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Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29/05/2007 - 27/11/2008
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Principles of method if other than guideline:
The single cell gel electrophoresis (SCGE) assay, also known as the "comet assay", is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakage in mammalian cells. The purpose of the in vivo Comet assay following the alkaline version (pH > 13) developed by Singh et al. (1988), is to identify those agents, which induce DNA damage such as single or double DNA strand breaks (SSB or DSB), alkali-labile sites, DNA-DNA / DNA-protein cross-linking and SSB associated with incomplete excision repair sites. The advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage. The purpose of this study is to assess the genotoxic activity of the test compound in one or several target organs under these experimental conditions.
GLP compliance:
yes
Type of assay:
other: comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyrocatechol
EC Number:
204-427-5
EC Name:
Pyrocatechol
Cas Number:
120-80-9
Molecular formula:
C6H6O2
IUPAC Name:
pyrocatechol

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, saint-Germain-sur-l'Arbresle; France
- Age at study initiation: 5 to 10 week old
- Weight at study initiation: 156 to 192 g
- Housing: animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by stainless steel netted lid, in which they were placed in groups of 3 or 2 by random distribution. The animals are identified by numbered ear rings.
- Diet (e.g. ad libitum): the animals were not fasted at the treatment time.
- Water (e.g. ad libitum): no data
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): 20times per hour
- Photoperiod (hrs dark / hrs light): light 12h a day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: distilled water
Details on exposure:
- Rats were treated with cathecol by gavage with standard volume of 10 mL/kg
- Catechol was administered in solution in distilled water, which were prepared before use. The test compound was administerd using two treatments at 24 hours interval.
- The negative control rats received distilled  water.
- the positive control rats received MNNG or dimethylhydrazine
- 3 to 6 hours after second treatment animals were sacrified and cells of the target organs were isolated.
Duration of treatment / exposure:
Two treatments at 24 hour interval and sacrifice 3 to 6 hours after the second treatment.
Frequency of treatment:
twice
Post exposure period:
no
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- For stomach : MNNG, 20 mg/kg/day
- For duodenum : Dimethylhydrazine, 20 mg/kg/day

Examinations

Tissues and cell types examined:
Stomach and duodenum
Details of tissue and slide preparation:
1) ISOALTION OF CELLS:
The abdominal surface of the animal is rinsed with 70% (v/v) ethanol and a 'V' shaped incision is made from the centre of the lower abdomen to the rib cage. The skin and muscles are removed to reveal the abdominal cavity.

STOMACH:
The stomach is removed, opened and rinsed with calcium- and magnesium-free phosphate buffer saline (PBS). The forestomach is discarded and the cells of gastric mucosa are isolated by enzymatic digestion following Burlinson et al. (1989) and Brault et al. (1999) as described hereafter:

The gastric mucosa is incubated in calcium- and magnesium-free Hank’s balanced salt solution (HBSS) containing 50 U/mL protease at 37 °C for 30 minutes in the oven. After the first incubation period, the mucosa is then flushed with the incubation medium to remove cells. The cell suspensions are centrifuged at 350 g for 5 minutes. The cell pellets are resuspended in HBSS containing 0.25 % dispase II and the suspensions are incubated at 37 °C for 15 minutes in a hot-bath.
After the second incubation period, 0.5 mL of fetal calf serum is added and the cell suspensions are filtered through a 150 µm nylon filter. The cell suspensions are then centrifuged at 350 g for 5 minutes and the cells are resuspended in HBSS.

DUODENUM:
The totality of the duodenum is removed and rinsed with calcium- and magnesium-free phosphate buffer saline (PBS). Cells of duodenum are isolated by enzymatic digestion following Evans et al. (1992) as described hereafter.

Firstly, a ligature is tightened at one end of each duodenum sample. Then, a mix of collagenase XI and dispase I (300 U/ml, 0.02 %) is directly injected into the sample of duodenum using a syringe and a second ligature is placed on the other end of the sample of duodenum. The sample of duodenum filled with the enzymatic mix is then incubated for 30 minutes at 37 °C in a hot-bath, in 10 mL of HBSS. After the incubation period, around 0.2 mL of fetal calf serum is added. The sample of duodenum is then opened and carefully scrapped in order to facilitate the cell dissociation. After that, the cell suspension is centrifuged 5 minutes at 150 g. The cell pellets are resuspended in HBSS.

