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Genetic toxicity in vitro

Description of key information

Three in vitro genotoxicity studies were carried out: the Ames test (also using the E coli strain), a chromosome aberration test, and a mouse lymphoma assay. All 3 tests did not show genotoxic effects.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
bacteria, other: Salmonella typhimurium strains TA98, 100, 1535, 1537 and Escherichia coli strain WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix.
Test concentrations with justification for top dose:
Concentration range in the main test (with and without metabolic activation): 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Solvent: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ENNG, 9-AA, 4-NQO
Remarks:
without S9-Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
Five concentrations of the test material were assayed in triplicate against
each tester strain, using the direct plate incorporation method in
accordance with the standard methods for mutagenicity tests using bacteria.

Known al iquots (0.1 ml) of one of the bacterial suspensions were
dispensed into sets of sterile test tubes followed by 2.0 ml of molten
trace histidine/tryptophan supplemented top agar at 45°C, 0.1 ml
of the appropriately diluted test material or vehicle control and
either 0.5 ml of the 59 liver microsome mix or phosphate buffer.
The contents of each test tube were mixed and equally distributed
onto the surface of Vogel-Bonner Minimal agar plates (one tube per
plate). This procedure was repeated, in triplicate, for each bacterial
strain and for each concentration of test material with and without
59-mix.

All of the plates were incubated at 37°C for approximately 48 hours
and the frequency of revertant colonies assessed using a Domino
colony counter. Due to the potentially volatile nature of the test
material, all of the plates were sealed in stainless steel containers
(one dose group per container) for the duration of the incubation
period.

The second experiment was performed using methodology as described for
experiment 1, using fresh bacterial cultures, test material and control
solutions in triplicate.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have
induced a dose-related and statistically(S) significant increase in mutation rate
(of at least twice the spontaneous reversion rate) in one or more strains of
bacteria in the presence and/or absence of the 59 microsomal enzymes in both
experiments at sub-toxic dose levels. In the event of the two experiments
giving conflicting or equivocal results, then a third experiment may be
performed to confirm the correct response. To be considered negative the
number of induced revertants compared to spontaneous revertants should be
less than twofold at each dose level employed, the intervals of which should
be between two and five fold and extend to the limits imposed by toxicity,
solUbility or up to the maximum recommended dose of 5000 tJg/plate. In this
case the limiting factor was the maximum recommended dose.
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
Observations:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: main test
Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

This study was conducted to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers (1, 2, 3) and Garner et al (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material. This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. This method also conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471, Method B14 in EC Commission Directive 92/69/EEC and the USA, EPA (TSCA) guidelines. A copy of the Certificate of Compliance with GLP, issued by the UK Department of Health, is included as Appendix IV.

The substance was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - December 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Chinese Hamster Lung cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 156.25 -5000 µg/ml
Concentration range in the main test (without metabolic activation): 1.22 -40 µg/ml
Vehicle / solvent:
Acetone
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Expression time:
Experiment 1:
Without S9-mix: 12 hours continuous
With S9-mix: 4 exposure , a wash and a
further 8 hours treatment free.

Experiment 2:
Without S9-mix: 12, 24, 48 hours continuous,
6 hours and 18 hrs treatment free
With S9-mix: 6hrs treatment, 18 treatment free
4 hours treatment 8 hr treatmentfree

Selection time:
See above.

Fixation time:
Two hours before harvest time
Evaluation criteria:
Many experiments with the CHL cell line have established a range of aberration
frequencies acceptable for control cultures, these are commonly in the range of
o to 3 % cells with aberrations (lshidate, 1987), Data Book of Chromosomal
Aberration Test In Vitro, (Revised Edition).
A positive response was recorded for a particular treatment if the % cells with
aberratipns (gaps included) was equal to or exceeded 10%, an equivocal
response was recorded for values between 5 and 10% and a negative response
for values less than 5%. For polyploid cells, an incidence greater than 10% is
generally recorded as positive.
However, consideration is given to a number of factors, such as the frequency
of chromosome exchange events which are comparatively rare in control
cultures, and the ultimate designation must rely upon experience and sound
scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).
Species / strain:
mammalian cell line, other: CHL cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 312.5 µg/ml)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mammalian cell line, other: CHL cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 9.77 µg/ml)
Vehicle controls validity:
valid
Positive controls validity:
not valid

Chromosome Aberration Test - Experiment 1: The test material showed the expected level of toxicity similar to that seen in the preliminary toxicity study. These data show a dose-related increase in toxicity in the with-59 exposure group and approximately 50% mitotic inhibition was achieved at 1250 ug/ml. In the exposure group without-59 the toxicity response curve was very steep, there being little evidence of toxicity at 19.53 ug/ml and total mitotic inhibition at 29.3 ug/ml. The vehicle control cultures gave values of chromosome aberrations within the expected range. The positive control cultures gave significant increases in the frequency of aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. The test material was seen to induce no statistically significant increases in the frequency of cells with aberrations. The test material did not induce a significant increase in the number of polyploid cells at any dose level in either of the treatment cases.

