2,4-dinitroanisole

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:

08 February - 28 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

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

The animals of the vehicle control group were inadvertently dosed with distilled water rather than arachis oil at time point zero. For the second dose at the 24-hour time-point the animals were correctly dosed with arachis oil. Since both these vehicles are acceptable for use in the Comet assay and the laboratory historical control data includes data from studies where these vehicles have been used independently it is considered that this error will have no impact on the validity, integrity or the result of the study.

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

1

Reference substance name:

2,4-dinitroanisole

EC Number:

204-310-9

EC Name:

2,4-dinitroanisole

Cas Number:

119-27-7

Molecular formula:

C7H6N2O5

IUPAC Name:

2,4-dinitroanisole

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring Nobel AS batch 55503625
- Expiration date of the lot/batch: 28/04/2019
- Purity test date: 100%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: arachis oil
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was freshly prepared as a suspension at the appropriate concentration in arachis oil
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Physical state / Appearence : Pale yellow powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han (HsdRccHan WIST)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: 183.4 to 208.1g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: solid floor polypropylene cages with woodflake bedding
- Diet : ad libitum
- Water :ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70% relative humidity
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used:arachis oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 10; 5; 2,5mg/mL
- Amount of vehicle (if gavage or dermal): 10mL
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity: treated as 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:



DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

Duration of treatment / exposure:

24h

Frequency of treatment:

Rats were dosed twice with a 24-hour interval

Post exposure period:

4 hours after the second exposure

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 males
3 males for positive control
8 males for vehicule control

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

N-nitroso-N-methylurea (MNU)
- Justification for choice of positive control : MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.
- Route of administration: oral route
- Doses / concentrations: 25 mg/kg

Examinations

Tissues and cell types examined:

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 dioxide asphyxiation).
Samples of liver and glandular stomach were obtained from each animal for comet processing.

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 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:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Glandular Stomach – A small section of the glandular 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 glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining tissue into a small volume of stomach buffer.
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.

DETAILS OF SLIDE PREPARATION:
Approximately 30 μL of the cell suspension 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 mix was immediately covered with a glass cover slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the cover slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled 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 to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
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 and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides (A and B replicates) were scored and the remaining slides ( C and D replicates) were stored as backup slides.

METHOD OF ANALYSIS:
The slides were coded prior to scoring to allow “blind” scoring. The slides were stained just prior to analysis for comets. To each dry slide, 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, i.e. Comet IV.
Two slides for each tissue per animal were scored with a maximum of 75 cells per slide giving an accumulative total of 150 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-points are percentage tail DNA (%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 non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

OTHER:

Evaluation criteria:

The following criteria will be 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 analyzed.
• The highest dose level selected meets the requirements of the guideline and the study plan.

Statistics:

A comparison was made between the vehicle control groups and the positive control groups. The individual slide score data for the percentage tail intensity and median percentage tail intensity was compared using a Students t-test with a √1+x transformation. Comparisons between the vehicle control groups and the test item dose groups were also made when it was considered that there was a marked increase over the vehicle control value.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING TOXICITY STUDY
A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The initial dose selected for use in this study, 300 mg/kg was based on toxicity information supplied.
In animals dosed with test item there were premature deaths at 150, 200 and 300 mg/kg. The female animal at 150 mg/kg was killed in extremis after the second dose was administered due to the severity of the clinical signs which were hunched posture, lethargy ataxia, decreased respiratory rate and labored respiration.
The following clinical signs were observed in some of the animals dosed with 100 mg/kg: hunched posture and lethargy. No clinical signs were observed in animals dosed with 50 mg/kg.
Where possible, bone marrow slides were prepared from the range-finding experiments for quantitative assessment.
The quantitative assessment revealed that there was no marked bone marrow toxicity observed at 100 mg/kg, however the presence of clinical signs was considered to give an indication of systematic absorption of the test item had occurred.
Based on the above data the Maximum Tolerated Dose (MTD) of the test item, 100 mg/kg, was selected for use in the main test, with 50 and 25 mg/kg as the lower dose levels. It was considered that there was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test.



RESULTS OF DEFINITIVE STUDY: COMET ASSAY

Mortality Data and Clinical Observations:
In the main test there were three premature deaths in the 100 mg/kg dose group. One animal was found dead prior to the second dose but had exhibited no clinical signs up to that point. The other two animals were found dead after the second dose administration. One of these animals had exhibited no clinical signs and the other had exhibited hunched posture and ataxia after the second dose administration. The following clinical signs were observed in some of the remaining animals: hunched posture and ataxia. No clinical signs were observed in the animals dosed at 50 mg/kg or 25 mg/kg. The loss of three animals in the 100 mg/kg dose group resulted in the dose group having less than the recommended number in the OECD 489 guideline of 5 animals. However, with no marked evidence of a dose related response it is considered that the purpose and integrity of the study was not affected.