For both tissues: the proportion of viable cells was determined with the help of a Malassez haemocytometer using Trypan blue technique before preparing slides to be assessed for DNA fragmentation.

2) COMET ASSAY

The Comet assay is performed under alkaline conditions essentially following the procedure of Singh et al. (1988). At least three slides are prepared for each animal and there was 4 animals per group, i.e. at least 12 slides per type of treatment (negative or positive control and two dose levels). There was 50 cells per slide that are randomly scored, i.e. 150 cells per animal.

The essential steps of comet assay are successively, layering of cells mixed with low melting point agarose (over coated glass microscope slides), lysis (to lyse the cell and nuclear membranes and other proteins), unwinding of DNA, electrophoresis, neutralization, staining and scoring.

2.1) Dried slides preparation (pre-layering)

Conventional slides are dipped in a 1.5 % normal melting point agarose in PBS while it was hot. After gently remove, underside of slides was wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry.

2.2) Slide preparation

Before use, a volume of 85 µl of 0.8% of Normal Agarose (NA) was added on microscope slide pre-layered with 1.5% of NA and cover with a glass coverslip. Slides were placed on a slide tray resting on the ice packs until the agarose layer hardens (3 to 5 minutes). Around 3 x 104 cells of the different concentrations tested were mixed with 75 µl of 0.5% of Low Melting Point Agarose (LMPA) kept at 37 °C and added on microscope slide after gentle slide off the coverslip. They were then covered with a new glass coverslip. Slides were placed on a slide tray on ice packs for 3 to 5 minutes.

2.3) Lysis at pH=10

After the top layer of agarose has solidified, the glass coverslips are removed and the slides are immersed for at least 1 hour at + 4 °C in the dark in a lysing solution consisting of 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, to which 1% Triton X-100 and 10% DMSO are freshly added (pH adjusted to 10 with NaOH).

2.4) Unwinding, electrophoresis and staining

The slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer (1 mM EDTA and 300 mM NaOH, pH > 13) to around 0.25 cm above the slides. In order to avoid excessive variation across the groups for each electrophoretic run, for each animal, only one of the quadriplicate slides is precessed in each run. The cells were exposed to the alkali for 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for 20 minutes at 0-4°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted sheltered from the daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for 5 minutes with 0.4 M tris (pH 7.5) and the DNA was exposed for 5 minutes to absolute ethanol in order to preserve all the comet assay slides. Subsequently, the slides were airdried and then stored at room temperature until they were scored for DNA migration.
Just prior to scoring, the DNA was stained using Propidium Iodide (20 µg/mL distilled water; 30 µL/slide).

2.5) Image analysis

Slides were examined at 250 x magnification using a fluorescent microscope (Leica Microscopy and Scientific Instruments Group, Heerbrugg, Switzerland) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated CCD camera to Comet Image Analysis System, version 4.0 software (Kinetic Imaging Ltd, Liverpool, UK).

At least three slides were prepared for each animal and there was 4 animals per group, i.e. at least 12 slides per type of treatment (negative or positive control and two dose levels).

2.6) Tail parameters

Olive Tail Moment (OTM) preconised by Olive (1993) was used to evaluate DNA damage. The OTM, expressed in arbitrary units, was calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in µm (B. Hellman et al., 1995; E. Rojas et al., 1999). The tail length was measured between the edge of comet head and the end of the comet tail.

A major advantage of using the OTM as an index of DNA damage was that both the amount of damaged DNA and the distance of migration of the genetic material in the tail were represented by a single number (J. Ashby et al., 1995).


Evaluation criteria:
Cytotoxicity:
Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue vital dye exclusion technique.
In accordance with a recognized group of scientists, the decrease in the viability should not be more than 30 % when compared to the concurent control. Cell viability in the target tissue that is below 70 % of that in the control animals may thus be considered excessive.
Therefore, according to the data obtained from the cytotoxicity assessment, doses are actually selected for genotoxicity assessment (two dose levels are chosen for each structured organ).
At least 1.2 x 105 viable cells are required for proceeding to slides preparation (4 slides with 3 x 104 viable cells per slide).

