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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
ASTA Medica AG
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material: Nicotinamide

Test animals

Species:
mouse
Strain:
other: BOR: NMRI, (SPF Han)
Sex:
male/female
Details on test animals and environmental conditions:
MAIN TEST

TEST ANIMALS
- Age at study initiation: 5 weeks
- Weight at study initiation: males 27 - 34 g, females: 24 - 29 g
- Assigned to test groups randomly: yes, under following basis: animals were placed into the different groups using computer generated random numbers.
- Fasting period before study: 16 hours
- Housing: Macrolon cages, type II
- Diet: Standard diet ad libitum, ssniff(R) M, "Special diet for Mice", supplied by SSNIFF Spezialdiaeten GmbH, D-4770 Soest
- Water: Ad libitum
- Acclimation period: 5 days under test conditions with veterinary care.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 6 a.m. - 6 p.m. CET artificial lighting, 6 p.m. - 6 a.m. CET darkness/natural light

IN-LIFE DATES
From 13 May 1991 to 15 May 1991

REPEAT TEST

TEST ANIMALS
- Source: Vinkelmann, Versuchstierzucht GmbH & Co.KG., D-4799 Borchen, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males 28 - 36 g, females: 21 - 27 g
- Assigned to test groups randomly: yes, under following basis: animals were placed into the different groups using computer generated random numbers.
- Fasting period before study: 16 hours
- Housing: Macrolon cages, type II
- Diet: Standard diet ad libitum, ssniff(R) M, "Special diet for Mice", supplied by SSNIFF Spezialdiaeten GmbH, D-4770 Soest
- Water: Ad libitum
- Acclimation period: 5 days under test conditions with veterinary care.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 21.5
- Humidity (%): 58 - 72
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 6 a.m. - 6 p.m. CET artificial lighting, 6 p.m. - 6 a.m. CET darkness/natural light

IN-LIFE DATES
From 09 Nov. 1992 to 11 Nov. 1992

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
MAIN AND REPEAT TESTS
- Vehicle(s)/solvent(s) used: aqua ad injectabilia.
- Administration Volume: 10.0 mL/kg body weight
Details on exposure:
Single intraperitoneal administration
Post exposure period:
The animals were observed for the first 4 to 6 hours after administration for the occurrence of toxicity symptoms; on days 2, 3, and 4 only once daily in the morning.
At 24-, 48- and 72-hour intervals after treatment, 6 mice per sex and control group and 7 mice per sex of the test material group were sacrificed to prepare bone marrow smears.
Doses / concentrations
Remarks:
Doses / Concentrations:
1470 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
MAIN TEST
Three groups of mice, the negative and positive control groups containing each 18 males and 18 females and the test material group with 21 males and 21 females.

REPEAT TEST
Three groups of mice, the negative and positive control groups containing each 12 males and 12 females and the test material group containing 7 mice per sex and sampling time for each of the three doses used (total in the test material group: 42 males and 42 females).
Control animals:
yes, concurrent vehicle
Positive control(s):
MAIN AND REPEAT TEST
- Positive control material: Cyclophosphamide
- Route of administration: Single intraperitoneal administration
- Doses / concentrations: 51.1 mg/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow was removed from the femurs.
Details of tissue and slide preparation:
MAIN TEST

CRITERIA FOR DOSE SELECTION:
An orientating study of the acute toxicity of Nicotinamide in mice after single intraperitoneal administration with the test substance batch used in the present experiment was performed. In this study 2150 mg/kg body weight (b.w.) in males and females caused only slight toxicity symptoms but deaths occurred in all animals (2 males and 2 females) within the first 24 hours after administration. 1470 mg/kg caused slight toxicity symptoms but no death. Therefore, 1470 mg/kg b.w. in males and females were considered to be the maximum tolerated dose in respect of the micronucleus test.

STUDY PROCEDURE:
Three groups of mice, the negative and positive control groups containing each 18 males and 18 females and the test material group with 21 males and 21 females each received a single intraperitoneal administration (diet withdrawal: 16 h before treatment).
Group 1, the negative control group, received physiological saline solution (0.9 %).
Group 2, the test material group, received 1470 mg/kg body weight (males and females) of
the test material. The test material was freshly dissolved in Ampuwa(R), aqua ad injectabilia.
Group 3, the positive control group, received Cyclophosphamide (51.1 mg/kg body weight) dissolved in physiological saline solution (0.9%).

