4-tert-butylpyrocatechol

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:

26 April 2019 - 26 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Adopted 29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylpyrocatechol

EC Number:

202-653-9

EC Name:

4-tert-butylpyrocatechol

Cas Number:

98-29-3

Molecular formula:

C10H14O2

IUPAC Name:

4-tert-butylbenzene-1,2-diol

Test material form:

solid

Details on test material:

CAS N°: 98-29-3
CE N°: 202-653-9.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- 4-tert-butylpyrocatechol (CAS n°: 98-29-3)
- Source: manufacturer.
- Purity: higher than 98%. No correction factor required.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light.
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: Vehicle: Elix water. Stability of test item in Elix water: Stability for at least 5 hours at room temperature under normal laboratory light conditions, for at least 9 days in the refrigerator and for at least 3 weeks in the freezer (≤-15°C) is confirmed over the concentration range 1 to 200 mg/mL, Project 517569. Homogeneity: the criteria for acceptability was a relative standard deviation (RSD) of concentrations of < 10% for each group. The formulations of Group 2 (100 mg/kg b.w) and Group 4 (400 mg/kg b.w) were homogeneous (i.e. coefficient of variation ≤10%).
- Solubility and stability of the test material in the vehicle: A solubility test was performed based on visual assessment. 4-tert-butylpyrocatechol was suspended (white suspension) in Elix water (Millipore Corp., Bedford, MA., USA). The specific gravity of Elix water is 1.0 g/mL. 4-tert-butylpyrocatechol concentrations were dosed within 3.5 hours after preparation. Solubility in the vehicle ( Elix water): 4.2 g/L at 20°C.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none.
- Preliminary purification step (if any): none.
- Final concentration of a dissolved solid, stock liquid or gel: none.
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): none.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat is the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain. These rats are recommended by
international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The animals were provided by Charles River, Sulzfeld, Germany.
- Age at study initiation: Young adult animals were selected, 6 weeks old at the start of treatment.
- Weight at study initiation: The body weights of the rats at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights was 148 ± 9.1 g and the range 134 – 166 g.
- Assigned to test groups randomly: [no/yes, under following basis: ] yes. The animals were allocated at random to the treatment groups.
- Fasting period before study: A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat).
- Housing: The animals were housed in room number A0.02 and A0.013 (dose-range finding study) or A0.13 (main study). Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water (e.g. ad libitum): The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Other: On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health. Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study. The total number of animals used in the dose-range finding study was 12 and in the main study 28. In the main study 5 male rats were treated per sampling time in each treatment group.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 18 to 24°C. The actual daily mean temperature during the study period was 20 to 21°C.
- Humidity (%): Relative target humidity of 40 to 70%. The actual daily mean relative humidity was 49 to 68%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.

IN-LIFE DATES: From:30 April 2019 To: 06 June 2019.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Elix water (Millipore Corp., Bedford, MA., USA). The test item was suspended in Elix water.
- Justification for choice of solvent/vehicle: First vehicle to be used if possible according to the OECD Guideline.

Details on exposure:

- A dose-range finding was done in order to select the Maximum Tolerated Dose (MTD). In this dose-range finding study 3 males and 3 females were dosed once daily via oral gavage with 500 mg 4-tert-butylpyrocatechol per kg body weight for a maximum of three days and 3 males and 3 females were dosed once daily via oral gavage with 400 mg 4-tert-butylpyrocatechol per kg body weight for a maximum of three days. The observation period after dosing was one to three days. Based on the results of the dose-range finding study test concentrations of 400 mg/kg/day was selected as maximum dose for the main test (maximum tolerated dose). Since there were no substantial differences based on the dose-range finding study in toxicity between sexes, only males were used in the main study.

