Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-01 to 2011-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to a recognised guideline and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
4 rather than 5 males were available for analysis at the scheduled termination date
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fishes. Test data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-3, Agricultural Production Bureau, November 24, 2000
Qualifier:
according to
Guideline:
other: Official notice of J MHLW, METI and ME (21 November 2003): YAKUSHOKUHATSU No.1121002 SEIKYOKU No.2 KANPOKIHATSU No. 031121002
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-phenylmaleimide
- Physical state: yellow solid
- Analytical purity: 99.8%
- Impurities (identity and concentrations): not stated
- Isomers composition: not stated
- Lot/batch No.: 1A09
- Expiration date of the lot/batch: 31 DEC 2012
- Stability under test conditions: not stated
- Storage condition of test material: room temperature in the dark
- Other:

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at (main) study initiation: both males and females were ca 40 days old
- Weight at study initiation: males 26.4g to 32.0g; females 21.5g to 25.2g
- Assigned to test groups randomly: yes
- Fasting period before study: none
- Housing: in cages and maintained in a controlled environment, with the thermostat and
relative humidity target ranges set at 19 to 23°C and 40 to 70% respectively
- Diet (e.g. ad libitum): pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) given ad libitum
- Water (e.g. ad libitum): tap water given ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 16 - 18 March 2011 (assumes test substance was administered over two consecutive days).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: none given
- Concentration of test material in vehicle: 0 mg/mL, 1.875 mg/mL, 3.75 mg/mL, 7.5 mg/mL and 11.25 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg/day
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of the test substance were prepared in Corn oil obtained from Sigma, batch
number 058K0070.

DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable.
- Mixing appropriate amounts with (Type of food): not applicable.
- Storage temperature of food: not applicable.
Duration of treatment / exposure:
There were two treatments 24 hours apart. The animals were sacrificed 24 hours after the second dose.
Frequency of treatment:
At 0 and 24 hours.
Post exposure period:
24 hours.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
112.5 mg/kg/day (7 females)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
75.0 mg/kg/day (5 males and 5 females)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
18.75 mg/kg/day (5 males)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
37.5 mg/kg/day (5 males and 5 females)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0 mg/kg/day (control) (5 males and 5 females)
Basis:
nominal conc.
No. of animals per sex per dose:
Please see above.
Control animals:
yes, concurrent vehicle
Positive control(s):
positive control: mitomycin C
- Justification for choice of positive control(s): commonly used in micronucleus studies and recommanded in the OECD guideline.
- Route of administration: oral gavage
- Doses / concentrations: 20 mL/kg

Examinations

Tissues and cell types examined:
Tissues: bone marrow from femurs
Cell types: polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: a preliminary test was run to determine the highest dosage which would be expected to elicit signs of toxicity
without producing extreme clinical signs or having a significant effect on survival.

DETAILS OF SLIDE PREPARATION (FIXATION AND STAINING):
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry.
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

MICROSCOPIC EXAMINATION: Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

METHOD OF ANALYSIS: there was no supporting analytical chemistry

SLIDE PREPARATION: The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides.
Evaluation criteria:
A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes for the treatment group compared with the vehicle control group (p<0.01); individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group and where these values fall within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.01).
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests, for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

Group 1 vehicle control
Group 2 males 18.75 mg/kg/day; females 37.5 mg/kg/day
Group 3 males 37.5 mg/kg/day; females 75 mg/kg/day
Group 4 males 75 mg/kg/day; females 112.5 mg/kg/day
group 5 positive control
For micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3.
Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 5. Huntingdon Life Sciences PLZ0043 16
Statistical significance was declared at the 1% level for all tests.
The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
one male mouse died, and numerous clinical signs have been observed.
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 75 - 150 mg/kg/day
- Solubility: not stated
- Clinical signs of toxicity in test animals: AQ preliminary toxicity (range-finding) test was conducted to determine the maaximum tolerated dose of the test material. Males and females were dosed at 75, 112.5 and 150 m/kg/day, then observed for 48 hours after dosing. Clinical signs and mortalities were recorded. At the end of the observation period surviving animals were killed. Excessive clinical signs were seen at 150 mg/kg/day and so 112.5 mg/kg/day was chosen as the highest concentration for the main study.
- Evidence of cytotoxicity in tissue analyzed: None
- Rationale for exposure: At the request of the sponsor both male and female animals were used in the micronucleus test. As a consequence of the substantial difference in toxicity observed between the sexes and the clinical signs observed dose levels selected for the micronucleus test were 18.75, 37.5 and 75 mg/kg/day in male animals and 37.5, 75 and 112.5 mg/kg/day in female animals.
- Harvest times: 48 hours after first dose
- High dose with and without activation: not done

