Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative for mutagenicity in vitro in an Ames test and in a Mouse Lymphoma Assay.

REACH Annex VIII requires performing “in vitro cytogenicity study in mammalian cells or in vitro micronucleus study”.  The study can be waived when “adequate data from an in vivo cytogenicity test are available”. For this substance an in vivo cytogenicity test is not available. However, available data from other peroxyesters (with varying alkyl and ester groups) tested in an in vivo cytogenicity studies show a negative result, irrespective of the positive outcome of the available in vitro studies. Furthermore, an evaluation of the peroxyesters was performed with the OECD toolbox. Based on profiling results for DNA binding and mutagenicity alerts, no cytogenicity is expected. Therefore, because in vivo data of all peroxyesters tested is negative and the fact that this is supported by the OECD toolbox assessment, read across to the available in vivo studies can be applied and consequently the in vitro cytogenicity testing can be waived for this substance. Further studies with this substance are scientifically unjustified. For further details see below.


Link to relevant study records

Referenceopen allclose all

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:
29 April 1986 - 29 May 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP. Deviation from the current guideline: With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Each tester strain contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat).
gal mutation in the galactose metabolism.
chl : mutation in nitrate reductase.
bio : defective biotin synthesis.
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).
Additional strain / cell type characteristics:
other: TA1537 histidine mutation: hisC3076, Frameshift, TA98 histidine mutation: hisD3052/R-factor, Frameshift, TA1535: hisG46, Base-pair substitutions, TA100: hisG46/R-factor, Base-pair substitutions. R-factor =plasmid pKM101 (increases error-prone DNA repair).
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Each tester strain contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat).
gal mutation in the galactose metabolism.
chl : mutation in nitrate reductase.
bio : defective biotin synthesis.
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).
Additional strain / cell type characteristics:
other: histidine mutation: hisD3052, Frameshift
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary test (TA100): 0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
Experiment 1, 2, 3, 4: 100, 333, 1000, 3333, 5000 µg/pkate (with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol absolute 0.1 ml
- Justification for choice of solvent/vehicle: soluble+stable in vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol absolute 0.1 ml
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate with independent repeat

NUMBER OF CELLS EVALUATED: revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

DETERMINATION OF CYTOTOXICITY
- Method: % survival
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the Ames test if:
a) It induces at least a 2-fold and statistically significant (student's t-test, p< 0.05) increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. Moreover, the positive response should be dose-rela'ted. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.
Statistics:
See above.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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
Conclusions:
Interpretation of results (migrated information):
negative

Negative in the Ames Salmonella/microsome preincubation test.
Executive summary:

The test substance was tested in the Ames Salmonella/microsome preincubation test up to the limit of toxicity (5 mg/plate). The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535; TA1537; TA1538; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as nonmutagenic in this test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
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:
2011-08-02 until 2011-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
according to OECD 476 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I / 4 hours treatment
without S9 mix: 2.2, 4.4, 8.8, 17.5, 35.0, 70.0, 140.0 nL/mL
with S9 mix: 17.5, 35.0, 70.0, 140.0, 280.0, 420.0, 560.0 nL/mL
Experiment II / 24 hours treatment
without S9 mix: 4.4, 8.8, 17.5, 26.3, 35.0, 52.5, 70.0 nL/mL
Experiment II / 4 hours treatment:
with S9 mix: 17.5, 35.0, 70.0, 140.0, 280.0, 420.0, 560.0 nL/mL

Following the expression phase of 48 hours the cultures (printed in bold letters) at the two highest concentrations of 70 and 140 nL/mL in experiment I without metabolic activation and at 420 and 560 nL/mL in experiment II with metabolic activation were not continued due to exceedingly severe cytotoxic effects. In experiment I the cultures at 17.5 and 35 nL/mL with metabolic activation and at 4.4 and 8.8 nL/mL in experiment II without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.30 in the solvent control versus pH 7.39 at 4.5 µL/mL)
- Effects of osmolality: Not increased (380 in the solvent control versus 350 at 4.5 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: Main experiment: Phase separation occurred at 140 nL test item/mL and above in the presence of metabolic activation.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment.
The highest concentration should be 10 mM, but not higher than 5 mg/mL or 5 µL/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50% are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20%.
The dose calculation was based on the volume of the liquid test item rather than the weight to constantly keep the temperature of the test item below 5 °C. The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Test item concentrations between 0.035 µL/mL and 4.5 µL/mL (equal to approximately 10 mM) were used with respect to the molecular weight and the purity of the test item (70.5%, preliminary information at the start of the experiment).
Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 0.035 µL/mL and above without metabolic activation and at 0.56 µL/mL and above with metabolic activation following 4 hours treatment. Following 24 hours treatment without metabolic activation the RSG was reduced <50% at 23.4 µg/mL. After 24h treatment cytotoxic effects were noted at 0.035 µL/mL and above without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Phase separation was observed by the unaided eye at 0.140 µL/mL and above in the parts following 4 hours treatment. After 24 hours treatment phase separation occurred at the maximum concentration of 0.070 µg/mL.
Therefore, the maximum concentration of the main experiments was adjusted according to the toxicity and solubility data of the pre-experiment. To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations. The individual concentrations were spaced by a factor of 2. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 35.0 nL/mL and above without metabolic activation (both cultures) and at 420 nL/mL (culture I) and 70.0 nL/mL (culture II) with metabolic activation. In the second experiment cytotoxic effects as described were noted at 70.0 nL/mL without metabolic activation (both cultures) and at 140 nL/mL with metabolic activation (culture I). The recommended cytotoxic range of approximately 10-20% relative total growth was covered without metabolic activation. In the presence of metabolic activation the maximum concentration was limited by phase separation noted in the first and second experiment at 140 µg/mL and above.
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Y cells
Remarks:
Migrated from field 'Test system'.

