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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames tests

Three tests are available and well performed, GLP studies, quoted Klimisch K1 (Stankowski, 1989; Debets, 1985, May, 1990). All of them are considered as key studies. In these tests, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were used, but no strain TA 102 or E. Coli WP2 uvrA.

In the first Ames test (Stankowski, 1989), performed on the five strains, test was weakly positive especially on TA98 and TA100 in presence of S9. In the second test (Debets, 1985), also performed on the five strains, similar results were observed (“positive” results instead of “weakly positive” results). In the third test (May, 1990), only 4 strains were used (no use of TA 1538). Dose-related increases in reversion to prototrophy were obtained in strains TA98, TA100 and TA1537 in the presence of S9 mix only.

In conclusion, TBEC shown mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in different Ames test.

 

Mouse Lymphoma Assay

TBEC was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method (Salvador, 2012). The study was designed to comply with OECD 476 guideline and GLP compliant. The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolizing system. The cells were exposed to the test item for a short treatment time (3 hours) at the following dose levels: without S9: 70.0, 58.3, 48.6, 40.5, 33.8 and 28.1 µg/ml; with S9: 100.8, 84.0, 70.0, 58.3, 48.6 and 40.5. In the absence of S9 metabolic activation, moderate toxicity was observed at the highest dose level tested (70.0mg/ml) reducing the relative total growth (%RTG) to 19% of the concurrent negative control value. Dose-related toxicity covering the range from the maximum to no relevant toxicity was seen over the selected dose levels. In the presence of S9 metabolic activation, severe toxicity was observed at the highest dose level tested (100.8mg/ml) reducing RTG to 6% of the concurrent negative control value. Dose-related toxicity covering the range from the maximum (RTG=20%) to a slight toxicity (RTG= 68%) was seen over the remaining concentrations. Both in the absence and presence of S9 metabolic activation, a statistically significant increase in mutation frequency (p<1%) was observed at all dose levels tested and the induced mutant frequencies were higher than the global evaluation factor (GEF). In addition, a significant dose-relationship was indicated by the linear trend analysis (p<0.1%). Since clear positive results were obtained, no additional experiment was performed. It was concluded that TBEC induces mutation at the TK locus of L5178Y mouse lymphoma cells in vitro, under the reported experimental conditions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study but without raw data.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 1535, 1537, 1538, 98 and 100
Details on mammalian cell type (if applicable):
strains regularly checked for their histidine requirement, ampicillin resistance (TA98 and TA100) and the frequency of spontaneous revertants
Metabolic activation:
with and without
Metabolic activation system:
S9 (following intraperitoneal injection of adult male Wistar rats of Aroclor 1524 (500 mg/kg bw) in corn oil, rats were killed by decapitation, livers were removed and the supernactant was extracted.)
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 5000 µg/plate
Vehicle / solvent:
DMSO excepting for positive controls: DMS for TA100, TA98 and TA1538 without activation, and for all strains with activation.
Water for TA1535 and TA1537.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: TA 1535: sodium azide; TA 100: methylmethanesulfonate; TA 98 and TA 1538: 4-nitro-o-phenylene-diamine; TA1537: 9-aminoacridine /with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

EXPOSURE DURATION: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3
Species / strain:
other: all strains
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA1535, TA1537, TA1538
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA98 and TA100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: yes, but only for TA98 and from 3333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

  Mean number of revertant (His+), colonies /3 replicate plates WITHOUT S9
Dose (µg/plate) TA1535 TA1537 TA1538 TA98 TA100
100 7+-3 7+-4 5+-1 30+-5 125+-13
333 9+-3 5+-1 4+-1 25+-5 99+-25
1000 9+-2 6+-3 7+-3 25+-4 85+-25
3333 9+-1 9+-4 7+-3 29+-2 92+-10
5000 11+-7 10+-2 7+-3 33+-7 87+-15
Solvent control 7+-3 8+-3 7+-2 17+-2 85+-26
Positive control 320+-22 1282+-138 1163+-32 860+-141 775+-33
  Mean number of revertant (His+), colonies /3 replicate plates WITH S9
Dose (µg/plate) TA1535 TA1537 TA1538 TA98 TA100
100 10+-0 14+-2 16+-3 54+-7 168+-10
333 8+-1 (b) 20+-7(b) 17+-1(b) 90+-10 223+-23(b)
1000 0(d) 0(d) 0(d) 26+-3 334+-30(b)
3333** 0(d) 0(d) 0(d) 9*(d) 354+-211(c )
5000** 0(d) 0(d) 0(d) 14*(d) 316+-23(d)
Solvent control 8+-1 10+-1 16+-2 32+-4 91+-2
Positive control 170+-17 71+-7 1106+-22 992+-80 1787+-140
b) slightly reduced background bacterial lawn
c) moderately reduced background bacterial lawn
d) background bacterial lawn extremely reduced or absent
* 2 plates with no bacterial growth
Conclusions:
TBEC shown mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of TBEC to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA1538, TA 98, TA 100) was evaluated in accordance with the international guidelines (OECD 471, 1986) and in compliance with the Principles of Good Laboratory Practice.

TBEC was tested in two independent experiments, with and without a metabolic activation system, according to direct plate incorporation method, without and with S9.

 

Bacterias were exposed to TBEC at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 0, 100, 333, 1000, 3333, 5000 µg/plate. After 48 hours of incubation at 37°C, the revertant colonies were scored.

 

No cytotoxicity and no noteworth increase in the number of revertants was noted without S9.

With S9, Cytotoxicity was clearly observed from 1000 µg/plate for strains TA1535, TA1537, TA1538.

TBEC produced dose-related increases in the number of revertants of testers strains TA98 and TA100 in the presence of S9.

In conclusion, under these experimental conditions, TBEC shown mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The Salmonella strains were checked by testing for growth inhibition in spot tests. Further checks were conducted to confirm strain sensitivities to mutagens known to induce different types of mutational event (2-aminofluorene, 9-aminoacridine, sodium azide) , and to antibiotic (ampicillin).
Metabolic activation:
with and without
Metabolic activation system:
S9 (young male CD rats, with intraperitoneal injection of Aroclor 500 mg/kg in corn oil, in order to induce microsomal enzyme activity, levers were removed 5 days after treatment)
Test concentrations with justification for top dose:
First test (with and without S9): 0, 50, 158, 500, 1580 and 5000 µg/plate
Second test (with and without S9): 100, 158, 250, 500, 1580 µg/plate (to avoid excessive toxicity observed in the first test)
Third test (performed in order to confirm results of the previous tests and only with S9): 100, 158, 250, 500, 1580 µg/plate
Vehicle / solvent:
DMSO (for all strains and for all positive controls excepted sodium azide, which was dissolved in water).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: depending on the strains: Benzo-a-pyrene, 2-nitrofluorene , 2-amino-anthracene, 9-aminoacridine, and sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium):

Toxicity of test material was shown by absence or thinning of the background lawn of the background lawn.

Evaluation criteria:
fter 48 hours incubation, numbers of revertant colonies were counted manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
other: TA98, TA 100 and TA1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: Inhibition of growth was observed from 1580 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Inhibition of growth was observed from 1580 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

  First test TA98  
  Revertant colony counts  
  Without S9 With S9
50 25+-1 37+-2
158 29+-1 80+-8
500 26+-2 47+-1
1580 26+-1 1+-1 (c )
5000 25+-3 0(d)
Benzo-a-pyrene 27+-2 201+-10
2-nitrofluorene 209+-22 /
DMSO 25+-3 34+-5
  Second test TA98  
  Without S9 With S9
100 26+-1 34+-4
158 24+-0 76+-7
250 25+-2 73+-9
500 25+-4 63+-22
1580 25+-2 5+-2(c)
Benzo-a-pyrene 28+-2 351+-14
2-nitrofluorene 245+-6 /
DMSO 24+-1 33+-5
  Third test TA98  
  Without S9 With S9
100 / 68+-3
158 / 78+-2
250 / 82+-12
500 / 86+-3
1580 / 2+-2
Benzo-a-pyrene / 320+-9
DMSO / 42+-4

c Thinning of the background lawn of non-revertant cells was observed
d the background lawn of non-revertant cells was absent

  First test TA100  
  Revertant colony counts  
  Without S9 With S9
50 108+-1 109+-4
158 111+-1 147+-22
500 108+-3 158+-9
1580 112+-4 90+-1(c)
5000 111+-1 0+-0 (d)
Benzo-a-pyrene 110+-2 415+-6
Sodium azide 640+-12 /
DMSO 104+-7 /
  Second test TA100  
  Without S9 With S9
100 104+-5 141+-9
158 103+-0 167+-5
250 105+-4 182+-23
500 110+-1 150+-8
1580 104+-3 94+-9 (c)
Benzo-a-pyrene 110+-1 496+-6
Sodium azide 563+-18 /
DMSO 108+-6 /
  Third test TA100  
  Without S9 With S9
100 / 175+-7
158 / 196+-17
250 / 280+-15
500 / 294+-59
1580 / 123+-5(c )
Benzo-a-pyrene / 426+-23
DMSO / 120+-6

  First test TA1535  
  Revertant colony counts  
  Without S9 With S9
50 13+-1 14+-2
158 14+-2 14+-1
500 13+-2 12+-2
1580 12+-1 1+-1(c )
5000 13+-2 0+-0(d)
2-amino-anthracene 12+-2 395+-8
Sodium azide 657+-2 /
DMSO 13+-2 /
  Second test TA1535  
  Without S9 With S9
100 13+-1 15+-2
158 12+-0 14+-2
250 14+-2 12+-2
500 13+-1 8+-2
1580 12+-2 1+-1(c )
2-amino-anthracene 10+-1 358+-16
Sodium azide 316+-17 /
DMSO 13+-1 /
  Third test TA1535  
  Without S9 With S9
100 / 17+-2
158 / 15+-5
250 / 18+-2
500 / 15+-3
1580 / 13+-1
2-amino-anthracene / 255+-40
DMSO / 18+-2

