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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 23rd to June 11th, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene]
EC Number:
306-832-3
EC Name:
1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene]
Cas Number:
97416-84-7
Molecular formula:
C23H24Br8O2
IUPAC Name:
1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene]
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.Prior to use cells were clensed of pre-existing mutants. Cultures of the cells are grown in RPMI 1640 minimal medium supplemented with 10 % horse serum heat-inactivated at 56 °C for 20 minutes before use (Complete medium 10 %). The incubations are at 37 °C in a 5 % carbon dioxide atmosphere (100 % nominal relative humidity).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 liver tissue fraction
Test concentrations with justification for top dose:
Assay n. 1:
S9 mix: (-/+);
Treatement time: 3 hours;
Dose levels: 625, 198, 62.5, 19.8 and 6.25 µg/ml

Assay n. 2:
S9 mix: (-);
Treatement time: 24 hours;
Dose levels: 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml

Assay n. 2:
S9 mix: (+);
Treatement time: 3 hours;
Dose levels: 625, 313, 156, 78.1 and 39.1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance is soluble in DMS up to the maximum concentration tested (625 µg/ml)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylmethanesulphonate (absence of S9 metabolism) batch no.: 76296KJ obtained from Sigma Chemical Co.; benzo(a)pyrene (presence of S9 metabolism) batch no.: 129K1892 obtained from Sigma Chemical Co.
Details on test system and experimental conditions:
METHOD OF APPLICATION:

DURATION
- Incubation: at 37 °C in a 5 % carbon dioxide atmosphere (100 % nominal relative humidity).
- Exposure duration: 3 and 24 hours.
- Expression time (cells in growth medium): During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2 x 10^5 cells/ml.

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 2x10^5 cells/ml
Evaluation criteria:
induced mutant frequency
Statistics:
Statistical analyses will be performed according to UKEMS guidelines

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: see below for further information
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: other: TK +/-
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

CYTOTOXICITY TEST

Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 625 μg/ml and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 μg/ml. Upon addition of the test item to the cultures, precipitate was noted at 625 and 313 μg/ml. At the end of the 3 hour treatment period, precipitate was observed at the same dose levels and opacity of the treatment medium was observed at 156 and 78.1 μg/ml. At the end of the 24 hour treatment period, precipitate was observed at 625 μg/mL and opacity was observed at 313 and 156 μg/ml.

In the absence of S9 metabolic activation, using the 3 hour treatment time,dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 30 %, 50 % and 65 % of the concurrent negative control value, while no toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 23 %, 28 % and 79 % of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), slight toxicity was observed at the five highest dose levels. No toxicity was observed over the remaining dose levels tested.

 

MUTATION ASSAYS

Results obtained are presented in the following table:

 

Assay No.

S9

Growth Factor

Cloning efficiency (%)

1

-

15

119

2

-

24

111

 

The study was accepted as valid.

 

Survival after treatment

In the first assay, in the absence of S9 metabolic activation, dose related toxicity was observed: the RTG was reduced to 29 % at the highest dose level compared to the concurrent negative control value, and was reduced to 42 % and 68 % at the two lower dose levels. In the presence of S9 metabolic activation, slight toxicity was observed at the highest concentration.

In the second assay, using a long treatment time in the absence of S9 metabolic activation, toxicity from slight to marked was observed at the three highest dose levels. The RTG was reduced to 19 %, 32 % and 73 % compared to the concurrent negative control value. In the presence of S9 metabolic activation, toxicity was observed at the dose level of 313mg/ml and not at 625mg/ml; this event is probably due to the different solubilisation of the test item in the treatment medium (presence of precipitate).

 

Mutation results

No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test item at any concentration level. For the negative and positive controls, the number of wells containing small colonies and those containing large colonies were reported. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

 

OSMOLALITY AND pH

The pH values and osmolality of the post-treatment media were determined The addition of the test item solution did not have any obvious effect on the osmolality of the treatment medium.

A slight decrease of pH was observed at all dose levels tested in the absence of S9 metabolism. However, since this decrease was within the physiological values, it is not considered to affect the test system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro, in the absence or presence of S9 metabolic activation, under the experimental conditions.
Executive summary:

Method

The test item AP 1300 S was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 

Observation

AP 1300 S was found to be soluble in dimethylsulfoxide (DMSO) at the concentration of 62.5 mg/ml, corresponding to 625 μg/ml in the final treatment medium. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation,

the test item was assayed at a maximum dose level of 625 μg/m and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 μg/ml.

In the absence of S9 metabolic activation, using the 3 hour treatment time, dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 30 %, 50 % and 65 % of the concurrent negative control value. Using the 24 hour treatment time, dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 23 %, 28 %

and 79 % of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested.

Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), slight toxicity was observed at higher dose levels. By the end of treatment time, precipitation of the test item or opacity of the treatment medium were observed at the four or three highest dose levels.

Based on the results obtained in the preliminary trial, two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels below:

 

Assay n. 1:

S9 mix: (-/+);

Treatement time: 3 hours;

Dose levels: 625, 198, 62.5, 19.8 and 6.25 µg/ml

 

Assay n. 2:

S9 mix: (-);

Treatement time: 24 hours;

Dose levels: 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml

 

Assay n. 2:

S9 mix: (+);

Treatement time: 3 hours;

Dose levels: 625, 313, 156, 78.1 and 39.1

 

The dose levels of the second experiment in the presence of S9 metabolism were selected on the basis of the results of the first experiment in order to focus on the highest concentrations that could be tested.

 

Results

No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.