Summary Table

      relative mutant   relative mutant  
  conc. nL S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment     culture I culture II
Solv. control with ethanol - 100.0  76 202 100.0 142 268
Pos. control with MMS 19.5 µg/mL -  44.6 261 202  23.0 611 268
Test item   2.2 -  81.0  80 202  69.3 106 268
Test item   4.4 -  84.1 109 202 118.2  87 268
Test item   8.8 - 103.5  99 202 101.2 100 268
Test item  17.5 -  84.1  53 202  60.9  76 268
Test item  35.0 -  6.0 132 202  14.5  51 268
Test item  70.0 - culture was not continued# culture was not continued#
Test item  140.0 - culture was not continued# culture was not continued#
       
Solv. control with ethanol + 100.0  68 194 100.0  58 184
Pos. control with CPA 3.0 µg/mL +  48.4 271 194  37.4 277 184
Pos. control with CPA 4.5 µg/mL  +   30.5 360 194  38.2 220 184
Test item  17.5  +  culture was not continued## culture was not continued##
Test item  35.0  +  culture was not continued## culture was not continued##
Test item  70.0  +   80.7 105 194  35.3 112 184
Test item 140 (p)  +   51.6  96 194  21.1  94 184
Test item 280 (p)  +   91.9  51 194  28.7  87 184
Test item 420 (p)  +   46.0 121 194  35.0 109 184
Test item 560 (p)  +   34.8 103 194  30.9 106 184
Experiment II / 24 h treatment     culture I culture II
Solv. control with ethanol - 100.0  83 209 100.0 100 226
Pos. control with MMS 13.0 µg/mL -  38.8 376 209  32.2 672 226
Test item   4.4 - culture was not continued## culture was not continued##
Test item   8.8 - culture was not continued## culture was not continued##
Test item  17.5 - 102.7  94 209 126.5  84 226
Test item  26.3 - 101.1  90 209 106.0 103 226
Test item  35.0 -  87.3  82 209 100.9 130 226
Test item  52.5 - 101.0  62 209 115.1 110 226
Test item  70.0 -  30.0  89 209  31.1 107 226
Experiment II / 4 h treatment     culture I culture II
Solv. control with ethanol + 100.0  86 212 100.0  67 193
Pos. control with CPA 3.0 µg/mL +  37.9 281 212  49.6 252 193
Pos. control with CPA 4.5 µg/mL +  19.4 495 212  47.7 262 193
Test item  17.5 +  63.4 120 212 111.6  73 193
Test item  35.0 +  84.7  86 212 114.8  88 193
Test item  70.0 +  51.4 111 212 112.8  76 193
Test item 140 (p) +  49.6 133 212 116.9  58 193
Test item 280 (p) +  49.8 112 212 100.3  51 193
Test item 420 (p) + culture was not continued### culture was not continued###
Test item 560 (p) + culture was not continued### culture was not continued###

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#    culture was not continued due to exceedingly severe cytotoxic effects
##
  culture was not continued since a minimum of only 4 analysable concentrations is required

###
 culture was not continued to avoid analysis of too many insoluble concentrations
p
    phase separation




Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS 51240-95-0) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours and in the presence of metabolic activation with a treatment period of 4 hours.

The experimental part of the second main experiment without metabolic activation was terminated prior to the generation of any data on mutagenicity since the cytotoxic range was not reached even at the highest concentrations. The dose range was adjusted and the experimental part was repeated under identical general experimental conditions. The data of the repeat experiment are included in experiment II.

The main experiments were evaluated at the following concentrations with and without metabolic activation:

Experiment I

without S9 mix:                                 2.2; 4.4; 8.8; 17.5; and 35.0 nL/mL
with S9 mix:                                  70.0; 140; 280.0; 420.0; 560.0 nL/mL

Experiment II

without S9 mix:                           17.5; 26.3; 35.0; 52.5; and 70.0 nL/mL
with S9 mix:                            17.5; 35.0; 70.0; 140.0; and 280.0 nL/mL

Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 35.0 nL/mL and above without metabolic activation (both cultures) and at 420 nL/mL (culture I) and 70.0 nL/mL (culture II) with metabolic activation. In the second experiment cytotoxic effects as described were noted at 70.0 nL/mL without metabolic activation (both cultures) and at 140 nL/mL with metabolic activation (culture I). The recommended cytotoxic range of approximately 10-20% relative total growth was covered without metabolic activation. In the presence of metabolic activation the maximum concentration was limited by phase separation noted in the first and second experiment at 140 µg/mL and above.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control in any of the experimental parts.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of the first experiment with metabolic activation.This trend however, was judged as irrelevant since it actually was reciprocal, going down versus increasing concentrations.

In this study the range of the solvent controls was from 58 up to 142 mutant colonies per 106cells; the range of the groups treated with the test item was from 51 up to 133 mutant colonies per 106cells.

The cloning efficiency exceeded the upper limit of 120% in the first culture of the first experiment with and without metabolic activation. The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range. Cloning efficiency values above 100% occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. The aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations. The positive control of the second culture of the second experiment with metabolic activation missed the acceptance criterion of at least 150 induced small colonies per 106cells by just 8 colonies per 106cells at 4.5 µg/mL (142 compared to 150). The data are judged as acceptable however, since the total colonies of the positive controls show a substantial increase compared to the solvent control with 252 and 262 colonies per 106cells compared to 67 colonies per 106cells.

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

Genetic toxicity in vivo

Description of key information

The substance is negative for mutagenicity in vitro in an Ames test and in a Mouse Lymphoma Assay. Therefore performing further in vivo mutagenicity studies with this substance is not required.