Acceptance criteria for result:
A study is accepted if both following criteria are fulfilled:

- In the solvent control group, the OTM median must be lower than 8.
- In the positive control groups, the OTM median must be statistically increased compared to the control group.

A compound is found to demonstrate genotoxic properties against the target organ if it results in a statistically significant increase in the OTM median compared with the negative control group and if the genotoxicity detected shows a dose-effect relationship.

A compound is found to have no genotoxic effect on the target organ if it does not comply with any of the criteria listed above.

If neither situation occurs, the results are discussed case by case and another independent study may be implemented after modifying the dose range taking into account all available relevant data. Any complementary assay will be the subject of a new study plan.

The criteria are not absolute but do constitute an aid to decision, which will make it possible to reach a conclusion in most cases.
Statistics:
Statistical analysis using non-parametric tests: Since the OTM frequencies and other tail parameters do not follow a gaussian distribution (E. Bauer et al., 1998), the non-parametric Kruskall-Wallis test was used to display a possible dose-effect relationship. Moreover the statistical significance of differences in the median values between each group versus the control group was determined with the non-parametric Mann-Whitney U-test.

Statistical analysis using parametric tests: First, for each assay, a box plot on Olive Tail Moment over group was realized in order to show the distribution per slide per animal. The statistical test for treatment effect was a trend-test using linear contrast in the treatment group: an estimation of the differences between each treated group and the negative control group was provided with its associated 95 % confidence interval. This statistical method was particularly well adapted for interpretation of in vivo data with possible inter-individual variations.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The in vivo comet assay performed under alkaline conditions, i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in the OFA Sprague Dawley male rats, stomach and duodenum, after two treatments by oral route at 3 dose levels (the maximum tolerated dose, MTD, 50 and 25% MTD), followed by one expression time of 3 to 6 hours after the last treatment. The test CATECHOL was dissolved in distilled water up to a maximum concentration of 80 mg/mL (toxicity assay) or 40 mg/mL (genotoxicity assay) and administered at a dose volume of 10 mL/kg, giving final doses of 800 and 400 mg/kg, respectively.
The different inferior dilutions were also performed with distilled water. Regarding the positive reference substance, both MNNG and dimethylhydrazine were dissolved in distilled water and administered under a dose volume of 10 mL/kg by oral route.

PRELIMINARY TEST RESULT

Results of the toxicity test by the oral route in OFA Sprague Dawley male rats (preliminary and confirmatory assays):
In this toxicity assay, two groups of 4 male rats were dosed orally twice at 800 and 400 mg/kg/day.
The maximum tolerated dose of CATECHOL was set at 400 mg/kg/day (x2) by oral route in OFA Sprague Dawley male rats. Indeed, the dose of 800 mg/kg/day (x2) elicited strong clinical signs, as described below and at the dose of 500 mg/kg/day (x2), 3 animals out of 4 died 25 min after the first
treatment. In return, the dose of 400 mg/kg/day (x2) induced no clinical signs.
DOSE OF 400 MG/KG/DAY (X2)
The dose level of 400 mg/kg/day (x2) induced no death or clinical signs in the four male rats after the 1st or the 2nd treatment.
DOSE OF 800 MG/KG/DAY (X2)
The first treatment at the high dose level of 800 mg/kg/day (x2) induced very strong clonic convulsions in the four male rats, 15 min to 30 min after the first treatment. For ethical reasons, the animals were euthanasied and no second treatment was performed.
In order to confirm that the dose of 400 mg/kg/day (x2) is the actual maximum tolerated dose in OFA Sprague Dawley male rats, a 2nd toxicity assay with a third intermediary dose of 500 mg/kg/day (x2) was implemented.
DOSE OF 500 MG/KG/DAY (X2)
The first treatment at the dose level of 500 mg/kg/day (x2) induced strong clonic convulsions in the four male rats, 15 min to 30 min after the first treatment. Three animals out of 4 died and for ethical reasons, the 4th animal was euthanasied and no second treatment was performed.
Under these conditions, the dose of 400 mg/kg/day (x2) was retained as the maximum dose to be tested in the comet assay. Two inferior doses were also tested, i.e. 200 and 100 mg/kg/day (x2).

