Registration Dossier

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.10.2008 - 11.11.2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD ) and in compliance with GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Reference for the In vivo Comet assay:

Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P,
et al. Recommendations for conducting the in vivo alkaline Comet assay.
Mutagenesis 2003;18(1):45-51.

Smith CC, Adkins DJ, Martin EA, O'Donovan MR. Recommendations for
design of the rat comet assay. Mutagenesis 2008;23(3):233-240.
GLP compliance:
Type of assay:
micronucleus assay

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): N-Methyl-4-nitroaniline
- Physical state: yellow to orange crystalline substance or powder
- Analytical purity: 99,3%
- Purity test date: 11.08.2008 date of CoA
- Lot/batch No.: 304269
- Expiration date of the lot/batch: 30.11.2008
- Storage condition of test material: At room temperature, in the dark (ambient humidity)

Test animals

other: Crl:WI(Han)
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Deutschland GmbH, Sulzfeld (Germany)
- Weight at study initiation: 223 – 246 g
- Fasting period before study: no
- Housing: groups of 2-5 animals in Noryl cages, type V
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4 days

- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 to 75 %
- Air changes (per hr): Ca 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 h

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used:0.5 % hydroxyethylcellulose (Natrosol® 250 HX)
- Amount of vehicle (if gavage or dermal): 10 mL/kg (negative controls 20 mL/kg)
Duration of treatment / exposure:
The animals were treated with 100 and 300 mg/kg N-Methyl-4-nitroaniline 52, 28 and 4 h prior to necropsy
The negative vehicle control (0.5 % hydroxyethylcellulose) and positive control group (200 mg/kg ethyl methanesulfonate/EMS, 2 animals) were
treated concurrently twice 28 and 4 h prior to necropsy
Frequency of treatment:
3 x at interval of 24h
Doses / concentrationsopen allclose all
Doses / Concentrations:

nominal conc.
100 mg/kg bw
Doses / Concentrations:

nominal conc.
300 mg/kg bw
No. of animals per sex per dose:
5 male rats 100 mg/kg bw (includes 4 male rates for Comet assay)
5 male rats 300 mg/kg bw (includes 4 male rates for Comet assay)
2 male rats 200 mg/kg bw ethyl methanesulfonate/EMS (positive control) for Comet assay and Micronucleus test
5 male rats 0 mg/kg bw (negative control) for Comet assay and Micronucleus test
Control animals:
yes, concurrent vehicle
Positive control(s):
- Route of administration: gavage
- Doses / concentrations: 200mg/kg


Tissues and cell types examined:
MICRONUCLEUS ASSAY: Anucleated erythrocytic cells of bone marrow of one femur
COMET ASSAY: DNA of liver, stomach and kidney
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 and 48 h post administration for the micronucleus assay and 2-6 h post administration (p.a.) for the comet assay depending on the expected maximum plasma levels

Evaluation criteria:
The assays are considered valid when a sufficient high number of animals is available for
analysis and the negative and positive control groups show the expected response.

Micronucleus assay: The criterion for a positive result is a statistically significant, dosedependent
increase in the frequency of micronucleated PCE in treated animals as compared
with the negative vehicle control. Additionally, historical control frequencies obtained in
similar experiments using this rat strain are taken into consideration.

Comet assay: The dose-dependent and statistical significant increase of the OTM in the
target organ liver as well as DNA damage level scores for stomach and kidney compared to
the negative control is indicative for a primary DNA damage.
Micronucleus assay: The statistical analysis of the data was performed using the Fisher-
Pitman permutation test. This method takes into account the characteristics of the
micronucleus assay (the animal is the experimental unit, small absolute incidences with nonnormal
distributions). All comparisons were made to the vehicle control. Mean and p values
are given in the summary table. A probability of p<0.05 was considered statistically

Comet assay:
No statistical analysis of the data was performed for the liver because the
values from the treated animals were in the same order compared to the negative controls. A
statistical evaluation was performed separately for kidney and stomach. A score for each slide
was built by multiplying the number of nuclei classified to an individual level with a rating
for this level (namely 0=no damage, 1=low damage, 2=medium damage, 3=high damage).
The statistical evaluation took into account these scores as target variable. For descriptive
considerations the number of cells/slide, the mean, the minimum and maximum values of the
scores across the slides were calculated for each treatment group. An analysis of variance
including the factor treatment group as fix and the factor animal as random effect was
performed. The error term of the analysis of variance was taken as an estimate for the
common variation. One-sided (increasing) t-tests were used to compare both the positive
control as well as the dose groups of the test item with the negative control. For the
investigation of a dose dependency (trend) the same analysis of variance was calculated on
the negative control and the two doses of the test item adding an F-test for linear contrasts
(-1, 0, 1). The evaluation was performed using the software package SAS 8.2 (SAS Institute
Inc., Cary, USA).