An in vivo study for cytogenicity is not available for the substance itself but weight of the evidence for similar substances, which are all negative in vivo, warrants the conclusion that this substance is also negative. Therefore performing further in vivo cytogenicity studies with this substance is scientifically unjustified. For further details see below.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic and germ cell study: gene mutation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 2003 - 23 February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: on the day of treatment, the animals were approximately 6 weeks old.
- Weight at study initiation: no data
- Assigned to test groups randomly: yes, under following basis: by sex
- Fasting period before study: no
- Housing: by groups in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum, A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge, France).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 14 January 2003 - 23 February 2003
Route of administration:
intraperitoneal
Vehicle:
The vehicle was corn oil, batch Nos. 81K2204 and 062k0006 (Sigma, Saint-Quentin-Fallavier, France).
Details on exposure:
Route for the vehicle and the test substance: at the request of the Sponsor, the intraperitoneal route was used,
• Volume: 10 mL/kg,
• CPA: oral route, one treatment
Frequency of treatment:
Two treatments separated by 24 hours
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 except for the high dose group where 8 animals were used
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA)
- Justification for choice of positive control(s): guideline recommendation
- Route of administration: Oral
- Doses / concentrations: Batch No. OD203A (Laboratoire Asta Médica, Mérignac, France) dissolved in distilled water at a concentration of 5 mg/mL. The preparation was stored at -20°C and thawed immediately before use.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. Except for piloerection noted in both males and females, no clinical signs were noted. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day. The two other selected dose-levels for the main test were 500 and 1000 mg/kg/day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 after the last treatment bone marrow samples were taken

DETAILS OF SLIDE PREPARATION: At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS: For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford.
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell et al., 1989) (d), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to
determine the χ2 value (Lovell et al., 1989) (d). When there was significant within-group heterogeneity, then that group was compared with the
control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969) (e). The student "t" test was used for the PE/NE ratio comparison. Probability values of p ≤ 0.05 was considered as significant.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No clinical signs and no mortality were observed in the animals of both sexes given 500 and 1000 mg/kg/day. Piloerection was noted in both males and females at 2000 mg/kg/day.
For both males and females, the mean values of MPE in the groups treated with the test item, were equivalent to those of the vehicle control group.
The PE/NE ratio was significantly (p < 0.001) lower when compared to that of the vehicle control group, in the males treated at 2000 mg/kg/day, showing that the bone marrow cells were effectively exposed to the test item.
Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.
Conclusions:
Interpretation of results: Negative
Under our experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.
Executive summary:

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female mice received two intraperitoneal treatments at dose-levels of 500, 1000 or 2000 mg/kg/day, at a 24-hour interval.

One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day.

The two other selected dose-levels for the main test were 500 and 1000 mg/kg/day. For both males and females, the mean values of MPE in the groups treated with the test item, were equivalent to those of the vehicle control group. The PE/NE ratio was significantly lower when compared to that of the vehicle control group, in the males treated at 2000 mg/kg/day, showing that the bone marrow cells were effectively exposed to the test item.

Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under our experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells

after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
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.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The relative humidity recorded in the animal room was sometimes outside the target range specified in the protocol. This minor deviation was not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Number: . 12 male and 12 female mice for the preliminary toxicity test
56 mice: 28 males and 28 females for the cytogenetic study.
Strain: Swiss leo: OFI (lOPS Caw).
Reason for this choice: rodent species generally accepted by regulatory authorities for this type
of study.
Breeder: Iffa Credo, I'Arbresle, France.
Age: on the day of treatment, the animals were approximately 6 weeks old.
Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination
to ensure that they were in good clinical conditions.
Acclimatisation: at least 5 days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex'
Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.

Environmental conditions
Upon their arrival at CIT, the animals were housed in an animal room, with the following
. environmental conditions:
· temperature: 21 ± 2°C,
· relative humidity: 30 to 70%,
· light/dark cycle: 12 hl12 h (07:00 - 19:00),
· ventilation: about 12 cycles/hour of filtered non-recycled fresh air.
The housing conditions (temperature, relative humidity, light/dark cycle and ventilation) were
checked regularly. .
The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved
sawdust (SICSA, 94142 Alfortville, France).
Bacteriological and chemical analysis of the sawdust, including the detection of possible
contaminants (pesticides, heavy metals) of the sawdust are performed by the suppliers.

Food and water
All animals had free access to A04 C pelleted maintenance diet (UAR, 91360 ViIlemoisson-surOrge,
France) and tap water (filtered using a 0.22 micron filter) ..
Each batch of food was analysed (composition and contaminants) by the supplier.
Bacteriological and chemical analysis of water, including the detection of possible contaminants
(pesticides, heavy metals and nitrosarnines) are performed regularly by extemallaboratories. The
results of these analyses are archived at CIT.
No contaminants are known to be present in the diet, drinking water or bedding material at
levels which may be expected to interfere with or prejudice the outcome of the study.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Administration
· Route for the vehicle and the test substance: oral, since it is a possible route of exposure in man
· Frequency: two treatments separated by 24 hours,
· Volume: 10 m/kg,
· CPA: oral route, one treatment.
The quantity of each substance administered to each animal was adjusted according to the most
recently recorded body weight.
Frequency of treatment:
Two treatments separated by 24 hours
Post exposure period:
The animals of the treated and vehicle control groups were killed 24 hours after the last
treatment and the animals of the positive control group were killed 24 hours after the single
treatment.
Remarks:
Doses / Concentrations:
62.5, 125 or 250 mg/kg/day (expressed as active material), at a 24-hour interval
Basis:
other: percent active
No. of animals per sex per dose:
In the main study, three groups of five male and five female mice.
Control animals:
yes, concurrent vehicle
Positive control(s):
One group of five males and five females received the positive control test substance
(cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
Tissues and cell types examined:
Bone marrow smears were then prepared. For each animal, the number of the micronuc1eated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE)
erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Details of tissue and slide preparation:
At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the 'animals were removed and the bone marrow was eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. All the slides were coded for scoring:

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochrornatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell and colI., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the x2 value (Lovell and colI., 1989). When there was Significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969).
The student "t" test was used for the PE/NE ratio comparison. Probability values of p < 0.05 was considered as significant.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In order to select the top dose-level for the cytogenetic study, 2000, 1000, 500 and 250 mg/kg/day were administered, to three males and three females. The interval between each administration was 24 hours.
At 2000 and 1000 mg/kg/day, all animals were found dead 24 hours after the first treatment. At 500 mglkg/day, hypoactivity and/or piloerection were noted in 113 males and 1/3 females, 2 and 6 hours after the second treatment. These two animals were found dead 24 hours after the second treatment.
At 250 mglkg/day, piloerection was observed in all animals 6 hours after the second treatment and persisted 18 hours later. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since observable toxic effects were noted, the top dose-level was based on the toxicity level, such that a higher dose-level was expected to induce lethality. . .
Consequently, 250 mg/kg/day was selected as the top dose-level. The two other dose-levels were 125 and 62.5 mg/kg/day.

