OO-tert-butyl monoperoxymaleate

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:

2022-04-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted July 29, 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

OO-tert-butyl monoperoxymaleate

EC Number:

217-691-1

EC Name:

OO-tert-butyl monoperoxymaleate

Cas Number:

1931-62-0

Molecular formula:

C8H12O5

IUPAC Name:

(2Z)-4-(tert-butylperoxy)-4-oxobut-2-enoic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pergan GmbH, batch no.: 1330431-01
- Purity of active component: 37% (w/w); contains phlegmatizer Triacetin at concentrations ≤60% (w/w)
- Expiry Date: 25 November 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8°C, protected from light.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was diluted in PEG400. The test item formulations were prepared freshly on each administration day. The prepared formulations were transferred to BSL Munich protected from light together with the control formulations under consideration of stability.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rats have been used for many years as standard experimental animals in genotoxicity investigations. In addition, they have been used as experimental animal in the toxicological safety characterization of the test item. Data from such experiments are useful for the design of the alkaline Comet Assay and interpretation of its findings.
According to OECD TG 489, healthy young adult rodents are recommended and rats being routinely used in this test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 7-9 weeks old
- Weight at study initiation: interval within 20% of the mean weight
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: The animals are kept in groups of 2-3 animals/sex/group/cage in IVCs (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water: ad libitum, tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Test item (OO-tert-butyl monoperoxymaleate) and additional control (Triacetin) were dissolved in polyethylene glycol (PEG 400).
The positive control ethyl methanesulfonate was dissolved in physiol. saline (0.9% NaCl);
- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 12/30/60 mg/ml for the 120/300/600 mg/kg bw/day dose groups, respectively (administration volume 10 ml/kg bw).
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw.
- Lot/batch no.: PEG 400: Sigma-Aldrich BCCG1486 (pre-experiment), BCCG9382 (Main Study); physiological saline: B.Braun; Batch: 13QLP251

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was diluted in PEG400. The vehicle was chosen according to its relative non-toxicity for the animals. On sponsors request no correction factor for purity was used in dose calculations. All animals received a single volume orally of 10 mL/kg bw.

- As the test item contained the phlegmatizer Triacetin, Triacetin was additionally used as control item. The final concentration for Triacetin was determined by the sponsor after the dose range finding with 360 mg/kg bw and was based on the concentration of the highest dose group (600 mg/kg bw) of the test material. Triacetin was weighed into a tared plastic vial on a precision balance and the vehicle was added.

The test item formulations were prepared freshly on each administration day. The prepared formulations were transferred to BSL Munich protected from light together with the control formulations under consideration of stability.

Duration of treatment / exposure:

Test item, control (PEG 400) and additional control (Triacetin): Two consecutive days
Positive control (ethyl methanesulfonate): Once

Frequency of treatment:

daily

Post exposure period:

4 hours after last treatment, animals were sacrificed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control (PEG 400)

Doses / concentrations 2

Dose / conc.:

120 mg/kg bw/day

Remarks:

Test item (37% (w/w) active component)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Test item (37% (w/w) active component)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day

Remarks:

Test item (37% (w/w) active component)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

other: Phlegmatizer Triacetin at 360 mg/kg bw/day

Positive control(s):

ethylmethanesulfonate
- Justification for choice of positive control(s): Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control.
- Route of administration: oral
- Doses / concentrations: 250 mg/kg bw

Examinations

Tissues and cell types examined:

Four hours after the last administration of the test item, samples of liver, glandular stomach, duodenum and gonads were taken. The liver, glandular stomach, and duodenum were analyzed for DNA strand breaks, while comet slides for male gonadal calls were prepared for later analysis for DNA strand breaks in case of positive results observed on somatic cells. All samples were kept in ice-cold mincing buffer and directly prepared. One part of the liver, glandular stomach and duodenum were preserved in 10% neutral-buffered formalin for histopathological evaluation.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Due to the severity of clinical signs (reduced spontaneous activity, hunched posture, piloerection and half eyes closed) and histopathological effects observed in a dose range finding study, a dose of 600 mg/kg bw was identified as the maximum tolerated dose (MTD) and selected as the highest dose (HD). Additional details on the results of the range-finding study are provided in section "Results and discussion" under "Additional information on results".
The dose level of the phlegmatizer Triacetin which was used as an additional control, was based on the high dose level of test material (600 mg/kg bw/day) and maximum content of Triacetin (60% (w/w)) in test item formulation.