- In the main study male animals were dosed by oral gavage with vehicle or with 100, 200 and 400 mg 4-tert-butylpyrocatechol per kg body weight for three consecutive days. The rats were dosed twice with the positive control Ethyl Methane Sulfonate (EMS) by oral gavage (oral intubation with a plastic gavage needle). In total 5 treatment groups were used, each consisting of 5 animals (males).
The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=45 h, respectively. The positive control was administered at t=24 h and t=45 h. The animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia at approximately t=48-49 h.


PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. 4-tert-butylpyrocatechol was suspended (white suspension) in Elix water (Millipore Corp., Bedford, MA., USA). The specific gravity of Elix water is 1.0 g/mL. 4-tert-butylpyrocatechol concentrations were dosed within 3.5 hours after preparation.

ANALYTICAL METHOD:
Analyses were performed on a single occasion during the treatment period according to a validated method (ABL Analytical work instruction (AWI) 4248, entitled: 4-tertbutylpyrocatechol in formulations using LC-DAD, validated in ABL validation study no.17115). The test site study number ABL no. 19149 was used for data collections and reporting. The concentrations analyzed in the formulations of Groups 2, 3 and 4 (samples prepared for use on 04 June 2019) were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 male rats per group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate (EMS) (CAS n°: 62-50-0) (EMS, Sigma Aldrich, Steinheim, German).
- Justification for choice of positive control(s): One of the positive control according to the OECD guideline.
- Route of administration: Oral (gavage).
- Doses / concentrations: At 200 mg/kg body weight dissolved in physiological saline. The dosing volume was 10 mL/kg body weight. EMS was used within 2 hours after preparation.

Examinations

Tissues and cell types examined:

Three tissues were examined: liver, stomach and duodenum. These three organs were isolated and single cell suspensions from liver, stomach and duodenum were made followed by comet slide preparation as described in the following field.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
As descibed below, selection of an adequate dose-range for the Comet main test was based on a dose-range finding study. Based on the dose-range finding study, three doses were selected for the main study. The highest dose was 400 mg/kg body weight (limit dose). The lower dose levels were 50% (200 mg/kg b.w - mid dose) and 25% of the high dose (100 mg/kg b.w - low dose).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):

Approximately 3 hours after the third treatment with the test compound or vehicle and second treatment with EMS liver, stomach and duodenum were collected/isolated and examined for DNA damage with the alkaline Comet assay.

ISOLATION CELLS:
Liver:
The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Stomach:
This isolation method for glandular stomach is based on the JACVAM Comet validation study. The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The forestomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)). The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia was gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis. The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Duodenum:
This isolation method for duodenum is based on the JACVAM Comet validation study. The duodenum was cut open and washed free from food using Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia was gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded. The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

DETAILS OF SLIDE PREPARATION:
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 12-25 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

LYSIS, ELECTROPHORESIS AND STAINING OF THE SLIDES:
The cells on the slides were overnight (approximately 17 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 or 30 (liver) minutes under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 to 6 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 to 6 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

SAMPLING, FIXATION AND STORAGE OF TISSUE FOR HISTOTECHNOLOGY AND HISTOPATHOLOGY:
Part of the liver, duodenum and glandular stomach from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
As animals dosed with the doses of 400 and 200 mg/kg/day showed slight, but statistically significant increases in Tail intensity in stomach and duodenum after scoring of the first 150 cells, histopathology of these doses and the vehicle was performed.

Histotechnology: The tissues were processed, embedded in paraffin wax, cut to slides at a thickness of 2-4 micrometers, and stained with haematoxylin and eosin.
Histopathology: Histopathology was performed on the tissues (stomach and duodenum) of the male animals in the main study that are treated with vehicle and 400 and 200 mg/kg bw/day. A peer review on the histopathology data was performed by a second pathologist.

METHOD OF ANALYSIS:
Comet scoring: To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample, as this resulted in equivocal results for stomach and duodenum 150 additional cells were evaluated. On a few slides, one of the agarose circles was damaged, therefore an agarose circle from the second backup slide was used for scoring.

The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.