  First test TA1537  
  Revertant colony counts  
  Without S9 With S9
50 7+-2 7+-1
158 6+-2 14+-5
500 8+-1 29+-9
1580 7+-2 0(c )
5000 7+-1 0(d )
Benzo-a-pyrene 5+-1 140+-7
9-Aminoacridine 584+-25 /
DMSO   /
  Second test TA1537  
  Without S9 With S9
100 7+-2 11+-2
158 7+-1 14+-2
250 7+-2 20+-6
500 7+-1 21+-8
1580 9+-1 0+-0(c )
Benzo-a-pyrene 5+-1 190+-8
9-Aminoacridine 316+-13 /
DMSO 5+-1 /
  Third test TA1537  
  Without S9 With S9
100 / 9+-0
158 / 15+-4
250 / 18+-5
500 / 15+-3
1580 / 2+-1
Benzo-a-pyrene / 128+-10
DMSO / 6+-2
Conclusions:
TBEC exhibited mutagenic activity, inducing frameshift mutations following metabolic activation.
Executive summary:

The potential of TBEC to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100) was evaluated in accordance with the international guidelines (OECD 471, 1986) and in compliance with the Principles of Good Laboratory Practice.

TBEC was tested in two independent experiments, with and without a metabolic activation system, according to direct plate incorporation (the first experiment and the second without S9 mix)

 

Bacterias were exposed to TBEC at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test: in the first test (with and without S9): 0, 50, 158, 500, 1580 and 5000 µg/plate; in the second test (with and without S9): 0, 100, 158, 250, 500, 1580 µg/plate (to avoid excessive toxicity observed in the first test) and in the third test (performed in order to confirm results of the previous tests and only with S9): 0, 100, 158, 250, 500, 1580 µg/plate

 

Cytotoxicity was observed from 1580 µg/plate in all strains (D5 µg/plate and above) with metabolic activation.

Dose-related increases in reversion to prototrophy were obtained in strains TA98, TA100 and TA1537 in the presence of S9 mix only.

 In conclusion, TBEC shown mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, performed according to Ames method.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhymurium TA1535, 1537, 1538, 98, 100
Details on mammalian cell type (if applicable):
All tester strains were checked for the presence of the appropriate genetic markers on approximately a monthly basis
Metabolic activation:
with and without
Metabolic activation system:
included 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors
Test concentrations with justification for top dose:
167, 500, 1670, 5000, 7500 and 10 000 µg/plate (no toxicity in the screening test, however oily droplets were observed at doses of 1670 and 5000 µg/plate)
Vehicle / solvent:
- All required dilutions were made with acetone, Lot #611610, supplied by Mallinckrodt, Inc. (Paris, KY)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: in the absence of S9 : TA1535 and TA100 - sodium azide; TA1537- 9-aminoacridine ; TA1538 and TA98 - 2-nitrofluorene ; in the presence of S9: 2-Anthramine
Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independant revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value.
Statistics:
Statistical analyses were performed using the the program developed by Snee and Irr (1981) with significance established at the
95% confidence limit. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as
the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA98, TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA1537, TA1538
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
slightly positive in one test
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA98 and TA100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TBEC was found to be incompletely soluble at doses 1670 µg/plate. Inhibited growth was observed in all tester strains at a dose of 10,000 ug/plate without S9.
In contrast, inhibited growth or complete toxicity was observed in all strains at doses 500 and/or 1670 ug/plate with S9. Revertant frequencies for all doses of TBEC in all strains without S9, and in strains TA1535 and TA1538 with S9, approximated or were less than those observed in the concurrent negative control cultures. However, statistically significant, dose-dependent increases in revertant frequencies, to approximately 3.0-, 1.8 - and 3.6-fold control values, were observed in strains TA1537, TA98 and TA100, respectively, at doses of 167 and/or 500 µg/plate with S9.

Therefore, TBEC was re-evaluated in all five tester strains at doses of 5.00, 16.7, 50.0, 167, 500, 1000 and 1670 ug/plate with S9. Inhibited growth was again observed in all strains at doses 500 ug/plate. Revertant frequencies for all doses of TBEC in strains TA1535, TA1537 and TA1538 approximated or were less than those observed in the concurrent negative controls. In contrast, statistically significant, dose-dependent increases in revertant frequencies, to approximately 1.8- and 2.2-fold those of the concurrent negative controls, were observed in strains TA98 and TA100, respectively, at doses up to 500 ug/plate.
All positive and negative control values in both assays were within acceptable limits.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of the three tests

  FIRST TEST            
  Dose level (µg/plate) S9? TA1535 TA1537 TA1538 TA98 TA100
Acetone   without 13(2) 8(3) 13(2) 40(4) 110(9)
Acetone   with 21(4) 10(2) 24(9) 52(4) 99(9)
TBEC 167 without 15 (3) 7(1) 14(0) 42(9) 110(6)
TBEC 500 without 18(4) 14(6) 14(4) 41(2) 120(8)
TBEC 1670 without 13(4) 12(3) 15(4) 41(4) 110(15)
TBEC 5000 without 15(2) 10(3) 14(2) 39(5) 100(9)
TBEC 7500 without 17(4) 10(3) 17(4) 43(16) 115(16)
TBEC 10000 without 15(1) 7(2) 11(6) 44(7) 88(22)
TBEC 167 with 9(3) 19(5) 19(9) 93(3) 246(21)
TBEC 500 with 2(2) 30(12)* 6(3) 77(14) 351(47)*
TBEC 1670 with _ 0(1) _ _ 75(151)
TBEC 5000 with _ _ _ _ _
TBEC 7500 with _ _ _ _ _
TBEC 10000 with _ _ _ _ _
               
  SECOND TEST S9? TA1535 TA1537 TA1538 TA98 TA100
Acetone   with 22(8) 15(5) 22(8) 52(8) 129(16)
TBEC 5 with 30(8) 18(2) 25(5) 45(12) 136(3)
TBEC 16,7 with 20(8) 19(2) 22(3) 61(5) 115(7)
TBEC 5 with 22(4) 15(4) 27(2) 82(4) 113(1)
TBEC 167 with 18(3) 20(4) 22(5) 92(19) 203(45)
TBEC 500 with 15(3) 21(5) 10(2) 1(1) 288(24)
TBEC 1000 with 0(0) 8(3) 1(2) 0(0) 179(36)
TBEC 1670 with 0(0) 0(0) 0(0)   104(40)
               
               
  (SD)            
  *positive response            
Conclusions:
The results for TBEC were weakly positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.
Executive summary:

In a GLP Ames test, TBEC was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 167, 500, 1670, 5000, 7500 and 10,000 µg/plate. Six dose levels of TBEC were evaluated with and without S9 in the event of unacceptably high toxicity and/or insolubility at the higher dose levels evaluated in the mutation assay. The test article was found to be incompletely soluble at doses 1670 µg/plate. Inhibited growth was observed in all tester strains at a dose of 10,000 ug/plate without S9.

In contrast, inhibited growth or complete toxicity was observed in all strains at doses 500 and/or 1670 ug/plate with S9. Revertant frequencies for all doses of TBEC in all strains without S9, and in strains TA1535 and TA1538 with S9, approximated or were less than those observed in the concurrent negative control cultures. However, statistically significant, dose-dependent increases in revertant frequencies, to approximately 3.0-, 1.8 - and 3.6-fold control values, were observed in strains TA1537, TA98 and TA100, respectively, at doses of 167 and/or 500 µg/plate with S9.

Therefore, TBEC was re-evaluated in all five tester strains at doses of 5.00, 16.7, 50.0, 167, 500, 1000 and 1670 ug/plate with S9. Inhibited growth was again observed in all strains at doses 500 ug/plate. Revertant frequencies for all doses of TBEC in strains TA1535, TA1537 and TA1538 approximated or were less than those observed in the concurrent negative controls.In contrast, statistically significant, dose-dependent increases in revertant frequencies, to approximately 1.8- and 2.2-fold those of the concurrent negative controls, were observed in strains TA98 and TA100, respectively, at doses up to 500 ug/plate.

All positive and negative control values in both assays were within acceptable limits.

Therefore, the results for TBEC were weakly positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2011-January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; coherence between data, results and conclusions
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
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Minimal medium A

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 IU/ml + Penicillin G 50.000 units/ml
F 68 Pluronic

Minimal medium B

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 units/ml + Penicillin G 50.000 units/ml

Complete medium (5%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium (10%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium A (20%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium B (20%)

Minimal medium B
Horse serum (heat-inactivated)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment without S9 mix:
70.0, 58.3, 48.6, 40.5, 33.8 and 28.1 µg/ml (3-hour treatment)

Experiment with S9 mix;
100.8, 84.0, 70.0, 58.3, 48.6 and 40.5 µg/ml (3-hour treatment)
Vehicle / solvent:
Vehicle used: dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
(without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
(with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: fluctuation method in medium

DURATION
- Exposure duration: 3 hour in the absence and presence of S9 metabolism
- Expression time (cells in growth medium): Two days after treatment (48 hours)
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: 1.6 cells/well plated in each of 96-well plates (two)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

DETERMINATION OF MUTATION
- Method: Induced mutant frequency (IMF)

Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:

(i) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126 x 10-6) at one or more doses.