DETAILS OF SLIDE PREPARATION:
All mice were sacrificed by CO2 overdose. Both femurs were removed from each mouse and the bone marrow cells flushed into a labelled centrifuge tube with approximately 1.5 ml of fetal calf serum (Art. No. 210463, Boehringer Mannheim, D-6800 Mannheim, Germany). The tubes were centrifuged at approx. 180 x g for 5 minutes (Eppendorf Zentrifuge 5415, Netheler & Hinz GmbH, D-2000 Hamburg, Germany), after which the supernatant serum was discarded and the bone marrow cells suspended upon a thin layer of serum. A small drop of the marrow serum suspension was smeared on a slide, which was identified by study number, animal number, species, sex, and date of preparation, and allowed to dry overnight.
At least two slides per animal were prepared. The following day, the smears were stained using the panoptic stain method developed by Pappenheim with May Grünwald and Giemsa solution.

METHOD OF ANALYSIS:
For each sampling time the bone marrow smears from the first 5 surviving animals per sex and group were used for evaluation. One slide per animal was examined. The remaining smears of each sex and group per interval were evaluated if macroscopic examination of the first smears revealed technical imperfections which precluded accurate microscopic analysis.
From each animal one thousand polychromatic erythrocytes (PCE) were scored under the microscope (magnification 650-1000 x, C. ZEISS, D-7082 Oberkochen, Germany) for the incidence of polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was calculated, based on 1000 erythrocytes (PCE + NCE) scored per slide, as a measure of the toxic efficacy of the test material.

REPEAT TEST

CRITERIA FOR DOSE SELECTION:
The test was performed with three doses of Nicotinamide:
A: 681 mg/kg (males and females)
B: 1000 mg/kg (males and females)
C: 1470 mg/kg (males and females)
Dose C of Nicotinamide is equal to the dose of the main test to prove whether the finding (significant increase in micronucleated polychromatic erythrocytes versus the respective control animals) is reproducible. Doses A and B are considered suitable for detecting a possible dose dependency of the finding.

STUDY PROCEDURE:
Three groups of mice, the negative and positive control groups containing each 12 males and 12 females and the test material group containing 7 mice per sex and sampling time for each of the three doses used (total in the test material group: 42 males and 42 females) each received a single intraperitoneal administration (diet withdrawal: 16 h before treatment).
Group 1, the negative control group, received physiological saline solution (0.9 %).
Group 2 (A,B,C), the test material group, received the following doses of Nicotinamide/kg body weight: A: 681 mg/kg; B: 1000 mg/kg; C: 1470 mg/kg.
The test material was given freshly dissolved in aqua ad injectabilia.
Group 3, the positive control group, received Cyclophosphamide (51.1 mg/kg body weight) dissolved.

DETAILS OF SILIDE PREPARATION:
All mice were sacrificed by CO2 overdose. Both femurs were removed from each mouse and the bone marrow cells flushed into a labelled centrifuge tube with approximately 1.5 ml of fetal calf serum (Art. No. 210463, Boehringer Mannheim, D-6800 Mannheim, Germany). The tubes were centrifuged at approx. 180 x g for 5 minutes (Eppendorf Zentrifuge 5415, Netheler & Hinz GmbH, D-2000 Hamburg, Germany), after which the supernatant serum was discarded and the bone marrow cells suspended upon a thin layer of serum. A small drop of the marrow serum suspension was smeared on a slide, which was identified by study number, animal number, species, sex, and date of preparation, and allowed to dry overnight.
At least two slides per animal were prepared.
The following day, the smears were stained using the panoptic stain method developed by Pappenheim. Before evaluation, the slides were coded. So they could not be assigned to animals of a specific group or sampling time during the microscopical examination.

