2-(butylnitroamino)ethyl nitrate

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:

05 December 2017 - 01 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

1

Reference substance name:

2-(butylnitroamino)ethyl nitrate

EC Number:

279-976-7

EC Name:

2-(butylnitroamino)ethyl nitrate

Cas Number:

82486-82-6

Molecular formula:

C6H13N3O5

IUPAC Name:

butyl(nitro)[2-(nitrooxy)ethyl]amine

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

male Wistar Han™ (HsdRccHan™WIST) rats were supplied by Envigo (UK).

Sex:

male

Details on test animals or test system and environmental conditions:

At the start of the main test the males weighed 169.8 g to 201.1 g and were approximately 7 to 12 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 animal weights, with means and standard deviations are presented in Table 1 and also included in Tables 14 to 18.
The animals were housed in groups of up to five 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 temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. 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:

arachis oil

Details on exposure:

A range-finding test was performed to find suitable dose levels of the test item following a triple oral administration at approximately 0, 24 and 44 hours. All surviving animals were dosed three times at (approximately) 0, 24 and 44 hours at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its body weight at the time of the initial dosing.
Animals were observed approximately 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
Where possible bone marrow was sampled from the animals of the range finding test and bone marrow smears were prepared and scored for PCE/NCE ratio as an indicator of any toxicity to bone marrow.

Duration of treatment / exposure:

0, 24 and 44 hours via the oral route with the test item at 500, 250 or 125 mg/kg.

Frequency of treatment:

3 times

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 for butyl-nena, 7 for vehicle control, 3 for positive controls

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide and N-Nitroso-N-methylurea

Examinations

Tissues and cell types examined:

Samples of liver, glandular stomach and bone marrow were obtained from each animal. With the exception of the cyclophosphamide positive control group where only bone marrow samples were taken, and the N-nitroso-N-methyl urea positive control group where only liver and glandular stomach samples were taken,
Sub-samples of the liver and glandular stomach taken from the vehicle control animals and the dose group animals were processed and preserved for possible histopathology investigations. Assessment of cytotoxicity by histopathology may be conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.

Details of tissue and 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, 30 µL was mixed with 270 µL of molten 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this cell suspension/agarose mix placed onto a pre coated slide. Four slide gels per animal for each tissue, two gels per pre-prepared agarose slide were prepared. The cell suspension/agarose mix was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow the gel to solidify. All the comet slides were processed through the subsequent procedure. Once the LMP agarose has set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. Ater the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5), to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period had finished electrophoresis was performed at approximately 0.7 V/cm (calculated between the electrodes), for 20 minutes. The buffer in the bath was maintained at a low temperature (approximately 2-10 °C) during the electrophoresis period. The temperature of the electrophoresis solution at the start of unwinding, the start of electrophoresis, and the end of electrophoresis was recorded. The voltage and current at the start and end of the electrophoresis period was recorded. The aim is 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 rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry. Once dry the slides were stored prior to staining and scoring. Two of the four processed slides were scored (A and B replicates) and the remaining two slides (C and D replicates) were stored as backup slides.
A 15 mL centrifuge tube was filled with approximately 3 mL of fetal 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 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 scoring of micronuclei and two reserved as spares.
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:

Evaluation and Interpretation of the Comet 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.

Interpretation of the Micronucleus Results:
A comparison will be made between the incidence of micronucleated polychromatic erythrocytes (PCE) occurring in each of the Treatment Groups, with the Negative Control Group. A result will be considered positive if a dose-related, statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (PCE) is demonstrated at either of the Treatment Group kill times.

Statistics:

For the Micronucleus Test statistical analysis of MN/4000 PCE and PCE/NCE ratio data was performed where necessary using a t test following a ¹(x+1) transformation of the data. (Lovell et al, 1989).
For the Comet Test comparisons were made between vehicle control groups and the dose groups and the positive control groups in the percentage tail intensity and median percentage tail intensity using a t test following a ¹(x+1) transformation of the individual slide data.

Results and discussion

Test results

Test results 1

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: preliminary test

Remarks:

preliminary test

Test results 2

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

comet assay

Test results 3

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

micronucleus test

Applicant's summary and conclusion

Conclusions:

In the Comet Test, the test item, Butyl-NENA induced dose related, statistically significant increases in the percentage tail intensity and median percentage tail intensity values in the liver. The test item is considered to be able to induce DNA strand breakage to the liver in vivo under the conditions of the test. There was a small but statistically significant increase in the percentage tail intensity and median percentage tail intensity in the glandular stomach.However, since the increase is within the historical control range for a vehicle it is considered to be of no biological relevance. The test item is considered to be unable to induce DNA strand breakage to the glandular stomach in vivo under the conditions of the test.

In the Micronucleus Test, the test item, Butyl-NENA induced dose related, statistically significant increases in the frequency of micronuclei and was considered to be mutagenic under the conditions of the test.