tert-butyl peroxypivalate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-03 to 2021-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 2017/735, Annex Part B, B.62: In vivo Mammalian Alkaline Comet Assay

Version / remarks:

2017-02-14

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl peroxypivalate

EC Number:

213-147-2

EC Name:

tert-butyl peroxypivalate

Cas Number:

927-07-1

Molecular formula:

C9H18O3

IUPAC Name:

tert-butyl 2,2-dimethylpropaneperoxoate

additive 1

Reference substance name:

2,2,4,6,6-pentamethylheptane

EC Number:

236-757-0

EC Name:

2,2,4,6,6-pentamethylheptane

Cas Number:

13475-82-6

Molecular formula:

C12H26

IUPAC Name:

isododecane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HAN:WIST

Details on species / strain selection:

Rats are routinely tested in this test and the chosen Wistar rat strain was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at the test laboratory.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90, Hungary
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 254-286 g, the weight variation in animals involved at the start of the study did not exceed ± 20 %.
- Assigned to test groups randomly: All animals were sorted according to body weight by computer and grouped according to weight ranges.
- Fasting period before study: Animals were not fasted before treatment.
- Housing: 3 animals/cage (treatment+vehicle groups), 2 animals/ cage (positive control) in Type III polypropylene/polycarbonate cages with certified laboratory wood bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3-23.8
- Humidity (%): 43-65
- Air changes (per hr): More than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From September 8th to September 09th

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Sunflower oil
- Justification for choice of vehicle: Based on the information about previously performed studies with the test item and based on a 14-Day Oral Gavage Dose Range Finding Study (see section 7.5.1).
- Concentration of test material in vehicle: 300, 150 and 75 mg/mL
- Amount of vehicle: 5 mL (test groups and negative control) and 10 mL (positive control)
- Batch No: 8005514002

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The treatment solutions were prepared freshly before each treatment.

Duration of treatment / exposure:

27-28 hours (The test item was administered by oral gavage twice: Once on the day 0 and 24 hours thereafter. The negative control animals were treated concurrently with the vehicle, only. The positive control animals were treated by oral gavage once during the experiment on the day 1. Samples were taken 3-4 hours after the second treatment.)

Frequency of treatment:

Treatment and vehicle control groups were treated twice (once on day 0 and 24 hours thereafter) and the positive control group was treated once on day 1.

Post exposure period:

Samples were taken 3-4 hours after the last treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

The measured concentration values were slightly higher (9-15 % higher) than the nominal values

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

The measured concentration values were slightly higher (9-15 % higher) than the nominal values

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

The measured concentration values were slightly higher (9-15 % higher) than the nominal values

No. of animals per sex per dose:

6 male animals per test item dose or vehicle control groups and 4 male animals in the positive control group. Only samples of 5 animals per dose and vehicle group and samples of 3 animals of the positive control group were analyzed.

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate (EMS)
- Route of administration: Oral by gavage
- Doses / concentrations: 200 mg/kg bw

Examinations

Tissues and cell types examined:

Stomach, duodenum and liver tissues

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
The sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hour after the last treatment. In this particular test and based on the experience of the laboratory over the last years, the sampling was performed 3-4 hours after the second treatment.

DETAILS OF SLIDE PREPARATION:
Conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. Thereafter the underside of the slides was wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and were allowed to dry. Before use, a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA and covered with a glass coverslip. The slides were placed on a tray until the agarose hardened (~ 5 minutes). After the cell isolations, each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter, a cell suspension (100 or 65 μL) containing ~104 – 105 cells was added on the microscope slide after gentle slide off the coverslip. The microscope slides were covered with a new coverslip. After the LMPA cell mixture hardened, an additional 70 μL of N MA was dropped on the microscope slide after a gentle slide off the (second) coverslip and a new coverslip was laid on the slide. After the repeated NMA layer hardened the coverslip was removed. After the top layer of agarose solidified and the last glass coverslip was removed, the slides were immersed in chilled lysing solution overnight at 2-8 °C in the dark. After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This rinsing procedure was performed in electrophoresis buffer. The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The slides were left in the electrophoresis tank for 30 min for the DNA to unwind. Thereafter, the electrophoresis was conducted for 30 min by applying a constant voltage of 0.7 V/cm and an electric current of about 300 mA (actual values: 270-302 mA). After electrophoresis, the slides were removed from the electrophoresis unit, were covered with neutralization solution, allowed to stand covered for about 5 minutes, thereafter blotted and covered again with neutralization solution. This procedure was repeated twice. Subsequently, the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides. The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 2 μg/mL Ethidium bromide.

