Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro: Positivefor mutation, with and without metabolic activation: Chromosomal Aberrations (CHO); Mouse Lymphoma Forward Mutation Assay.Mixed results: S. typhimurium TA100, 1537 and TA98 positive in one assay, with and without S9; negative in another assay, with and without S9; SCE (CHO) - positive without S9 in two assays, positive with S9 in one/negative in another Negative Results:S. typhimurium TA1538 and TA 1535, with and without activation.;

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March, 1985 - July, 1988
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:
A detailed presentation of the technique for detecting chromosomal aberrations is found in Galloway et al. (1985, 1987).
GLP compliance:
yes
Remarks:
NTP follows GLP as a matter of policy.
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from Arochlor 1254 induced SD rats
Test concentrations with justification for top dose:
0.0, 5.0, 10.0 ug/ml
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
Study performed at Environmental Health Research & Testing. A detailed presentation of the technique for detecting chromosomal aberrations is found in Galloway et al. (1985, 1987). Briefly, Chinese hamster ovary cells were incubated with study compound or solvent (dimethylsulfoxide). Cells were arrested in first metaphase by addition of colcemid and harvested by mitotic shake off, fixed, and stainedin 6% Giemsa.

In the absence of S9, cells were incubated with study compound or solvent for 8-10 h at 37° C. Cells were then washed and fresh medium containing colcemid was added for an additional 2-3 h followed by harvest.

In the presence of S9, cells were incubated with study compound or solvent for 2 h at 37°C. Cells were then washed, medium was added, and incubation was continued for 8-10 h. Colcemid was added for the last 2-3 h of incubation before harvest. S9 was from the livers of Aroclor 1254-induced male Sprague Dawley rats.

Because of significant chemical-induced cell cycle delay, incubation time prior to addition of colcemid was lengthened to provide sufficient metaphases at harvest.
Evaluation criteria:
Not stated in source report.
Statistics:
Not stated in source report.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
See pp B-5 & B-6 in source report.

See pp B-5 & B-6 in source report.

Conclusions:
t-BP-induced chromosomal aberrations in Chinese hamster ovary cells in vitro, with and without S9.
Executive summary:

t-BP-induced chromosomal aberrations in Chinese hamster ovary cells in vitro, with and without S9.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
No GLP statement or indication of intent to follow GLP. Test substance identification does not indicate purity, however, the study report does provide a Lot. No. (PW6406). No detail regarding animal source or husbandry.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to evaluate t-butyl peroxybenzoate for the potential to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells when tested with and without metabolic activation.

The Mouse Lymphoma Assay is a short-term test for screening compounds for potential genetic activity. This test utilizes a mammalian cell line as a target to measure forward mutational events. This system has been shown to be sensitive and capable of detecting the activity of a wide range of chemical classes, some of which are not detected in the AmesTest. (Clive, D., et al., Mutation Research 59: 61-108, 1979).

The L5178Y mouse lymphoma cell line is presumed to be diploid in nature and three TK phenotypes have been recognized: TK+/+, TK+/-, and TK-/-.The TK+/+ and TK+/- cells are sensitive to trifluorothymidine (TFT) and resistant to a solution of thymidine, hypoxanthine, methotrexate and glycine (THMG). The TK-/- phenotype exhibits reverse sensitivity and resistance patterns. The heterozygous TK+/- phenotype is used as the target cell in this test system.

When TK+/- heterozygous phenotype cells are exposed to agents that can alter DNA, one of the possible consequences of this alteration is the induction of forward mutations which result in a change from TK+/- to the TK-/- phenotypes. This assay measures the induction of the TK-/-phenotype as its endpoint.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
T/K
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The in vitro system species is identified as L5178Y heterozygousTK+/- mouse lymphoma cells, subline 3.7.2 C which were received from
Dr. Donald Clive, Research Triangle Park, North Carolina. Stocks ofthese cells were frozen and stored in liquid nitrogen until used. Prior to use in the assay, cells are routinely cleansed of spontaneous mutants by overnight treatment with methotrexate, thymidine, hypoxanthine and glycine. The culture used for the toxicity assay was thawed on 6/25/84 and cleansed on 7/5/84 and the culture used for the mutation assay was thawed on 7/10/84and cleansed on 7/19/84.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
3, 5, 7, 10 & 15 ug/ml w/o S9
5, 7, 10, 15 & 22 ug/ml w S(
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate (w/o S9); 3-methylchoanthrene (w S9)
Details on test system and experimental conditions:
Toxicity Test
An initial toxicity test was performed with and without metabolic
activation using dose levels ranging; from 1000 to 3 ug/m1. Due to
less than 90% cell survival at all but the lowest dose, a second
toxicity test was required in order to establish a dose range for the
forward mutation assay. The dose levels for the second toxicity test
ranged from 100 to 0.3 ug/ml. The toxicity test was performed by
adding 0.1 ml of the solvent, medium control, 100x positive control,
or 100x test .agent to the appropriate 50 ml centrifuge tubes
containing 6.0 x 106 TK+/- cells in 6 ml of cell culture medium.
An additional 4 ml of S-9 mix were added to the tubes for metabolic
activation; four ml of FlOP were added to the nonactivated tubes.
The cells were exposed to the chemical for four hours, washed twice,
resuspended in 20 ml FlOP, and incubated overnight.
On the day following treatment, all cultures were set back to
0.3xl06 cells per ml when necessary and incubated until the
following day. On the second post treatment day the cell density was
again determined for each culture and relative growth was determined.
The dose range for the forward mutation assay was based on the
second toxicity test. The high dose level (50 ug/ml) was chosen to
demonstrate a high level of toxicity. Seven subsequent dilutions
were made to yield dose levels of approximately 34, 22, 15, 10, 7, 5
and 3 ug/ml.