MAIN EXPERIMENT
At least 5 male rats per dose were treated orally twice with 400, 200 and 100 mg/kg/day (x2) CATECHOL for the in vivo comet assay on stomach and duodenum. The 2 highest doses of 400 and 200 mg/kg/day (x2), giving acceptable cell viability using the Trypan blue vital dye exclusion technique, i.e. >70%, were analysed first. At sponsor’s request, the 3rd dose of 100 mg/kg/day (x2), also presenting a cell viability above 70%, was analyzed and the results presented in the Final report. The test results are summarized in Tables.
The historical data for negative control and positive control were constituted with an assay after two treatments followed by one sampling time, 3 to 6 hours after the last treatment.
Significant increases in the mean OTM median values were noted in the groups treated with MNNG and dimethylhydrazine, demonstrating the sensitivity of the animal strain used to specific clastogenic agents on stomach and duodenum. Thus, the validity criteria for the test were fulfilled and the test was valid. The weight homogeneity of the animals used in this test after random-distribution was verified by comparing the weight mean of the treatment groups with that of the control group (Student t-test). There was no statistically significant difference between the weights of animals treated with the test item and those of control rats.

The results of the cellular viability determination upon the Trypan blue exclusion method are presented: the calculated relative viabilities for stomach and duodenum cells were superior to 70 %.

However, it is to be noted that very strong clonic convulsions were observed 5 minutes after oral administration of Catechol at the dose of 400 mg/kg/day (x2), leading to one death between 30 minutes and 2 hours after the 1st treatment. Furthermore, the 4 animals were trembling after the 2nd treatment at 400 mg/kg/day. The animals treated with the two inferior doses of 200 and 100 mg/kg/day elicited no clinical signs.
Furthermore, the observation of slides during image analysis of stomach and duodenum cells, showed a very low cell density at the two highest doses analysed of 400 and 200 mg/kg/day (x2). This low cell density indicates the presence of cell lysis that could not be identified with the Trypan blue vital dye exclusion technique nor with the measurement of ghost cells.

GENOTOXICITY IN RAT STOMACH CELLS
No statistically significant increase in the OTM medians was observed in rat stomach cells treated with the three doses of Catechol, i.e. 400 – 200 and 100 mg/kg/day (x2). Indeed, the values of median OTM for 600 cells were of 0.81 – 0.35 and 1.13 at 400 – 200 and 100 mg/kg/day (x2), respectively, vs. 2.26 in the negative control group. The OTM medians at the three tested doses were under the value of OTM median for the control group.
The F test (F of Snedecor) showed a statistically significant difference between group OTM variance (p<0.0001). Due to this non-homogeneity of variance between OTM groups, statistical analysis was performed using non-parametric tests.
The non-parametric statistical assessment allowed to display a significant dose-response relationship (Kruskall-Wallis, p<0.0001).
The pair wise analysis using Mann-Whitney showed statistically significant decreases in the median OTM of the three tested doses of 400 – 200 and 100 mg/kg/day (x2), vs. the negative control (Mann-Whitney, p<0.0001). Nevertheless, this decrease had no meaning in terms of genotoxicity.
Regarding the percentages of ghost cells, no increase compared to the negative control was observed.
Under these conditions, Catéchol was not considered as a DNA strand breaks and/or alkali-labile sites inducer on stomach cells in the rat.