Evaluation criteria:

Comet scoring.

ACCEPTABILITY CRITERIA:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
c) Adequate numbers of cells and doses have been analysed.
d) The highest test dose is the MTD or 2000 mg/kg/day.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.

1) A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the negative historical control data range.

2) A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Since the Dunnett’s or Welch t test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Negative controls validity:

valid

Remarks:

Water.

Positive controls validity:

valid

Remarks:

EMS.

Remarks on result:

other:

Remarks:

Three tissues were examined: llver, duodenum and stomach. The tail intensity (%) in liver cells is the following: vehicle control = 1.37 +/- 0.35 ; TBC (100 mg/kg) = 1.41 +/- 0.21 ; TBC (200 mg/kg) = 1.39 +/- 0.49 ; TBC (400 mg/kg) = 1.31 +/- 0.17 and positive control (EMS - 200 mg/kg) = 76.83 +/- 5.69. The tail intensity (%) in duodenum cells is the following: vehicle control = 4.86 +/- 1.28 ; TBC (100 mg/kg) = 5.47 +/- 2.82 ; TBC (200 mg/kg) = 7.51 +/- 1.55 ; TBC (400 mg/kg) = 5.69 +/- 1.70 and positive control (EMS - 200 mg/kg) = 44.04 +/- 4.83. The tail intensity (%) in stomach cells is the following: vehicle control = 3.24 +/- 0.64 ; TBC (100 mg/kg) = 3.33 +/- 0.53 ; TBC (200 mg/kg) = 4.86 +/- 1.62 ; TBC (400 mg/kg) = 4.65 +/- 0.88 and positive control (EMS - 200 mg/kg) = 51.36 +/- 4.94.

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 400 mg/kg b.w.
- Clinical signs of toxicity in test animals: Females: lethargy, ataxia, slow breathing, gurgling breathing, ventral recumbency, rough coat and hunched posture were observed at the dose level of 500 mg/kg b.w. Lethargy, rough coat, ataxia and hunched posture were observed at the dose level of 400 mg/kg b.w. Males: lethargy, slow breathing, ataxia, ventral recumbency, rough coat, hunched posture and no reaction to stimulus were observed at the dose level of 500 mg/kg b.w. Lethargy, ataxia, ventral recumbency, rough coat, hunched posture, slow breathing and gurgling breathing were observed at the dose level of 400 mg/kg b.w. See table 1 in the field below 'Any other information on results incl. tables'.
- Other: Based on the results of the dose-range finding study dose levels of 100, 200 and 400 mg/kg body weight were selected as appropriate doses for the main test. Since there were no substantial differences based on the dose-range finding study in toxicity between sexes, only males were used in the main study.

RESULTS OF DEFINITIVE STUDY
- Mortablity and clinical signs: The animals of the groups treated with the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. There was no mortality in all the groups treated with 4-tert-butylpyrocatechol. Within the first hour after dosing all animals of the group treated with 400 mg/kg body weight were lethargic, showed ataxia, rough coat and showed hunched postures. One animal in the group treated with 200 mg/kg body weight showed gurgling breathing and a hunched posture. Two animals in the group treated with 100 mg/kg body weight showed gurgling breathing. For more details, see table 2 in the field below 'Any other information on results incl. tables'.

- Body weight: See table 3 in the field below 'Any other information on results incl. tables'.