(ii) There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration.
Statistics:
Dunnett test. Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

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 specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No precipitate was noted upon addition of the test item to the cultures in all treatment series and by the end of treatment incubation period. The addition of the test item solutions did not have any obvious effect on the osmolality or pH of the treatment medium.

RANGE-FINDING/SCREENING STUDIES (Cytotoxicity test):
On the basis of the solubility results, a cytotoxicity assay was performed in the absence and presence of S9 metabolic activation, at a maximum dose level of 2460 µg/ml and at a wide range of lower dose levels: 1230, 615, 308, 154, 76.9, 38.4, 19.2 and 9.61 µg/ml.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived to treatment or insufficient number of cells recovered after treatment at the five higher dose levels tested. Severe toxicity was observed at a concentration of 76.9 µg/ml. Slight toxicity was observed at the next lower dose level (38.4 µg/ml). No toxicity was observed over the remaining concentrations tested.
Using the 24 hour treatment time, no cells survived to treatment or insufficient number of cells recovered after treatment at the six higher dose levels. Severe toxicity was seen at 38.4 µg/ml , while mild toxicity was observed at 19.2 µg/ml. No relevant toxicity was observed at the lowest dose level (9.61 µg/ml).
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived to treatment or insufficient number of cells recovered after treatment at the five higher dose levels. Mild toxicity was noted at the next lower concentration of 76.9 µg/ml, while no relevant toxicity was observed over the remaining dose levels tested.

COMPARISON WITH HISTORICAL CONTROL DATA:

Solvent and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

ADDITIONAL INFORMATION ON MUTAGENICITY:

Based on the results obtained in the cytotoxicity trial, a main assay for mutation to 5 trifluorothymidine resistance was performed using the dose levels described in the following table:


Assay No.: S9 Treatment
time (hours) Dose levels (µg/ml)
1 - 3 70.0, 58.3, 48.6, 40.5, 33.8 and 28.1
1 + 3 100.8, 84.0, 70.0, 58.3, 48.6 and 40.5

A statistically and biologically significant increase in mutation frequency was observed at all dose levels tested both in the absence and presence of S9 metabolic activation. In addition, a significant dose-relationship was indicated by the linear trend analysis (p<0.1%) and both small and large colonies frequencies were increased, in the absence and presence of S9 metabolic activation.

Colony sizing for positive test item concentrations indicated a shift towards the small colony mutants which represent large genetic changes frequently visible as chromosome aberrations.
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

The cells were exposed to the test item for a short treatment time (3 hours).Since clear positive results were obtained, no additional experiment was performed.

After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 105cells/ml. The cultures were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2 x 105cells/ml.

Plating for 5-trifluorothymidine resistance

After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/ml) and an estimated 2 x 103cells were plated in each well of four 96-well plates.

Plates were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability

After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Results

A main assay for mutation to trifluorothymidine resistance was performed using the dose levels described in the following table:

Assay No.:

 

 

S9

Treatment

time (hours)

Dose levels (ug/ml)

1

-

3

70.0, 58.3, 48.6, 40.5, 33.8 and 28.1

1

+

3

100.8, 84.0, 70.0, 58.3, 48.6 and 40.5

 

Solvent and positive control cultures were included in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10-6viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the negative control cultures and the control growth factor over 2 days fell within the range. The study was accepted as valid.

Survival after treatment

In the absence of S9 metabolic activation, moderate toxicity was observed at the highest dose level tested (70.0mg/ml) reducing the relative total growth (%RTG) to 19% of the concurrent negative control value. Dose-related toxicity covering the range from the maximum to no relevant toxicity was seen over the selected dose levels. In the presence of S9 metabolic activation, severe toxicity was observed at the highest dose level tested (100.8mg/ml) reducing RTG to 6% of the concurrent negative control value. Dose-related toxicity covering the range from the maximum (RTG=20%) to a slight toxicity (RTG= 68%) was seen over the remaining concentrations.

Both in the absence and presence of S9 metabolic activation, a statistically significant increase in mutation frequency (p<1%) was observed at all dose levels tested and the induced mutant frequencies were higher than the global evaluation factor (GEF).In addition, a significant dose-relationship was indicated by the linear trend analysis (p<0.1%).

It is concluded that TBECinduces mutation at the TK locus of L5178Y mouse lymphoma cellsin vitro,under the reported experimental conditions.

 

Conclusions:
TBEC induces mutation at the TK locus of L5178Y mouse lymphoma cells in vitro with and without S9, under the reported experimental conditions.
Executive summary:

TBEC was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

The study was designed to comply with   OECD Guideline for the testing of chemicals No. 476 (adopted July 1997).

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

The cells were exposed to the test item for a short treatment time (3 hours) at the following dose levels:  without S9: 70.0, 58.3, 48.6, 40.5, 33.8 and 28.1 µg/ml; with S9:  100.8, 84.0, 70.0, 58.3, 48.6 and 40.5.

In the absence of S9 metabolic activation, moderate toxicity was observed at the highest dose level tested (70.0mg/ml) reducing the relative total growth (%RTG) to 19% of the concurrent negative control value. Dose-related toxicity covering the range from the maximum to no relevant toxicity was seen over the selected dose levels. In the presence of S9 metabolic activation, severe toxicity was observed at the highest dose level tested (100.8mg/ml) reducing RTG to 6% of the concurrent negative control value. Dose-related toxicity covering the range from the maximum (RTG=20%) to a slight toxicity (RTG= 68%) was seen over the remaining concentrations.

Both in the absence and presence of S9 metabolic activation, a statistically significant increase in mutation frequency (p<1%) was observed at all dose levels tested and the induced mutant frequencies were higher than the global evaluation factor (GEF).In addition, a significant dose-relationship was indicated by the linear trend analysis (p<0.1%).

Since clear positive results were obtained, no additional experiment was performed.

It was concluded that TBEC induces mutation at the TK locus of L5178Y mouse lymphoma cellsin vitro, under the reported experimental conditions.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus assays

TBEC was tested in two independents mammalian erythrocyte micronucleus tests (OECD 474, GLP) which are both quoted Klimisch 1 and are used as key studies (Gudi, 1999; Hadouk, 2002).

 

In the first test (Gudi, 1999), mice were dosed orally with 300, 600 or 1200 mg/kg body weight. Reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls, which suggests test article availability in bone marrow. 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.

 

In the second test (Hadouk, 2002), mice received two oral treatments of TBEC at dose-levels of 500, 1000 or 2000 mg/kg/day, at a 24-hour interval. For both males and females, the mean values of MPE as well as the PE/(PE+NE) ratio in the groups treated with the test item, were equivalent to those of the vehicle group. It was not possible to say whether the substance was available in the bone marrow, nevertheless as the highest dose of 2000 mg/kg x 2 was used, the test is regarded as valid.

The results of the two in vivo assays indicate that TBEC does not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. 

 

Comet assay

The evaluation of the genotoxic potential of TBEC was done through the in vivo comet assay performed under alkaline conditions,i.e.pH > 13 (Alkaline Single Cell Gel Electrophoresis, OECD TG no. 485) in both male and female OFA Sprague Dawley rats, in isolated glandular stomach, liver, duodenum and kidney cells (Simar, 2018).

A Dose Range Finding assaywas performed with 4 groups of 3 male and 3 female rats receiving 2 daily treatments at 24-hour intervals at the doses of 2000, 1000, 250 and 0 mg/kg/day per osfor thedetermination of the MTD (Maximal Tolerated Dose) and the assessment of the cytotoxicity of the test item on the glandular stomach of all animals by both a histological analysis and the halo test.

A slight stereotypy (slight head movements and chewing) was observed in all male and female rats 15 minutes after the first and the second treatment with 2000 mg/kg. Difficulty to breath for 1 male 15 minutes after the first treatment at this dose level was also noticed. No other clinical signs were noted whatever the period of observation or at both 1000 and 250 mg/kg/day (x2). Otherwise, during the sampling of stomachs for histological assessment and the halo assay (see below), it was noted that the stomachs from the highest dose group (2000 mg/kg) were larger than the ones from the other treatment groups.

The histological assessment of a portion of the stomach (glandular part and forestomach) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of toxicity (i.e.assessment of necrosis). As well, the halo assay performed on a portion of the stomach (glandular part only) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of either necrotic or apoptotic cells when compared to the concurrent vehicle control.

From the overall results obtained in the dose range finding assay (i.e.clinical assessment, histological assessment of stomach cells and halo test), the highest dose of 2000 mg/ kg/ day (x2) was retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2) per oswere also tested.

For the comet assay, groups of male and females rats (5/sex for the controls and low and mid dose levels, and 7/sex for the top dose level) were treated twice at 24-hour interval by oral route (gavage) with the test item at dose levels of 2000, 1000 and 500 mg/kg/day in corn oil, or with methylmethane sulfonate at 100 mg/kg/day in sterile water as positive control, or the vehicle corn oil under 10 ml/kg as negative control. All animals were sacrificed following one expression time of 3 to 6 hours after the last treatment. One hundred and fifty cells (50 cells/slide, 3 slides /animal) observed per animal.

The determination of TBEC in treatment formulations was performed by a GLP- compliant laboratory using a validated analyticin treatment formulations used in the main assays were satisfactory with less than 10% deviation from target concentrations.

The mean of medians of percentage of DNA in tail of the concurrent negative controls were within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).

In all organs, the concurrent positive control induced statistically significant increases of percentage of DNA in tail compared with the negative control. These values are comparable to the historical positive control data with 2 exceptions in male and female duodenum with values of 20.08 and 20.49%, respectively vs. 28.45 – 73.93%.

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed glandular stomach, duodenum, liver and kidney cells at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male and female rats.