GENOTOXICITY IN RAT DUODENUM CELLS
Statistically significant increases in the OTM medians were observed in duodenum cells from rats, treated with the doses of 200 and 100 mg/kg/day (x2) of Catéchol. Indeed, the values of median OTM for 600 cells (4 animals tested per group) reached 3.38 and 4.64 at the two doses of 200 and 100 mg/kg/day (x2), respectively, vs. 1.48 in the negative control group (Tables 3 and 10). At these two dose-levels of 200 and 100 mg/kg/day (x2), two animals out of the four treated, presented particularly strong increases in the median OTM, with values reaching 6.07 and 4.21 at the dose of 200 mg/kg/day (x2) and 9.73 and 7.29 at the dose of 100 mg/kg/day (x2) (for 150 cells observed by animal, Table 10). In return, the dose of 400 mg/kg/day (x2) did not induce a statistically significant increase in the median OTM, with a value of 1.28. This value was under the value of OTM median for the control group.
It is noteworthy that the highest median OTM was observed at the lowest dose tested of 100 mg/kg/day (x2). The decrease in the values of median OTM at the two upper doses of 400 and 200 mg/kg/day (x2), compared to the dose of 100 mg/kg/day (x2), is most probably related to toxicity of the test item Catechol, consistent with the clinical signs described above, i.e. strong clonic convulsions were observed 5 minutes after oral administration of Catechol at the dose of 400 mg/kg/day (x2), leading to one death between 30 minutes and 2 hours after the 1st treatment and the 4 animals were trembling after the 2nd treatment at 400 mg/kg/day. In return, no clinical signs were observed after the treatments at 200 and 100 mg/kg.
Furthermore, the cytotoxicity evaluated with the Trypan blue vital exclusion method and the measurement of the percentage of ghost cells during image analysis, did not permit to evaluate the potential cytotoxicity at the three doses tested.
The bell-shaped curve effect observed is considered as an indicator of the genotoxic activity of Catechol. No dose without genotoxic effect could be determined at a concentration lower than 100 mg/kg/day (x2). Therefore, it would be useful to implement a complementary assay in order to investigate if Catechol induces a genotoxic effect at doses lower than 100 mg/kg/day (x2) and to determine the highest dose under 100 mg/kg/day (x2) without genotoxic activity.
Regarding the statistical assessment, the F test (F of Snedecor) showed a statistically significant difference between group OTM variance (p<0.0001). Due to this non-homogeneity of variance between OTM groups, statistical analysis was performed using non-parametric tests.
The non-parametric statistical assessment allowed to display a significant dose-response relationship
(Kruskall-Wallis, p<0.0001). It is noteworthy that this is a reverse dose-effect relationship, i.e. there is a decrease in the median OTM when increasing the doses of Catechol.
The pair wise analysis using Mann-Whitney showed statistically significant increases in the median OTM of the two tested doses of 200 and 100 mg/kg/day (x2), vs. the negative control. In return, the dose of 400 mg/kg/day (x2) did not induce a statistically significant increase in the median OTM.
Regarding the percentages of ghost cells, no increase compared to the negative control was observed.
Under these conditions, Catechol was considered as a DNA strand breaks and/or alkali-labile sites inducer on duodenum cells in the rat.
HYPOTHESIS ON THE GENOTOXICITY OF CATECHOL
The results obtained in the in vivo comet assay on stomach in presence of CATECHOL, i.e.:
• no statistically significant increases in DNA strand breaks at non-lethal dose on rat stomach cells after oral administration.
• statistically significant decreases in the median OTM of the three tested doses and displaying significant reverse dose-response relationship
• absence of signs of cytotoxicity at the three doses tested with the Trypan blue vital exclusion method
• no significant increase in the measurement of the percentage of ghost cells on the slides
• very low cell density observed during image analysis indicating a probable cell lysis due to cytotoxicity and /or highly damaged cells with loss of information and in duodenum, i.e. :
• statistically significant increases in DNA strand breaks, with the highest increase of median OTM the lowest dose tested, decrease in the values of median OTM at the two upper doses and statistically significant increases at the highest dose tested
• displaying a significant reverse dose-response relationship, decrease in the median OTM when increasing the doses of Catechol, i.e. a bell-shaped curve response.
• absence of signs of cytotoxicity at the three doses tested with the Trypan blue vital exclusion method
• no significant increase in the measurement of the percentage of ghost cells on the slides
• very low cell density observed during image analysis indicating a probable cell lysis due to cytotoxicity and /or highly damaged cells with loss of information lead to different hypothesis.