METHOD OF ANALYSIS
The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Olympus BH-2) equipped with an appropriate excitation filter (TRITC) and with an Alpha DCM 510B CMOS camera. For image analysis, the Andor Kinetic Imaging Komet 6.0 (Andor Technology) was used. For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment and per vehicle control and 450 per positive control). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection.

Evaluation criteria:

The test chemical is clearly negative if:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
• there is no concentration-related increase when evaluated with an appropriate trend test;
• all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administrations;
• direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

The test chemical is clearly positive if:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
• the increase is dose-related when evaluated with an appropriate trend test;
• any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administrations;

There is no requirement for verification of clearly negative or positive response.

Statistics:

The statistical significance of % tail DNA values, tail length, OTM values and number of ghost cells was calculated using the appropriate statistical method, using SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the intergroup comparisons were performed using Mann-Whitney U-test.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Body weight reduction, strongly reduced activity, piloerection, increased respiration rate, general lethargy, strong diarrhoea, narrow palpebral fissure, incoordination, prone position, cyanosis (2000 mg/kg bw); reduced activity, increased respiration frequency, incoordination, general lethargy, abdominal distension, piloerection (1800 mg/kg bw) (1800 mg/kg bw); General lethargy, piloerection, at one animal reduced activity, increased respiration frequency (1500 mg/kg bw)

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: According to OECD guideline 489, a dose where some signs of toxicity but no severe pain or suffering occurs shoud be selected as highest dose. In the highest test dose used in this assay, animals showed some behavioural changes and clinical signs (piloerection, decreased activity, increased respiration rate, diarrheoa, unstable motion) one hour after the second treatment and symptoms were intensified just before sacrifice. At this time point, also animals in the 750 mg/kg bw group showed piloerection, decreased activity and increased respiration (2 animals). These symptoms were in line with the requirements of the OECD guideline and thus, dose selection was considered appropriate. The oral route was considered to be the most relevant exposure route and the glandular stomach, the duodenum and the liver were selected as the tissues of interest as most exposed organs by this exposure route.
- Statistical evaluation: The statistical evaluation of data (% tail DNA, tail length and OTM) of stomach and duodenum samples did not show statistically significant differences from that of the vehicle control in any case. In the case of liver samples, no statistically significant differences were obtained between the mean median % tail DNA value of the vehicle control and each dose. The % tail DNA values showed a slightly increasing tendency with the increasing doses, but the linear trend analysis did not show significance, consequently no clear dose related increase in % tail DNA values was obtained. The effect ratio (ratio indicated an increase of means of % tail DNA of each dose over the vehicle control value) values were in the range of 1.02-1.20, showing a slight change that was no significant from a mutagenicity point of view.