F. Forward Mutation Assay
The eight 100x concentrations of the test compound were prepared to
contain the test dose in 0.1 ml volumes. The highest final concentration
used for the mutation assay with and without activation was
50 ug/ml. Duplicate cultures for each test compound concentration
and triplicate cultures of positive, solvent and medium controls were
dosed as follows:
Six million precleansed cells in
added to 50 ml centrifuge tubes.
added to the tubes not requiring
six ml of cell culture medium were
An additional four ml of F0 were
activation, and four ml of the S-9
mix were added to those tubes being tested with metabolic activation.
Next, 0.1 ml of either the 100x test chemical dilution, 100x positive
control, solvent or media control was added to the appropriate tubes.
Each tube was mixed, gassed with a mixture of 5% CO2 in air, sealed
and incubated at 37.5° + .5°C on a revolving roller drum for four
hours. Following this incubation, the tubes were centrifuged and the
treatment solutions decanted. The cells were washed twice with FlOP
and resuspended in 20 ml FlOP after the second wash. The tube
cultures were then gassed and reincubated as described above for a
two-day expression time. Growth of the cells was monitored at one
day postexposure and the cultures adjusted to .3 x 106 cells/ml, if
necessary, At the end of the expression period, a sample from each
of the cultures to be cloned was centrifuged, and the cells
resuspended at 1.0 x 106 viable cells/ml in FlOP.
Five concentrations of the test compound with and without metabolic
activation were used for cloning.
Approximately 1.0 x 106 cells were plated in each of three selective
medium plates containing trifluorothymidine (TFT), and approximately
200 cells were cloned in each of three nonselective plates for each
test and control tube. After incubation, the mutant colonies (TK-/-)
were counted on the selective TFT-containing plates; similarly,
colonies in the non-selective medium plates were counted.
Due to contamination in the positive control (MeA) plates, it was
necessary to repeat activated portion of this study. The data
pertaining to activation that follow are the result of this retest.
Evaluation criteria:
A test chemical will be considered positive if a dose-related
response is obtained in I'lnicn the mutation frequencies of at
least two test concentrations are at least two-fold higher than
the mutation frequency of the solvent control.

Mutation frequencies of the test compound will be calculated for
dose levels with 10% or greater cell survival.
Statistics:
none reported
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The five highest dose levels with a relative percent suspension growth of at least 10% were subjected to TFT selection. The doses selected with metabolic activation ranged from 22 to 5 ug/ml and without metabolic activation from 15 to 3 ug/ml.

The test material, t-butyl peroxybenzoate, induced a greater than two-fold increase in the mean mutation frequency with duplicate doses of the high dose level (22 ug/ml) with metabolic activation. A greater than two-fold increase was produced in a dose related response at the three high doses (15, 10 and 7 ug/ml) in the absence of metabolic activation. The spontaneous mutation frequencies and the levels of activity of the positive controls in this assay confirmed the sensitivity of the test.