1/ The test item Catechol seems to induce excessive fragmentation of DNA. This extreme DNA fragmentation may be caused by:
- DNA strand breaks, single or double DNA strand breaks (SSB or DSB).
- Another possible mechanism could be DNA fragilization through the formation of alkali-labile sites. In such cases, the formation of apurinic or apyrimidic sites by excision of damaged bases by a DNA glycosylase, may alter and fragilize DNA. The alkali-labile sites are stable up to a pH of 12.5 but are
eliminated at a pH of 13, as in the in vivo rodent alkaline assay, causing DNA strand breaks (Eastman & Barry, 1992).
The difference of genotoxic activity observable in stomach and in duodenum cells may be related to the fact that the concentration of Catechol in contact with the tested organ is higher in the stomach than in the duodenum.
The observation of the slides at the two highest doses tested of 400 and 200 mg/kg/day (x2) during image analysis showed a total absence of ghost cells, as well as a very low cell density, that may be related to the total lysis of the cells after DNA fragmentation. The strong pulverization of DNA hindered correct staining of the DNA and thus observation and scoring of comet cells. The limit of detection has been trespassed.
Furthermore, the lowest dose tested of 100 mg/kg/day (x2), inducing a genotoxic activity in the comet
assay, presented a normal cell density on the slides during image analysis.
The absence of signs of cytotoxicity at the three doses tested using techniques such as Trypan blue vital exclusion method and the measurement of the percentage of ghost cells on the slides may be due to the fact that some ghost cells are not visible on the slides. Indeed, as stated by Burlinson (2007), for certain chemicals, the enhanced DNA migration is not observed in the in vivo rodent alkaline comet assay, despite the presence of necrosis or apoptosis in the target organs. In that case, the cytotoxicity is directly exerted by Catechol.

2/ The test item Catechol is most probably an oxidizing agent, producing free radicals damaging
DNA. In order to show that reactive oxygen species may be involved in the genotoxic activity of Catechol, an in vivo rodent alkaline (pH>13) comet assay adding a specific endonuclease enzyme, e.g. formamidopyrimidine-DNA glycosylase (Fpg), could be performed at lower doses than the ones tested in the current study.
The Fpg enzyme presents a DNA glycosylase activity, associated with an AP lyase activity leading to the formation of AP sites in DNA followed by the formation of single strand DNA breaks. In a normal cell, DNA repair is achieved by the action of other enzymes (DNA polymerase and DNA ligase). Under the conditions of the Comet assay, these enzymes are present until the sacrifice of animals but are absent during the Unwinding, Electrophoresis and staining steps of the Comet Assay. The adjunction of Fpg during the Comet assay allows to increase the number of breaks in DNA induced through an oxidizing mechanism and leads to an increase in comet response in case of DNA oxidative damage induced by Catechol. The expected results after treatment with Catechol and addition of Fpg, would be an increase in median OTM values, compared to the OTM values of cells exposed to Catechol alone, without addition of Fpg enzyme.
Nevertheless, performing this new comet assay will not modify the results and conclusions of the current study.
The decrease in the median OTM in stomach and duodenum cells when increasing the doses of Catechol and the reverse dose-response relationship indicate the possible existence of a bell-shape curved. As described by Burlinson et Al (2007), in some cases it is also possible to detect a decrease in DNA migration, as observed with Catechol in stomach and duodenum cells, due to:
- the loss of heavily damaged or dying cells during sample processing or electrophoresis, and /or secondary toxicity induced by Catechol.
- The downturn phenomenon in the dose-response curve may also be attributed to an altered bioavailability at higher dose levels.
Due to the bell-shape curve effect of the genotoxic activity of Catechol, it is not excluded that Catechol may display genotoxic activity both in stomach and duodenum cells at dose-levels lower than 100 mg/kg/day (x2).

CONTROL OF CONCENTRATION IN DOSING FORMULATIONS
A satisfactory agreement was observed between the actual and nominal concentrations of CATECHOL in treatment solutions used in the in vivo comet assay performed on stomach and duodenum. Indeed, the deviations from nominal concentrations were within an acceptable range of ±10%. Furthermore, solutions of CATECHOL can be considered as stable at –18°C, during two months.

Any other information on results incl. tables

Table: In vivo comet assay isolated rat stomach cells recapitulative table

 

In vivo Comet assay in isolated rat stomach cells

groups

compound

doses in mg/kg/day (x2)

OTM Median for 600 cells (1)

F Snedecor (homogeneity of variances)

NON PARAMETRIC statistical evaluation

NON PARAMETRIC statistical evaluation

Relative ratio of ghost cells (4)

 

 

 

 

 

p kruskall-wallis (2)

P Mann-whitney (3)

 

solvent control

distilled water

0

2.26

p< 0.0001

p< 0.0001

-

-

treated

catechol

400

0.81

p< 0.0001

p< 0.0001

p< 0.0001

0.54

treated

catechol

200

0.35

p< 0.0001

p< 0.0001

p< 0.0001

0.59

treated

catechol

100

1.13

p< 0.0001

p< 0.0001

p< 0.0001

0.96

Positive control

MNNG

20 mg/kg/day (x1)

5.37

-

-

p< 0.0001

0.95

 

A statistically significant linear trend in nuclear fragmentation, excluding positive control was revealed

by the Kruskall-Wallis test. The analysis showed a statistically significant dose-related decrease in the

median OTM at the three tested doses of 400 – 200 and 100 mg/kg/day (x2). The trend had hence no

meaning in terms of genotoxicity.