Results and discussion

Test results
no effects
The incidence of “hedgehogs”, indicating toxicity was unaffected in N-Methyl-4-nitroaniline treated rats
Vehicle controls valid:
Negative controls valid:
not examined
Positive controls valid:
Additional information on results:
CRITERIA FOR DOSE SELECTION: The doses were based on a non-GLP pretest with male rats. Two rats given 2000 mg/kg died
overnight after the first dosing. These rats showed lethargy, irregular breath, ruffled fur and
yellow colored urine. Reduced body weight and motor activity, as well as ruffled fur, yellow
colored urine and red-stained beddings were also seen in rats given 1000 mg/kg and
500 mg/kg (raw data). Therefore, based on these results, a top dose of 300 mg/kg was chosen
for the main study and a three-fold lower dose of 100 mg/kg at which no clinical signs of
toxicity were expected. These two dose levels allow the detection of dose-dependent effects.

Any other information on results incl. tables

Clinical Signs of Toxicity and Mortality:

The high dose animals showed apathy and ruffled fur within 1 hour after administration and these effects lasted for several hours. The effects were less pronounced after the 2nd and no longer observed after the 3rd administration. The low dose animals only showed ruffled fur after the first administration. All animals completed the study.


Micronucleus Induction:

N-Methyl-4-nitroaniline did not induce a treatment-related increase in the mean MNE % (0.14-0.16 %) compared with the concurrent negative vehicle control (0.18 %). All these values were within the historical control range (0.07 - 0.20 %) obtained in this laboratory with this rat strain. As expected, the positive control EMS caused a statistically significant increase of MNE % (0.80 %).


N-Methyl-4-nitroaniline induced slight, though statistically not significant myelotoxicity with

a dose-dependent decrease in the mean MNE % of 23.8 and 21.5 in rats given 100 and

300 mg/kg p.o., respectively, compared with the concurrent vehicle control (29.4 %). The

historical control range in this rat strain is 18.3-43.5 %. EMS (200 mg/kg p.o.) did not affect

the number of PCE (25.0 %).


N-Methyl-4-nitroaniline did not increase the mean OTM values in the liver after 100 mg/kg and 300 mg/kg N-Methyl-4-nitroaniline given three times p.o. compared to the negative vehicle control. The mean OTM values were 0.18, 0.20, and 0.21 for control, low- and high dose, respectively. The %DNA in tail values for the liver were 2.36, 2.48, and 2.60 for control, low- and high-dose, respectively. All these values were well within our historical control range in the liver observed in the last two years. Therefore, neither a statistical analysis nor a histopathological evaluation was considered necessary.

The positive control EMS induced a mean OTM values of 0.41 compared to the negative control with a mean OTM of 0.18, i.e. two-fold higher. The historical control range for the positive control methyl methanesulfonate (110 mg/kg p.o.) established under the same setting as in this study usually showed an average increase by 8-fold. All values for the negative vehicle control were well within the historical control range. Therefore, the validity of this study is given.

N-Methyl-4-nitroaniline did not increase the DNA damage level scores in the stomach and

kidney. The DNA damage level scores in the stomach were 37.9, 31.3, and 44.6 for negative

vehicle control, low- and high dose, respectively, and in the kidney 21.8, 17.8, and 19.5.

The positive control EMS induced a statistically significant increase in DNA damage scores with 143.0 in the stomach and 111.0 in the kidney compared to the negative vehicle control.

The incidence of “hedgehogs”, indicating toxicity was unaffected in N-Methyl-4-nitroaniline treated rats with estimated mean percentage of ~3.3-3.5 % in the liver (control 2.4 %), ~7.8- 8.1 % in the stomach (control ~9.1 %), and ~3.6-5.3 % in the kidney (control 8.3 %). The values for “hedgehogs” in rats given EMS were slightly increased with ~4.5 %, ~22.8 %, and ~11.3 % in the liver, stomach and kidney, respectively, compared to control.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
N-Methyl-4-nitroaniline did not induce micronuclei in the bone marrow of rats treated orally
with 100 and 300 mg/kg N-Methyl-4-nitroaniline. Furthermore, it did not induce damage
(single strand breaks and alkaline-labile sites) in the liver, stomach, and kidney as assessed by
the comet assay.
In terms of risk assessment, N-Methyl-4-nitroaniline induced a clear positive result in the
Ames test which is known to be oversensitive to aromatic nitro compounds. However,
N-Methyl-4-nitroaniline did not show clastogenicity and DNA reactivity in biologically
relevant in vivo assays when tested up to toxic doses. Therefore, N-Methyl-4-nitroaniline is
considered not to be genotoxic based on this weight of evidence approach.