Under the conditions of this assay. t-butyl peroxybenzoate is considered to be mutagenic.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
positive

Under the conditions of this assay, t-butyl peroxybenzoate was found to be mutagenic in the L5178Y Mouse Lymphoma Forward Mutation Assay.
Executive summary:

Mouse lymphoma cells were treated with eight dose levels of t-butyl peroxybenzoate ranging from 50 to 3 ug/ml. Duplicate doses at 22 ug/ml in the presence of metabolic activation and 15, 10 and 7 ug/ml in the absence of metabolic activation induced a mean mutation frequency greater than two-fold higher than the solvent control. Both positive and negative controls in this assay confirmed the sensitivity of the test system. Under the conditions of this assay, t-butyl peroxybenzoate was found to be mutagenic in the L5178Y Mouse Lymphoma Forward Mutation Assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March, 1985 - July, 1988
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:
No guidelines are cited in the source document. Study performed at Case Western Reserve University. The detailed protocol and these data are presented in Mortelmans et al. (1986). Cells and study compound or solvent (dimethylsulfoxide) were incubated in the absence of exogenous metabolic activation (-S9) or with Aroclor 1254-induced S9 from male Syrian hamster liver or male Sprague Dawley rat liver. High dose was limited by toxicity or solubility, but did not exceed 10 mg/plate; 0 mg/plate dose is the solvent control.
GLP compliance:
yes
Remarks:
NTP follows GLP as a matter of policy.
Type of assay:
bacterial reverse mutation assay
Target gene:
his+
Test concentrations with justification for top dose:
0.0, 1.0, 3.3, 10.0, [16.0, 33.0, 66.7, 100.0] ug/plate**

**The data tables in the source report list these concentrations as "mg/plate. However, Normal protocols do not include concentrations this high. Moreover, the data summary in the source report lists these concentrations as ug/plate, consistent with normal protocols.
Vehicle / solvent:
dimethlysulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Remarks:
2-aminoanthracene; 4-nitro-O-phenylenediamine; sodium azide; 9-aminoacridine [see attached study report for details]
Details on test system and experimental conditions:
Not provided in source report. Details were provided in the original literature reference: Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., and Zeiger, E. (1986) Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ. Mutagen. 8 (Suppl 7), 1-119.

"All chemicals were initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mgjplate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity. As a rule, at least one toxic dose was incorporated into the first mutagenicity test; the
repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached."

"All chemicals were assayed for mutagenicity in the preincubation assay [Haworth et d, 19831. To each of 13 X 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.5 ml (EGG) or 2.0 ml (CWR, SFU) of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 19561 in 15 X 100-mm plastic petri dishes (Falcon Muta-Assay, 1028 [EGG, SRII, and Fisher Scientific plates [CWR]). When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr. Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. AU assays were repeated (as described above) no less than 1 wk after completion of the initial test."
Evaluation criteria:
Not provided in source report. Details were provided in the original literature reference: Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., and Zeiger, E. (1986) Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ. Mutagen. 8 (Suppl 7), 1-119.

"The criteria used for data evaluation were the same as those described previously [Haworth et al, 19831, and are summarized as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible;
or when the response was of insufficient magnitude to support a determination of mutagenicity. The initial determination of mutagenic, nonmutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer (E.Z.). The chemicals were decoded by the chemical repository (Radian Corporation) only after the mutagenicity or nonmutagenicity of the chemicals had been determined."
Statistics:
Not provided in source report.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: tested up to either toxicity limit or limit of solubility
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: tested up to either toxicity limit or limit of solubility
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: tested up to either toxicity limit or limit of solubility
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: tested up to either toxicity limit or limit of solubility
Vehicle controls validity:
valid
Positive controls validity:
valid

See data tables below.

Conclusions:
t-BP was found to be mutagenic in Salmonella typhimurium strains TA100, TA1537, and TA98, with and without metabolic activation.
Executive summary:

t-BP was found to be mutagenic in Salmonella typhimurium strains TA100, TA1537, and TA98, with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No GLP statement or indication of intent to follow GLP. Test substance identification does not indicate purity. No detail regarding animal source or husbandry.
Qualifier:
equivalent or similar to
Guideline:
other: Ames, et. al., 1981
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA1538
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
500.00, 166.70, 55.60, 18.50, 6.20 and 0.00 ug/plate
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 9-Aminoacridine; 2-Nitrofluorene; 2-Aminoanthracene
Details on test system and experimental conditions:
Mutational Test
Direct exposure of the auxotrophic ~ typhimurium strains to the test compound was accomplished by the plate incorporation assay. Five concentrations of the test agent were evaluated in triplicate against the five bacterial strains with and without the metabolic activation system.
Approximately 0.2 ml of the test material weighing 200 mg were mixed with 3.8 ml of the solvent to contain approximately 50 mg/ml. This solution was subsequently diluted in the same solvent to 500, 166.7, 55.6, 18.5, and 6.2 ug/0.1 ml.