No clinical signs and no mortality were observed in the animals of both sexes given 125 and 62.5 mg/kg/day. At 250 mg/kg/day, piloerection was noted in 3/5 males as well as in 1/5 females from the main group. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test substance, were equivalent to those of the vehicle group. The mean values of MPE as wen as the PE/NE ratio for the vehicle control groups were consistent with our historical data. For one female from the positive control group (corresponding to slide 26) no significant increase in the frequency of MPE was noted. However since for the remaining nine animals cyclophosphamide induced a clear increase in the frequency of MPE and since a very significant increase (p < 0.001) in the mean frequency was noted, this was considered as indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under our experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).

Conclusions:
Interpretation of results: Negative
Under our experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hourinterval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).
Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss leo: OFI (lOPS Caw) mice received two oral treatments of the substance at dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material), at a 24-hour interval. One group of five males and five females received the vehicle (com oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test substance (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since observable toxic effects were noted in the preliminary test, the top dose-level was based on the toxicity level, such that a higher dose-level was expected to induce lethality. Consequently, 250 mg/kg/day was selected as the top dose-level. The two other dose-levels were 125 and 62.5 mg/kg/day. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test substance, were equivalent to those of the vehicle group. The mean values of MPE as well as the PE/NE ratio for the vehicle control groups were consistent with our historical data. For one female from the positive control group, no significant increase in the frequency of MPE was noted. However since for the remaining nine animals cyclophosphamide induced a clear increase in the frequency of MPE and since a very significant increase (p <0.001) in the mean frequency was noted, this was· considered as indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under our experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-12-03 to 2003-01-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Directive 2000/32/EC
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian vivo Micronucleus Test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: male: 32.4 g; female: 26.3 g
- Fasting period before study: no
- Housing: Group of five animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material. Paper bedding was provided as nest material.
- Diet: Free access to standard pelleted laboratory animal diet.
- Water: Free access to tap-water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2°C
- Humidity: 30-70 %
- Air changes: 15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light and 12 hours dark per day

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
The mice received a single intraperitoneal injection of a tolerated (high), an intermediate and a low dose of the test item.
Duration of treatment / exposure:
Single treatment
Frequency of treatment:
Single treatment
Post exposure period:
The animals were sacrificed by cervical dislocation 24 or 48 hours after dosing.
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
No. of animals per sex per dose:
5 mice
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide dissolved in physiological saline
- Route of administration: single intraperitoneal injectin
- Doses / concentrations: 50 mg salt/kg bw
Tissues and cell types examined:
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a dose range finding study 14 animals were dosed intraperitoneally with 2000, 1500 and 1000 mg/kg bw.

TREATMENT AND SAMPLING TIMES:
The mice received a single intraperitoneal injection of a tolerated (high), an intermediate and a low dose of the test item. The animals were sacrificed by cervical dislocation 24 or 48 hours after dosing.

DETAILS OF SLIDE PREPARATION:
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with serum by aspiration with the remaining serum. Two slides were prepared per animal.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. At frist the slids were screened at a magnification of 100 x for regions of suitable technical quality. Slides were scored at magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:
Equivocal results should be clasified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups seperately.
A test subsatnce is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups seperately.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
Wilcoxon Rank Sum Test, two-sided test at p< 0.05
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a dose range finding study 14 animals were dosed intraperitoneally with 2000, 1500 and 1000 mg/kg bw. Based on the results of this dose range finding study dose levels of 1000, 500 and 250 mg/kg bw were selected as appropriate doses for the Micronucleus Test.

RESULTS OF DEFINITIVE STUDY
- Mortality and systemic toxic signs:
The animals of the groups treated with 500 and 250 mg test item/kg bw and the animals of the negative and positive control groups showed no abnormalities.
During the first hour after dosing seven (3 male and 4 female) animals of the groups treated with 1000 mg/kg bw were lethargic. All other animals showed no reaction to treatment. Within 18 hours after dosing all animals of the groups treated with 1000 mg/kg bw were lethargic and had a hunched posture, one animal also had a rough coat. Within 42 hours after dosing four (2 male and 2 female) animals had a rough coat and a hunched posture, all animals recovered from the treatment.
-Micronucleated polychromatic erythrocytes:
The mean number of micronucleated polychromatic erythrocytes scored in the test item treated groups were compared with the correponding solvent control group.
No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item.
- Ratio polychromatic to normochromatic erythrocytes:
The animals of the groups, which were, treated with the test item and the negative control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphaminde showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.
Conclusions:
It is concluded that tert-butyl peroxyneodecanoate is not mutagenic in the micronucleus test under the experimental conditions.
Executive summary:

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erthrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 1000 mg/kg bw, one group was dosed with 500 mg/kg bw and one group was dosed with 250 mg/kg bw. After dosing the animals of the dose level of 1000 mg/kg bw showed the following toxic signs: lethary, rough coat and a hunched posture. The animals of the dose levels of 500 and 250 mg/kg bw showed no abnormalities after dosing.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide at 50 mg/kg bw served as positive control.

Bone marrow of the groups treated with the test item was sampled 24 or 48 hours after dosing. Bone marrow from the negtive control group was harvested at 24 hours after dosing only and bone marrow from the positive control group harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a staistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item.

The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic erythrocytes compared to the vehicle controls.

Based on the clinical signs observed at the highest dose (lethargy, hunched posture and rough coat) it is assumed that the substance is taken up in the body and is systhemically available.

It is therefore concluded that this test is valid and that tert-butyl peroxyneodecanoate is not mutagenic in the micronucleus test under the experimental conditions.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-12-21 and 2002-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Qualifier:
according to
Guideline:
other: ICH Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Drawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 27.6 - 31.5 g (male), 23.5 - 28.1 g (female)
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: certified laboratory rodent chow (Harlan TEKLAD certified Rodent 7012C); ad libitum
- Water: tap water (Washington Suburban Sanitary Commission, Potomac Plant); ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle: corn oil
- Justification for choice of solvent/vehicle: based on a solubility determination of the test article and compatibility of the vehicle with the test system
Details on exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered at a dose volume of 20 mL/kg bw.
Frequency of treatment:
single intraperitoneal injection
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg)
Tissues and cell types examined:
- Bone marrow cells were examined microscopically for micronucleated polychromatic erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection based on a pilot toxicity study followed by a toxicity study.