In conclusion, TBEC induced no biologically significant increases in DNA strand breaks. Therefore, TBEC has no genotoxic activity in glandular stomach, duodenum, liver and kidney cells of male and female Sprague Dawley rats.

 

DNA damages and gene mutations assay

The ability of TBEC to produce DNA damage as 8-OH-dG formation, CAA->CTA transversions in codon 61 of the mouse Ha-ras gene and sustained epidermal hyperplasia was evaluated when administered to female Sencar mice topically twice weekly for four weeks (Slaga, 1997; Hanausek et al., 2004). Epidermal and dermal hyperplasia and total dermal cellularity were measured at day 2 and day 4 after last dosing. 8-OH-dG/dG ratio was determined by HPLC/ECD using DNA isolated from frozen skins. Ha-ras gene mutation was determined in DNA isolated from 25 paraffin sections (8µm) using MSP-32P assay. When applied repetitively to the dorsal skin of Sencar mice in doses of 10, 100 and 200 µmol/mouse, TBEC caused an increase in skin cellularity. No increase of 8 -OH-dG and no Ha-ras gene mutation were detected in DNA isolated from the epidermis after repetitive applications of 200 µmol/mouse TBEC. The positive control, DMBA (100 nmoles), effectively induced DNA damage (8-OH-dG), Ha-ras gene mutation, epidermal hyperplasia and dermal hyperplasia.

Link to relevant study records

Referenceopen allclose all

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
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 5-10 weeks old
- Weight at study initiation: 171 g and 223 g in males and between 174 g and 210 g in females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: three or two animals per cage.
- Diet (e.g. ad libitum): A04C-10 from SAFE (batch 16216).
- Water: drinking water, softened by reverse osmosis and filtered on 0.22 ìm membrane, was provided ad libitum.
- Acclimation period: 7 or 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 200, 100, 50 or 25 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The stability of the test item in the vehicle was 21 days at ambient temperature.
Preparations for treatments in the dose-range finding were performed just before use.
The concentration of LUPEROX® TBEC in dosing formulations were controlled analytically.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Post exposure period:
Tissue samples were taken 2 to 6 hours after the last treatment.
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
group 4
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
group 5
No. of animals per sex per dose:
4 groups of 5/sex, and one group of 7/sex (highest dose)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Methylmethane sulfonate
- Justification for choice of positive control(s): historical data
- Route of administration: oral, 24 hours and 2 to 6 hours before sacrifice.
- Doses / concentrations: 100 mg/kg/day
Tissues and cell types examined:
Isolated glandular stomach, liver, duodenum and kidney cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A Dose Range Finding assay was performed with 4 groups of 3 male and 3 female rats receiving 2 daily treatments at 24-hour intervals at the doses of 2000, 1000, 250 and 0 mg/kg/day per os for the determination of the MTD (Maximal Tolerated Dose) and the assessment of the cytotoxicity of the test item on the glandular stomach of all animals by both a histological analysis and the halo test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment took the form of 2 successive administrations at 24-hour intervals by oral route (gavage). Samples were taken 2 to 6 hours after the last treatment.
The animals were treated according to the experimental program below:

GROUP NUMBER – TREATMENT PER DAY (x2) NUMBER OF ANIMALS
Males Females
1. Vehicle alone (negative control), PO 5 5
2. Methylmethane sulfonate 100 mg/kg/day PO (x2) 5 5
3. Test item: 2000 mg/kg/day (x2)a, PO 5 (+2)b 5 (+2)b
4. Test item: 1000 mg/kg/day (x2) , PO 5 5
5. Test item: 500 mg/kg/day (x2) , PO 5 5
PO: per os (oral)
a As the maximum tolerated dose was equal or higher than 2000 mg/kg, this dose was administered in line with OECD (2016) and HAYASHI et al. (2007).
b Two animals per gender studied in the high-dose group were treated, in parallel. They were sacrificed but were not examined as no mortality was observed in the five first treated animals.

DETAILS OF SLIDE PREPARATION:
- Tissue sampling and cell isolation
The 5 males and the 5 females1 of each group were assigned for cell isolation and assessed for DNA fragmentation.
Individual animals were anaesthetized with isoflurane and exsanguined.
A 'V' shaped incision will be made from the center of the lower abdomen to the rib cage. The skin and muscles will be removed to reveal the abdominal cavity.
A portion of the Glandular Stomach, Liver, Duodenum and one of the 2 Kidneys were removed and washed in the cold mincing buffer until as much blood as possible has been removed (see also § 9.2 for histological assessment). The portion was minced with a pair of fine scissors to release the cells. The cell suspension were stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Moreover, a part of the stomach cell suspension from each animal (control and treated) was smeared and fixed on 2 labelled slides for eventual subsequent cytological evaluation (see § 9.3).
Cells were enumerated on a hemocytometer, and an adequate number of cells was harvested from each cell suspension for proceeding to slides preparation with 20 x 103 cells per slide whatever the organ assessed.
Single cell preparations were done within one hour after animal sacrifice.
- Protocol for the Comet assay
Dried slides preparation (pre-layering)
Conventional slides were dipped in hot 1.5 % normal melting point agarose in PBS. After gentle removal, the underside of the slides were wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry
- Slide preparation
Before use, a volume of 85 ìL of 0.8% of Normal Agarose (NA) was added on the microscope slide pre-layered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardens (3 to 5 minutes). The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 ìL/slide) kept at ca. 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Four slides per animal were prepared for the Comet assay.
Lysis
After the top layer of agarose has solidified, the glass coverslips were removed and the slides were immersed for one night at ca. + 4 °C in the dark in a lysing solution.
Unwinding, electrophoresis and staining
After this incubation period, the slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer (pH > 13) to around 0.25 cm above the slides. In order to avoid excessive variation across the groups during each electrophoretic run, only one of the replicate slides were processed in each run for each animal (DNA – unwinding and electrophoresis). The cells were exposed to the alkali for 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for 20 minutes at <10°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted protected from daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for 5 minutes with 0.4 M Tris (pH 7.5) and the DNA was exposed for 5 minutes to absolute ethanol in order to preserve all the Comet assay slides. Subsequently, the slides were airdried and then stored at room temperature until they were scored for DNA migration.
- DNA staining and image analysis
Slides were coded by a person not involved in the reading (coding sheets are presented in Appendix No. 7)
Just prior to scoring, the DNA was stained using propidium iodide (final concentration of 20 ìg/mL sterile water; 25 ìL/slide).
Slides were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS - DM 2000, Heerbrugg, Switzerland), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to the Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK).
For all groups three slides were analysed with 50 nuclei per slide randomly scored. The five animals were used, i.e. 15 slides per group, 750 analysed nuclei per group.
- Scoring and parameters
According to the OECD guideline (489, 2016), the percentage of DNA in tail (percent tail intensity) are considered as the most relevant DNA damage parameter. This parameter appears to be the most linearly related to dose (B. Burlinson et al., 2007). Then, the median of the percentage of DNA in tail measured in the different nuclei for each slide and for each animal were calculated, considering the animal as statistical unit (D. Lovell and T. Omori, 2008). Finally the mean of medians obtained for the different animals was calculated for each group.
In addition, each slide was also examined for presence of hedgehogs. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and apoptosis (therefore possible indicator of toxicity). A hedgehog results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. Hedgehogs were excluded from image analysis data collection and calculation of percent tail intensity. Their frequency might be useful for data interpretation. Statistical evaluation was done with the X2 test to determine significance of differences in group values between each group versus the vehicle control.
In the 2000, 1000 and 500 mg/kg/day (x2) treated groups the frequencies of hedgehogs were inferior to 50% whatever the organ assessed when compared to the relative vehicle control, in both male and female rats.
Otherwise, only a slight statistically significant increase in the percent hedgehogs was noted in the stomach of female rat at the intermediary dose of 1000 mg/kg/day(x2) with 2.51% of hedgehogs vs. 0.78% in the relative control group. It was without any incidence for the assessment of data for genotoxicity.
- Acceptance criteria for results of the Comet assay
A study is accepted if the following criteria are fulfilled:
- The mean of medians of percentage of DNA in tail of the concurrent negative controls should be within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).
- The concurrent positive controls should induce means of medians of percentage of DNA in tail that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent negative control.
- The appropriate number of doses and cells must be analyzed.
Moreover:
- In the vehicle group, an eventual increase in the frequency of hedgehogs must not be >50%.
The validity criteria for the test were considered as fulfilled with 2 exceptions regarding the results for positive control in male and female duodenum. Test was validated.
Evaluation criteria:
For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent negative control,
- This increase is dose-related when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.
A test item is considered clearly negative if:
- none of the test doses exhibits a statistically significant increase compared with the concurrent negative control,
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are within the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.
Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.
Sex:
male/female
Genotoxicity:
negative
Remarks:
glandular stomach, liver, duodenum and kidney cells
Toxicity:
no effects
Remarks:
but tested up to the limit dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
A slight stereotypy (slight head movements and chewing) was observed in all male and female rats 15 minutes after the first and the second treatment with 2000 mg/kg. Difficulty to breath for 1 male 15 minutes after the first treatment at this dose level was also noticed. No other clinical signs were noted whatever the period of observation or at both 1000 and 250 mg/kg/day (x2). Otherwise, during the sampling of stomachs for histological assessment and the halo assay (see below), it was noted that the stomachs from the highest dose group (2000 mg/kg) were larger than the ones from the other treatment groups.
- Evidence of cytotoxicity in tissue analyzed:
The histological assessment of a portion of the stomach (glandular part and forestomach) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of toxicity (i.e. assessment of necrosis).
As well, the halo assay performed on a portion of the stomach (glandular part only) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of either necrotic or apoptotic cells when compared to the concurrent vehicle control.
- Rationale for exposure:
From the overall results obtained in the dose range finding assay (i.e. clinical assessment, histological assessment of stomach cells and halo test), the highest dose of 2000 mg/ kg/ day (x2) was retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2) per os were also tested.