To each 2.5 ml of complete top agar, 0.1 ml of an overnight broth culture of each tester strain, 0.1 ml of the appropriate dilution of the test agent or diluent and 0.5 ml of the S-9 mix for the activated tests were added. The contents of each tube were thoroughly mixed and poured onto the VBE minimal agar plates. Positive control chemicals were treated in a similar manner. Plates were gently rotated and tilted to assure uniform distribution of the top agar, allowed to harden on an even surface for approximately one hour, inverted, and placed in a dark 37~0.5 C incubator. After two days, the colonies (revertants to histidine prototrophy) in both test and control plates were counted. Due to high spontaneous reversion frequencies with strain TA100, this portion of the assay was repeated. Accordingly ml of the test material were mixed with 3.8 ml of Disband diluted as described for the repeat test with strain TA100 both in the presence and absence of metabolic activation.
Evaluation criteria:
Before an agent is reported to be either active or inactive in the Salmonella/microsomal assay, the following criteria must be met:
1. Demonstration of toxicity of the chemical for the bacterial strain(s), unless this is not possible due to a limited solubility of the test compound
2. The spontaneous revertants for each strain must be within acceptable limits.
3. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls.
4. Positive mutagens must give at least 2x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.
All criteria noted above must be met before results with an unknown agent can be evaluated. A chemical that exhibits a positive dose response over three concentrations with the smallest of these increases equal to twice the solvent control is considered to be mutagenic in the S. typhimurium assay.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material is not mutagenic in this test system.
Executive summary:

The test compound with and without activation did not induce a significant increase in the number of revertant colonies over that shown in the solvent control plates for strains TA1535, TA1537, TA1538, TA100, TA98. Diagnostic controls confirm the sensitivity and responsiveness of the tester strains to detect genetic interaction with known mutagenic agents.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Incomplete study report (literally, portions missing); inadequate reporting of test conditions, including controls; no characterization of test substance; non-GLP
Qualifier:
no guideline followed
Principles of method if other than guideline:
Capacity to induce his+ revertants in modified Salmonella typhimurium (TA 98 and 100, 1535, 1537, 1538,)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his+
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9; Arochlor induced rat
Test concentrations with justification for top dose:
not reported
Vehicle / solvent:
DMSO; acetone
Remarks:
none reported, but implied by report of results
Details on test system and experimental conditions:
"Upon receipt from the sponsor the samples were stored in a refrigerator at 4°C. A description of the experimental procedure is presented as an appendix to the report.* Bacterial survival was determined by plating a 10E-5 dilution instead of the undiluted bacterial culture in top agar with extra histidine to allow growth of all, viable bacteria (his+ and his-). For the rest the procedure used was identical with the Ames test procedure. In the first experiment Trigonox C was tested in DMSO; in the following experiments it was tested in acetone instead of in DMSO. All dilutions were prepared immediately before use. The metabolic activation properties of the various S-9 batches used in the present study are given in table 2 of the appendix on page A-6)."*

*apparently there is more of the report than was available at time of this RS preparation.
Evaluation criteria:
not reported
Statistics:
none reported
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Additional information on results:
Incorporation of the test product with the various bacterial tester strains induced a dose-related increase in the numbers of his+ revertants with strains TA 1537, TA 98 and TA 100 in the absence of the S-9 mix in each of three independent experiments.The results obtained with TA 1535 and with TA 100 in the presence of the S-9 mix were rather variable, while those obtained with TA 1538 did not show any evidence of an increase in the numbers of his+ revertants upon treatment with Trigonox C. At the higher dose levels the test product was rather toxic for the bacteria as indicated in the tables.* Although the toxic and mutagenic concentrations were very close and even overlapped each other, the results obtained with TA 1537, TA 98 and TA 100 are considered to be indicative of mutagenic activity. Size and appearance of the revertant colonies on control and test plates were fully comparable, and from a random test it appeared that the colonies taken from the test plates were really his + revertants.

*apparently there is more of the report than was available at time of this RS preparation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation ;variable with activation

Mutagenic in a Salmonella reverse mutation assay without activation.
Executive summary:

"The mutagenic activity of Trigonox C was examined in the Salmonella/microsome mutagenicity test, using a set of five histidine requiring mutants of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and liver homogenate of Aroclor-induced rats. Incorporation of Trigonox C with TA 1537 TA 98 and TA 100 induced a dose-related, reproducible increase in the numbers of his+ revertants at doses that were very close to or even overlapped the dose levels that influenced bacterial survival. The results obtained with strains TA 1537 and TA 98 were variable. It was concluded that the results obtained with Trigonox C were indicative of mutagenic activity."