TREATMENT AND SAMPLING TIMES: Bone marrow cells were collected after 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenmald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Bone marrow cells, polychromatic and normochromatic erythrocytes were analyzed for the presence of micronuclei. For analysis slides were coded using a random number table. Using medium magnification (10x40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10x100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total reythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:
The test article was considered to induce positive results if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay is recommended. The test article is considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a valid test - mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the positive control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group.
Statistics:
According to Kasten-Bowman Tables
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no toxicity was observed up to 1200 mg/kg which is the highest dose used in the micronucleus study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Pilot Toxicity Study: Mortality and clinical signs included prostation were observed in males and females at the highest dose of 2000 mg/kg. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg and in males and females at 2000 mg/kg. Ataxia, paralysis and irregular brathing were noted in males and females at 2000 mg/kg.In addition, pilorection was observed in males at 10 mg/kg.

Toxicity Study: Mortality was observed at concentrations of 1400 mg/kg and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg.

Definitive study - no mortality was observed.
Clinical signs including lethargy and piloerection were noted in 300, 600, and 1200 mg/kg dose groups. Slight reductions (up to 4%) in the ratio of polychromatic to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. No significant increase in micronucleated polychromatic erythrocytes (PCEs) were observed in any test substance treated group (0.7 or less per 1000 PCEs). A significant increase (p<0.05) in micronucleated polychromatic erythrocytes was observed in the positive control group (~ 20 per 1000 PCEs). The validity criteria were met.
Conclusions:
Interpretation of results: Negative
The test substance (50% active ingredient) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Hence, the test substance was concluded to be negative in the micronucleus in vivo test.
Executive summary:

The test article (50% active ingredient) was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess toxicity of the test article and set dose levels for the definitive study consisted of a pilot study followed by a toxicity study. The second phase, the definitive micronucleus study, was designed to evaluate the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. In both phases of the study, test and control articles were administered at a dose volume of 20 mL/kg body weight by a single intraperitoneal injection.

The test article was soluble in corn oil at 200 mg/mL, the maximum concnetration tested in the solubility test. All test article dosing formulations were delivered to the test system as light yellow solutions.

Cyclophosphamide monhydrate (CP) was used as the positive control article at a dose of 50 mg/kg bw.

In the pilot toxicity study, male mice (2/group) were dosed with 1, 10, 100 or 1000 mg test article/kg body weight and male and female mice (5/sex/group) were dosed with 2000 mg test article/kg body weight. Mortality was observed in 4/5 males and 5/5 females at 2000 mg/kg bw. Clinical signs immediately following dose administration included: prostration in males and females at 2000 mg/kg bw. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg bw and in males and females at 2000 mg/kg bw. Ataxia, paralysis and irregular breathing were noted in males and females at 2000 mg/kg bw. In addition, pilorection was observed in males at 10 mg/kg bw.

In the toxicity assay, male and female mice (5/sex/group) were dosed with 1200, 1400, 1600 or 1800 mg test article/kg body weight. Mortality was observed at concentrations of 1400 mg/kg bw and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg bw.

In the definitive micronucleus study, male and female mice (5/sex/group) were dosed with 300, 600 or 1200 mg test article/kg body weight, vehicle or positive control article. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy and pilorection in males and females at 300, 600 and 1200 mg/kg bw. Bone marrow cells were collected 24 and 48 hours after treatment. Slight reductions (up to 4%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. These reductions suggest that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman).

The validity critria for the positive control (Cyclophosphamide monohydrate) and the negative control (corn oil) were met.

It can be concluded that the test substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. The test substance was negative in the mouse micronucleus assay.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-05-03 until 2000-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP compliance
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Swiss Ico: OF1 (IOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Crédo, l'Arbresle, France.
- Age at study initiation: approximately 6 weeks old
- Assigned to test groups randomly: yes, under following basis: upon arrival, the animals were randomly allocated to the groups by sex.
- Housing: in polycarbonate cages
- Diet: A04 C pelleted maintenance diet (UAR, 91360 Villemoisson-sur-Orge, France)
- Water: tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ±2°C
- Humidity: 30 to 70 %
- Air changes: about 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod: 12 h/12 h (07:00 - 19:00)
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in the vehicle (corn oil) in order to achieve the concentrations, expressed as active material, of 50, 100 or 200 mg/mL (corresponding to 66.31, 132.63 or 265.25 mg/mL of the test substance as supplied respectively) and then homogenized using a magnetic stirrer. The preparations were made immediately before use.
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
two treatments separated by 24 hours
Post exposure period:
None
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPA), dissolved in distilled water at a concentration of 5 mg/mL.
Tissues and cell types examined:
erythrocytes (evaluation of micronucleated polychromatic erythrocytes) in bone marrow of male and female mice
Details of tissue and slide preparation:
- At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. All the slides were coded for scoring

- For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocytes ratio was established by scoring a total of 1000 erythrocytes (PE + NE)
Evaluation criteria:
must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value, the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the %2 value.
When there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test. The student "t" test was used for the PE/NE ratio comparison. Probability values of p < 0.05 was considered as significant.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At 2000 mg/kg bw/day, piloerection and/or hypoactivity were noted in almost all animals 2 and 24 hours after the last treatment. One female from the supplementary group was found dead 24 hours after the last treatment.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg bw/day were administered, to three males and three females. The interval between each administration was 24 hours. Hypoactivity was noted in all animals 2 and 6 hours following the second treatment. Piloerection was also observed in 2/3 males 2 hours after the second treatment and persisted until sacrifice. In addition, half-closed eyes were noted in all animals 6 hours following the second treatment and persisted in 2/3 males and 1/3 females 18 hours later.
Since no mortality was noted in the preliminary test performed at 2000 mg/kg bw/day therefore this dose-level was selected as the highest dose-level for treatment.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: the mean values of MPE in the groups treated with the test substance, were equivalent to those of the vehicle group
- Ratio of PCE/NCE: the PE/NE ratio was significantly (p < 0.05) lower when compared to that of the vehicle group, in the groups given 2000 mg/kg bw/day (for both males and females) or 1000 mg/kg bw/day (for females only), showing that the bone marrow cells were effectively exposed to the test substance

- Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

- The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Conclusions:
Interpretation of results: Negative
Under the study´s experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg bw/day.
Executive summary:

In this study the test substance was examined for its potential to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice according to OECD guideline No. 474 and EU Method B.12.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received two oral treatments of the test substance at dose-levels of 500,1000 or 2000 mg/kg bw/day, at a 24-hour interval. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test substance (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg bw.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

For both males and females, the mean values of MPE in the groups treated with the test substance, were equivalent to those of the vehicle group. The PE/NE ratio was significantly (p < 0.05) lower when compared to that of the vehicle group, in the groups given 2000 mg/kg bw/day (for both males and females) or 1000 mg/kg bw/day (for females only), showing that the bone marrow cells were effectively exposed to the test substance. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under the experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 500,1000 or 2000 mg/kg bw/day.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non GLP; Both doses by far exceeded the max. doses recommended by current OECD Guideline (474)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Both doses by far exceeded the max. doses recommended by current OECD Guideline (474)
Principles of method if other than guideline:
NA
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Males: 27 - 35 g; Females: 24 - 29.9 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): ad libitum, TEKLAD Certified Rodent Chow 7012C
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23°C
- Humidity (%): 50 ± 20°C

Route of administration:
oral: gavage
Vehicle:
Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at a concentration of 500 mg/mL, the maximum concentration tested.
Details on exposure:
NA
Duration of treatment / exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral gavage at a constant volume of 20 mL/kg bw. Mice were observed after dose administration for clinical signs of chemical effect.
Frequency of treatment:
24, 48, 72 hrs after dose administration
Post exposure period:
None
Remarks:
Doses / Concentrations:
20 mL/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP) will be administrated as the positive control at a dose of 60 mg/kg. CP will be administrated by the same route as the test article.
Tissues and cell types examined:
The micronucleus study evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.
Details of tissue and slide preparation:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1979; Mackey and Mac Gregor, 1979)
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration (p>0.05, Kastenbaum-Bowman).

No remarks

Conclusions:
Interpretation of results: Negative
Under the conditions of the assay described in this report, tert.-Butylperoxy- 2-ethylhexanoat did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The test article, tert.-Butylperoxy- 2-ethylhexanoat, was tested in the mouse micronucleus assay. In the study, test and control articles were administered in a constant volume of 20 mL/kg bw by a single oral gavage.

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at a concentration of 500 mg/mL, the maximum concentration tested.

In the pilot assay (toxicity test), male mice were dosed with 1, 10, 100 or 1000 mg test article/kg bw and male and female mice were dosed with 5000 mg/kg bw. Mortality occurred an 1/5 males and 3/5 females at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in male mice at 1000 mg/kg and in male and female mice at 5000 mg/kg, prostration and crusty eyes in one male mouse and diarrhoea in one female mouse at 5000 mg/kg. Treatment-related mortality was observed to be less than 50 % in the pilot toxicity study. However, in females the mortality was estimated to be 60% at 5000 mg/kg. Therefore, the high dose in the micronucleus study for males was set at 5000 mg/kg. For females the high dose was set at 4000 mg/kg which was estimated to be approximately 80 % of 5000 mg/kg. Both doses by far exceeded the maximum doses recommended by current OECD guideline.

In the micronucleus assay, male mice were dosed with 1250, 2500 or 5000 mg/kg and female with 1000, 2000 or 4000 mg/kg bw of tert.-Butylperoxy- 2-ethylhexanoat. Mortality occurred in 1 /20 male mice at 5000 mg/kg and 2 /25 female mice at 4000 mg/kg. Clinical signs following dose administration included: lethargy in male and female mice at all dose levels, diarrhoea in male mice at 2500 and 5000 mg/kg and in female mice at 2000 and 4000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Moderate reductions (up to 36 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the vehicle control groups. A reduction of 36 % suggests bioavailability of the test article to the target organ, the bone marrow.

Under the conditions of the assay described in this report, tert.-Butylperoxy- 2-ethylhexanoat did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow even at pronounced systemically toxic doses and was concluded to be negative in the micronucleus test using male and female ICR mice.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well executed and reported study subjected to peer review and conducted according to modern standards, including GLP.
Principles of method if other than guideline:
Smears were prepared from peripheral blood samples obtained by cardiac puncture from dosed and control mice exposed to t-BP for 13-weeks [via gavage] at the time of terminal kill. Slides were stained with Hoechst 333258/pyronin Y (MacGregor et al., 1983). At least 10000 NCE and 2000 PCE from each animal were scored for micronuclei.
GLP compliance:
yes
Remarks:
The t-BP studies were performed in compliance with FDA Good Laboratory Practices regulations (21 CFR 58). The Quality Assurance Unit of Battelle Columbus Laboratories performed audits and inspections of protocols, procedures, data, and reports throughout
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
B6C3F1 mice used in the 13-week study were produced under strict barrier conditions at Simonsen Laboratories, Inc. (Gilroy, CA). Animals were progeny of defined, microflora-associated parents that were transferred from isolators to barrier-maintained rooms. Rats and mice were shipped to the study laboratory at 4 to 5 weeks of age, quarantined there for 11 days, and placed on study at approximately 6 weeks of age. Blood samples were collected and the sera analyzed for viral titers from 5 animals per sex at study start and termination in the 13-week studies. Data from 12 viral screens performed in mice showed that there were no positive antibody titers (Boorman et al., 1986; Rao et al., 1989). Diet: NIH 07 pelleted feed and water, ad libitum. Animal Room Environment: Temp: 68-75°F; relative humidity: 35-65%; fluorescent light 12 h/d; 12-15 room air changes/h. Time Held Before Study: 11 d. Age When Placed on Study: 6 wks
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
t-butyl perbenzoate: 0, 30, 60, 125, 250, 500 mg t-butyl perbenzoate per kg body weight in deionized water by gavage.