METHOD OF ANALYSIS:
For each sampling time the bone marrow smears from the first 5 animals per group and sex (and per dose in group 2) were used for evaluation. One slide per animal was examined. The remaining smears of each group per interval were evaluated if macroscopic examination of the first smears revealed technical imperfections which precluded accurate microscopic analysis. From each animal one thousand polychromatic erythrocytes (PCE) were scored under the microscope (magnification 650-1000 x, C. ZEISS, D-7082 Oberkochen, Germany) for the incidence of polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was calculated, based on 1000 erythrocytes (PCE + NCE) scored per slide, as a measure of the toxic efficacy of the test material.
Evaluation criteria:
MAIN AND REPEAT TEST
If a test material produced no statistically significant and reproducible positive response at anyone of the test points compared to the negative control group, it is considered non-mutagenic in this system (significance level: 5 %; p <= 0.05).
Statistics:
MAIN AND REPEAT TEST
The frequencies of polychromatic erythrocytes with micronuclei of the test material group and of the positive control group were compared with those of the negative control group at each sampling time.
A Poisson test was applied. The data from each treatment group for each sex as well as for both sexes combined, were compared with the respective negative control group data using a VAX 8200 computer (Digital Equipment Corp., D-8000 Muenchen, Germany). The values of the first 5 animals of each sex and sampling time were used for statistical evaluation.

Results and discussion

Test resultsopen allclose all
Sex:
male/female
Genotoxicity:
ambiguous
Remarks:
MAIN TEST
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
male/female
Genotoxicity:
negative
Remarks:
REPEAT TEST
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF MAIN TEST
Clinical examination: Animals of the test material group exhibited clinical signs as moderate to severe hypokinesia (21/21 males, 21121 females), slight to severe tremor (7/21 males, 9121 females), slight clonic convulsions (1/21 females), decrease of muscle tone with abdominal position (1/21 males, 1121 females), ptosis (21/21 males, 21121 females), lacrimation (11/21 females), ruffled fur (2121 males, 13/21 females), and restrained gait (1/21 females).
No test material group animal died.
Negative and positive control group animals did not show any abnormal clinical signs.

Evaluation of bone marrow: After single intraperitoneal administration of the test material at 1470 mg/kg (males and females) body weight no statistically significant test material-related increase of micronucleated polychromatic erythrocytes was observed in either male or female animals, when compared with corresponding negative control group animals at the 24-hour sampling time. The same was true for females at the 48-hour sampling time, for males at the 72-hour sampling time, as well as for both sexes combined at the 24- and 72-hour sampling times. A statistically significant increase in the number of micronucleated polychromatic erythrocytes of test material group animals compared to the respective negative controls was present in males at the 48-hour sampling time (p = 0.011), in females at the 72-hour sampling time (p = 0.021), and in both sexes combined at the 48-hour sampling time (p = 0.006).
A clear reduction in PCEINCE ratio was present in several test material group animals, when compared with the corresponding negative control group animals, indicating a toxic effect of the test material on bone marrow. The positive control group animals receiving cyclophosphamide exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes, except females at the 72-hour sampling time, and thus validated the test system. The dose used for this reference mutagen had a toxic effect on bone marrow as shown by the reduced PCE/NCE ratio at 48 and 72 hours after administration.

Conclusion main test: Under the experimental conditions reported, Nicotinamide is considered not to induce chromosome mutations in mice by damage to the chromosomes or to the mitotic apparatus at 24 hours after the animals had received a single intraperitoneal dose of 1470 mg/kg body weight (males and females). However a clastogenic effect cannot be excluded from the present data as shown by a statistically significant increase in micronucleated polychromatic erythrocytes in males at the 48-hour sampling time, in females at the 72-hour sampling time, and in males and females combined at the 48-hour sampling time.
To proof whether these latter findings indicate a potential mutagenic activity of the test material, according to the test guidelines, a second test (repetition test) was performed to examine the reproducibility of the statistically significant positive response at the 48- and 72-hour sampling times (males resp. females) and to assess a possible dose relation in the increase of micronucleated polychromatic erythrocytes.

RESULTS OF REPEAT TEST
Clinical examination: Animals of the test material group exhibited clinical signs as slight hypokinesia (at 681 mg/kg body weight 2/14 males, 1/14 females; at 1000 mg/kg body weight 6/14 males, 9/14 females; at 1470 mg/kg body weight 1/14 males), moderate hypokinesia (at 1000 mg/kg body weight 3/14 males, 2/14 females; at 1470 mg/kg body weight 4/14 males, 3/14 females), severe hypokinesia (at 1000 mg/kg body weight 1/14 males, 1/14 females; at 1470 mg/kg body weight 9/14 males, 11/14 females), ptosis (at 1000 mg/kg body weight 7/14 males, 9/14 females; at 1470 mg/kg body weight 14/14 males, 14/14 females), lacrimation (at 1000 mg/kg body weight 2/14 females; at 1470 mg/kg body weight 2/14 females), slight tremor (at 1470 mg/kg body weight 5/14 males, 6/14 females), moderate tremor (at 1470 mg/kg body weight 1/14 males, 2/14 females), severe tremor (at 1470 mg/kg body weight 1/14 females), moderate clonic convulsions (at 1470 mg/kg body weight 1/14 males), decrease of muscle tone with abdominal position (at 1470 mg/kg body weight 1/14 males, 1/14 females), cyanosis (at 1470 mg/kg body weight 1/14 females), and paralysis of hind leg (at 1470 mg/kg body weight 1/14 females).
No test material group animal died.
Negative and positive control group animals did not show any abnormal clinical signs.