RESULTS OF DEFINITIVE STUDY
The mean of medians of percentage of DNA in tail of the concurrent negative controls were within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).
In all organs, the concurrent positive control induced statistically significant increases of percentage of DNA in tail compared with the negative control. These values are comparable to the historical positive control data with 2 exceptions in male and female duodenum with values of 20.08 and 20.49%, respectively vs. 28.45 – 73.93%.
No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in Isolated glandular stomach, liver, duodenum and kidney cells of OFA Sprague Dawley male and female rats.
Conclusions:
LUPEROX® TBEC is not genotoxic in glandular stomach, liver, duodenum and kidney from male and female rats treated once a day for 2 days with up to 2000 mg/kg.
Executive summary:

The evaluation of the genotoxic potential of LUPEROX® TBEC (98.2 %w/w of OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate) was done through the in vivo comet assay performed under alkaline conditions,i.e.pH > 13 (Alkaline Single Cell Gel Electrophoresis, OECD TG no. 485) in both male and female OFA Sprague Dawley rats, in isolated glandular stomach, liver, duodenum and kidney cells.

A Dose Range Finding assaywas performed with 4 groups of 3 male and 3 female rats receiving 2 daily treatments at 24-hour intervals at the doses of 2000, 1000, 250 and 0 mg/kg/day per os for the determination of the MTD (Maximal Tolerated Dose) and the assessment of the cytotoxicity of the test item on the glandular stomach of all animals by both a histological analysis and the halo test.

A slight stereotypy (slight head movements and chewing) was observed in all male and female rats 15 minutes after the first and the second treatment with 2000 mg/kg. Difficulty to breath for 1 male 15 minutes after the first treatment at this dose level was also noticed. No other clinical signs were noted whatever the period of observation or at both 1000 and 250 mg/kg/day (x2). Otherwise, during the sampling of stomachs for histological assessment and the halo assay (see below), it was noted that the stomachs from the highest dose group (2000 mg/kg) were larger than the ones from the other treatment groups.

The histological assessment of a portion of the stomach (glandular part and forestomach) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of toxicity (i.e.assessment of necrosis). As well, the halo assay performed on a portion of the stomach (glandular part only) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of either necrotic or apoptotic cells when compared to the concurrent vehicle control.

From the overall results obtained in the dose range finding assay (i.e.clinical assessment, histological assessment of stomach cells and halo test), the highest dose of 2000 mg/ kg/ day (x2) was retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2)per oswere also tested.

For the comet assay, groups of male and females rats (5/sex for the controls and low and mid dose levels, and 7/sex for the top dose level) were treated twice at 24-hour interval by oral route (gavage) with the test item at dose levels of 2000, 1000 and 500 mg/kg/day in corn oil, or with methylmethane sulfonate at 100 mg/kg/day in sterile water as positive control, or the vehicle corn oil under 10 ml/kg as negative control. All animals were sacrificed following one expression time of 3 to 6 hours after the last treatment. One hundred and fifty cells (50 cells/slide, 3 slides /animal) observed per animal.

The determination of LUPEROX® TBEC in treatment formulations was performed by a GLP- compliant laboratory using a validated analyticin treatment formulations used in the main assays were satisfactory with less than 10% deviation from target concentrations.

The mean of medians of percentage of DNA in tail of the concurrent negative controls were within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).

In all organs, the concurrent positive control induced statistically significant increases of percentage of DNA in tail compared with the negative control. These values are comparable to the historical positive control data with 2 exceptions in male and female duodenum with values of 20.08 and 20.49%, respectively vs. 28.45 – 73.93%.

 

Glandular stomach cells

 

in vivo COMET ASSAY IN ISOLATED RAT MALE GLANDULAR STOMACH CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

4.48

  

 

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

2.50

<0.05

0.50

N.S.

1000

2.50

<0.05

1.35

N.S.

500

2.73

<0.05

1.64

N.S.

Positive control

Methylmethane Sulfonate

 

100

39.39

-

<0.01

2.24

<0.01

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male rats.

Statistically significant decreases in the mean of medians of percentage of DNA in tail were noted at all doses. This effect was considered to be without biologically significance and any incidence in terms of genotoxic hazard.

The test item was thus not genotoxic toward the glandular stomach cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE GLANDULAR STOMACH CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

1.93

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

2.10

N.S.

1.15

N.S.

1000

2.01

N.S.

3.22

<0.01

500

2.23

N.S.

1.82

N.S.

Positive control

Methylmethane Sulfonate

 

100

43.17

-

<0.01

3.75

<0.01

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus not genotoxic toward the glandular stomach cells in female rat.

 

Liver cells

 

in vivo COMET ASSAY IN ISOLATED RAT MALE LIVER CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

0.29

  

 

<0.05

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.13

N.S.

0.38

N.S.

1000

0.16

N.S.

0.38

N.S.

500

0.45

N.S.

0.75

N.S.

Positive control

Methylmethane Sulfonate

 

100

25.52

-

<0.01

0.61

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male rats. A statistically dose-dependent effect was noted but was considered without biologically significance.

The test item was thus considered as not genotoxic toward the liver cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE LIVER CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

0.51

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.17

N.S.

2.01

N.S.

1000

0.23

N.S.

1.99

N.S.

500

0.31

N.S.

2.66

N.S.

Positive control

Methylmethane Sulfonate

 

100

24.34

-

<0.01

2.65

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus not genotoxic toward the liver cells in female rat.

 

Duodenum cells

 

in vivo COMET ASSAY IN ISOLATED RAT MALE DUODENUM CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

2.54

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

3.33

N.S.

0.65

N.S.

1000

2.41

N.S.

0.73

N.S.

500

4.54

N.S.

1.06

N.S.

Positive control

Methylmethane Sulfonate

 

100

20.08

-

<0.01

1.32

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male rats.

The test item was thus not genotoxic toward the duodenum cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE DUODENUM CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

2.83

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

2.24

N.S.

0.88

N.S.

1000

2.72

N.S.

0.83

N.S.

500

1.99

N.S.

0.44

N.S.

Positive control

Methylmethane Sulfonate

 

100

20.49

-

<0.01

0.89

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus not genotoxic toward the duodenum cells in female rat.

 

Kidney

 

in vivo COMET ASSAY IN ISOLATED RAT MALE KIDNEY CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

1.05

 

  

<0.05

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.31

<0.01

0.67

N.S.

1000

0.41

N.S.

0.89

N.S.

500

0.77

N.S.

0.82

N.S.

Positive control

Methylmethane Sulfonate

 

100

24.14

-

<0.01

0.18

<0.001

N.S.: not statistically significant at the threshold p=0.05

 

A statistically and dose-related significant decrease in the mean of medians of percentage of DNA in tail was observed at the highest tested doses of 2000 mg/kg/day (x2) in OFA Sprague Dawley male rats. This effect was considered to be without biological significance.

The test item was thus considered as not genotoxic toward the kidney cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE KIDNEY CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

0.43

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.33

N.S.

1.44

N.S.

1000

1.36

N.S.

2.27

N.S.

500

3.20

N.S.

1.98

N.S.

Positive control

Methylmethane Sulfonate

 

100

24.04

-

<0.01

1.71

N.S.

N.S.: not statistically significant at the threshold p=0.05

 

No statistically significant decrease or increase in the mean of medians of percentage of DNA in tail was observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus considered as not genotoxic toward the kidney cells in female rat.

 

In conclusion, LUPEROX® TBEC (98.2 %w/w of OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate) induced no biologically significant increase in DNA strand breaks. Therefore, LUPEROX® TBEC has no genotoxic activity in glandular stomach, liver, duodenum and kidney cells of male and female Sprague Dawley rats.

 

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:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
ICR mice were obtained from Harlan Sprague Dawley, Inc., Madison. At the initiation of the study, the mice were 6 to 8 weeks old. Animal body weights recorded at randomization were within the following ranges: Pilot study: males, 29.0 - 32.2 g, Females, 24.0 - 28.1 g; Toxicity study: Males, 27.8 - 35.1 g, Females, 22.6 - 25.6 g, Males, 28.1 - 35.5 g, Females, 23.0 - 30.1 g
Mice were obtained from a source monitored for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were quarantined for no less than 5 days after receipt. The mice were observed each working day for signs of illness, unusual food and water consumption, and other conditions of poor health. The animals were judged to be healthy prior to utilization in the assay.

Housing: controlled environment of 74±6°F, 50±20% relative humidity, and a 12 hour light dark cycle.
Mice of the same sex were housed up to five per cage in plastic autoclavable cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material.
Food and water ad libitum. There were no contaminants in the feed or water which were considered to have influenced the results of the study.

Mice were obtained from a source monitored for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were quarantined for no less than 5 days after receipt. The mice were observed each working day for signs of illness, unusual food and water consumption, and other conditions of poor health. The animals were judged to be healthy prior to utilization in the assay.





Route of administration:
intraperitoneal
Vehicle:
Vehicle: corn oil (CAS number 8001-30-1) obtained from Sigma Chemical Company.
Details on exposure:
20 mL/kg body weight
Duration of treatment / exposure:
Single intraperitoneal exposure
Frequency of treatment:
once
Post exposure period:
24 to 48 hours
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
10 for vehicle group, 5 for low-dose and mid dose group, 10 for high dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes: cyclophosphamide
Tissues and cell types examined:
bone marrow, erythrocytes
Details of tissue and slide preparation:
At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by C02 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 a capped 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 slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Evaluation criteria:
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.