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: other: SCE
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
No GLP statement or indication of intent to follow GLP. Test substance identification does not indicate purity, however, the study report does provide a Lot. No. (PP1523). No detail regarding animal source or husbandry.
Qualifier:
no guideline followed
Principles of method if other than guideline:
exposure of cultured CHO cells to graded doses of test substance with subsequent measurement of the incidence of SCEs
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster Ovary cells CHO, K-l, Number CCL61 were obtained fromthe American Type Culture Collection. CHO cells are a permanent cell line with an average modal number of 20, and an average cycling timeof 10-14 hours. CHO cells are maintained as a monolayer in Ham's F-12 medium with 10% fetal calf serum (FCS) and frozen stocks are held in a liquid nitrogen storage tank.The cultures used for the mutation assay were thawed on September 18, 1984 and used at passage Px+6.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
5200, 520, 52, 5.2, 0.52, and 0.052 ug/ml
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate and cyclophosphamide
Details on test system and experimental conditions:
Toxicity Testing
Six concentrations of t-8utyl Peroxybenzoate were prepared to contain the test dose in a 0.025 ml volume. The highest dose tested both with and without metabolic activation was 5,200 ug/ml with subsequent concentrations of 520, 52, 5.2, 0.52, and 0.052ug/ml. Sixteen 25 cm2 tissue culture flasks were seeded with 3.0xl05 CHO cells in 5 ml Ham's F-l2 medium. Twenty-four hours after culture initiation, the medium, 5-9 mix, and test solutions were dispensed into the appropriate flasks. Each flask was mixed, tightly capped, and incubated at 37+0.5°C. Following a four-hour exposure, each flask was rinsed twice witha balanced salt solution, 5 ml Ham's F-12 medium added to each flask, and the flasks incubated again at 37+0.5°C. Approximately 24 hours after the flasks were rinsed, cell countswere performed on each flask using a Coulter Counter.

SCE Assay
1. Design and Dosing
Six concentrations of t-Butyl Peroxybenzoate were prepared to contain the test dose in a 0.025 ml volume. The highest dose tested without metabolic activation was 52 ug/ml with the subsequent concentration of 18, 5.2, 1.8 and 0.52 ug/ml. The highest dose tested with metabolic activation was 18 ug/ml with subsequent concentrations of 5.2, 1.8, 0.52 and 0.18 ug/ml. Thirty-two 25 cm2 tissue culture flasks were seeded with3.0xl05 CHO cells in 5 ml Ham's F-12 medium. Twenty four hours after culture initiation, the medium, S-9 mix, and test solutions were dispensed into the appropriate flasks in duplicate. Each flask was mixed, tightly capped, and incubated at 37+0.5°C. Following a two-hour exposure, each flask was rinsed twice with a balanced salt solution, 5 ml Ham's F-12 medium containing 5-bromodeoxyuridine (Brdu) added to each flask, and the flasks incubated again at 37+0.5°C. Each flask was wrapped in aluminum foil to exclude light. Approximately 24 hours after the flasks were rinsed, colcemid (0.2 ug/ml) was added to all flasks to arrest cells in metaphase.

2. Preparation of CHO Chromosomes
Two hours after the addition of colcemid metaphases were collected by mitotic shake-off. Each flask was firmly tapped and the media was poured into an appropriately labelled 15 ml centrifuge tube. The cells were washed once in 0.075 M KC1, and washed twice in an acetic alcohol fixative (3:1, methanol: acetic washed acid) .

3. Preparation of Slides
Two slides were prepared for each treatment group, one from each duplicate flask. Slides were prepared by dropping the cells ontoclean wet slides and air drying.

4. Slide Staining
Slides were stained for 10 minutes in Hoechst 33258 (5 ug/ml) in phosphate buffer (pH 6.8) and rinsed in glass distilled H20.Coverslips were mounted with the same buffer and the slide exposed to a black light for approximately 1 hour. Coverslips were removed and the slides stained in 2% Giemsa in buffer, rinsed twice, and air dried. Each slide was mounted with a glass coverslip using Coverbond®.

5. Slide Randomization
Flask numbers on each slide were covered with masking tape and each was assigned a temporary slide number. The coding was performed by an individual not involved in the scoring of the slides.

6. Slide Analysis
Fifty cells in the metaphase stage of mitosis were scored whenever possible at each dose level for the number of sister chromatid exchanges (SCE's). Results are presented as the number of SCE's per cell, and the number of SCE's per chromosome.

Statistics:
All data were analyzed by analysis of variance (ANOVA) with individual group comparisons performed by a one-tailed student's t- test.
The data were analyzed at the 99% confidence interval (p < .01).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of this study, t-Butyl Peroxybenzoate is considered to be mutagenic in this test system.
Executive summary:

The exposure of CHO cells to 52, 18, 5.2, 1.8, 0.52, and 0.18 ug/ml of t-Butyl Peroxybenzoate produced statistically significant increases in the frequency of sister chromatid exchanges (SCE) at 52, 18, 5.2, and 1.8 ug/ml without metabolic activation, and at 18 and 5.2 ug/m1 with activation. Therefore, under the conditions of this study, t-Butyl Peroxybenzoate is considered to be mutagenic in this test system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In Vivo Negative: Mouse Micronucleus Assay and Comet Assay.