Chromosome Aberration Test - Experiment 2: The test material showed the expected level of toxicity similar to that seen in the preliminary cytotoxicity study and Experi ment 1. In most cases the maximum evaluated dose level achieved an approximate 50% inhibition of mitotic index. The vehicle control cultures gave values of chromosome aberrations within the expected range. The positive control cultures, except cyclophosphamide without S9 treatment, gave significant increases in the frequency of cells with aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. Cyclophosphamide is used in the absence of activation as a control for the with-activation treatment group. The test material was seen to induce no statistically significant or dose-related increases in the frequency of cells with aberrations. The test material did not induce a significant increase In the number of polyploid cells at any dose level in any of the six treatment cases.

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce any statistically significant or dose-related increases
in the frequency of cells with aberrations. The test material was shown to be toxic
to CHL cells in vitro in all six treatment cases, with a very steep dose response curve.
The test material, INITIATOR D-129, was shown to be non-clastogenic to CHL cells
in vitro.
Executive summary:

This study was designed to assess the potential chromosomal mutagenicity of a test material, on the metaphase chromosomes of the Chinese Hamster lung (CHl) cell line according to the requirements of the japanese New Chemical Substance law (MITI) and the updated Annex V B 10 Method.

Duplicate cultures of Chinese hamster lung (CHL) cells were treated with the test material at a minimum of four dose levels, in each treatment case together with vehicle and positive controls in each of two experiments. In Experiment 1 a single cell-harvest time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of metabolic activation (followed by 18 hours treatment-free incubation); 4 hours exposure with the addition of metabolic activation (followed by 8 hours treatment-free incubation); 12 hours continuous exposure, 24 hours continuous exposure and 48 hours continuous exposure. The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 2.5 and 2500 ug/ml depending on the particular treatment regime used; the continuous exposures were more toxic than the pulse exposures.

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.

The positive control treatments gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the frequency of cells with aberrations in either Experiment 1 or in Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases. The test material was shown to be non-clastogenic to CHL cells in vitro.

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:
August-October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/-3.7.2C mouse lymphoma cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphtoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
with metabolic acitivation:
test 1: 1-387 µg/mL, test 2: 1-350 µg/mL
without metabolic activation:
test 1: 1-66 µg/mL, test 2: 5-53 µg/mL, test 3: 10-56 µg/mL
Vehicle / solvent:
acetone
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
no
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Exposure duration:
with S9-mix: 3 h
without S9-mix: 3 and 24 h

Expression time: 2 days

Selection time: 11 or 12 days

Seletive medium: trifluorthymidine (TFT)

Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated
for its biological relevance including a comparison of the results with the historical control data range.
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induced at least a three-fold increase in the mutation frequency compared to the solvent control
in a dose-dependent manner; or
b) In case a positive result was repeated, the positive response should be reproducible in at least
one repeated experiment.
c) In addition, the observed increase should be biologically relevant and was compared with the
historical control data range.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutation frequency of at least three-fold compared
to the solvent controL
b) The results were confirmed in an independently repeated test.
A test substance is considered equivocal (questionable) in the mutation assay if:
- No clear conclusion for positive or negative result could be made after an additional confirmation
study.
Statistics:
The experimental results were not subjected to statistical analysis.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 44-50 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(at 333 µg/mL in a preliminary test)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation occurred at 40 µg/mL

The test article precipitated in the exposure medium at concentrations of 100 ug/ml and above. The test article was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 ug/ml.