DETAILS OF SLIDE PREPARATION:
- Preparation of single cells:
A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL low-melting agarose (LMA) 0.6% (w/v) in PBS solution.
The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. The duodenum was cut open into two halves. One half of the duodenum was minced with a pair of scissors, the other one was kept for histopathology. The cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
The stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
The end of the epithelial capsule of the gonad was punctured to squeeze out the seminiferous tubules. Without pressure the tubules were flattened with a tissue roller in D-PBS until it became cloudy with released cells. The cell suspension was kept on ice for 1-2 minutes until accidentally collected tubules settled on the bottom of the tube. The cell suspension was transferred to a new tube leaving the settled tubules behind and was centrifuged at 11,000 x g for 30 sec at 4°C. The supernatant was discarded carefully without disturbing the pellet, which was diluted in Mincing buffer. A volume of 30 μL of the supernatant was pipetted into a tube and mixed with 270 μL LMA solution.

The cell pellets (150 cells/animal/tissue) were shortly put on ice and were directly used for preparing comet slides.

The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min at 4°C. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min at 4°C. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order.
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated.
Cells were classified into three potential categories scorable, non-scorable and “hedgehog” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail are considered to be heavily damaged cells). To avoid artefacts only scorable cells and at least 150 cells per sample were scored, if available. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus [5]. The hedgehogs were manually classified by the evaluator.

Evaluation criteria:

Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.
Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.

Statistics:

All slides, including those of positive and vehicle controls were independently coded before microscopic analysis and subsequently scored blinded. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.
For each tissue type, the mean of the individual animal means was then determined to give a group mean. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control (PEG400).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Dose-related clinical effects in MD (reduced spontaneous activity) and HD group (reduced spontaneous activity, piloerection, half eyes closure, hunched posture) and histopathological effects in HD group (stomach erosion/necrosis, hepatocyte hypertrophy).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-600 mg/kg bw
- Solubility: The test item was diluted in PEG400, after the use of an aqueous solution/suspension was not possible.
- Clinical signs of toxicity in test animals:
In the pre-experiment, one male and one female rat received a single dose of 500 mg/kg bw orally by gavage on two consecutive days.
The male rat showed reduced spontaneous activity and prone position 30 minutes after the first administration of the test item. The symptoms lasted until 2 h and were gone after 4 h. After the second administration, piloerection and hunched posture were visible after 2 h, but vanished afterwards. The female animal showed reduced spontaneous activity 1 h after the first administration of the test item and reduced spontaneous activity and prone position after 2 h. No clinical signs were observed after 4 h. Two hours after the second administration, reduced spontaneous activity, hunched posture and piloerection were visible but were not present after 4 h.
Based on these observations, the amount of the test item was enhanced to 600 mg/kg bw/d. Three male and three female rats received a single dose of 600 mg/kg bw orally on two consecutive days.
The first male rat showed reduced spontaneous activity and hunched posture 30 minutes after the first administration of the test item, reduced spontaneous activity, prone position and piloerection after 1 h and reduced spontaneous activity and piloerection after 2 h. At 4 h of the observation period, piloerection was still present. After the second administration, reduced spontaneous activity, hunched posture and piloerection were observed after 1 h. These symptoms in addition with half eyes closed were still visible after 4 h of the last administration. The second male rat showed reduced spontaneous activity 30 minutes after the first administration of the test item, while reduced spontaneous activity and prone position was present in the third male rat. Reduced spontaneous activity, prone position and piloerection was observed in both male rats after 1 h and reduced spontaneous activity and piloerection after 2 h till 4 h of the observation period. Thirty minutes after the second administration, the two male rats showed reduced spontaneous activity, hunched posture, piloerection and half eyes closed were observed. Piloerection was still visible after 4 h.
The first female animal showed reduced spontaneous activity and hunched posture 30 minutes after the first administration of the test item, reduced spontaneous activity, prone position and piloerection after 1 h and reduced spontaneous activity and piloerection after 2 h. After 4 h piloerection was still present. One hour after the second administration, reduced spontaneous activity and piloerection were observed. Piloerection, hunched posture and half eyes closed were present after 4 h of the observation period. The other two female rats showed reduced spontaneous activity, prone position and piloerection 1 h after the first administration of the test item and reduced spontaneous activity, hunched posture and piloerection after 2 h. After 4 h of the observation period, piloerection and reduced spontaneous activity were still present. One hour after the second administration, reduced spontaneous activity, hunched posture, piloerection and half eyes closed were observed. Piloerection was still visible after 4 h.
- Evidence of cytotoxicity in tissue analysed: In the glandular stomach, mucosal erosion (4/6 animals), necrosis/apoptosis of epithelial cells at the tips of the gastric pits (6/6 animals), and mixed cell infiltrates (1/6 animals) were observed in different animals that received 600 mg/kg bw test item. Minimal centrilobular hypertrophy was observed in one animal that received 500 mg/kg bw test item and in one animal that received 600 mg/kg bw test item. In addition, minimal, single hepatocytes necrosis/apoptosis were observed in one animal that received 600 mg/kg bw test item.
- Rationale for exposure: Based on the results obtained in the dose-range finding study, the main study was conducted with male rats, as no gender specific differences were determined, and 600 mg/kg bw was chosen as highest dose in the main experiment. Due to the observed severity of clinical signs and the incidence and severity of histopathological effects (for more information, please refer to Table 5 and Table 6 in section "Any other information on results incl. tables"), this dose represents the MTD.