RESULTS OF DEFINITIVE STUDY
- The micronucleus test was carried out in male and female animals.
Animals were treated with N-phenylmaleimide at dose levels of 18.75, 37.5 and 75 mg/kg/day in male animals and 37.5, 75 and 112.5 mg/kg/day in female animals.
No clinical signs of toxicity were observed in male or female animals in the vehicle control group and positive control group or female animals administered N-phenylmaleimide at 37.5 or 75.0 mg/kg/day over the duration of the test.
At 18.75 mg/kg/day clinical signs of toxicity observed in male animals included underactive behaviour, piloerection, rales breathing and hunched posture.
All animals survived until scheduled termination.
At 37.5 mg/kg/day clinical signs of toxicity observed in male animals included underactive behaviour, piloerection, rales breathing, hunched posture, ungroomed coat, irregular breathing, both eyelids partially closed, swollen and dark abdomen, slow breathing and pallor skin colour, one male animal was found dead on Day 3.
At 75.0 mg/kg/day clinical signs of toxicity observed in male animals included underactive behaviour, piloerection, rales breathing, hunched posture, ungroomed coat, irregular breathing, both eyelids partially closed, swollen and dark abdomen and salivation. One male animal was found dead on day 3.
At 112.5 mg/kg/day clinical signs of toxicity observed in female animals included underactive behaviour, piloerection, rales breathing, ungroomed coat, irregular breathing, salivation and overactive behaviour. One female animal was found dead on Day 2 and one female animal was found dead on Day 3. Some incidences of bodyweight loss were observed in all groups including controls during the test. The post mortem examinations did not find any signs of mis-dosing.

- Ratio of PCE/NCE (for Micronucleus assay):

The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male or female animals. Mitomycin C caused a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01) in male and female animals. The test substance did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male or female animals. The test substance did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female animals. Mitomycin C did not cause a statistically significant decrease in the proportion of polychromatic erythrocytes in male or female animals.

Any other information on results incl. tables

SUMMARY OF RESULTS

Male Data

 Sampling time after 2nd dose  Treatment Dose (mg/kg/day)  Proportion of PCE (%)

#

 Incidence MPCE (mean)

#

 24 hours  Vehicle  -  54.9  1.0
    N-phenylmaleimide  18.75  49.5  0.8
    N-phenylmaleimide  37.5  40.5  2.3
    N-phenylmaleimide  75.0  53.6  0.8
   Mitomycin C  12(a)  50.4  63.66**
         

Female Data

 Sampling time after 2nd dose  Treatment Dose (mg/kg/day)  Proportion of PCE (%) 

#

 Incidence MPCE (mean)

#

 24 hours  Vehicle  -  54.6  0.4
    N-phenylmaleimide  37.5  61.4  1.4
    N-phenylmaleimide 75.0  55.6  1.2
    N-phenylmaleimide  112.5  50.2  1.8
   Mitomycin C  12 (a)  53.0  71.6**
         

Vehicle: Corn oil

PCE: Polychromatic erythrocytes

MPCE: Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined

(a): Positive control dosed once only 24 hours prior to termination

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate non-parametric method of analysis based on permutation (one-sided probabilities): ** p<0.01 significant

otherwise p>0.01 not significant

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
N-phenylmaleimide did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in either male or female CD-1 mice when administered orally by gavage in this in vivo test procedure.