3,4-dihydroxybenzonitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

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:

29 July 2016

Deviations:

yes

Remarks:

Temporary deviations from the maximum level of target humidity occurred. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzonitrile

EC Number:

241-367-9

EC Name:

3,4-dihydroxybenzonitrile

Cas Number:

17345-61-8

Molecular formula:

C7H5NO2

IUPAC Name:

3,4-dihydroxybenzonitrile

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Strain: Crl: WI(Han) Condition: Outbred, SPF-Quality

Details on species / strain selection:

The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Sex: dose finding was done with both sexes, the main test with males.
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food was supplied during the night before last dosing (approximately 7 g/rat) until maximum 4 hours after administration of the test item.
- Housing:
Caging: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group were housed together.
The animals were housed in room number A0.03.
Cage Identification: Colour-coded cage card indicating Test Facility Study No., group, animal number(s).
- Diet (e.g. ad libitum):
SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany (pellets).
Ad libitum, except during designated procedures.
- Water (e.g. ad libitum):
Municipal tap water.
Freely available to each animal via water bottles.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 24 Aug 2021 (study initiation) To: 28 Oct 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Propylene glycol.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test item.
The test item was dissolved in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of PG is 1.04 g/mL. Test item concentrations were treated with ultra-sonic waves to obtain a colourless solution. Test item concentrations were dosed within 3 hours after preparation.
Any residual volumes were discarded.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Daily: oral gavage on day 1 and 2

Post exposure period:

None: Approximately 3-4 hours after the last treatment with the test item, vehicle or EMS Liver, Glandular Stomach and Duodenum were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

187.5 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

No. of animals per sex per dose:

5-8 (3 additional animals in highest doses may be used to compensate for possible deaths)

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate (EMS, Sigma Aldrich, Steinheim, Germany)
- Justification for choice of positive control(s): Guideline positive control
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg body weight dissolved in physiological saline

Examinations

Tissues and cell types examined:

Approximately 3-4 hours after the last treatment with the test item, vehicle or EMS Liver, Glandular Stomach and Duodenum were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Details of tissue and slide preparation:

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.
Isolation of glandular stomach cells
This isolation method for glandular stomach is based on the JACVAM Comet validation study (Ref 4).
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 fore- stomach 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 were 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 glandular 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.
Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study (Ref 4).
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 duodeunum was cut open and the surface epithelia of the glandular epithelia were 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.

Preparation of slides:
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 12-35 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 (16-17.5 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 (glandular stomach and duodenum) or 30 (liver) 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 (glandular stomach and duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4.0-5.7°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5-6 minutes. The slides were subsequently immersed for 5-7 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 4-6 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, stomach and duodenum 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). No histotechnology and histopathology was needed.

Evaluation criteria:

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.
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. The occurrence of hedgehogs was scored in all treatment groups and the control. There was no effect of the test item on Hedgehogs, no hedgehogs were found in any of the slides

Statistics:

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

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical observations were made in the groups treated with 500 mg test item /kg body weight. No mortality was observed.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In the highest dose group, unexpected deaths were observed (6 out of 8 animals), and therefore this groups could not be analyzed. An additional study was performed at 500 mg/kg.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Results of the main study 1

Additional information on results:

Dose-range finding study:
In the dose-range finding test, male and female animals dosed with up to 1000 and 750 mg test item per kilogram body weight showed treatment related clinical signs or mortality. Male animals were more affected than females. Based on the results of the dose-range finding study dose levels of 187.5, 375 and 750 mg/kg body weight were selected as appropriate doses for male animals for the main test.


Main study 1:
Mortality: The animals of the groups treated with 187.5 mg test item/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Clinical observations were made in the groups treated with 375 and 750 mg test item /kg body weight. In the highest dose group, unexpected deaths were observed (6 out of 8 animals), and therefore this groups could not be analyzed. An additional study was performed at 500 mg/kg, which was determined to be the MTD for both males and females.
Comet Slide Analysis:
No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male treated animals (up to 375 mg/kg bw) compared to the vehicle treated animals. The slides of the 2 surviving animals at 750 mg/kg bw were not analyzed as this dose was considered above the MTD.
The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 3.77 ± 0.45% (mean ± SD), 5.88 ± 0.93% (mean ± SD) and 9.78 ± 4.35% (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 73.67 ± 1.38% (mean ± SD; p<0.001 Students t test;), 49.40 ± 12.0% (mean ± SD; p<0.001 Students t test;) and 61.96 ± 3.16% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) were analyzed. However, the highest test dose was higher than the MTD, due to the observed deaths. Therefore, an additional study was performed with 500 mg/kg bw as the MTD.

Main Study 2:
Mortality and Toxic Signs: The animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Clinical observations (lethargy and ataxia) were made in the groups treated with 500 mg test item /kg body weight.
Comet Slide Analysis in the Additional Study: No statistically significant increase in the mean Tail Intensity (%) was observed in Liver and Duodenum cells of test item treated male treated animals compared to the vehicle treated animals.
The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 4.13 ± 0.49% (mean ± SD), 6.73 ± 1.33% (mean ± SD) and 5.76 ± 1.46% (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 82.24 ± 3.69% (mean ± SD; p<0.001 Students t test;), 59.73 ± 2.25% (mean ± SD; p<0.001 Students t test;) and 61.98 ± 2.76% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

Applicant's summary and conclusion

Conclusions:

The comet assay is valid and 3,4-dihydroxybenzonitrile is not genotoxic in the Comet assay in Liver, Duodenum and Stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 500 mg/kg (the maximum tolerated dose 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 3,4-dihydroxybenzonitrile 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 Wistar Han rat was 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.

The study procedures described in this report were based on the most recent OECD guideline.

Batch CH02906 / CQ lot 019233401 of the test item was a white powder. The test item was dissolved in propylene glycol.

Based on the results of the dose-range finding test, initially a study with one sex (males) was performed. In the main study male animals were dosed with vehicle (propylene glycol), test item (at 187.5, 375 and 750 mg/kg body weight) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male animals compared to the vehicle treated animals.

The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 3.77 ± 0.45% (mean ± SD), 5.88 ± 0.93% (mean ± SD) and 9.78 ± 4.35% (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 73.67 ± 1.38% (mean ± SD; p<0.001 Students t test;),

49.40 ± 12.0% (mean ± SD; p<0.001 Students t test;) and 61.96 ± 3.16% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

Adequate numbers of cells and doses were analysed. However, unexpected deaths were observed at the highest dose level (6 out of 8 animals). Therefore, the Maximum Tolerated Dose was lowered from 750 mg/kg body weight to 500 mg/kg body weight, and this dose group was analyzed in an additional study in males. This was considered the MTD for both males and females and consequently only one sex was used.

In the additional study, no statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male animals compared to the vehicle treated animals.

The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 4.13 ± 0.49% (mean ± SD), 6.73 ± 1.33% (mean ± SD) and 5.76 ± 1.46% (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 82.24 ± 3.69% (mean ± SD; p<0.001 Students t test;), 59.73 ± 2.25% (mean ± SD; p<0.001 Students t test;) and 61.98 ± 2.76% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

In conclusion, the test is valid and 3,4-dihydroxybenzonitrile is not genotoxic in the Comet assay in Liver and Glandular Stomach and Duodenum cells when sampled approximately
3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.