- Comet slide analysis: Negative control: the mean Tail Intensity in liver, duodenum and stomach cells of vehicle-treated rats was 1.37 ± 0.35% (mean ± SD), 4.86 ± 1.28% (mean ± SD) and 3.24 ± 0.64% (mean ± SD) respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. Positive control: the positive control EMS induced a significant increase and showed a mean Tail Intensity of 77 ± 5.69% (mean ± SD), 44 ± 4.83% and 51 ± 4.94% (mean ± SD) in liver, duodenum and stomach cells, respectively. The mean positive control Tail Intensity for stomach and duodenum was within the 95% control limits of the distribution of the historical positive control database. For the liver this value was just below the lower limit however as a 56 fold significant increase was induced this did not impact the results. Adequate numbers of cells (150 cells for liver and 300 cells for duodenum and stomach) and doses were analysed and the highest test dose was the MTD. Hence, the assay was concluded acceptable assay. Test item: no biologically relevant statistically significant increase in the mean Tail Intensity (%) was observed in liver and duodenum cells of 4-tert-butylpyrocatechol treated male animals compared to the vehicle treated animals. In stomach cells a slight, but statistically significant increase of the Tail Intensity (%) was found at the high dose, however this did not exceed the 95% confidence interval for the historical negative controls and is therefore not biologically relevant. See tables 4, 5 and 6 in the field below 'Any other information on results incl. tables'.

- Formulation analysis: Formulation analysis was performed to determine the accuracy of preparation of the test item in formulations. The concentrations analyzed in the formulations of the high dose, mid dose and low dose were in agreement with target concentrations. No test substance was detected in the vehicle control.

- Histopathology: Histopathology showed morphologic alterations following the administration of 4-tertbutylpyrocatechol to male Wistar (Han) rats present in the stomach and duodenum at 200 and 400 mg/kg/day. Findings representing cell death were only present at 400 mg/kg/day in 1/5 rats in duodenum and 4/5 rats in stomach.
Stomach: Erosion/ulcer glandular and/or non-glandular was present in 4/5 rats treated at 400 mg/kg/day up to moderate degree. Inflammation, mixed cell glandular and/or non-glandular was present in 3/5 rats treated at 400 mg/kg/day up to moderate degree. Hemorrhage glandular was present in 1/5 rats treated at 400 mg/kg/day at slight degree. Hyperplasia squamous cell non-glandular was present in 1/5 rats treated at 200 mg/kg/day at slight degree.
Duodenum: Erosion/ulcer mucosa, was present in 1/5 rats treated at 400 mg/kg/day at minimal degree. Vacuolation Goblet cell was present in 1/5 rats treated at 200 mg/kg/day at slight degree.
Of these findings, the erosion/ulcer in the stomach and duodenum were the only findings representing cell death.

- Statistical evaluation:
Test item:
Comparison vehicle control and test item groups by using the Dunnett’s test (p<0.05, onesided):
No statistically significant differences, except in the highest dose group of stomach cells.
The Cochran Armitage Trend test showed a positive trend in stomach cells.

Positive control:
Comparison vehicle control group and positive control group by using the Student’s t test (p<0.05, one sided):
The positive control increased the Tail Intensity significantly (p<0.001) compared to the vehicle in liver, stomach and duodenum cells.

Any other information on results incl. tables

Tables of results:

Table 1: Mortality and Toxic Signs after Treatment with 4-tert-butylpyrocatechol in the Dose-range Finding Study

Group

Sex

Animal Number

Dose mg/kg

Toxic signs*

day 1 within … hours after dosing

day 2

day 2 within … hours after dosing

day 3

day 3 within … hours after dosing

2.5 hrs

2.5 hrs

2.5 hrs

A

Male

1

500

F, P, W, G

J

F,C,N,J

N,J

N,J

A

Female

2

500

F, P, W

J

F,C,N,J

F,N,J

F,N,J

A

Male

3

500

F,P,W,G

F,N,J

F,C,N,J,P

F,N,J

F,G,P,W

A

Male

4

500

F,P,W,G

F,N,J

F,W,N,J,P

F,N,J

F,C,N,J,P

A

Female

5

500

F, P, W,N

F,J,X

F,N,J,X

F,J,X

F,N,J

A

Female

6

500

F, P, W,N

F,J,X

F,N,J

F,J

F,N,J

B

Male

7

400

F,N,J,C

B

F,W,P

N,J

F,C,N,J

B

Female

8

400

F,N,J,C

B

F,C,N,J

N,J

F,C,N,J

B

Male

9

400

F,C

B

F,C,N,J

F,N,J,X

F,C,N,J,X

B

Male

10

400

F,C

B

F,C

B

F,J

B

Female

11

400

F,C,N,J

B

F,C,N,J

B

F,C,J

B

Female

12

400

F,C,N,J

B

F,C,N,J

X

F,C,J

 

