Carbonohydrazide

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:

The study was performed between 9 January 2017 and 6 June 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 489, adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Carbonohydrazide

EC Number:

207-837-2

EC Name:

Carbonohydrazide

Cas Number:

497-18-7

Molecular formula:

CH6N4O

IUPAC Name:

carbonohydrazide

Details on test material:

Identification: Carbohydrazide
CAS number: 497-18-7
EC number: 207-837-2
Molecular formula: CH6N4O
Physical state/Appearance: White solid
Batch: 20160425
Purity: 99.58%
Expiry Date: 24 April 2018
Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

Identification: Carbohydrazide
CAS number: 497-18-7
EC number: 207-837-2
Molecular formula: CH6N4O
Physical state/Appearance: White solid
Batch: 20160425
Purity: 99.58%
Expiry Date: 24 April 2018
Storage Conditions: Room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HsdRCCHan™WIST

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo (UK). At the start of the main test the males weighed 191.6 g to 216.8 g, and were approximately eight to ten weeks old. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study. The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels.

The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle used was distilled water. The vehicle was used as supplied.

Details on exposure:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION:
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in distilled water and dosed within 2 hours of preparation. The stability and homogeneity of the test item formulations was previously determined and results showed the formulations to be stable for at least twenty three days.

PROCEDURE:
Range-Finder:
To find suitable dose levels of the test item for the main test, a range-finding study at dose levels of 300, 600, 720 and 900 mg/mL was performed. One male and one female per dose level was dosed twiced within 24 hours by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing. Additionally two additional males were treated at 720 mg/kg, blood sampled and the plasma frozen prior to analysis for proof of absorption.

MAIN TEST:
In animals dosed with test item in the Range-Finding study at 900 mg/kg, there was one premature death in one female. 900 mg/kg was therefore considered to be unsuitable. Based on the clinical observations in the Range Finder, 720 mg/kg was selected as the maximum tolerated dose (MTD) and dose levels of 180, 360 and 720 mg/mL were used for the main test. Groups each of seven males were dosed twice with a 24 hour interval via the oral route by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing. Control animals were treated in an identical manner with N-Nitroso-N-methylurea (positive control) or distilled water (negative control, vehicle alone).

Duration of treatment / exposure:

Animals were dosed twice with a 24 hours interval and killed approximately 4 hours following the second administration.

Frequency of treatment:

Twice within 24 hours

Post exposure period:

4 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control (distilled water)

Doses / concentrations 2

Dose / conc.:

720 mg/kg bw/day

Remarks:

Test Item (Carbohydrazide)

Doses / concentrations 3

Dose / conc.:

360 mg/kg bw/day

Remarks:

Test Item (Carbohydrazide)

Doses / concentrations 4

Dose / conc.:

180 mg/kg bw/day

Remarks:

Test Item (Carbohydrazide)

No. of animals per sex per dose:

Vehicle Control (distilled water): 7 male rats
Positive Control (N-Nitroso-N-methylurea): 5 male rats
Test Item (Carbohydrazide; all dose levels): 7 male rats

Control animals:

yes, concurrent vehicle

Positive control(s):

Identification: N-Nitroso-N-Methylurea (MNU)
Physical state/Appearance: Pale orange solid
Supplier’s Lot Number: 4486-027
Purity: 90% (corrected for in formulation)
Expiry: 05 December 2018
Storage Conditions: Approximately -20 °C in the dark
Solvent: Distilled water

Examinations

Tissues and cell types examined:

COMET ASSAY:
Samples of liver and glandular stomach were obtained from each animal.

PROOF OF ABSORPTION:
Blood from the animals of the MTD dose group (720 mg/kg/day) was sampled.
Bone marrow was taken from the animals of the vehicle and MTD dose group.