RESULTS OF DEFINITIVE STUDY (Detailed tabular results for each group of animals treated with the test substance, vehicle and positive controls and each tissue are provided in Tables 1 and 2 in section "Any other information on results incl. tables")
- Appropriateness of dose levels and route: Based on the clinical signs observed in the HD and MD groups in the main experiment (All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, half eyes closure and hunched posture, after the first administration of the test item, which were also present after the second administration. On both days of treatment, all animals treated with 300 mg/kg bw (MD) showed reduced spontaneous activity 4 h after administration of the test item, while rats treated with 120 mg/kg bw (LD) showed no clinical signs, for more information, please refer to Table 7 in section "Any other information on results incl. tables") and the histopathological evaluation (for more information, please refer to Table 8 in section "Any other information on results incl. tables"), dose groups were selected to range from the MTD which is represented by the HD group (600 mg/kg bw/day) to a dose without any evidence of adverse effects in the LD group (120 mg/kg bw/day).
- Statistical evaluation: A statistically significant increase in induced DNA strand breaks was determined for the positive control group of each organ. No significances compared to the vehicle control (PEG400) were determined for the incidence of induced DNA-strand breaks in any of the dose groups of each organ. A concentration-related increase was noted in male liver cells as the tail intensity value for the highest concentration was increased compared to the lower concentrations tested. However, this positive trend test was not regarded as biologically relevant as the concentration was not significantly increased compared to the vehicle control groups and the tail intensities of all groups were in the historical control range (for more information on historical control data, please refer to Table 4 in section "Any other information on results incl. tables") of the test facility. No concentration-related increase was noted in glandular stomach and duodenum cells of male rats.

Any other information on results incl. tables

Table 1: Summary of Mean Tail Intensities [%] in Liver, Glandular Stomach and Duodenum

Dose	Mean Tail Intensity [%]			Clinical Signs
Group	Liver	Glandular Stomach	Duodenum
PC	13.73*	7.70*	6.94*	None
VC1	0.96	2.89	2.72	None
VC2	0.66	2.97	1.24	None
LD	0.79	1.87	1.3	None
MD	1.25	2.16	2.16	Reduced spontaneous activity
HD	1.4	1.7	3.45	Reduced spontaneous activity, piloerection, half eyelid closure and hunched posture
Statistically significant trend	Yes°	No	No	/
Historic Control Range	Negative Control: 0.07% – 3.82% Positive Control: 2.81% – 28.08%	Negative Control: 1.30% - 5.46% Positive Control: 5.99% – 34.62%	Negative Control: 0.89% - 4.06% Positive Control: 1.77% – 30.75%

*      significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

°      a trend test (One-way ANOVA) revealed a statistically significant increase, which was not considered as biologically relevant.

VC1:	Vehicle Control (PEG400)	PC:	Positive Control (Ethyl methanesulfonate: 250 mg/kg bw)
LD:	Low Dose	MD:	Mid Dose
HD:	High Dose	VC2:	Vehicle Control (Triacetin)

Table 2: Detailed Results for Tail Intensity in Liver, Glandular Stomach and Duodenum cells

