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Genetic toxicity: in vitro

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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: Apparently well conducted GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

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:
tert-pentyl peroxypivalate
EC Number:
249-530-6
EC Name:
tert-pentyl peroxypivalate
Cas Number:
29240-17-3
Molecular formula:
C10H20O3
IUPAC Name:
tert-pentyl peroxypivalate
additive 1
Reference substance name:
Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated
EC Number:
297-629-8
EC Name:
Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated
Cas Number:
93685-81-5
Molecular formula:
not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
isododecane
Test material form:
other: colorless liquid
Details on test material:
· name:
- protocol and labelling: t-AMYL PEROXYPIVALATE LUPEROX 554M75
· batch number:
- protocol and labelling: 646-9803-702 (13/03/98)
· description:
- at receipt and on the test article description: colourless liquid
· container: one plastic flask
· date of receipt: 16 December 1998
· storage conditions: at -20°C and protected from light
· active material content: 75% in isododecane
· expiry date: June 1999.

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiments without S9 mix:
The selected treatment-levels were as follows:
156.25,312.5,625, 1250 and 2500 ug/plate: for the TA 1535 strain in the first experiment as
well as for the TA 102 strain in the second experiment,
312.5,625, 1250,2500 and 3125 ug/plate: for all strains (except for the TA 1535 strain) in the
first experiment,
78.125, 156.25,312.5,625 and 1250 ug/plate: for all strains (except for the TA 102 strain) in
the second experiment.

Experiments with S9 mix:
The selected treatment-levels were as follows:
156.25,312.5,625, 1250 and 2500 ug/plate: for the TA 1535 strain in the first experiment as
well as for all tester strains (except for the TA 98 strain) in the second experiment,
312.5,625,1250,2500 and 3125 ug/plate: for all strains (except for the TA 1535 strain) in the
first experiment,
78.125, 156.25, 312.5, 625 and 1250 ug/plate: for the T A 98 strain in the second experiment.
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
The five strains of Salmonella typhimurium (Ames et al., 1975; Maron and Ames, 1983):
TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by B.N. Ames' Laboratory
(University of California, Berkeley, USA). They are stored in a cryoprotective medium (1 ml
nutrient broth and 0.09 ml dimethylsulfoxide) in a liquid nitrogen container.
The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken
under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth
was then placed under agitation in an incubator at 37°C for about 14 hours, to produce bacterial
suspensions.
Each strain derived from Salmonella typhimurium L T 2 contains one mutation in the histidine.
operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic substances, further mutations have been
added:
· the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of
the bacteria and increases permeability to large molecules that do not penetrate the nonnal
bacteria cell wall,
· the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which
renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
· the addition of the plasmid pKM 101 (conferring ampicillin resistance) to strains TA 98,
TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
· in addition, the pAQ1 tetracycline resistant plasmidic factor has been added to the TA 102
strain.


Evaluation criteria:
Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per
plate was scored. The individual results and the mean number of revertants, with the
corresponding standard deviation and ratio (mutants obtained in the presence of the test
substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
. the number of revertants in the vehicle controls is consistent with our historical data
(appendix 2),
. the number of revertants in the positive controls is higher than that of the vehicle controls and is
consistent with our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls,
in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive
result. Reference to historical data, or other considerations of biological relevance may also be
taken into account in the evaluation of the data obtained.
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
The test substance was freely soluble in the vehicle (ethanol) at 100 mg/ml.
Consequently, with a maximum dose volume of 50 ug/plate, the dose-levels were: 10, 100,500,
1000,2500 and 5000 ug/plate.
A slight to moderate emulsion was observed in the Petri plates when scoring the revertants at
dose-levels: greater than or equal to 2500 ug/plate.

Without S9 mix, a slight toxicity was induced in the TA 98 and TA 102 strains at 5000 ug/plate.
In the TA 100 strain, a slight to strong toxicity was noted at dose-levels greater than or equal to 2500 ug/plate.

With S9 mix, the test substance was totally toxic at 5000 ug/plate in the three tester strains. In
addition, a slight toxicity was induced at 2500 ug/plate in both the TA 98 and TA 100 strains.
In the TA 102 strain, a 3 and a 7 fold increase in the number of revertants was noted at dose levels
of 1000 and 2500 ug/plate, respectively.

MUTAGENICITY EXPERIMENTS
The number of revertints of the vehicle and positive controls was as specified in the acceptance
criteria. The study was therefore considered valid.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level
was based on the level of toxicity, according to the criteria specified in the international
guidelines.

Experiments without S9 mix:
The selected treatment-levels were as follows:
156.25,312.5,625, 1250 and 2500 ug/plate: for the TA 1535 strain in the first experiment as
well as for the TA 102 strain in the second experiment,
312.5,625, 1250,2500 and 3125 ug/plate: for all strains (except for the TA 1535 strain) in the
first experiment,
78.125, 156.25,312.5,625 and 1250 ug/plate: for all strains (except for the TA 102 strain) in
the second experiment.

A slight emulsion was generally noted at dose-levels > =1250 ug/plate.
In the first experiment, a slight toxicity was noted in the TA 1535 and TA 102 strains at dose levels
> = 50 ug/plate and >= 2500 ug/plate, respectively.
In the three remaining tester strains, a slight to strong toxicity was noted at dose-levels
>= 312.5 ug/plate.

In the second experiment, a slight to moderate toxicity was noted in the TA 102 and TA 100
strains (at the highest dose-level) as well as in the TA 1537 strain (at dose-levels
>= 312.5 ug/plate).
The test substance did not induce any significant increase in the number of revertants, in both
experiments, in any of the five strains.

