1-isoctyloxycarbonyl ethylated 2,4,6 tris (2,4- hydroxyphenyl) -1,3,5 triazine derivatives

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline conform study, purity of test item not mentioned

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

his, trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvr-def., rfa-def. R-factor

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from aroclor induced rat liver

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

TA 1535, T A I 00

Positive control substance:

other: sodium azide, NaN3

Remarks:

without metabolic activation

Controls 2

Positive controls:

yes

Remarks:

TA1537, TA98

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

without metabolic activation

Controls 3

Positive controls:

yes

Remarks:

WP2 uvrA

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 4

Positive controls:

yes

Remarks:

all strains

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in overlay agar (plate incorporation)

DURATION
Precultures
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml
Erienmeyer flasks containing 20 ml nutrient medium. A solution of 20 pi ampicillin
(25 pg/ml) was added to the strains TA 98 and TA 100. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

SELECTION AGENT (mutation assays): ampicillin

NUMBER OF CELLS EVALUATED: colonies were counted using the AUTOCOUNT

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of
revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or
thrice (strains TA 1535 and TA 1537) the colony count ofthe corresponding solvent control
is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically
relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second
experiment. However, whenever the colony counts remain within the historical range of
negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effects in TA 1535/1537/98 at 1000 or 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Additional information on results:

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains
TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for
toxicity and mutation induction with three plates each. The experimental conditions in this
pre-experiment were the same as described below for the experiment I (plate incorporation
test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a
clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

In the pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. The
pre-experiment is reported as part of experiment I since no relevant toxic effects were
observed and 5000 pg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations
were tested:
33; 100; 333; 1000; 2500; and 5000 pg/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed nonnal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Minor toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, were observed in strain TA 1535 at 1000 and 5000 µg/plate with metabolic activation and in strain TA 1537 at 2500 and 5000 pg/plate with and without metabolic activation in experiment I. In experiment II, a minor toxic effect was also observed in strain TA 98 at 5000 µg/plate with metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome ofthe strains used.