direct plate assay
Experiment 2: pre-incubation assay. Test item and bacterial culture were pre-incubated for 30 minutes by 70 rpm at 37°C, either with or without S9-mix.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were monitored.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Rationale for test conditions:

The dose ranges in the main tests were based on the results of dose range finding test in TA100 and WP2uvrA.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 μg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1:
- Dose range finding: Precipitation was observed at concentrations of 1600 and 5000 μg/plate.
- First mutation experiment: Precipitation was observed at concentrations of 1600 and 5000 μg/plate.
Experiment 2:
- Precipitation of Broomdex on the plates was observed at the top dose of 5000 μg/plate.


RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 1600 μg/plate and above in the absence of S9-mix. In tester strain WP2uvrA, no toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate


COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative control values were within the laboratory historical control data ranges, except the response for TA98, in the absence and presence of S9-mix, in the first experiment, which was below the historical control range. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (9 revertant colonies) when compared against relevant historical control data (10 revertant colonies), the validity of the test was considered to be not affected.
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

In the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay performed according to ICH guideline and GLP principles, Broomdex was found not to be mutagenic with or without metabolic activation when tested up to and including 5000 μg/plate.

Executive summary:

The mutagenic activity of broomdex was evaluated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay according to ICH guideline and in compliance with GLP principles. All bacterial strains showed negative responses up to 5000 μg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Cytotoxicity was only observed in tester strain TA100 in the absence of S9-mix at concentrations of 1600 and 5000 μg/plate. Precipitation of Broomdex on the plates was observed at concentrations of 1600 and 5000 μg/plate. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Broomdex is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.