Experiments with S9 mix:
The selected treatment-levels were as follows:
156.25,312.5,625, 1250 and 2500 ug/plate: for the TA 1535 strain in the first experiment as
well as for all tester strains (except for the TA 98 strain) in the second experiment,
312.5,625,1250,2500 and 3125 ug/plate: for all strains (except for the TA 1535 strain) in the
first experiment.
78.125, 156.25, 312.5, 625 and 1250 ug/plate: for the T A 98 strain in the second experiment.
A slight emulsion was generally noted at dose-levels >= 1250 ug/plate.
In the first experiment, the test substance was slightly to totally toxic in the TA 1537, TA 100
and TA 102 strains (at doses >= 2500 ug/plate) as well as in the TA 98 strain (at doses
>= 1250 ug/plate).

In the second experiment, the test substance was slightly to totally toxic at doses >= 625 ug/plate
in the TA 1537 and TA 100 strains. A slight to moderate (for the TA 98 strain) and a slight to
strong (for the TA 1535 strain) toxicity was noted at dose-levels >= 625 and >= 1250 ug/plate,
respectively. In the TA 102 strain, a slight toxicity was noted at 2500 ug/plate.
In the first experiment, a 2 fold increase in revertants was noted in the T A 98 strain
at 625 ug/plate but not at higher dose-levels where toxicity was induced. The second experiment
was performed using the preincubation method with this strain and no noteworthy increase in
revertants was noted. Therefore the previous 2 fold increase in revertants was not considered as
biologically relevant.

In the TA 102 strain in the first experiment, a 2.2-3.76 fold increase in the number of revertants
was induced at dose-levels of between 312.5 and 1250 ug/plate. No increase was noted at higher
dose-levels where toxicity could have masked the mutagenic effect.
This result confirmed the increase previously noted in the preliminary test.

In the second experiment, repeated under the same experimental conditions (direct plate
incorporation method), a significant increase in revertants was reproduced, even if a lower effect
was noted: up to 2.1 fold the vehicle control value at 1250 ug/plate.

Applicant's summary and conclusion

Conclusions:
positive with metabolic activation

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on the TA 102 strain of Salmonella typhimurium, with S9 mix.
Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce reverse mutation in Salmonella typhimurium.

A preliminary toxicity test was performed to define the dose-levels of the substance to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except the second with S9 mix (excluding the TA 102 strain), which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test substance was dissolved in ethanol.

Experiments without S9 mix: The selected treatment-levels were as follows: 156.25,312.5,625, 1250 and 2500 ug/plate: for the TA 1535 strain in the first experiment as well as for the TA 102 strain in the second experiment. 312.5,625, 1250,2500 and 3125 ug/plate: for all strains (except for the TA 1535 strain) in the first experiment, 78.125, 156.25,312.5,625 and 1250 ug/plate: for all strains (except for the TA 102 strain) in the second experiment. A slight emulsion was generally noted at dose-levels > =1250 ug/plate. In the first experiment, a slight toxicity was noted in the TA 1535 and TA 102 strains at dose levels > = 50 ug/plate and >= 2500 ug/plate, respectively. In the three remaining tester strains, a slight to strong toxicity was noted at dose-levels >= 312.5 ug/plate. . In the second experiment, a slight to moderate toxicity was noted in the TA 102 and TA 100 strains (at the highest dose-level) as well as in the TA 1537 strain (at dose-levels >= 312.5 ug/plate). The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the five strains. Experiments with S9 mix: The selected treatment-levels were as follows: 156.25,312.5,625, 1250 and 2500 ug/plate: for the TA 1535 strain in the first experiment as well as for all tester strains (except for the TA 98 strain) in the second experiment, 312.5,625,1250,2500 and 3125 ug/plate: for all strains (except for the TA 1535 strain) in the first experiment, 78.125, 156.25, 312.5, 625 and 1250 ug/plate: for the T A 98 strain in the second experiment. A slight emulsion was generally noted at dose-levels >= 1250 ug/plate. In the first experiment, the test substance was slightly to totally toxic in the TA 1537, TA 100 and TA 102 strains (at doses >= 2500 ug/plate) as well as in the TA 98 strain (at doses >= 1250 ug/plate). In the second experiment, the test substance was slightly to totally toxic at doses >= 625 ug/plate in the TA 1537 and TA 100 strains. A slight to moderate (for the TA 98 strain) and a slight to strong (for the TA 1535 strain) toxicity was noted at dose-levels >= 625 and >= 1250 ug/plate, respectively. In the TA 102 strain, a slight toxicity was noted at 2500 ug/plate. In the first experiment, a 2 fold increase in revertants was noted in the T A 98 strain at 625 ug/plate but not at higher dose-levels where toxicity was induced. The second experiment was performed using the preincubation method with this strain and no noteworthy increase in revertants was noted. Therefore the previous 2 fold increase in revertants was not considered as biologically relevant. In the TA 102 strain in the first experiment, a 2.2-3.76 fold increase in the number of revertants was induced at dose-levels of between 312.5 and 1250 ug/plate. No increase was noted at higher dose-levels where toxicity could have masked the mutagenic effect. This result confirmed the increase previously noted in the preliminary test. In the second experiment, repeated under the same experimental conditions (direct plate incorporation method), a significant increase in revertants was reproduced, even if a lower effect was noted: up to 2.1 fold the vehicle control value at 1250 ug/plate.

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on the TA 102 strain of Salmonella typhimurium, with S9 mix.