Link to relevant study records

Referenceopen allclose all

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 (migrated information): 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 cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is given in Annex 1 of the study report.
Identification: tert-butyl perbenzoate (CAS# 614-45-9)
Chemical Name: tert-butyl peroxybenzoate
EC Name: tert-butyl perbenzoate
Product Name: Trigonox C
Physical state/Appearance: Pale yellow liquid
CAS Number: 614-45-9
Lot/Batch Number: 1802465400
Purity: 98.8% (Not corrected for in the formulations)
Expiry Date: 01 April 2028
Storage Conditions: Room temperature in the dark
Intended use/Application: Polymerization initiator
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 185.5-224.1 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing:The animals were housed in groups of no more than five by sex in solid-floor polypropylene cages with woodflake bedding.
The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). These enrichment items were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Diet/water (e.g. ad libitum): Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.
The diet and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: april 2018
Route of administration:
oral: gavage
Vehicle:
Identification:Corn oil
Supplier:Sigma-Aldrich
Supplier’s Lot Number:MKBZ9899V
Physical state/ Appearance:Amber colored liquid
Purity:Treated as 100%
Expiry Date:10 January 2022
Storage Conditions:Room temperature

The total dose volume was 10 ml/kg and the volume administered to each animal was calculated according to its body weight at the time of the initial dosing
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in Corn oil.
Determination by analysis of the concentration, homogeneity and stability of the test item preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.
Duration of treatment / exposure:
dosed twice with a 24 hour interval
Frequency of treatment:
twice
Post exposure period:
4 hours
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle Control (Corn oil)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Positive Control (N-Nitroso-N-methylurea)
Dose / conc.:
1 600 mg/kg bw/day (nominal)
Remarks:
tert-butyl perbenzoate (CAS# 614-45-9)
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
tert-butyl perbenzoate (CAS# 614-45-9)
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
tert-butyl perbenzoate (CAS# 614-45-9)
No. of animals per sex per dose:
7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-methylurea
Tissues and cell types examined:
Sub-samples of the liver and glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology may be conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.
The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:
Glandular Stomach – A small section of the glandular stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining tissue into a small volume of stomach buffer.
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex following a double oral administration at zero and 24 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

The RF doses were: 2000, 1600 and 1000 mg/kg bw. Both males and females were dosed. All animals were dosed twice 24 hours apart at the appropriate dose level by gavage using a rubber cannula attached to a graduated syringe. Animals were observed one hour after each dosing and immediately prior to termination, 28 hours after the start of the test. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
Bone marrow was sampled from the animals of the final range finding test (1600 mg/kg) and bone marrow smears were prepared and scored for PCE/NCE ratio as an indicator of any toxicity to bone marrow and to demonstrate absorption of the test item. The quantitative assessment revealed that there was no marked bone marrow toxicity observed at 1600 mg/kg, however the presence of clinical signs was considered to give an indication that systematic absorption of the test item had occurred.

Based on the data from a range finding study the maximum tolerated dose (MTD) of the test item, 1600 mg/kg, was selected for use in the main test, with 800 and 400 mg/kg as the lower dose levels. Although the animals survived the 2000 mg/kg dose level in the range finding test, the clinical signs were considered too severe for a main experiment whereas 1600 mg/kg was considered to be acceptable. It was considered that there was no marked difference in clinical signs between the male and female animals in the range finding experiments and therefore only male animals were used for the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The Main Comet Test was performed using male rats only. Groups each of seven male rats, were dosed twice with a 24 hour interval via the oral route with the test item at 1600, 800 or 400 mg/kg. The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 4 hours following the second administration, 28 hours after the start of the test. In addition, two further groups of rats were included in the study; one group (seven male rats) was dosed twice with a 24-hour interval via the oral route with the vehicle alone (Corn oil) and a second group (three male rats) was dosed twice orally with a 24-hour interval with N-Nitroso-N-methylurea (MNU). MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive controls were killed approximately 4 hours following the second administration, 28 hours after the start of the test. All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

DETAILS OF SLIDE PREPARATION:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass cover slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the cover slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.

