Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of a Ames test indicate that DNAN is mutagen to bacteria as it has caused a dose-dependent increase in the number of revertants in all 5 tester strains over the different concentrations in a reproducible manner. Furthermore, tested compound is direct mutagen causing AT and GC base-pair substitution and frameshift mutagen as there was no difference in the number of revertants between the activated (S9+) and non-activated (S9-) groups.

In a Chromosome aberration test in Human Lymphocytes in vitro, Envigo Research Limited., 2018, DNAN was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberration either in the presence or absence of metabolic activation at any dose level in any of the exposure groups.

Even though the in vitro resuls indicated that tested compound is mutagen to bacteria as it has caused a dose dependent increase in the number of revertants in all 5 tester strains, the negative in vivo test allows to conclude that DNAN is not genotoxic.

Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 October, 2017 - 09 February, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not applicable
Remarks:
No analysis was carried out to determine the homogeneity, concentration and stability of the test formulation. it was formulated within é hours of it being applied to the test system. It is assumed that it was stable for this duration.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55503625
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 100%
- Physical state / Apparence : Pale yellow powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

- Solubility and stability of the test substance in the solvent/vehicle:


TREATMENT OF TEST MATERIAL PRIOR TO TESTING


- Final dilution of a dissolved solid, stock liquid or gel: The test item is soluble in DMS O


OTHER SPECIFICS:
Target gene:
Chromosome aberrations
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Peripheral circulation of a non-smoking volonteer who had been previously screened for suitability
- Suitability of cells: done
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: 18-35 years old, 1 male and 2 females
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Celles were grown in Eagle's minimal essential medium with HEPES buffer (MEM),
supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10ù foetal bovine serum (FBS) at approximately 37°C with 5% CO2 in humidified air.
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
The dose level used in the Main Experiment were selected using data from the Preliminary Toxicity test where the results indicated that the maximum
concentration should be limited on both precipitate and toxicity, depending on the exposure group.

Group Final concentration of the test item DNAN (µg/mL)
4(20)-hour without S9 0, 40, 80, 160, 320, 560, 640, 960
4(20)-hour with S9 0, 40, 80, 160, 320, 560, 640, 960
24-hour without S9 0, 10, 20, 60, 80, 120, 240


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not souble in aqueous media
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 48 hours incubation
- Exposure duration: 4-hours exposure in the presence of an induce rat liver homogenate metaolizing system (S9), 4 hours exposure in the absence of metabolic activation (S9) and a 24-hour exposure in the absence of metabolic activation.
- Expression time (cells in growth medium): 20-hour expression period

- Fixation time (start of exposure up to fixation or harvest of cells):



SPINDLE INHIBITOR (cytogenetic assays): Mitosis was arrested by addition of demecolcine 2,5 hours before the required harvest time



NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: the cells (from the KCL suspension) were fixed into fresh methanl/glacial
acetic acid. The fixative was changed at least 3 times and the cells stored at approx. 4°C to ensure complete fixation prior to slide preparation.
The lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and re-suspension in a small aount of fixative. Several
drops of this suspension were dropped onto clean, wet microscope slide and left to air dry. Each sllide was permanently labeled with the appropriate
identification data.

NUMBER OF CELLS EVALUATED: 2000 lymphocyte cell nuclei

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate.



DETERMINATION OF CYTOTOXICITY
- Method: precipitation and mitotic index. A total of 2000 lymphocyte cell nuclei were counted and the number of the cells in metaphase recorded and expressed as the mitotic index ans as a percentage of the vehicle control value.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


- OTHER:
Rationale for test conditions:
The following criteria were used to determine a valid assay :
- The frequency of cells with structural chromosome aberrations in the vehicle control cultures was within the laboratory historical control data range
- All the positive control chemicals induced a positive response and demonstrated the validity of the experiment and the integrity of the S9-mix
- The stuyd was performed using all 3 exposure conditions using a top concentration which meets the requirements of the current testing guideline
- The required number of cells and concentrations were analyzed
Evaluation criteria:
A test item can be considered to be clearly negative if, any of the experimental conditions examined :
- The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data
- No toxicology or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis
- There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if :
- The number of cells with strucutral chromosome aberrations is outside the range of the laboratory historical control data
- At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the conccurent negative control
- The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using a Fisher's Exact Test (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells aberrations excludings gaps is less than 0.05 when compared to
its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproductible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
As the criteria required to valid the assay are provided, the assay was considered as valid.
The test item did not induce any statistically significant increases the the frequency of cells with aberrations either in the absence or presence of metabolic activation at any dose level in any of the exposure groups.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure group.
Remarks on result:
other: Valid results
Conclusions:
The test item is considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

A Chromosome aberration test in Human Lymphocytes in vitro, Bowles A., 2018, has been performed according to the 473 OECD guideline.

Duplicate culture of human lymphocytes, treated with the test item were evaluated for chormosome aberrations at 3 dose levels, together with vehicle and positive controls, at 3 exposure conditions : 4 hours exposure in the presence of an induce rat liver homogenate metabolizing system (S9), 4 hours expposure in the absence of metabolic activation (S9) both with a 20-hour expression period and a 24 -hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity test where the results indicated that the maximum concentration should be limited on both precipitate and toxicity, depending on the exposure group (0, 40, 80, 160, 320, 560, 640, 960 µg/mL : concentrations tested for the 4 -hour exposure either with and without S9 and 0, 10, 20, 40, 60, 80 120, 240 µg/mL: concentrations used for the 24 -hour exposure without S9).

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberration either in the presence or absence of metabolic activation at any dose level in any of the exposure groups.

The test item, DNAN was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a Comet assay, performed according to the OECD 489 Guideline, 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.

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

Link to relevant study records
Reference
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
Qualifier:
according to
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. certificate)
Type of assay:
mammalian comet assay
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
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han (HsdRccHan WIST)
Sex:
male
Details on test animals 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:
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
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
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
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.
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.

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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Not genotoxic according to ECHA (Chapter R.7a. Endpoint specific guidance) guidance figure R.7.7 -1, regarding to the Annex VIII of REACH.