Any other information on results incl. tables

Table 1: Tail DNA % of Medians per Slide, Means per Animal, per Dose (Stomach)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	Tail DNA %
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Mean Median of 750 cells)
Sunflower oil (Helianthi annui oleum raffinatum) (x2)	109	109S1	50	150	18.78	12.54	13.70   SD: 4.92
		109S2	50		9.51
		109S3	50		9.33
	114	114S1	50	150	7.98	7.22
		114S2	50		8.31
		114S3	50		5.37
	118	118S1	50	150	8.53	11.57
		118S2	50		12.10
		118S3	50		14.08
	122	122S1	50	150	17.25	17.76
		122S2	50		15.61
		122S3	50		20.44
	129	129S1	50	150	20.49	19.41
		129S2	50		18.28
		129S3	50		19.46
TBPPI-75-AL [375 mg/kg body weight/day (x2)]	104	104S1	50	150	15.82	18.47	15.13   SD: 3.91 NS
		104S2	50		20.83
		104S3	50		18.77
	113	113S1	50	150	18.21	14.34
		113S2	50		14.46
		113S3	50		10.34
	115	115S1	50	150	15.50	14.83
		115S2	50		12.22
		115S3	50		16.78
	116	116S1	50	150	9.68	9.17
		116S2	50		8.28
		116S3	50		9.55
	128	128S1	50	150	13.85	18.84
		128S2	50		20.84
		128S3	50		21.84
TBPPI-75-AL [750 mg/kg body weight/day (x2)]	110	110S1	50	150	14.15	14.33	16.50   SD: 3.92 NS
		110S2	50		13.82
		110S3	50		15.04
	111	111S1	50	150	12.99	14.96
		111S2	50		16.69
		111S3	50		15.19
	125	125S1	50	150	11.55	13.33
		125S2	50		18.10
		125S3	50		10.34
	126	126S1	50	150	23.96	16.72
		126S2	50		12.69
		126S3	50		13.52
	127	127S1	50	150	16.70	23.16
		127S2	50		21.11
		127S3	50		31.69

Table 1 (continued): Tail DNA % of Medians per Slide, Means per Animal, per Dose (Stomach)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	Tail DNA %
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Mean Median of 750 cells)^1)
TBPPI-75-AL [1500 mg/kg body weight/day (x2)]	106	106S1	50	150	15.61	15.94	14.08   SD: 1.78 NS
		106S2	50		15.65
		106S3	50		16.57
	108	108S1	50	150	15.85	13.84
		108S2	50		15.53
		108S3	50		10.15
	119	119S1	50	150	10.01	14.97
		119S2	50		19.78
		119S3	50		15.12
	120	120S1	50	150	9.12	11.21
		120S2	50		10.57
		120S3	50		13.95
	132	132S1	50	150	16.58	14.46
		132S2	50		11.09
		132S3	50		15.71
EMS (200 mg/kg body weight/day) x1	103	103S1	50	150	41.84	41.22	43.36   SD: 5.54   **
		103S2	50		41.00
		103S3	50		40.82
	107	107S1	50	150	48.09	49.65
		107S2	50		51.09
		107S3	50		49.77
	121	121S1	50	150	38.20	39.21
		121S2	50		38.21
		121S3	50		41.23

EMS     :    Ethyl methanesulfonate

SD       :    Standard deviation

¹⁾        :    In the case of EMS positive control 450 cells/tissue.

 

Statistically not significant: NS

Statistically significant:

*      : p<0.05

**    : p<0.01

Duncan's multiple range test

 

 

Table 2: Tail DNA % of Medians per Slide, Means per Animal, per Dose (Duodenum)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	Tail DNA %
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Mean Median of 750 cells)
Sunflower oil (Helianthi annui oleum raffinatum) (x2)	109	109D1	50	150	17.31	11.60	12.82   SD: 2.60   -
		109D2	50		6.38
		109D3	50		11.11
	114	114D1	50	150	10.82	9.63
		114D2	50		8.95
		114D3	50		9.12
	118	118D1	50	150	12.35	11.86
		118D2	50		9.42
		118D3	50		13.82
	122	122D1	50	150	11.13	15.40
		122D2	50		21.77
		122D3	50		13.29
	129	129D1	50	150	12.97	15.63
		129D2	50		12.78
		129D3	50		21.16
TBPPI-75-AL [375 mg/kg body weight/day (x2)]	104	104D1	50	150	18.59	17.31	12.31   SD: 3.35 NS
		104D2	50		15.08
		104D3	50		18.27
	113	113D1	50	150	11.34	11.85
		113D2	50		10.48
		113D3	50		13.72
	115	115D1	50	150	13.78	13.39
		115D2	50		17.52
		115D3	50		8.87
	116	116D1	50	150	11.30	10.67
		116D2	50		8.80
		116D3	50		11.90
	128	128D1	50	150	8.06	8.32
		128D2	50		9.86
		128D3	50		7.05
TBPPI-75-AL [750 mg/kg body weight/day (x2)]	110	110D1	50	150	13.20	10.32	12.94   SD: 4.44 NS
		110D2	50		9.67
		110D3	50		8.09
	111	111D1	50	150	10.78	10.69
		111D2	50		9.52
		111D3	50		11.76
	125	125D1	50	150	13.25	10.49
		125D2	50		10.58
		125D3	50		7.65
	126	126D1	50	150	17.06	12.45
		126D2	50		9.90
		126D3	50		10.41
	127	127D1	50	150	20.15	20.73
		127D2	50		19.90
		127D3	50		22.16