METHOD OF ANALYSIS:
The slides were coded prior to scoring to allow “blind” scoring. The slides are stained just prior to analysis for comets. To each dry slide, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV version 4.3.1.Two slide gels for each tissue per animal were scored with a maximum of 75 cells per slide gel giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell is not scored twice. The slide score data from each experiment was processed using the Excel macro program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups were made. The primary end-points are percentage tail DNA (%Tail intensity) and median percentage tail intensity. Each slide was assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

OTHER:
Histopathology
Samples of the liver and glandular stomach from the vehicle and test item animals were preserved in buffered 10% formalin.
Due to an increase in DNA damage being observed in the glandular stomach, the Sponsor requested histopathology be performed on the vehicle control group and the three dose groups (400, 800 and 1600 mg/kg). The preserved stomach tissue from these dose groups was dispatched to the test site for Histopathology Processing and Examination. The stomach was processed and stained with Hematoxylin and Eosin prior to the slides being read. Histological Processing and Examination was conducted in accordance with the Test Site Standard Operating Procedures.
Evaluation criteria:
The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Positive if:
• At least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control.
• The response is considered to be dose related
• The results are substantially outside the laboratory historical vehicle control range.
The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.
There is no requirement for verification of a clearly positive or negative response.
Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgment. This may require the scoring of additional slides to increase the number of cells and, therefore, add more power to the data. If this does not resolve the issue then the result will be given as equivocal or questionable, and may require the histopathological assessment of the tissues to see if cell toxicity may be the causative agent rather than any genotoxic mechanism.
Statistics:
A comparison was made between the vehicle control groups and the positive control groups. The individual slide score data for the percentage tail intensity and median percentage tail intensity was compared using a Students t-test with a √1+x transformation. Comparisons between the vehicle control groups and the test item dose groups were also made when it was considered that there was a marked increase over the vehicle control value.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
cytotoxicity was observed
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Mortality Data and Clinical Observations

There were no premature deaths seen in any of the test item dose groups. Clinical signs were observed in animals dosed with the test item at the MTD (1600 mg/kg), these included as follows: hunched posture, ataxia, splayed gait and labored respiration. Hunched posture was observed in two of the animals in the MTD/2 dose group (800 mg/kg) and there were no clinical observations seen in the animals of the MTD/4 dose group.

Evaluation of Comet Assay Slides

A summary of the results for each of the tissues of the Comet Assay, liver and glandular stomach, is given below in table 2. Individual slide data and animal data for each tissue are presented in Tables 3 to 12 (attcahed as pdf) with the means, medians and standard deviations calculated from the individual animal data and the group data.

The vehicle control group demonstrated percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the

liver and glandular stomach, comparable with the laboratory historical control range for these tissues. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

Two of the ‘A’ replicates in the positive control group of the glandular stomach, for animals 9 and 10 did not achieve the expected increases in the percentage tail intensity. The gels for these two replicates were on the same slide and it is concluded that the slide did not electrophorese properly. However, since the other slides in the group, including the ‘B’ replicates for animals 9 and 10, all demonstrated marked increases over the vehicle control group and the positive control group as a whole was statistically significant it is considered to be of no consequence.

There was no evidence of an increase in the liver percentage tail intensity or median percentage tail intensity in the test item animals compared to the concurrent vehicle control group.

The glandular stomach demonstrated statistically significant increases in percentage tail intensities and median percentage tail intensities in the test item groups when compared to the vehicle control group. The increases were not strictly dose related in that the MTD/2 dose group had higher values than the MTD dose group. There were particularly high values noted in certain animals which raised the overall mean for the groups. Animal 15 in the MTD group and animals 20, 22 and 24 in the MTD/2 group and animal 27 in the MTD/4 all had high percentage tail intensities compared with the rest of their group. It is considered that these animals were particularly sensitive to the effects of the test item. Although statistically significant, the mean and median percentage tail intensities for the 400 mg/kg (MTD/4) dose group and the 1600 mg/kg (MTD) were within the current laboratory historical control range for a vehicle.

Due to the increases in the percentage tail intensities observed in the glandular stomach, histology was performed on the retained glandular stomach tissue from the test item dose groups and the vehicle control group to ascertain whether the response was due to genotoxicity or cytotoxicity. The histopathology using Haematoxlin and Eosin staining demonstrated evidence of inflammatory and degenerative changes in the glandular stomach which were considered to be consistent with cytotoxicity.

There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. The hedgehog frequency data for each tissue is included in Tables 3 to 12.

Discussion

There was no evidence of an increase in the liver percentage tail intensity or median percentage tail intensity in the test item animals compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage in the liver in vivo under the conditions of the test. Statistically significant increases in the glandular stomach percentage tail intensities and median percentage tail intensities of the test item groups were demonstrated when compared to the vehicle control group. As a result of this increase, histology was performed at the Sponsors request on the preserved glandular stomach tissue from the test item dose groups and the vehicle control group to ascertain whether the response was due to genotoxicity or cytotoxicity. The histology results indicate that there was an increase in inflammatory cell infiltrate in the glandular region in all three treatment groups when compared to the control group. Ulceration and necrosis were observed in the animals of the MTD dose group. These observations are considered to demonstrate the irritant effects of the test item since the stomach is the first site of contact. The test item is also documented as being a skin irritant and a skin sensitizer. It is therefore considered that the increases in percentage tail intensity observed in the stomach are due to damaged or necrotic cells as a result of cytotoxicity rather than a true genotoxic mechanism.