All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated 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 unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control.
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 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 (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and 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:
Summary of bone Marrow Micronucleus Study Using Luperox TBEC 00-tert butyl 0 (2 ethylhexyl)monoperoxycarbonate
CAS # 34443-12-4:
Treatment Sex Time (hr) Number of mice PCE/Total Erythrocytes (Mean +-sd) Changes from control (%) Micronucleated Polychromatic number per 1000 PCEs (Mean +-sd) Erythrocytes numbers per PCEs scored
20 mL/kg corn oil M 24 5 0.47 ± 0.04 _ 0.2 ± 0.45 2 / 10000
20 mL/kg corn oil F 24 5 0.52 ± 0.03 _ 0.3 ± 0.45 3 / 10000
LuperoxTBEC 300 mg/kg M 24 5 0.48 ± 0.07 2 0.3 ± 0.27 3 / 10000
LuperoxTBEC 300 mg/kg F 24 5 0.55 ± 0.03 6 0.5 ± 0.00 5 / 10000
Luperox TBEC 600 mg/kg M 24 5 0.50 ± 0.04 6 0.4 ± 0.42 4 / 10000
Luperox TBEC 600 mg/kg F 24 5 0.51 ± 0.06 -2 0.2 ± 0.27 2 / 10000
Luperox TBEC 1200 mg/kg M 24 5 0.45 ± 0.05 -4 0.5 ± 0.35 5 / 10000
Luperox TBEC 1200 mg/kg F 24 5 0,5 ± 0.05 -4 0.5 ± 0.35 5 / 10000
CP 50 mg/kg M 24 5 0.47 ± 0.06 0 35.0 ± 7.19 * 350/10000
CP 50 mg/kg F 24 5 0.46 ± 0.09 -12 32.0 ± 9.25 * 320/10000
20 mL/kg corn oil M 48 5 0.53 ± 0.03 _ 0.2 ± 0.27 2 /10000
20 mL/kg corn oil F 48 5 0.56 ± 0.02 _ o. 1 ± 0.22 1 /10000
Luperox TBEC 1200 mg/kg M 48 5 0.49 ± 0.06 -8 0.2 ± 0.27 2/10000
Luperox TBEC 1200 mg/kg F 48 5 0.45 ± 0.03 -20 0.3 ± 0.45 3/10000



1*, ps0.05 (Kastenbaum-Bowman Tables)








Pilot toxicity study results:

 

Treatment

 

ClinicalObservation

 

Number of Animals with Clinical Signs/ Total

Number of Animals

Dosed

 

Number of Animals Died/ Total Number of Animals Dosed

 

Males

 

Females

 

Males

 

Females

 

Luperox TBEC,

1mg/kg

 

Normal

 

2/2

 

NA

 

0/2

 

NA

 

Luperox TBEC,

10mg/kg

 

Normal

 

2/2

 

NA

 

0/2

 

NA

Luperox TBEC,

100mg/kg

Normal

2/2

NA

0/2

NA

 

Luperox TBEC,

1000mg/kg

 

Lethargy

 

2/2

 

NA

 

0/2

 

NA

 

Luperox TBEC,

2000mg/kg

 

Lethargy

Piloerection Prostration

Irregular breathing

 

5/5

5/5

 

5/5

5/5

1/5

1/5

 

5/5

 

5/5

 

Treatment

 

ClinicalObservation

 

Number of Animals with Clinical Signs/ Total

Number of An1mals

Dosed

 

Number of Animals Died/Total Number of Animals Dosed

 

Males

 

Females

 

Males

 

Females

 

Luperox TBEC,

1200mg/kg

 

Lethargy

Piloerection

Diarrhea

 

5/5

5/5

2/5

 

5/5

5/5

2/5

 

0/5

 

0/5

 

Luperox TBEC,

1400mg/kg

 

Lethargy

Piloerection

Diarrhea

 

5/5

5/5

2/5

 

5/5

5/5

2/4

 

2/5

 

0/5

 

Luperox TBEC,

1600mg/kg

 

Lethargy

Piloerection

Diarrhea

Prostration

Irregular breathing

Crustyeyes

 

5/5

5/5

2/5

1/5

 

2/5

 

3/5

3/5

2/5

4/5

4/5

 

3/5

 

4/5

 

Luperox TBEC,

1800 mg/kg

 

Prostration

Irregular breathing

Diarrhea

Lethargy

Piloerection

Crustyeyes

 

5/5

5/5

3/5

2/5

2/5

 

5/5

5/5

2/5

 

2/5

 

5/5

 

5/5

Clinical Signs Following Dose Administration of Luperox TBEC

 

Treatment

 

ClinicalObservation

 

Number of Animals with Clinical Signs/ Total

Number of Animals

Dosed

 

Number of Animals Died/Total Number of Animals Dosed

 

Males

 

Females

 

Males

 

Females

 

CornOil,

20 mL/kg

 

Normal

 

10/10

 

10/10

 

0/10

 

0/10

 

Luperox  TBEC,

300 mg/kg

 

Lethargy

Piloerection

 

5/5

5/5

 

5/5

5/5

 

0/5

 

0/5

 

Luperox TBEC,

600 mg/kg

 

Lethargy

Piloerection

 

5/5

5/5

 

5/5

5/5

 

0/5

 

0/5

 

Luperox TBEC,

1200 mg/kg

 

Lethargy Piloerection

D1arrhea

 

15/15

15/15

6/15

 

15/15

15/15

7/15

 

0/15

 

0/15

 

CP,50mg/kg

 

Normal

 

5/5

 

5/5

 

0/5

 

0/5

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions, 00-tert butyl 0 (2 ethylhexyl) monoperoxycarbonate, CAS 34443-12-4, did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice.
Executive summary:

00-tert butyl 0 (2 ethylhexyl) monoperoxycarbonate was tested in a Mammalian Erythrocyte Micronucleus Test, according to a method similar to the OECD n° 474 Guideline and in compliance with the Principles of Good Laboratory Practice

In this micronucleus  assay, male and female  mice were dosed with 300, 600 or 1200 mg/kg body weight. No mortality was observed in any male or female mice during the course of the study.  Clinical signs following dose administration included: lethargy and piloerection in all male and female mice at ail dose levels and diarrhea in males and females at 1200 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in all male and female mice at all dose levels and diarrhea in males and females at 1200 mg/kg. 

 

Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes.  Reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls.  Reductions of 20% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in females at high dose at 48 hours which suggests test article availability in bone marrow.  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-Bov.man).

 

Reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls.  Reductions of 20% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in females at high dose at 48 hours which suggests test article availability in bone marrow.  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-Bov.man).

 

  

The results of the assay indicate that under the conditions described in this report, 00-tert butyl 0 (2 ethylhexyl) monoperoxycarbonate, CAS # 34443-12-4 did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. 

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Swiss Ico: OF1 (IOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
Animals
Reason for the choice of the strain: rodent species generally accepted by regulatory authorities for this type of study.
Breeder: Charles River Laboratories, l'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.
Acclimation: 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
. temperature: 22 ± 2°C,
. relative humidity: 30 to 70%,
. light/dark cycle: 12 h/12 h (07:00 - 19:00),
. ventilation: at least 12 cycles/hour of filtered non-recycled fresh air.

Food and water ad libitum
Each batch of food wass analysed by the supplier for composition and contaminant levels. Bacteriological and chemical analysis of water are performed regularly by external laboratories, These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
No contaminants were known to have been 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:
The vehicle was corn oil, batch No. 81K2204 (Sigma, 38297 Saint-Quentin-Fallavier, France). For the main test, the test item was suspended in the vehicle in order to achieve the concentrations of 50, 100 or 200 mg/mL and then homogenized using a magnetic stirrer. Using a treatment volume of 10 mL/kg, the target dose-levels were 500, 1000 or 2000 mg/kg/day.
Details on exposure:
. Route for the vehicle and the test item: oral, since it is a possible route of exposure in Man,
. Frequency: two treatments separated by 24 hours,
. Volume: 10 mL/kg,
. CPA: oral route, one treatment.
Duration of treatment / exposure:
two oral exposure
Frequency of treatment:
twice, at 24 hours of interval
Post exposure period:
24 hours afeter the last exposure, then the sampling was performed
Remarks:
Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Preliminary toxicity test: 3 males and 3 females mice were used.
Main cytogenetic test: 56 mice, 28 males and 28 females were used.: 5 males and 5 females for each dose level, excepting 2000 mg/kg (8 males and 8 females because one animal died; for that group the surviving supplementary animals were humanely killed and bone marrow smears were not prepared)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide dissolved in distilled water at a concentration of 5 mg/mL.
Tissues and cell types examined:
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).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: see preliminary test

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).
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 ¿2 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
Remarks:
Clinical signs at 1000 and 2000 mg/g, no decrease of the ratio PE/NE in females, slight decrease for male, but not statistically significant
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: with 3 male and 3 female; Clinical signs and any mortality were recorded for a period of 48 hours. 2000 mg/kg/day were administered twice.The interval between each administration was 24 hours. At this dose-level, hypoactivity, dyspnea, half-closed eyes and/or piloerection were noted but no mortality was induced. 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 selected for the main test was 2000mg/kg/day. The two other selected dose-levels were 1000 and 500 mg/kg/day.