 

Table: In vivo comet assay isolated rat duodenum cells recapitulative table

 

In vivo Comet assay in isolated rat duodenum cells

groups

compound

doses in mg/kg/day (x2)

OTM Median for 600 cells (1)

F Snedecor (homogeneity of variances)

NON PARAMETRIC statistical evaluation

NON PARAMETRIC statistical evaluation

Relative ratio of ghost cells (4)

 

 

 

 

 

p kruskall-wallis (2)

P Mann-whitney (3)

 

solvent control

distilled water

0

1.48

p< 0.0001

p< 0.0001

-

-

treated

catechol

400

1.28

p< 0.0001

p< 0.0001

N.S.

0.79

treated

catechol

200

3.38

p< 0.0001

p< 0.0001

p< 0.0001

0.94

treated

catechol

100

4.64

p< 0.0001

p< 0.0001

p< 0.0001

1.10

Positive control

Dimethylhydrazine

20 mg/kg/day (x1)

8.94

-

-

p< 0.0001

0.87

 

(1) for 450 cells in positive control

(2) total group without positive control

(3) OTM values obtained in treated group compared to OTM values obtained in solvent control group

(4) corresponds to the percentage of ghost cells per treated group/ percentage of ghost cell in negative control group

 A statistically significant linear trend in nuclear fragmentation, excluding positive control was revealed by the Kruskall-Wallis test.

Applicant's summary and conclusion

Conclusions:
Positive
Executive summary:

The test item CATECHOL (batch FPC0619301) provided by RHODIA was investigated by the means of the in vivo comet assay on stomach and duodenum, under alkaline conditions (SCGE) in the male OFA Sprague Dawley rats treated orally twice with 400, 200 and 100 mg/kg/day, with one sampling time 3 to 6 hours after the last treatment. Following the results of the toxicity assay, the maximum tolerated dose (MTD) determined was of 400 mg/kg/day. This dose was retained as the maximum dose to be tested, as well as two lower doses corresponding to MTD/2 and MTD/4. These two doses were not toxic, indeed, no clinical signs were observed. Under these experimental conditions, CATECHOL induced no statistically significant increases in DNA strand breaks at non-lethal dose on rat stomach cells after oral administration. CATECHOL is hence devoid of genotoxic activity on the stomach.

In return, CATECHOL induced statistically significant increases in DNA strand breaks at non-lethal doses on rat duodenum cells after oral administration, with the highest increase of median OTM at the lowest dose tested of 100 mg/kg/day (x2). Furthermore, the very low cell density observed at the two highest doses tested during image analysis, indicates a probable cell lysis due to cytotoxicity

and /or highly damaged cells with loss of information. Regarding the criteria described in paragraph 9, atest item is found to demonstrate genotoxic properties against the target organ if it results in a statistically significant increase in the OTM median compared with the negative control group and if the genotoxicity detected shows a dose-effect relationship. In fact the results showed a statistically significant increase in the median OTM, but with an inverse dose-effect relation, with a bell-shaped curve response. The test item Catechol was thus considered as genotoxic on rat duodenum cells. It would be useful to implement a complementary assay under the same experimental conditions on duodenum cells only, but using doses lower than 100 mg/kg/day (x2), in order to determine if there is a high dose without genotoxic effect under the dose level of 100 mg/kg/day (x2). Under these conditions, CATECHOL was considered as a DNA strand breaks and/or alkali-labile sites inducer on duodenum cells. A satisfactory agreement was observed between the actual and nominal concentrations of CATECHOL in treatment solutions used in the in vivo comet assay performed on stomach and duodenum. Indeed, the deviations from nominal concentrations were within an acceptable range of

±10%.

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