Table 2 Summary of Results for Percentage Tail Intensity, Median Percentage Tail Intensity and Percentage Hedgehogs for each Tissue

Liver

Dose Level

Group Mean % Hedgehogs

Group Mean of % Tail Intensity

Group Mean of Median % Tail Intensity

Vehicle
(Corn oil)

0

0.36

0.01

400 mg/kg (MTD/4)

0

0.38

0.01

800 mg/kg (MTD/2)

0

0.39

0.01

1600 mg/kg (MTD)

0

0.34

0

Positive control
(N-Nitroso-N-Methylurea)

0.15

19.59***

19.57***

Glandular Stomach

Dose Level

Group Mean % Hedgehogs

Group Mean of % Tail Intensity

Group Mean of Median % Tail Intensity

Vehicle
(Corn oil)

0

1.73

0.63

400 mg/kg (MTD/4)

0.99

7.13**

5.90***

800 mg/kg (MTD/2)

5.54

15.58**

14.88**

1600 mg/kg (MTD)

2.43

7.69***

5.98***

Positive control
(N-Nitroso-N-Methylurea)

12.38

18.21*

17.47*



***     P<0.001

**      P<0.01

*        P<0.05

 

 

Conclusions:
The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver. There were increases in the percentage tail intensity and median percentage tail intensity in the glandular stomach but these were considered to be due to cytotoxicity rather than a genotoxic mechanism.
Therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.
Executive summary:

The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Tice et al., 2000. The method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2016). The primary target tissues of the comet assay were liver and glandular stomach.

A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex. The Comet Assay Main Test was conducted at the Maximum Tolerated dose (MTD) 1600 mg/kg with 800 mg/kg and 400 mg/kg as the lower dose levels. Groups of male rats (seven per group) were dosed a 0 hours and 24-hours and were killed at 28 hours (4 hours after the second dose administration). In addition, two further groups of male rats were included in the study; one group (seven rats) were dosed via the oral route with the vehicle alone (corn oil) at 0 and 24 hours, and a second group (three male rats) were dosed orally with N-Nitroso-N-methylurea (MNU) at 0 and 24 hours to act as the positive control. The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 28 hours after the start of the test. The glandular stomach and liver were processed for comet slides. Samples of glandular stomach and liver were preserved in formalin for possible histopathology in the event of a positive response.

The presence of clinical signs indicated that systemic absorption had occurred. There was no evidence of an increase in the liver percentage tail intensity or median percentage tail intensity in the test item animals compared to the concurrent vehicle control group. Statistically significant increases in the glandular stomach percentage tail intensities and median percentage tail intensities of the test item groups were demonstrated when compared to the concurrent vehicle control group. As a result of this response histopathology was performed on the preserved stomach samples to see if there was any evidence of cytotoxicity which would indicate that the response was not due to a true genotoxic mechanism. The histopathology using Haematoxlin and Eosin staining demonstrated evidence of inflammatory and degenerative changes in the glandular stomach which were consistent with cytotoxicity. The histology results indicated that cytotoxicity was responsible for the increased percentage tail intensities in the glandular stomach.

The positive control group induced a marked increase in percentage tail intensity and median percentage tail intensity in the liver and glandular stomach indicating that the test method itself was operating as expected. The vehicle control groups had percentage tail intensity and median percentage tail intensity values within the expected range.

The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity values in the liver when compared to the concurrent vehicle control group. Although increases were seen in the percentage tail intensity and median percentage tail intensity values of the test item dose groups of the glandular stomach they were considered to be due to a cytotoxic mechanism based on the results of the histopathology. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

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

Additional information

Short description of key information:

In Vitro:

Positive for mutation, with and without metabolic activation: Chromosomal Aberations (CHO); Mouse Lymphoma Forward Mutation Assay.

Mixed results: S. typhimurim TA100, 1537 and TA98 positive in one assay, with and without S9; negative in another assay, with and without S9; SCE (CHO) - positive without S9 in two assays, positive with S9 in one/negative in another

Negative Results:S. typhimurium TA1538 and TA 1535, with and without activation.;

In Vivo

Negative: Mouse Micronucleus Assay and Comet Assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Positive with and without activation in two or more in vitro mammalian cell assays. However an in vivo micronucleus assay and comet assay are negative and no lesions were reported in a 13 -week repeat dose study.