Tail intensity in Liver Cells
Animal per Group	Mean of Medians
	PC	VC1	VC2	LD	MD	HD
I	14.19	0.8	0.67	1.17	0.69	1.45
II	14.81	1.11	0.47	0.57	1.57	1.3
III	19.93	0.7	0.52	0.93	1.49	1.11
IV	9.45	1.14	0.53	0.64	1.54	1.14
V	10.24	1.03	1.09	0.65	0.98	2
Group Mean:	13.73*	0.96	0.66	0.79	1.25	1.4
± SD:	4.19	0.19	0.25	0.25	0.4	0.36
Tail intensity in Glandular Stomach Cells
Animal per Group	Mean of Medians
	PC	VC1	VC2	LD	MD	HD
I	9	3.54	3.7	2.91	1.84	3.01
II	10.7	3.5	1.46	2.5	2.41	2.7
III	0.65	2.38	2.37	1.25	3.6	0.81
IV	11.59	2.72	5.75	2	1.95	1.06
V	6.56	2.31	1.59	0.7	1.01	0.9
Group Mean:	7.70*	2.89	2.97	1.87	2.16	1.7
± SD:	4.38	0.6	1.79	0.9	0.95	1.07
Tail intensity in Duodenum Cells
Animal per Group	Mean of Medians
	PC	VC1	VC2	LD	MD	HD
I	3.8	5.53	1.56	1.08	2.85	4.66
II	4.14	2.89	1.57	0.65	1.14	5.62
III	11.27	2.34	0.79	0.89	1.77	1.04
IV	9.85	1.93	0.71	0.89	1.96	0.58
V	5.65	0.93	1.55	3.01	3.07	5.37
Group Mean:	6.94*	2.72	1.24	1.3	2.16	3.45
± SD:	3.41	1.73	0.45	0.97	0.8	2.45
LD:	Low Dose
MD:	Mid Dose
HD:	High Dose
PC:	Positive Control (Ethyl methanesulfonate: 250 mg/kg bw)
SD:	Standard Deviation
VC1:	Vehicle Control (PEG400)
VC2:	Additional Control (Triactin)
* significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

Table 3: Linear Trend test - Statistical significance of test item treatment at the 5 % level (p < 0.05)

	Liver Cells	Glandular Stomach Cells	Duodenum Cells
Significance	+*	-	-
P Value	0.0159	0.7802	0.0532
+:	statistically significant trend test
+*:	statistically significant concentration-dependency was noted but considered as not biological relevant.
-:	statistically not significant trend test

Table 4: Laboratory Historical Control Data for tail intensity (%) (2017 – 2020)

Negative and Vehicle Controls
	tail intensity [%]
	liver	glandular stomach	duodenum
mean [%]	1.95	3.38	2.48
SD [%]	0.94	1.04	0.79
RSD [%]	48.11	30.72	31.96
min [%]	0.65	1.61	1.11
max [%]	3.97	4.95	3.81
LCL [%]	0.07	1.3	0.89
UCL [%]	3.82	5.46	4.06
n	20	12	14
Median	1.81	3.67	2.38
Positive Controls
	tail intensity [%]
	liver	glandular stomach	duodenum
mean [%]	15.45	20.31	16.26
SD [%]	6.32	7.16	7.24
RSD [%]	40.91	35.25	44.55
min [%]	5.66	10.72	8.21
max [%]	30.94	42.79	37.52
LCL [%]	2.81	5.99	1.77
UCL [%]	28.08	34.62	30.75
n	32	22	27
Median	15.23	17.34	14.94
mean:	Group mean tail intensity [%]
min.:	minimum tail intensity [%]
LCL:	lower control limit [%]
n:	number of assays
SD:	Standard Deviation [%]
max.:	maximum tail intensity [%]
UCL:	upper control limit [%]

Table 5: Clinical Signs observed in the Pre-Experiment at 600 mg/kg bw

Clinical Signs	Time post-administration / Sex
	(3 male and 3 female rats)
	0 h		0.5 h		1 h		2 h		4 h		24 h		24.5 h		25 h		28 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Dose level	600 mg/kg bw
Reduction of spontaneous activity	0	0	3	1	3	3	3	3	2	2	0	0	2	0	1	3	1	0
Piloerection	0	0	0	0	3	3	3	3	3	3	0	0	2	0	1	3	3	3
Half eyelid closure	0	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	1	1
Prone Position	0	0	1	0	3	3	0	0	0	0	0	0	0	0	0	0	0	0
Hunched posture	0	0	1	1	0	0	0	2	0	0	0	0	2	0	1	2	1	1

Table 6: Incidence and severity of relevant microscopic changes in the dose range finding experiment

Findings / Groups	500 mg/kg bw	600 mg/kg bw
Animals Affected / Mean Grade	2	6
Glandular stomach
Erosion	-	4/1.8
Necrosis / Apoptosis	-	6/1.0
Mixed cell infiltrates	-	1/2.0
Liver
Centrilobular hypertrophy	1/1.0	1/1.0
Mononuclear cell infiltrates	2/1.0	5/1.0