Table 2 (continued): Tail DNA % of Medians per Slide, Means per Animal, per Dose (Duodenum)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	Tail DNA %
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Mean Median of 750 cells)^1)
TBPPI-75-AL [1500 mg/kg body weight/day (x2)]	106	106D1	50	150	14.89	13.18	12.89   SD: 4.14 NS
		106D2	50		13.51
		106D3	50		11.16
	108	108D1	50	150	6.03	9.78
		108D2	50		12.61
		108D3	50		10.72
	119	119D1	50	150	23.16	19.55
		119D2	50		14.58
		119D3	50		20.91
	120	120D1	50	150	10.96	9.06
		120D2	50		8.86
		120D3	50		7.37
	132	132D1	50	150	16.55	12.86
		132D2	50		10.19
		132D3	50		11.85
EMS (200 mg/kg body weight/day) x1	103	103D1	50	150	30.62	28.16	39.01   SD: 10.04**
		103D2	50		28.54
		103D3	50		25.32
	107	107D1	50	150	51.92	47.97
		107D2	50		45.42
		107D3	50		46.57
	121	121D1	50	150	40.88	40.92
		121D2	50		43.85
		121D3	50		38.04

EMS     :    Ethyl methanesulfonate

SD       :    Standard deviation

¹⁾        :    In the case of EMS positive control 450 cells/tissue.

 

Statistically not significant: NS

Statistically significant:

*      : p<0.05

**    : p<0.01

Duncan's multiple range test

 

 

Table 3: Tail DNA % of Medians per Slide, Means per Animal, per Dose (Liver)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	Tail DNA %
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Mean Median of 750 cells)
Sunflower oil (Helianthi annui oleum raffinatum) (x2)	109	109L1	50	150	4.32	6.49	6.91   SD: 1.05   -
		109L2	50		7.25
		109L3	50		7.90
	114	114L1	50	150	6.29	5.80
		114L2	50		4.96
		114L3	50		6.16
	118	118L1	50	150	8.71	6.42
		118L2	50		4.86
		118L3	50		5.68
	122	122L1	50	150	7.31	7.31
		122L2	50		8.01
		122L3	50		6.62
	129	129L1	50	150	9.82	8.53
		129L2	50		6.54
		129L3	50		9.23
TBPPI-75-AL [375 mg/kg body weight/day (x2)]	104	104L1	50	150	5.40	6.20	7.06   SD: 0.61 NS
		104L2	50		6.69
		104L3	50		6.50
	113	113L1	50	150	7.97	7.75
		113L2	50		6.12
		113L3	50		9.16
	115	115L1	50	150	5.90	7.36
		115L2	50		5.68
		115L3	50		10.50
	116	116L1	50	150	8.29	6.71
		116L2	50		5.38
		116L3	50		6.45
	128	128L1	50	150	10.06	7.30
		128L2	50		4.30
		128L3	50		7.54
TBPPI-75-AL [750 mg/kg body weight/day (x2)]	110	110L1	50	150	6.16	5.02	7.93   SD: 2.97 NS
		110L2	50		5.01
		110L3	50		3.90
	111	111L1	50	150	9.20	6.88
		111L2	50		5.37
		111L3	50		6.09
	125	125L1	50	150	5.81	6.85
		125L2	50		9.18
		125L3	50		5.55
	126	126L1	50	150	7.63	8.01
		126L2	50		7.95
		126L3	50		8.46
	127	127L1	50	150	12.22	12.89
		127L2	50		12.14
		127L3	50		14.32