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 30, 60, 125, 250, 500 mg t-butyl perbenzoate per kg body weight
Basis:
other: as administered via gavage
No. of animals per sex per dose:
10 males; 10 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
None reported in source document.
Tissues and cell types examined:
Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the time of terminal kill. At least 10000 NCE and 2000 PCE from each animal were scored for micronuclei.
Details of tissue and slide preparation:
Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the time of terminal kill. At least 10000 NCE and 2000 PCE from each animal were scored for micronuclei.
Evaluation criteria:
Cochran-Armitage linear regression of proportions for PCE's or linear contrasts from Analysis of Variance for NCE's.
Statistics:
Cochran-Armitage linear regression of proportions for PCE's or linear contrasts from Analysis of Variance for NCE's.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Additional information on results:
Following 13-weeks of gavage exposure to t-Butyl Perbenzoate, via gavage, no significant elevation in the frequency of micronucleated erythrocytes was observed in peripheral blood samples taken from either male or female mice via cardiac puncture.

See p B-7 of source report.

Conclusions:
Interpretation of results: Negative
Following 13-weeks of gavage exposure to t-Butyl Perbenzoate, via gavage, no significant elevation in the frequency of micronucleated erythrocytes was observed in peripheral blood samples taken from either male or female mice via cardiac puncture.
Executive summary:

Following 13-weeks of gavage exposure to t-Butyl Perbenzoate, via gavage, no significant elevation in the frequency of micronucleated erythrocytes was observed in peripheral blood samples taken from either male or female mice via cardiac puncture.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: GLP, OECD study 474 (July 1997)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Crédo, I'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: individually in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum A04 C pelleted maintenance diet (SSNIFF Spezialdiät GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum drinking water filtered by a 0.22µ membrane
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 30 to 70
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Volume: 10 ml/kg
Duration of treatment / exposure:
2 administrations separated by 24 hours.
Frequency of treatment:
once daily for 2 days
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): commomly used in this test and recommanded by the OECD guideline
- Route of administration: oral
- Frequency: one administration only
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Femurs of the animals were removed and all the bone marrow was flushed out using fetal calf serum.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square
test value (Lovell and coll., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the x2 value (Lovell and coll., 1989).
When there was significant within-group heterogeneity, then that group was compared with the
control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969). The student 11t11 test was used for the PEINE ratio comparison.
Probability values of p0.05 was considered as significant.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
systemic toxicity
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (6 animals)
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. Hypoactivity and/or dyspnea and sometimes staggering gait were noted in all animals during at least the 4 hours following treatment. Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were killed by C02 inhalation in excess. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no mortality was noted, the top dose-level selected for the main test was 2000 mg/kg/day.
Conclusions:
Interpretation of results: Negative
In conclusion, t-amyl 2-Ethylhexaneperoxoate did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg.
Executive summary:

T-amyl 2-Ethylhexaneperoxoate was tested in a Mammalian Erythrocyte Micronucleus Test, according to the OECD n° 474 Guideline and EC 92/69/EEC B.12 guidelines in compliance with the Principles of Good Laboratory Practice.

In a bone marrow micronucleus assay, groups of 5 males and 5 females Swiss Ico:OF1 (IOPS Caw) mouse were treated by gavage with t-amyl 2-Ethylhexaneperoxoate (purity 96.5%) at doses of 0, 500, 1000, and 2000 mg/kg. 

One group of 5 males and 5 females received the vehicle under the same experimental conditions, and acted as control group. One group of 5 males and 5 females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

Bone marrow cells were harvested at 24 and/or 48 hours post-treatment. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

At 2000 mg/kg/day, dyspnea, hypoactivity, sedation, staggering gait and/or pi1oerection were noted in both males and females. At 24 hours following the second treatment only piloerection persisted in males.

The mean values of MPE as well as the PE/NE ratio in the groups treated with the test item were considered as equivalent to those of the vehicle control group.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.

Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under these experimental conditions, t-amyl 2-Ethylhexaneperoxoate did not induce any noteworthy increase in the number of micronucleated with structural chromosome aberration, both with and without S9 mix, in any experiment. 

In conclusion, t-amyl 2 -ethylhexaneperoxoated did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP; OECD guideline was followed
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
June 1989
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Spargue Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Pilot study: 28.2 - 30.6 g at randomization (males), 26.0 - 28.3 g at randomization (females); Toxicity study: 28.4 - 34.6 g at randomization (males), 25.5 - 29.4 g at randomization (females); Supplemental toxicity study: 29.0 - 35.3 g at randomization (males), 25.8 - 30.7 g at randomization (females); Micronucleus assay: 28.0 - 34.4 g at randomization (males), 26.0 - 29.3 g at randomization (females)
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material.
- Diet: free access to rodent chow
- Water: free access to tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C
- Humidity: 50 +/- 20 %
- Photoperiod: 12 hour light/dark cycle

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
none
Remarks:
Doses / Concentrations:
190, 380, 760 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
- Vehicle control and the low test dose: 15 animals
- High test dose: 20 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Doses / concentrations: 60 mg/kg
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study and a supplemental toxicity study.

TREATMENT AND SAMPLING TIMES: Test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examinedmicroscopically for micronucleated polychromatic erythrocytes.

DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to acapped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Scoring for micronuclei with microscope.
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<=0.05, Kastenbaum-Bowman Tables).
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed seperately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or uncofirmed positive and a repeat assay recommended. The test item was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000, 5000 mg test item/kg (total volume of 20 mL test ite/vehicle miture/kg bw
- Solubility: test item is insoluble in water; test item was soluble in corn oil at a maximum concentration of 500 mg/mL
- Clinical signs of toxicity in test animals: Mortality occurred within three days of dose administration as follows: 5/5 males and 5/5 females at 5000 mg/kg and 1/2 males at 1000 mg/kg. Clinical signs, which were noted within four hours or later after dose administration, included lethargy in male mice at 100 and 1000 mg/kg. All other animals appeared normal throughout the observation period.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The number of micronucleated polchromatic erythrocytes per 1000 polychromatic erythrocytes in test item-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of the dose level or bone marrow collection time.
- Ratio of PCE/NCE: Reductions of up to 28 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in test item-treated mice relative to their respective vehicle controls.
Conclusions:
Interpretation of results: Negative
Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

Tert-butyl peroxypivalate was tested in mouse micronucleus assay according OECD guideline no. 474 and EU method B.12. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study and a supplemental toxicity study. The second phase, the micronucleus study, evaluated the potential of the test item to increase the incidene of micronucleated polychromatic erythocytes in bone marrow of male and female mice. In both phases of the study, test and control items were administered in a constant volume of 20 mL/kg body weight by single intraperitoneal injection.