Main test: No clinical signs and no mortality were observed in the animals of both sexes given 500mg/kg/day. At 1000 mg/kg/day, half-closed eyes and piloerection were noted in females, 2 hours following the second treatment. At 2000 mg/kg/day, half-closed eyes, hypoactivity and/or piloerection were noted in both males and females. In addition, one female from the main group and two females from the supplementary animals died during the observation period.
Conclusions:
TBEC 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/day.
Executive summary:

TBEC was tested in anMammalian Erythrocyte Micronucleus Test, according to the OECD n° 474 Guideline guideline and in compliance with the Principles of Good Laboratory Practice.  

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  LUPEROX  TBEC  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 were killed 24 hours after the last 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 as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle 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.

Under  these  experimental  conditions,  TBEC 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/day.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Evaluation of DNA damage (8-OH-dG formation) and sustained epidermal hyperplasia when administered to female Sencar mice topically twice weekly for four weeks
GLP compliance:
yes
Type of assay:
other: 8-OH-dG formation
Species:
mouse
Strain:
Sencar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Cancer Institute Laboratories, Frederick, MD.
- Age at study initiation: 7 -9 weeks old
- Weight at study initiation:
- Assigned to test groups randomly: yes, under following basis: body weight
- Housing: up to 10 mice/cage.
- Diet: Rodent chow Wayne, ad libitum
- Water: Deionized or distilled water, ad libitum
- Acclimation period: 2-3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 4
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
Details on exposure:
Ten SENCAR female virgin mice per dose level were used, and three dose levels were used to compare hyperplasiogenic activity with 2 µg 12-0-tetradecanoyl phorbol-13-acetate (TPA); acetone served as a vehicle control for TPA. Applications were made twice weekly for four weeks, in 200 µL acetone. In addition, dimethylbenz[a]anthracene (DMBA) (100 nmols, 2x/wk for 4 wks), served as a positive control for sustained hyperplasia, 8-OH-dG formation and Ha-ras mutation.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
twice weekly
Post exposure period:
2 days
Dose / conc.:
10 other: µmol/mouse
Dose / conc.:
100 other: µmol/mouse
Dose / conc.:
200 other: µmol/mouse
No. of animals per sex per dose:
10
Control animals:
other: Acetone (Groups 4 and 38), ethanol (Group 40) and dimethyl phthalate (Group 41) were used as vehicle contrais. No Treatment animals (Groups 5 and 39) were used as additional negative controls.
Positive control(s):
7,12-dimethylbenzanthracene (DMBA) and 12-0-tetradecanoyl phorbol-13-acetate (TPA)
Tissues and cell types examined:
Skin cells
Details of tissue and slide preparation:
- Clinical Observations:
Twice daily, for evidence of mortality or moribundity.
- Physical Examinations:
Once prior to treatment and weekly thereafter.
- Body Weights:
Once prior to treatment and weekly thereafter.
- Necropsies
. Gross Necropsy
The necropsy will include examination of:
•Extemal body surface
•AH orifices
•Carcass cervical tissues and organs
•Thoracic, abdominal, and pelvic cavities and their viscera.
. Tissue Preservation:
The following tissues (when present) from each animal will be preserved in neutral-buffered formalin:
•Approximately 1 cm² of the dorsal skin
•Gross lesions and abnormalities.
The remaining portion of the dorsal skin from each animal will be quick frozen in liquid nitrogen:
- Histopathology
Approximately 1 cm² of dosed dorsal skin from each animal was preserved in formalin for slide preparation and the remainder of the dosed skin was rapidly frozen for isolation of DNA. Epidermal thickness was determined for each animal from at least 20 randomly selected sites of interfollicular epidermis, using formalin-fixed, paraffin-embedded sections (5 µm) stained with hematoxylin and eosin. Dermal cellularity was determined by light microscopie determination of the number of dermis-associated cells within 10 high magnification ( 1000 x) fields of fixed, stained sections of skin from each mouse. Dermal cellularity, considered a measure of inflammation is expressed as the mean for all mice in each treatment group. All dermis-associated cells were counted, including polymorphonuclear cells, lymphocytes, macrophages, fibroblasts, and mast cells.
- 8-OH-dG Determination
The 8-OH-dG content of DNA isolated from dosed mouse skin was used as a measure of treatment-related oxidative damage. DN A was isolated from freshly frozen skin of each mouse following non-phenol extraction and ethanol precipitation. Approximately 100 µg of isolated DNA was digested to nucleosides with nuclease P1 and alkali ne phosphatase. Quantitation of nucleosides was accomplished by high performance liquid chromatography (LC- 600/SPD-6A-CR4A HPLC System, Shimadzu Corp., Columbia, MA) (4,33,34) with electrochemical detection using an ESA system, (ESA, !ne., Chelmsford, MA). Normal bases were quantitated by liquid chromatography with U V detection. Data were expressed as pmol 8-OH-dG/10e5 pmol dG. Al! analyses were performed in duplicate or triplicate, with appropriate standard curves ta correlate area units or peak height with concentration. Skin from mice treated with DMBA (100 nmol, 2x/wk for 4 wks) served as the positive control and skin from vehicle­treated and untreated animals served as negative controls.
Statistics:
To determine which treatment groups differed from the control groups, a one-way analysis of variance was performed on each measured parameter (e.g., epidermal thickness). If the treatment means were determined to be significantly different (p<0.05), the means were compared using a Tukey honestly significant difference (HSD) ranked mean comparison test. If only one control was used, Dunnett's ranked means test would be more appropriate. However, with multiple controls, both positive and negative, use of the HSD is more appropriate. These post hoc tests were developed to reduce the number of equivocal positive differences to the Type I error from those differences which are seen when multiple t-tests are performed. The HSD results provide groups of treatments which are not significantly different, and these groups may overlap. Usually the treatment means are ordered from smallest to largest and lines are drawn to indicate the means that could not be distinguished (that are similar) at the desired significance level (p<0.05). Since the untreated and acetone-treated groups were not different, they were pooled to give a single limit. This limit is based upon a one-sided t-test and bas been calculated for both 2 and 4 day observations. The mean epithelial thickness values did not differ significantly from the mean values of acetone and untreated groups. The pooling of nontreated and acetone treated control groups is justified because there is no significant difference between the two. The pooling increases the number of degrees of freedom in the pooled controls while not significantly increasing the variance and standard deviations, and thus, increases the sensitivity of the test. The same approach was used for statistical analyses of HPLC data where pooled controls of nontreated and acetone treated groups were used since there was no statistical difference between the two. The same method was also applied to analyze total cellularity.
Sex:
female
Genotoxicity:
other: no increase of 8-OH-dG/dG ratios compared to acetone control
Toxicity:
yes
Remarks:
increase of total skin cellularity
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Body Weight and Food Consumption
The mean weights of the animals were analyzed at day 2 and day 4 after treatment by one-way analysis of variance (ANOVA). There were no significant differences in body weight, body weight gains, and food consumption between animals treated with TBEC and with vehicle or untreated control animals.

- Clinical Signs and Gross Necropsy
There were no observed abnormalities.

- Sustained Epidermal Hyperplasia
TBEC showed no hyperplastic effects at any of three doses tested (Tables 1 and 2).

- Dermal Cellularity
Total cellularity was considered as an important measure of inflammation caused by the test materials. DMBA (100 nmol), TPA and TBEC produced a significant increase in total cellularity. TBEC do not produce any significant level of inflammation.

- 8-Hydroxy-2'-deoxyguanosine Formation
Oxygen free radical attack on DNA may lead to strand breakage and/or formation of modified bases like thymine glycol, hydroxymethyluracil, or 8-hydroxy-2'-deoxyguanosine. Oxygen free radical damage to DNA may be repaired depending on the type and extent of the damage, but little is known
regarding the repair of 8-hydroxy-2'-deoxyguanosine in vivo. TBEC did not produced statistically significant increases in 8-0H-dG levels compared to control animals (Table 3). As expected, the positive control DMBA ( 100 nmol) produced elevated levels of 8-0H-dG.

Table 3. Average 8 -OH-dG/dG Ratios x 10e5 ± S.D. Determined by HPLC/ECD

 

Group No.

Treatmenl

Dose

Ratio 8 -OH-dG/dG

X 10e5

± S.D.

1

DMBA

100 nmol

9.96*

2.0

2

DMBA

10 nmol

2.21

2.5

3

TPA

 

2µgl

5.60

1.9

4

ACT

-

4.16

0.4

5

NT

-

2.20

1.3

33

TBEC

10 µmol

1.66

2.0

34

100 µmol

2.49

0.8

35

200 µmol

1.57

2.0

 

Calf Thymus

-

2.24

0.2

*lndicate statistically significant increase over controls (P <0.05)

 

Conclusions:
Interpretation of results (migrated information): negative no increase of 8-OH-dG/dG ratios
Executive summary:

The ability of t-Butyl O-(2-ethyl-hexylmonoperoxycarbonate (TBEC) to produce DNA damage, as 8-Hydroxy-2'-deoxyguanosine (8 -OH-dG), formation and sustained epidermal hyperplasia was evaluated when administered to female Sencar mice topically twice weekly for four weeks. Epidermal and dermal hyperplasia and total dermal cellularity were measured at day 2 and day 4 after last dosing. 8-OH-dG/dG ratio was determined by HPLC/ECD using DNA isolated from frozen skins. When applied repetitively, to the dorsal skin of Sencar mice in doses of 10, 100 and 200 µmol/mouse, TBEC caused an increase in dermal cellularity over the threshold. No increase (compared to acetone controls) of 8-OH-dG was detected in DNA isolated from the epidermis after repetitive applications of 200 µmol/mouse TBEC. The results indicate that no 8-OH-dG is formed in vivo in cellular DNA upon treatment with the peroxide. The positive control, DMBA (100 nmoles), effectively induced DNA damage (8-OH-dG), epidermal hyperplasia and dermal hyperplasia.