Table 3 (continued): Tail DNA % of Medians per Slide, Means per Animal, per Dose (Liver)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	Tail DNA %
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Mean Median of 750 cells)^1)
TBPPI-75-AL [1500 mg/kg body weight/day (x2)]	106	106L1	50	150	7.09	7.69	8.27   SD: 0.91 NS
		106L2	50		10.19
		106L3	50		5.79
	108	108L1	50	150	8.87	9.15
		108L2	50		9.20
		108L3	50		9.38
	119	119L1	50	150	7.78	7.66
		119L2	50		6.34
		119L3	50		8.86
	120	120L1	50	150	12.10	9.37
		120L2	50		8.65
		120L3	50		7.38
	132	132L1	50	150	8.18	7.50
		132L2	50		8.71
		132L3	50		5.62
EMS (200 mg/kg body weight/day) x1	103	103L1	50	150	24.22	31.50	38.88   SD: 8.27*
		103L2	50		24.90
		103L3	50		45.37
	107	107L1	50	150	37.29	37.32
		107L2	50		36.80
		107L3	50		37.89
	121	121L1	50	150	45.74	47.82
		121L2	50		49.21
		121L3	50		48.50

EMS     :    Ethyl methanesulfonate

SD       :    Standard deviation

¹⁾        :    In the case of EMS positive control 450 cells/tissue.

 

Statistically not significant: NS

Statistically significant:

*      : p<0.05

**    : p<0.01

Mann-Whitney U-test Versus Control

 

 

Table 4: Historical Control Data for Stomach Cells (C-Charts)

	Historical control data for stomach cells (C-chart)
	Sunflower oil (Negative (vehicle) control)
	% DNA in Tail	Tail length	OTM
Mean	10.67	41.17	3.62
Lower confidence interval	1.79	4.92	0.00
Upper confidence interval	19.55	77.42	7.41
SD	3.20	13.06	1.37
n	5	5	5
	Historical control data for stomach cells (C-chart)
	Positive control (Ethyl methanesulfonate)
	% DNA in Tail	Tail length	OTM
Mean	34.42	112.20	17.73
Lower confidence interval	16.58	49.05	0.00
Upper confidence interval	52.26	175.36	38.46
SD	7.89	27.92	9.16
n	10	10	10

n              : number of experiments

OTM         : Olive Tail Moment=(Tail.mean - Head.mean) x Tail%DNA/100

SD           : Standard deviation

Remark: At the C-chart calculations in the case of sunflower oil control the results of five experiments were taken into consideration. The lower confidence intervals at OTM calculations, due to the nature of these calculations, were negative. Instead of the negative values, zero was incorporated into the table. Furthermore, in the case of Ethyl methanesulfonate positive control at the OTM calculations instead of the negative lower confidence interval value, the table contains zero.

 

Table 5: Historical Control Data for Duodenum Cells (C-Charts)

	Historical control data for duodenum cells (C-chart)
	Sunflower oil (Negative (vehicle) control)
	% DNA in Tail	Tail length	OTM
Mean	11.52	46.55	3.71
Lower confidence interval	1.09	2.02	0.00
Upper confidence interval	21.94	91.09	8.06
SD	3.28	14.00	1.36
n	4	4	4
	Historical control data for duodenum cells (C-chart)
	Positive control (Ethyl methanesulfonate)
	% DNA in Tail	Tail length	OTM
Mean	30.54	100.82	13.56
Lower confidence interval	14.36	33.71	4.29
Upper confidence interval	46.73	167.94	22.84
SD	6.29	26.10	3.61
n	6	6	6

n              : number of experiments

OTM         : Olive Tail Moment=(Tail.mean - Head.mean) x Tail%DNA/100

SD           : Standard deviation

Remark: At the C-chart calculations in the case of sunflower oil negative control four experiments were taken into consideration. The lower confidence intervals at OTM calculations, due to the nature of these calculations, were negative. Instead of the negative values zero was incorporated into the table.