Details of tissue and slide preparation:

TISSUE SAMPLE REQUIREMENTS:
Humane euthanasia was performed on the animals at the end of the exposure period, using a method that did not affect the integrity of the required tissues (carbon monoxide asphyxiation). Samples of liver and glandular stomach were obtained from each animal. Blood was also sampled from the animals of the MTD dose group, processed and the plasma frozen for subsequent analysis to prove absorption. Bone marrow was taken from the animals of the vehicle and MTD dose groups and processed for bone marrow slides. These were then assessed for the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) to give an indication of toxicity and further evidence for absorption of the test item.

Sub-samples of the liver and glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology is conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.

The remaining tissue samples were processed to provide single cell suspensions, providing sufficient cells for scoring, for the Comet Assay as follows:

Liver - A small piece of liver was excised (approximately 1 cm³) and washed in liver buffer (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.

Glandular Stomach – The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by further scraping of the exposed tissue.

The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed as quickly as possible using ice-cold buffers to maintain the tissues and cell preparations at low temperature.

SLIDE PREPARATION:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature prior to the start of the experiment. Prior to use in the study the slides were labelled for animal number, project number and tissue type.

Once the cell suspensions had been obtained, approximately 30 μL was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify. All of the slides went through the subsequent processing:

Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight.

After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis unit, which was filled with chilled electrophoresis buffer, until the slide surface was just covered. The slides were then left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period had finished the slides were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for approximately 20 minutes. The buffer in the bath was maintained at low temperature (approximately 2 - 10 °C) during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis. The voltage and current at the start and end of the electrophoresis period was recorded. The aim was to induce sufficient migration of the DNA so that minimal sized Comets are produced in the nuclei of vehicle control cells.

At the end of the electrophoresis period the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer (0.4M Tris pH 7.5) and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer performed twice more. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry. Once dry the slides were stored prior to scoring which was performed from the 11 May 2017 to the 25 May 2017. Two of the four processed slide gels were scored and the remaining slides were stored as backup slides.

COMET SCORING:
The processed Comet slides were coded to allow “blind” scoring using a computer generated code and stained just prior to analysis for comets. To each dry slide gel, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program.

Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per animal. Care was taken to guarantee that a cell was not scored twice. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1. Comparisons between the vehicle control group response and that of the test item dose groups was made. The primary end-point was percentage DNA in the tail (percentage Tail intensity and median percentage Tail intensity).

Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or nonexistent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

BONE MARROW SLIDE PREPARATION:
A 15 mL centrifuge tube was filled with approximately 2-3 mL of foetal bovine serum (FBS) and, using a suitable hypodermic needle mounted on a 2 mL syringe, a portion of the serum was withdrawn from the centrifuge tube into the syringe. The needle was pushed a few millimeters into the bone marrow canal and the marrow from 1 femur was flushed into the serum in the centrifuge tube.

Approximately 1 mL of the bone marrow suspension was transferred to a microfuge tube and centrifuged at approximately 200 g for approximately 5 minutes and the supernatant was removed using a Pasteur pipette leaving sufficient serum to just cover the top of the cell pellet. The cells were mixed to give a homogenous suspension. A small drop of each bone marrow suspension was transferred to the frosted end of four glass microscope slides and smears were made and air-dried. Two slides were stained for assessment of the erythrocytes.

The smears were fixed in absolute methanol for 20 minutes and stained with May Grünwald stain and then counterstained with Giemsa stain and subsequently rinsed with pH 6.8 buffer solution for 3 minutes and then rinsed in distilled water. After air drying the smears had a cover slip applied using a mounting medium.

Evaluation criteria:

The mean and median percentage DNA for each slide was determined and the mean of the median value calculated for each animal. The mean of the individual animal means was calculated to give a group mean.

ACCEPTANCE CRITERIA:
The following criteria are used to determine a valid assay:
-The concurrent negative control is comparable with the laboratory historical negative control range.
- The positive controls induce responses that are comparable with those in the laboratory historical positive control range.
- Adequate numbers of cells and doses have been analysed.
- The highest dose level selected meets the requirements of the guideline and the study plan.