First mutagenicity test

Evaluation of toxicity In the absence of S9-mix, the dose levels of 77 to 111 ug/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, no toxicity was observed and all dose levels were evaluated. The dose levels selected to measure mutation frequencies at the TK-Iocus were: Without S9-mix: 1, 5, 11, 22, 33, 44, 55 and 66 ug/ml exposure medium. With S9-mix: 1, 11, 28, 55, 83, 111, 276 and 387 ug/ml exposure medium. In the absence of S9-mix, the relative total growth of the highest test sUbstance concentration was reduced by 93% compared to the total growth of the solvent controls. In the presence of S9-mix, no reduction of the relative total growth was observed up to the highest dose level tested. Evaluation of the mutagenicity No significant increase in the mutation frequency at the TK locus was observed after treatment with Trigonox 301 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Trigonox 301 treated cultures were comparable to the numbers of small and large colonies of the solvent controls. 8.3.2. Second mutagenicity test To obtain more information about the possible mutagenicity of Trigonox 301, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12% (v/v) S9-mix with a 3 hour treatment period. Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing. Without S9-mix: 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ug/ml exposure medium. With 12% (v/v) S9-mix: 1, 10, 25, 50, 75, 100, 250 and 350 ug/ml exposure medium. Evaluation of toxicity In the absence of S9-mix, no dose level was reached with a survival of 10 to 20%, this part of the experiment was repeated with the following concentrations: 5, 10, 20, 30, 40, 50, 53, 56, 60, 63, 66 and 70 ug/mL The dose levels of 56 to 70 ug/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, no toxicity was observed and all dose levels were evaluated. The dose levels selected to measure mutation frequencies at the TK-Iocus were: Without S9-mix: 5, 10, 20, 30, 40, 50 and 53 ug/ml exposure medium. With S9-mix: 1, 10, 25, 50, 75, 100, 250 and 350 ug/ml exposure medium. In the absence of S9-mix, the relative total growth of the highest test SUbstance concentration was reduced by 93% compared to the total growth of the solvent controls. In the presence of S9-mix, no reduction of the relative total growth was observed up to the highest dose level tested. Evaluation of mutagenicity No significant increase in the mutation frequency at the TK locus was observed after treatment with Trigonox 301 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Trigonox 301 treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Third mutagenicity test Since no dose level was reached with a survival of 10-20% and no dose level with intermediate toxicity in the absence of S9-mix in the second experiment, an additional mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose levels were selected for the third mutagenicity test: 5, 10, 20, 30, 35, 40, 43, 46, 50, 53, 56, 60, 63, 65 and 70 ug/ml exposure medium. Evaluation of toxicity Since the dose levels of 5 to 35 ug/ml showed no cytotoxicity, the lowest dose of 5 ug/ml was regarded not relevant for mutation frequency measurement. The dose levels of 43 to 53 ug/ml showed no remarkable difference in the cell growth. Therefore, the concentrations of 43 and 53 ug/ml were not selected for mutation frequency measurement. The concentrations of 60 to 70 ug/ml were not selected for mutation frequency measurement, since these concentrations were too toxic. The dose levels selected to measure mutation frequencies at the TK-Iocus were: 10, 20, 30, 35, 40, 46, 50 and 56 uglml exposure medium. The relative total growth of the highest test substance concentration was reduced by 75% compared to the total growth of the solvent controls.

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not mutagenic in the TK mutation test ystem under the experimental conditions
Executive summary:

Evaluation of the mutagenic activity of Trigonox 301 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat), This report describes the effects of Trigonox 301 on the induction of forward mutations at the thymidine-kinase locus (TK-Iocus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9 .. mix (rat liver S9-mix induced by a combination of phenobarbital and l1-naphthoflavone). The study procedures described in this report were based on the most recent OEeD and EEC guidelines. Batch DVT 0706516079 of Trigonox 301 was a clear colourless liquid with a purity of 41 %. The test substance was dissolved in acetone. In the first experiment, Trigonox 301 was tested up to concentrations of 66 and 387 ug/ml in the absence and presence of 8% (v/v) S9-mix, respectively, The incubation time was 3 hours. Trigonox 301 was tested up to the cytotoxic level of 93% in the absence of S9-mix. In the presence of S9-mix, no toxicity was observed, however Trigonox 301 was tested beyond the precipitating dose level of 44 ug/ml. In the second experiment, Trigonox 301 was tested up to concentrations of 53 and 350 ug/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Trigonox 301 was tested up to a cytotoxic level of 93% in the absence of S9-mix. In the presence of S9-mix, no toxicity was observed, however Trigonox 301 was tested beyond the preCipitating dose level of 40 iJg/ml. Since no dose level with a survival of 10-20% or with intermediate toxicity was reached in the absence of S9-mix in the second experiment, an additional mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Trigonox 301 was tested up to a concentration of 56 ug/ml in the absence S9-mix. The incubation time was 24 hours, Trigonox 301 was tested up to the cytotoxic level of 75%. Mutation frequencies in cultures treated with positive control chemicals were increased by 12-, 11- and 5A-fold for MMS in the absence of S9-mix, and by 16- and 13-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, Trigonox 301 did not induce a significant increase in the mutation frequency in the first experiment This result was confirmed in independent repeat experiments with modifications in the duration of treatment time. In the presence of S9-mix, Trigonox 301 did not induce a significant increase in the mutation frequency in the first experiment This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that Trigonox 301 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Additional information

Justification for classification or non-classification

Based on the absence of genotoxicity in 3 separate in vitro tests, the methylethylketone peroxide trimer (Trigonox 301) does not need classification for this endpoint, nor any in vivo genotoxicity testing.