*  Legend 'Mortality and toxic signs': B= No abnormalities, C = ataxia; F = lethargy; G = no reaction to stimulus;
J = hunched posture; N = rough coat; P =slow breathing;  W = ventral recumbency, X = gurgling breathing.

Table 2: Mortality and Toxic Signs after Treatment with 4-tert-butylpyrocatechol in the Main Finding Study

Group

Sex

Animal Number

Dose mg/kg

Toxic signs*

day 1 within … hours after dosing

day 2

day 2 within … hours after dosing

day 3

2.5 hrs

2.5 hrs

B

Male

18

100

B

B

B

B

B

Male

19

100

B

X

X

B

B

Male

20

100

B

B

B

B

B

Male

21

100

B

B

B

B

B

Male

22

100

B

X

X

X

C

Male

23

200

B

B

B

B

C

Male

24

200

B

XJ

XJ

XJ

C

Male

25

200

B

B

B

B

C

Male

26

200

B

B

B

B

C

Male

27

200

B

B

B

B

D

Male

28

400

FCJ

X

FJ

FJ

D

Male

29

400

FJ

X

FJ

FJ

D

Male

30

400

FJ

B

FJ

FJ

D

Male

31

400

FJ

J

FJ

FJ

D

Male

32

400

FJ

B

FNJX

FJX

D^2

Male

38

400

FJC

B

FJC

FJ

D^2

Male

39

400

FJC

B

FJ

FJ

D^2

Male

40

400

FJ

B

FJX

FJ

*  Legend 'Mortality and toxic signs': B= No abnormalities, C = ataxia; F = lethargy; J = hunched posture; N=rough coat; X = gurgling breathing

²Additional animals used to compensate for possible deaths (not analyzed in the study)

 

Table 3: Mean Body Weight Immediately Prior to Dosing with 4-tert-butylpyrocatechol and EMS in the Main Study.

Group code	Dose (mg/kg/bw)	Day 1 Body weight gram (Mean ± S.D.)			Day 2 Body weight gram (Mean ± S.D.)			Day 3 Body weight gram (Mean ± S.D.)
A	0	153.2	±	9.3	148.6	±	8.6	151.4	±	9.4
B	100	144.8	±	7.0	139.8	±	10.0	143.0	±	9.7
C	200	144.8	±	12.4	137.6	±	17.7	139.8	±	22.4
D	400	152.4	±	7.6	146.4	±	12.9	145.6	±	12.0
E	200 (EMS)	^1	±		146.8	±	13.7	150.0	±	12.9
D^2		144.3	±	5.9	140.7	±	4.7	138.0	±	7.8

¹Not dosed on day 1. Dosing with EMS was started on Day 2

²Additional animals used to compensate for possible deaths (not analyzed in the study)

 

Table 4: Overview Tail Intensity in liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	1.37	0.35
4-tert-butylpyrocatechol (100 mg/kg)	1.41	0.21
4-tert-butylpyrocatechol (200 mg/kg)	1.39	0.49
4-tert-butylpyrocatechol (400 mg/kg)	1.31	0.17
EMS (200 mg/kg)	76.83	5.69

 

Table 5: Overview Tail Intensity in duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	4.86	1.28
4-tert-butylpyrocatechol (100 mg/kg)	5.47	2.82
4-tert-butylpyrocatechol (200 mg/kg)	7.51	1.55
4-tert-butylpyrocatechol (400 mg/kg)	5.69	1.70
EMS (200 mg/kg)	44.04	4.83

 

 