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Principles of method if other than guideline:
Evaluation of Ha-ras mutations and sustained epidermal hyperplasia when administered to female Sencar mice topically twice weekly for four weeks
GLP compliance:
yes
Type of assay:
other: Ha-ras mutations
Species:
mouse
Strain:
Sencar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Cancer Institute Laboratories, Frederick, MD.
- Age at study initiation: 7 -9 weeks old
- Weight at study initiation:
- Assigned to test groups randomly: yes, under following basis: body weight
- Housing: up to 10 mice/cage.
- Diet: Rodent chow Wayne, ad libitum
- Water: Deionized or distilled water, ad libitum
- Acclimation period: 2-3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 4
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: acetone; ethanol; dimethyl phthalate
Details on exposure:
Ten SENCAR female virgin mice per dose level were used, and three dose levels were used to compare hyperplasiogenic activity with 2 µg 12-0-tetradecanoyl phorbol-13-acetate (TPA); acetone served as a vehicle control for TPA. Applications were made twice weekly for four weeks, in 200 µL acetone. In addition, dimethylbenz[a]anthracene (DMBA) (100 nmols, 2x/wk for 4 wks), served as a positive control for sustained hyperplasia, 8-OH-dG formation and Ha-ras mutation.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
twice weekly
Post exposure period:
2 days
Remarks:
Doses / Concentrations:
10, 100 and 200 µmol/mouse
Basis:

No. of animals per sex per dose:
10
Control animals:
other: Acetone (Groups 4 and 38), ethanol (Group 40) and dimethyl phthalate (Group 41) were used as vehicle controls. No Treatment animals (Groups 5 and 39) ere used as additional negative controls.
Positive control(s):
7,12-dimethylbenzanthracene (DMBA) and 12-0-tetradecanoyl phorbol-13-acetate (TPA)
Tissues and cell types examined:
Skin cells
Details of tissue and slide preparation:
- Clinical Observations:
Twice daily, for evidence of mortality or moribundity.
- Physical Examinations:
Once prior to treatment and weekly thereafter.
- Body Weights:
Once prior to treatment and weekly thereafter.
- Necropsies
. Gross Necropsy
The necropsy will include examination of:
•Extemal body surface
•AH orifices
•Carcass cervical tissues and organs
•Thoracic, abdominal, and pelvic cavities and their viscera.
. Tissue Preservation:
The following tissues (when present) from each animal will be preserved in neutral-buffered formalin:
•Approximately 1 cm² of the dorsal skin
•Gross lesions and abnormalities.
The remaining portion of the dorsal skin from each animal will be quick frozen in liquid nitrogen:
- Histopathology
Tissues will be embedded, sectioned, and stained with hematoxylin and eosin. Epithelial and dermal thickness will be determined visually from each skin by light microscopy. Photographs will be taken of all samples for documentation (see RSS "8-0H-dG formation" same section)
- Detection of c-Ha ras Mutations
Four days after the last administration, DNA isolated from freshly­frozen tissues of 5 mice per group was analyzed for mutations in codons 12 & 13, and codon 61 of c-Ha-ras by PCR analysis. 4-day specimens were used for this analysis because some of the 2-day DNAs were degraded and therefore not suitable for Ha-ras analysis. In codons 12 & 13 there is a Mnl 1 restriction site which spans the three nucleotides of codon 12 and the first nucleotide of
codon 13 in the wild type sequence. Mutations in any of these bases result in the loss of the Mnl 1 site. exon 1 (which contains codons 12 and 13) of the c-Ha-ras gene was amplified from genomic DNA using a Perkin-Elmer thermal cycler. The reaction was extracted with phenol­CHCl3 and the DNA was precipitated with ethanol. The DNA was then resuspended in enzyme buffer, and the PCR product was restricted with Mnl 1 and the digest electrophoresed on a 8% nondenaturing polyacrylamide gel. No loss of Mnl restriction site was observed and the conclusion was that there are no mutations in codons 12 and 13 in the tested material. DNA for Ha-ras analysis was also obtained from paraffin-embedded sections cut at 8 µ from samples collected two days after last dosing. The sections from each paraffin block were placed in microfuge tubes, deparaffinized with xylene and ethanol, centrifuged and resuspended in 5% chelex with proteinase K.
The first procedure used for Ha-ras codon 61 was derived from Nelson et al. The 3MSP61 mut reverse primer was designed so that its 3' end nucleotide (A) pairs with the middle nucleotide (underlined) of a CAA->CTA transversion in codon 61, and selectively amplifies mutated DNA under the conditions described below. The assay was based on the fact that Taq polymerase lacks 3' exonuclease activity and thus cannot repair a rnismatch at the 3' end of the annealed primer. The conditions of the assay depend on the reverse primer failing to anneal sufficiently to the wild type sequence so that extension does not occur. Using the same forward primer, one reaction was run with the reverse rnismatch primer (3MSP61mut) and another reaction was run with a reverse wild type primer (3MSP61 wt). This protocoJ detects only CAA->CTA transversion, mutations that are the most prevalent in codon 61 point mutations. An Xbal RFLP site (T/CTAGA) is created in this transversion. The reactions containing the mismatch products were run on 2% low melt agarose for subsequent purification and sequencing. The ratio of the amount of cut (wild type DNA) to uncut (mutated DNA) was determined by quantifying ethidium bromide staining intensity or 32p labeling. The DNA from the plasmid pHras61mut was used as a positive control sample. The plasmid pHras61 contains cloned exon 2 Ha-ras DNA from a Sencar mouse tumor. The cloned mutation was verified by DNA sequencing. The mutation is the CAA­>CTA transversion in codon 61 (located in exon 2) of the mouse Ha-ras gene. The following primers were used in this assay:
5MSP61 (23 mer) 5'-CTA AGC CTG TTG TTT TGC AGG AC-3'
3MSP61mut (20 mer) 5'-CAT GGC ACT ATA CTC TTC TA-3'
3MSP6l wt (20 mer) 5'-CAT GGC ACT ATA CTC TTC TT-3'.
The sequence of 3MSP61wt bas 2 or 3 mismatches from N-ras and K-ras sequences, respectively. The fragment size that is amplified is 110 bp.
Statistics:
To determine which treatment groups differed from the control groups, a one-way analysis of variance was performed on each measured parameter (e.g., epidermal thickness). If the treatment means were determined to be significantly different (p<0.05), the means were compared using a Tukey honestly significant difference (HSD) ranked mean comparison test. If only one control was used, Dunnett's ranked means test would be more appropriate. However, with multiple controls, both positive and negative, use of the HSD is more appropriate. These post hoc tests were developed to reduce the number of equivocal positive differences to the Type I error from those differences which are seen when multiple t-tests are performed. The HSD results provide groups of treatments which are not significantly different, and these groups may overlap. Usually the treatment means are ordered from smallest to largest and lines are drawn to indicate the means that could not be distinguished (that are similar) at the desired significance level (p<0.05). Since the untreated and acetone-treated groups were not different, they were pooled to give a single limit. This limit is based upon a one-sided t-test and bas been calculated for both 2 and 4 day observations. The mean epithelial thickness values did not differ significantly from the mean values of acetone and untreated groups. The pooling of nontreated and acetone treated control groups is justified because there is no significant difference between the two. The pooling increases the number of degrees of freedom in the pooled controls while not significantly increasing the variance and standard deviations, and thus, increases the sensitivity of the test. The same approach was used for statistical analyses of HPLC data where pooled controls of nontreated and acetone treated groups were used since there was no statistical difference between the two. The same method was also applied to analyze total cellularity.
Sex:
female
Genotoxicity:
negative
Remarks:
no c-Ha-ras proto-oncogene mutations
Toxicity:
yes
Remarks:
increased dermal cellularity
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Body Weight and Food Consumption
The mean weights of the animals were analyzed at day 2 and day 4 after treatment by one-way analysis of variance (ANOVA). There were no significant differences in body weight, body weight gains, and food consumption between animals treated with TBEC and with vehicle or untreated control animals.

- Clinical Signs and Gross Necropsy
There were no observed abnormalities.

- Sustained Epidermal Hyperplasia
TBEC showed no hyperplastic effects at any of three doses tested (Tables 1 and 2).

- Dermal Cellularity
Total cellularity was considered as an important measure of inflammation caused by the test materials. DMBA (100 nmol), TPA and TBEC produced a significant increase in total cellularity. TBEC do not produce any significant level of inflammation.

- Ha-ras Mutation Analysis
90% (9/10) of DMBA (100 nmol) samples showed Ha-ras mutations in this assay. TBEC dose level of 200 µmol/mouser didn'ts induce CAA->CTA transversions in codon 61 of the mouse Ha-ras gene using MSP-32P assay and DNA isolated from 25 paraffin sections (8 µm).
Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

The ability of TBEC to produce CAA->CTA transversions in codon 61 of the mouse Ha-ras gene and sustained epidermal hyperplasia was evaluated when administered to female Sencar mice topically twice weekly for four weeks. Epidermal and dermal hyperplasia and total dermal cellularity were measured at day 2 and day 4 after last dosing. Ha-ras gene mutation was determined in DNA isolated from 25 paraffin sections (8µm) using MSP-32P assay. No Ha-ras gene mutation was detected in DNA isolated from the epidermis 4 days after repetitive applications of 200 µmol/mouse TBEC. The positive control, DMBA (100 nmoles), effectively induced Ha-ras gene mutations, epidermal hyperplasia and dermal hyperplasia.

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

Additional information

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), TBEC is not classified for genotoxicity.