In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test item/kg body weight and male and female mice were dosed with 5000 mg/kg. Mortality was observed in 1/2 male mice at 1000 mg/kg and 5/5 male mice and 5/5 female mice at 5000 mg/kg. Clinical signs following dose administration included lethary in male mice at 100, 1000 mg/kg.

In the toxicity assay, male and female mice were dosed with 400, 800, 1200, or 2000 mg test item/kg body weight. Mortality was observed in 5/5 male mice and 5/5 female mice at 1200 and 2000 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 400 and 800 mg/kg and prostration in male and female mice at 1200 and 2000 mg/kg. Due to the lack of a test item dose level with an intermediate level of toxicity, a supplemental toxicity assay was performed.

In the supplemental toxicity assay, male and female mice were dosed with 900, or 1100 mg test item/kg body weight. mortality was observed in 2/5 male mice and 1/5 female mice at 900 mg/kg and 5/5 male mice and 5/5 female mice at 1100 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 900 and 1100 mg/kg and prostration in male mice at 900 mg/kg and in mice at 1100 mg/kg. The high dose for the micronucleus test was set at 760 mg/kg which was estimated to be approximately 80% of the LD50/3.

In the micronucleus assay, male and female mice were dosed with 190, 380 or 760 mg/kg body weight of tert-butyl peroxypivalate. Mortality was observed in 3/20 male and 1/20 female mice receiving 760 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at all test item dose levels. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examinedmicroscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 28 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some ofte test item-treated groups relative to the respective vehicle controls.

No significant increase in micronucleated polychromatic erythrocytes in test item-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration.

Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.

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

Additional information

Summary peroxyester CA in vitro and in vivo data:

Peroxyester

tert-butyl peroxy benzoate

tert-butyl peroxy-3,5,5-trimethyl-hexanoate

tert-butyl peroxy-2-ethyl hexanoate

tert-amyl peroxy-2-ethyl hexanoate

CAS

614-45-9

13122-18-4

3006-82-4

686-31-7

Smiles

CC(C)(C)OOC(=O)c1ccccc1

CC(CC(=O)OOC(C)(C)C)CC(C)(C)C

CCCCC(CC)C(=O)OOC(C)(C)C

CCCCC(CC)C(=O)OOC(C)(C)CC

Molecular formula

C11H14O3

C13H26O3

C12H24O3

C13H26O3

CA in vitro

Positive

Positive

w

w

CA in vivo

Negative

Negative

Negative

Negative

 

 

Peroxyester

tert-butyl peroxyneo decanoate

tert-butyl peroxy pivalate

tert-butyl peroxy diethylacetate

tert-amyl peroxyneo decanoate

CAS

26748-41-4

927-07-1

2550-33-6

68299-16-1

Smiles

CCC(C)(CCC(C)C)C(=O)OOC(C)(C)C

CC(C)(C)OOC(=O)C(C)(C)C

CCC(CC)C(=O)OOC(C)(C)C

CCC(C)(C)OOC(=O)CCCCCC(C)(C)C

Molecular formula

C14H28O3

C9H18O3

C10H20O3

C15H30O3

CA in vitro

w

w

w

w

CA in vivo

Negative

Negative

Negative*

Negative*

 

 

Peroxyester

tert-amyl peroxy pivalate

2,5-dimethyl-2,5-di(2-ethylhexanoyl peroxy)hexane

tert-butyl peroxy isobutyrate

tert-butyl peroxy acetate

CAS

29240-17-3

13052-09-0

109-13-7

107-71-1

Smiles

CCC(C)(C)OOC(=O)C(C)(C)C

CCCCC(CC)C(=O)OOC(C)(C)CCC(C)(C)OOC(=O)C(CC)CCCC

CC(C)C(=O)OOC(C)(C)C

CC(=O)OOC(C)(C)C

Molecular formula

C10H20O3

C24H46O6

C8H16O3

C6H12O3

CA in vitro

w

w

positive

w

CA in vivo

Negative

Negative

Negative

Negative

  w = waived due to availability in vivo data

* = robust study summary not available

 

QSAR results:

CAS Number

All peroxyesters

SMILES

All peroxyesters

Molecular Formula

All peroxyesters

Predefined substance type

Mono constituent

CAS Smiles relation

High

DNA alerts for AMES by OASIS

No alert found

DNA alerts for CA and MNT by OASIS

No alert found

DNA binding by OASIS

Radical;Radical >> Radical mechanism by ROS formation (indirect) or direct radical attack on DNA;Radical >> Radical mechanism by ROS formation (indirect) or direct radical attack on DNA >> Organic Peroxy Compounds

DNA binding by OECD

No alert found

Protein binding alerts for Chromosomal aberration by OASIS

No alert found

Protein binding by OASIS

Radical reactions;Radical reactions >> Free radical formation;Radical reactions >> Free radical formation >> Organic peroxy compounds

in vitro mutagenicity (Ames test) alerts by ISS

No alert found

in vivo mutagenicity (Micronucleus) alerts by ISS

H-acceptor-path3-H-acceptor

Mutagenicity (Ames test) model (CAESAR) 2.1.13 ) VEGA

NON-Mutagenic

Mutagenicity (Ames test) model (SarPy/IRFMN) 1.0.7 VEGA

NON-Mutagenic

Mutagenicity (Ames test) model (ISS) 1.0.2 VEGA

NON-Mutagenic (outside domain model)

Mutagenicity (Ames test) model (KNN/Read-Across) 1.0.0 VEGA

NON-Mutagenic (could be outside domain model)

Justification for classification or non-classification

Available data shows a negative outcome for genotoxicity based on these results, the data is conclusive but not sufficient for classification.