Table 6: Overview Tail Intensity in stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	3.24	0.64
4-tert-butylpyrocatechol (100 mg/kg)	3.33	0.53
4-tert-butylpyocatechol (200 mg/kg)	4.86	1.62
4-tert-butylpyocatechol (400 mg/kg)	4.65	0.88
EMS (200 mg/kg)	51.36	4.94

 

Applicant's summary and conclusion

Conclusions:

In conclusion, the test is valid and 4-tert-butylpyrocatechol is not genotoxic in the Comet assay in liver, duodenum and stomach when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for three consecutive days at the low (100 mg/kg), mid (200 mg/kg) and high (400 mg/kg) doses (400 mg/kg - corresponds to the maximum tolerated in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of 4-tert-butylpyrocatechol (TBC) when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in DNA strand breaks in liver, duodenum and stomach. The test item was suspended in Elix water.

In the dose-range finding study 3 males and 3 females were dosed once daily via oral gavage with 500 mg 4-tert-butylpyrocatechol per kg body weight for a maximum of three days and 3 males and 3 females were dosed once daily via oral gavage with 400 mg 4-tert-butylpyrocatechol per kg body weight for a maximum of three days. The animals showed several toxic signs. Since there were no substantial differences in toxicity between sexes only males were used in the main study. Based on the results of the dose-range finding study test concentrations of 400 mg/kg/day was selected as maximum dose for the main test (maximum tolerated dose).

In the main study male animals were dosed by oral gavage with vehicle or with 100, 200 and 400 mg 4-tert-butylpyrocatechol per kg body weight for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. The animals showed the following toxic signs after dosing with 4-tert-butylpyrocatechol whatever the administrated doses: ataxia, lethargy, hunched posture, rough coat and gurgling breathing. No mortality was observed after dosing with 4-tert-butylpyrocatechol. No treatment related clinical signs or mortality were noted in any animal treated with EMS (positive control) or control animals receiving vehicle. Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or 4-tert-butylpyrocatechol, the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and liver, stomach and duodenum were isolated. Single cell suspensions from liver, stomach and duodenum were made followed by comet slide preparation.

Results: The mean Tail Intensity in liver, duodenum and stomach cells of vehicle-treated rats was 1.37 ± 0.35% (mean ± SD), 4.86 ± 1.28% (mean ± SD) and 3.24 ± 0.64% (mean ± SD) respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 77 ± 5.69% (mean ± SD), 44 ± 4.83% and 51 ± 4.94% (mean ± SD) in liver, duodenum and stomach cells, respectively. The mean positive control Tail Intensity for stomach and duodenum was within the 95% control limits of the distribution of the historical positive control database. For the liver this value was just below the lower limit however as a 56 fold significant increase was induced this did not impact the results. Adequate numbers of cells and doses were analysed and the highest test dose was the MTD. Hence, the assay was concluded acceptable assay.

No biologically relevant statistically significant increase in the mean Tail Intensity (%) was observed in liver and duodenum cells of 4-tert-butylpyrocatechol treated male animals compared to the vehicle treated animals. In stomach cells a slight, but statistically significant increase of the Tail Intensity (%) was found at the high dose, however this did not exceed the 95% confidence interval for the historical negative controls and is therefore not biologically relevant.

Histopathology showed morphologic alterations following the administration of 4-tertbutylpyrocatechol to male Wistar (Han) rats present in the stomach and duodenum at 200 and 400 mg/kg/day. Findings representing cell death were only present at 400 mg/kg/day in 1/5 rats in duodenum and 4/5 rats in stomach.

In conclusion, the test is valid and 4-tert-butylpyrocatechol is not genotoxic in the Comet assay in liver, duodenum and stomach when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for three consecutive days at the low (100 mg/kg), mid (200 mg/kg) and high (400 mg/kg) doses (400 mg/kg - corresponds to the maximum tolerated in accordance with current regulatory guidelines) under the experimental conditions described in this report.