The vehicle control group demonstrated percentage tail intensities which were consistent with the current laboratory historical control range. Although the vehicle control group was inadvertently dosed with two different vehicles (distilled water and arachis oil) it is considered that this error has no impact on the outcome or integrity of the study. Both these vehicles are routinely used in the comet assay and the laboratory historical data includes data from studies where these vehicles have been used independently.
The positive control item (MNU) produced a statistically significant marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, with values which were 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.
There were no marked increases in percentage tail intensity or median percentage tail intensity for any of the test item dose levels in the glandular stomach which exceeded the current laboratory historical control range for a vehicle, confirming the test item did not induce DNA damage in the glandular stomach.
The liver did demonstrate a small but statistically significant increase in both the percentage tail intensity and the median percentage tail intensity at the maximum dose level (100 mg/kg). However, the increase in the mean percentage tail intensity for the group was within the current laboratory historical range for a vehicle and much of the increase could be attributed to one animal (animal 17) which had values which exceeded the current laboratory historical control range for a vehicle. The increase in median percentage tail intensity was marginally higher than the upper limit of the laboratory historical range for vehicle, but again this increase was mainly due to increases in one animal. Since the three requirements in the study plan to designate a positive response are not met we consider the increase in the liver at the maximum dose to be of no toxicological significance. The test item was very toxic and it is considered that any small increases are likely to be due to cytotoxicity rather than mutagenicity.
There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. The hedgehog frequency data for each tissue is included in Tables 3 to 12.
The test item was unexpectedly toxic in the main test at 100 mg/kg resulting in the premature death of three animals. It is considered that the remaining animals resulted in the group not meeting the minimum animal number of five per group as stated in the test guideline. However, the mean percentage tail intensity values for both tissues were within the historical control range and did not indicate any genotoxic activity and, therefore, it was considered there was no impact on the integrity or purpose of the study.

Any other information on results incl. tables

Mortality data for the Range-Finding Toxicity Test:

Dose Level (mg/kg)	Sex	Number of Animals Treated	Route	Deaths on Day
				0	1
300	male	1	oral	0	1
300	female	1	oral	0	1
200	male	1	oral	0	1
200	female	1	oral	0	1
150	male	1	oral	0	1
150	female	1	oral	0	1
100	male	1	oral	0	0
100	female	1	oral	0	0
50	male	1	oral	0	0
50	female	1	oral	0	0
100	male	2	oral	0	0
100	female	2	oral	0	0

Applicant's summary and conclusion

Conclusions:

The test item DNAN, did not induce any toxicologically significant increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

Executive summary:

A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex.

and GLP compliance, 7 male rats (Wistar) administered by oral gavage with 100, 50 and 25 mg/kg of DNAN at time 0 and 24 hours after the initial dosing. A further group of 5 rats were treated as the vehicle control group and a group of 3 rats were treated with as positive control, the N-Nitroso-N-methylurea. Animals were killed 4 hours after the second administration, at a sampling time of 28 hours. The glandular stomach and liver tissues were sampled and processed, the slides were then prepared prior to scoring for the presence of Comets.

The presence of clinical signs indicated that systemic absorption had occurred.

There was no evidence of an increase in the glandular stomach in the percentage tail intensity or median percentage tail intensity in the test item dose groups when compared to the concurrent vehicle control group.

The liver did demonstrate a small but statistically significant increase in both the percentage tail intensity and the median percentage tail intensity at the maximum dose level (100 mg/kg). However, the increase in the mean percentage tail intensity was within the current laboratory historical range for a vehicle and much of the increase could be attributed to one animal. The increase in median percentage tail intensity was marginally higher than the upper limit of the laboratory historical range for vehicle, but again this increase was mainly due to increases in one animal. As we do not meet the three requirements in the study plan to designate a positive response we consider this to be of no toxicological significance.

The positive control item produced a marked increase in the percentage tail intensity value in the liver and glandular stomach, indicating that the test method was working as expected. The vehicle control group for the liver and the glandular stomach had percentage tail intensity values which were consistent with the current laboratory historical range for a vehicle.

The test item, DNAN, did not induce any toxicologically significant increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.