EVALUATION AND INTERPRETATION OF RESULTS:
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Negative if:
-None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
-There is no evidence of a dose-related response
-The results are within the laboratory historical vehicle control range
-There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved

The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.

Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Positive if:
-At least one of the test doses exhibits a statistically significant increase compared to
the concurrent negative control.
-The response is considered to be dose related
-The results are substantially outside the laboratory historical vehicle control range.

The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.

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

Statistics:

Mean, medians and standard deviations were calculated from the individual animal data and the group data. As a clear response response was observed, further statistical analysis was not considered necessary.

If a less than clear response would have been observed, statistical analysis comparing vehicle control groups and each corresponding treatment group using individual slide data for the percentage tail intensity and median percentage tail intensity.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TOXICITY:
There were three premature deaths seen in the 720 mg/kg dose group which occurred shortly after the second dose. Clinical signs were observed in animals dosed with the test item at 720 mg/kg, these included as follows: hunched posture, splayed gait, ataxia, lethargy. No clinical signs were observed in the animals of the 360 mg/kg and 180 mg/kg dose groups.
The evaluation of bone marrow smears showed a reduction in the PCE/NCE ratio at 720 mg/kg when compared to the vehicle control group, demonstrating some toxicity in the bone marrow as a result of the test item.

POSITIVE AND NEGATIVE CONTROL DATA:
The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, comparable with the laboratory historical control range for these tissues. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

GENOTOXICITY:
There were no marked increases in percentage tail intensity for any of the test item dose levels in the glandular stomach or liver tissues which exceeded the current historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver or glandular stomach.
There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. High numbers of hedgehogs observed in the glandular stomach are characteristic of gastro-intestinal tract where there is a high turnover of cells.

PROOF OF ABSORPTION:
Carbohydrazide was detected in the plasma taken at termination of animals of the final range finding experiment using HPLC-MS-MS confirming absorption. The mean analytical recovery rate was R=103 % with R²= 0,9943.

Applicant's summary and conclusion

Conclusions:

The test item, Carbohydrazide, did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo, under the conditions of the test.

Executive summary:

Introduction

 

The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeet al., 2000. The method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2016).

The primary target tissues of the comet assay were liver and glandular stomach.

Blood was taken at termination of the animals of the final range finding experiment and the plasma analyzed for proof of absorption. Blood was also taken at termination of the MTD animals of the main test and the plasma frozen in case it was required for analysis for proof of absorption.

Bone marrow was sampled at termination from the vehicle and MTD animals of the main test and processed for bone marrow smears so that an assessment of the PCE/NCE ratio could be made.

 

Methods

 

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 720 mg/kg with 360 mg/kg and 180 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration, the glandular stomach and liver tissues sampled and processed, the slides were then prepared prior to scoring for the presence of Comets.

Further groups of rats were given a double oral dose of water (seven rats) or N-Nitroso-N-methylurea (5 rats), to serve as vehicle and positive controls respectively.

Bone marrow was taken from the MTD and the vehicle control animals and processed for bone marrow smears to assess the PCE to NCE ratio.

Results

 

The presence of clinical signs at the MTD dose level indicated that systemic absorption had occurred and the results of the plasma analysis demonstrated the presence of Carbohydrazide in the blood plasma. The assessment of the bone marrow slides from the vehicle and MTD dose groups also demonstrated some toxicity in the MTD dose group indicating that absorption had occurred.

The test item was tested up to the maximum tolerated dose level of 720 mg/kg using male animals only. Three premature deaths occurred in the main test at the 720 mg/kg dose level after the second dose was administered and before the 28 hour time point. There was no evidence of an increase in the percentage tail intensity or median percentage intensity in animals dosed with the test item dose groups when compared to the concurrent vehicle control group.

The positive control material produced a marked increase in the percentage tail intensity and median percentage tail intensity in both the liver and glandular stomach, indicating that the test method was working as expected.

 

Conclusion

 

The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissuesin vivo,under the conditions of the test.