Table 7: Clinical Signs observed in the Main Experiment

Clinical Signs	Time post-administration / Sex
	(5 male rats)
	0 h	0.5 h	1 h	2 h	3 h	4 h	24 h	24.5 h	25 h	26 h	27 h	28 h
	m	m	m	m	m	m	m	m	m	m	m	m
Dose level	120 mg/kg bw
No clinical signs observed	-	-	-	-	-	-	-	-	-	-	-	-
Dose level	300 mg/kg bw
Reduction of spontaneous activity	0	0	0	0	0	0	5	0	0	0	0	4
Dose level	600 mg/kg bw
Reduction of spontaneous activity	0	0	5	5	5	5	0	5	5	5	5	5
Piloerection	0	0	2	3	4	5	1	0	0	0	0	2
Half eyelid closure	0	0	0	0	0	2	0	0	0	0	0	1
Hunched posture	0	0	0	5	5	5	0	5	5	5	5	5

Table 8: Incidence and severity of relevant microscopic changes in the main experiment

Findings / Groups	Vehicle Control	Additional control group	LD	MD	HD	Positive control
	PEG400	360 mg/kg bw Triacetin	120 mg/kg bw	300 mg/kg bw	600 mg/kg bw	250 mg/kg bw EMS
Animal per Sex Affected / Mean Grade	5(M)	5(M)	5(M)	5(M)	7(M)	5(M)
Glandular stomach
Erosion	-	-	-	-	1/2.0	-
Necrosis / Apoptosis	-	-	-	-	3/1.0	-
Mixed cell infiltrates	1/1.0	2/2.0	1/2.0	-	1/2.0	-
Liver
Centrilobular hypertrophy	2/1.0	3/1.0	4/1.0	4/1.0	6/1.0	4/1.0

Applicant's summary and conclusion

Conclusions:

The test item O,O-tert-butyl monoperoxymaleate did not induce biologically relevant increases in DNA-strand breaks in any of the tissues evaluated (liver, glandular stomach, duodenum) after oral (gavage) administration up to the maximum tolerated dose (600 mg/kg bw/day) in male rats.
Therefore, the test item is considered to be non-DNA damaging under the experimental conditions of the in vivo mammalian Alkaline Comet Assay.

Executive summary:

In an in vivo mammalian Alkaline Comet Assay conducted according to OECD 489, the test material O,O-tert-Butyl monoperoxymaleate (purity: 37%) was administered to 5 male Crl(WI):Han rats/dose by oral gavage at dose levels of 120, 300 and 600 mg/kg bw/day in PEG 400 for two consecutive days.

No correction factor for purity was used in dose calculations. The phlegmatizer Triacetin was used as additional control item at a dose of 360 mg/kg bw/day (via oral gavage), based on the selected high dose level of the test material (600 mg/kg bw/day) and the maximum content of Triacetin (60% (w/w)) in the test item formulation.

There were no signs of toxicity in animals treated with 120 mg/kg bw/day. Animals in the 300 mg/kg bw/day group showed reduction of spontaneous activity, while animals of the high dose showed more severe clinical signs such as reduction of spontaneous activity, piloerection, half eyelid closure and hunched posture. No specific biologically-relevant effect on body weight was observed in any of the dose groups. Specific treatment-related histopathological effects were observed in the 600 mg/kg bw/day group, which included erosion and necrosis in the stomach and increased incidences of centrilobular hepatocyte hypertrophy in the liver. Based on these results, systemic availability of the substance can be reasonably assumed and the high dose level can be considered to represent the maximum tolerated dose (MTD).

Cells from the liver, glandular stomach and duodenum were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated.

The tail intensities of the vehicle control group consisting of PEG 400 was within the historical control limits and therefore accepted for addition to the laboratory control data set. The additional control group (Triacetin) was within historical control limits. The positive control ethyl methanesulfonate (250 mg/kg bw by oral gavage) induced a statistically significant increase in DNA damage for all evaluated organs within historical control limits of the positive control of the testing facility, thereby demonstrating the validity of the assay.

No biologically relevant increase of tail intensity was found after treatment with the test item in any of the dose groups and organs evaluated compared to the vehicle controls. Mean tail intensity values for the test item dose groups were within the range of the concurrent vehicle controls and within the historical control limits. A slight dose-related increase was observed for liver cells but considered not biologically relevant as the tail intensity values were not statistically significant on a single dose level and were within historical control range of the testing facility.

Based on these observations, the test item is considered to be non-DNA damaging under the experimental conditions of the in vivo mammalian Alkaline Comet Assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 489 for in vivo mammalian alkaline comet assay.