 

 

Table 6: Historical Control Data for Liver Cells (C-Charts)

	Historical control data for liver cells (C-chart)
	Sunflower oil (Negative (vehicle) control)
	% DNA in Tail	Tail length	OTM
Mean	5.86	26.41	1.85
Lower confidence interval	1.58	0.00	0.09
Upper confidence interval	10.15	53.98	3.61
SD	1.54	9.93	0.63
n	5	5	5
	Historical control data for liver cells (C-chart)
	Positive control (Ethyl methanesulfonate)
	% DNA in Tail	Tail length	OTM
Mean	25.51	110.51	12.90
Lower confidence interval	10.62	57.84	0.00
Upper confidence interval	40.39	163.19	25.85
SD	6.58	23.29	5.73
n	10	10	10

n              : number of experiments

OTM         : Olive Tail Moment=(Tail.mean - Head.mean) x Tail%DNA/100

SD           : Standard deviation

Remark: At the C-chart calculations in the case of sunflower oil control the results of five experiments were taken into consideration. The lower confidence intervals at the tail length values, due to the nature of these calculations, were negative. Instead of the negative values zero was incorporated into the tables. Furthermore, in the case of Ethyl methanesulfonate positive control at the OTM calculations instead of the negative lower confidence interval value, the table contains zero.

Applicant's summary and conclusion

Conclusions:

The test item was investigated by the means of the in vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions in the male WISTAR rats. The test item was administered twice via oral gavage at the dose levels 1500, 750 and 375 mg/kg body weight/day on two consecutive days. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item TBPPI-75- AL did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells. Concurrent controls confirmed the sensitivity and validity of the test. The investigated test item TBPPI-75-AL did not show genotoxic activity in the examined tissues in this Comet Assay.

Executive summary:

In a GLP compliant In Vivo Alkaline Comet Assay on the Rat Stomach, Duodenum and Liver according to OECD guideline 489, the clastogenic potential of the test item was investigated. Male Wistar rats were treated on two consecutive days with the test item at concentrations of 375, 750 and 1500 mg/kg bw/day by oral gavage. Sunflower oil was used as negative control and ethyl methanesulfonate (EMS) was used as positive control. Formulations were prepared freshly before each treatment. The test item was formulated in the vehicle in nominal concentrations of 300, 150 and 75 mg/mL. The test item in sunflower oil formulations was considered to be homogeneous. The measured concentration values were slightly higher (9-15 % higher), than the nominal values in both analytical occasions at all concentrations. The slightly higher concentration levels of treatments solutions (and in consequence doses) did not cause significantly different observations, clinical signs, symptoms at the investigated animals, than already noticed in the preliminary tests. The slightly higher measured concentrations were considered acceptable and the nominal concentration values of 300, 150 and 75 mg/mL (corresponding dose levels: 1500, 750 and 375 mg/kg body weight/day) were applied throughout the study. The animals of the test item dose groups and the negative control animals were treated by oral gavage twice, once on the day 0 and once 24 hours thereafter. The positive control animals were treated by oral gavage once during the experiment on day 1. 5 mL/kg body weight at the vehicle control and test item doses, and 10 mL/kg body weight at the positive control. 3-4 hours after the second treatment (test item treatments and vehicle control) and 3-4 hours after the treatment (positive control) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined using a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Comet Assay steps were the following: Embedding the cells; Lysis (pH=10); Unwinding (pH>13; for 30 min.); Electrophoresis (pH>13; for 30 min. at 25V and about 300 mA);Neutralisation (pH=7.5; 3 times for 5 min.) and Preservation (abs. ethanol for 5 min. and air dried) of slides. Prior to scoring, the DNA was stained with 2 μg/mL Ethidium bromide. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection. 6 animals in the test item treated groups and negative control group, respectively; 4 animals in the positive control group. 5 animals in the test item treated groups and negative control group, respectively; 3 animals in the positive control group. For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment and per vehicle control; 450 per positive control). All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 1500 mg/kg body weight/day and in the controls. During the treatment period, after the second treatment, unequivocal signs of test item toxicity, obvious behavioural changes and clinical signs that appeared in dose-related manner were noticed. Piloerection, decreased or strongly decreased activity, increased respiration rate were the most characteristic signs that were most obvious at the highest dose of 1500 mg/kg body weight/day. At the time of tissue isolation, normal appearance and anatomy of stomach, duodenum and liver was noticed at the vehicle control animals and at three animals of the positive control. Unequivocal signs of toxicity and local test item effects (e.g.: tympanites in the stomach or gastrointestinal tract, characteristic chemical smell at the stomach opening, characteristic stomach content: bedding material or significant volumes of liquid (assumed: water) were noticed at all test item treated groups. The intensity, degree of these toxic effects, observations showed a dose dependent tendency. Furthermore, at the highest dose of 1500 mg/kg body weight/day, a mosaic pattern was noticed on the liver in three animals. The average body weights slightly increased (by 0.19-1.32 %) in the negative control and in the treated dose groups of 375 and 750 mg/kg body weight/day when comparing the weight values measured on day 0 and just before the sacrifice, which is in the range of the expected increase for such exposure times. At the dose group of 1500 mg/kg body weight/day, on average 4.66 % weight reduction was noticed, also indicating a test item toxic effect (see above). At the positive control, on average 1.95 % weight reduction was noticed. In the cytotoxicity screening measurements (using Trypan blue dye exclusion method), no cytotoxicity was noticed in any test item and control item treatments in any target tissue. In the stomach, the percentage of ghost cells differed statistically significant from that of the of the vehicle control at the dose level of 750 mg/kg body weight/day. The percentage of ghost cells was ~12 % in the vehicle control group and ~18 % at 750 mg/kg body weight/day. The percentage of ghost cells at 750 mg/kg body weight/day was slightly above the laboratory’s historical control data range for stomach preparations. However, dose dependent increase was not established in the percentage of ghost cells at the stomach samples, (i. e. at 1500 mg/kg body weight/day the ghost cell percentage was 16 %, within the historical control data range and not statistically significant and the linear trend analysis did not show significance). The slightly higher ghost cell frequency was not accompanied unequivocally with increased DNA migration. Therefore, the slightly higher percentage of ghost cells was considered acceptable and being within the biological variability range of the test. At the examined test item treated groups, the number of ghost cells in the duodenum samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control. In the liver, the percentage of ghost cells (the mean value was 2 % at the vehicle control and 4 % at 1500 mg/kg body weight/day) differed statistically significant from that of the vehicle control at the highest dose level of 1500 mg/kg body weight/day. The ghost cell percentages for liver preparations (means and individual values) remained well within the laboratory’s historical control data range in all doses; however, the percentage of ghost cells showed a dose related increase (also confirmed by linear trend analysis). The slightly higher frequency of ghost cells was not accompanied with unequivocally increased DNA migration: the linear trend analysis of changes of % tail DNA, tail length and OTM values did not show significance, consequently no clear dose related increase was obtained at these parameters. Therefore, the relatively higher number of ghost cells in liver samples especially at the highest dose level was predominantly associated with toxic effects attributable to the test item, which was observed during macroscopic inspection (see above) of the tissue prior to cell isolation which also could have influence on the prepared tissue (cell suspension) quality. A statistically significant increase of ghost cells was noticed in all tissues after EMS treatment. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the literature increased frequency of ghost cells may also indicate cells with severe DNA damage (genotoxicity). Based on the mutagenicity results and the laboratory’s earlier experience, the relatively higher number of ghost cells at the positive control, EMS treatment are considered being a possible indicator of genotoxicity. The mean median % tail DNA values of each dose remained in the vehicle control range at the examined tissues, and the slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the highest dose of 1500 mg/kg body weight/day. The mean median % tail DNA values of the vehicle control and test item doses in the stomach, duodenum and liver samples fell within the corresponding historical control data ranges within the 95 % confidence intervals, C-charts. Additionally, the tail length values and Olive Tail Moment (OTM) of the vehicle control and each treatment were compared. The tail length values of the stomach, duodenum and liver samples did not differ statistically significantly from that of the vehicle control in whole examined dose range. The Olive Tail Moment values in the stomach, duodenum and liver of the test item treated groups did not differ statistically significant from that of the vehicle control. Additionally, all of the tail length and OTM values (vehicle control and dose groups) remained well within the established historical control data ranges, within the 95 % confidence intervals, C-charts. In summary, under the experimental conditions presented in this report, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells and is thus not considered clastogenic.