Evaluation of bone marrow: After single intraperitoneal administration of the test material in male and female animals at 681 and 1000 mg/kg body weight, respectively, no statistically significant test material-related increase of micronucleated polychromatic erythrocytes was observed, when compared with corresponding negative control group animals at the examined 48- and 72-hour sampling times. At 1470 mg/kg body weight no statistically significant increase in the number of micronucleated polychromatic erythrocytes was present in males at the 48-hour sampling time, as well as in both males and females at the 72-hour sampling time. In females of the 48-hour sampling time at 1470 mg/kg body weight a statistically significant (p = 0.006) increase in micronucleated polychromatic erythrocytes was present when compared to the respective negative controls. Due to this increase the same was true (p = 0.004) when analysis for animals administered 1470 mg/kg body weight was performed for both sexes combined at the 48-hour sampling time. Increased incidences of micronucleated polychromatic erythrocytes were found only in animals of the 1470 mg/kg body weight dose, where a high cytotoxic effect on bone marrow could be evidenced from a clear reduction in the PCE/NCE ratio (animal No. 760, male, 9%°' PCE/NCE = 0.70; animal No. 774, female, 7% °' PCE/NCE= 0.51; animal No. 775, female, 10%°' PCE/NCE = 0.22) (see Tables 6-10).
A reduction in PCE/NCE ratio was present in several test material treated mice at 1470 mg/kg body weight, but only in one female at 1000 mg/kg body weight, compared to negative control group mice, indicating a toxic effect of the test material on bone marrow.
The positive control group animals receiving cyclophosphamide exhibited a statistically significant increase in the number of micronucleated polychromatic erythrocytes, except males at the 72-hour sampling time, and thus validated the test system. The dose used for this reference mutagen had a toxic effect on bone marrow as shown by the reduced PCE/NCE ratio.

Evaluation and conclusion: Under the experimental conditions reported, the positive results of the main test, i.e. statistically significant increase of micronucleated polychromatic erythrocytes in males at the 48-hour sampling time and in females at the 72-hour sampling time, were not reproducible during the repetition test using the same dose of Nicotinamide, i.e. 1470 mg/kg body weight, as in the main test. Neither there was a dose related response at any test point when additionally doses of 681 and 1000 mg/kg body weight were used.
Nevertheless, during the repetition test a statistically significant increase of micronucleated polychromatic erythrocytes was present at a test point, where such a tendency was not observed during the main test, i.e. in females of the 48-hour sampling time at 1470 mg/kg body weight. In these females increased incidences of micronucleated polychromatic erythrocytes were found in animals with a clearly decreased ratio of PCE/NCE. The same phenomenon was present in one male of the 48-hour sampling time at 1470 mg/kg. These findings are to be considered as results of an exaggerated pharmacological action of the test material Nicotinamide. In so far, they are congruent with in vitro findings, where Nicotinamide increases the frequencies of SCEs when cytotoxic concentrations are used.
Nicotinamide, a vitamin, is part of the coenzyme NAD/NADP and in so far involved in many enzymatic reactions of the mammalian metabolism. So, for instance, high levels of Nicotinamide have an inhibitory effect on drug oxidation of type I substrates as N-nitrosomorpholine, the mutagenic activity of which is reduced in such a case. On the other hand, the mutagenic activity of N-nitrosomorpholine is increased by low levels of nicotinamide and NADP via inhibition of pyridine nucleotidase. Poly (ADP-ribose) synthetase is an enzyme which plays an important role in the DNA replication and repair. Nicotinamide as a precursor of the substrate of this enzyme, NAD, therefore is also needed in certain concentrations to guarantee sufficient DNA repair. On the other hand, in in vitro assays nicotinamide in high, cytotoxic concentrations proved to be an inhibitor of the poly (ADP-ribose) synthetase. As a result inhibitory effects on unscheduled DNA synthesis caused by nicotinamide could be seen. This inhibition of natural repair mechanisms at cytotoxic concentrations of nicotinamide is also considered the cause for increased SCE frequencies.
Inhibition of the poly (ADP-ribose) synthetase is also a result of a deficiency of nicotinamide leading to increased mutations in Chinese hamster V79 cells. Nicotinamide did not induce chromosomal aberrations in a Chinese hamster fibroblast cell line (CHL) and was negative in the Ames test. As the findings indicate Nicotinamide has no direct mutagenic potential, but at high, cytotoxic concentrations as well as in situations of a deficiency of Nicotinamide genotoxic effects may be observed, especially in in vitro assays. These effects are considered to be due to disturbances in NAD/NADP dependent enzyme systems, among which also enzymes responsible for DNA synthesis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant test material-related increase in the number of micronucleated polychromatic erythrocytes was present in either male or female mice at the 24-hour sampling time, in females at the 48-hour sampling time, in males at the 72-hour sampling time, and when analyzed both sexes combined at the 24- and 72-hour sampling times. The statistically significant positive response in males of the 48-hour sampling time and in females of the 72-hour sampling time of the main test could not be verified during the repetition test using two additional dose levels. Therefore, according to the criteria of assessment, Nicotinamide is non-mutagenic in the reported in vivo mouse micronucleus test.
Executive summary:

A study was carried out according to EU Method B.12 and OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test). The in vivo micronucleus test in mice was performed to assess the potential mutagenic activity, induced by Nicotinamide through damage to the chromosomes or to the mitotic apparatus, according to Schmid (1975) with the modifications of Salamone et al. (1980). The main test was performed with three groups, the negative and positive control groups with 18 males and 18 females each, and the test material group containing 21 males and 21 females. All animals were given a single intraperitoneal administration of 10.0 ml/kg body weight. The negative control group received physiological saline solution (0.9 %). The test material-group animals received the maximum tolerated dose in respect of the micronucleus test, i.e. 1470 mg/kg body weight (males and females) Nicotinamide. The test material was given dissolved in aqua ad injectabilia. The positive control group received 51.1 mg/kg body weight cyclophosphamide dissolved in physiological saline solution (0.9 %). Nicotinamide related toxic symptoms were registered in all test material group animals. The symptoms consisted in moderate to severe hypokinesia, slight to severe tremor, slight clonic convulsions, decrease of muscle tone, ptosis, lacrimation, ruffled fur, and restrained gait. No test material group animal died. Twenty-four (24), 48 and 72 hours after treatment, six mice per sex and control group and seven mice per sex of the test material group were sacrificed and bone marrow was removed from the femurs for examination. The first five animals each per sex and group were used for evaluation. One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test material by counting a total of 1000 erythrocytes. The incidences of polychromatic erythrocytes with micronuclei were statistically analyzed by means of a Poisson test. Estimation and testing were performed at each sampling time for each treatment group and for each sex, as well as for both sexes combined as compared with the respective negative control group data. After single intraperitoneal administration of the test material at 1470 mg/kg (males and females) body weight no statistically significant test material-related increase of micronucleated polychromatic erythrocytes was observed in either male or female animals, when compared with corresponding negative control group animals at the 24-hour sampling time. The same was true for females at the 48-hour sampling time, for males at the 72-hour sampling time, as well as for both sexes combined at the 24- and 72-hour sampling times. A statistically significant increase in the number of micronucleated polychromatic erythrocytes of test material group animals compared to the respective negative controls was present in males at the 48-hour sampling time, in females at the 72-hour sampling time, and in both sexes combined at the 48-hour sampling time. A clear reduction in PCE/NCE ratio was present in several test material group animals, when compared with the corresponding negative control group animals, indicating a toxic effect of the test material on bone marrow. The positive control group animals, which received cyclophosphamide, exhibited a significant increase in the number of micronucleated polychromatic erythrocytes, except females at the 72-hour sampling time, and thus validated the test system. To examine the reproducibility of the statistically significant positive response at the 48- and 72-hour sampling times and to assess a possible dose relation in the increase of micronucleated polychromatic erythrocytes, a second test (repetition test) was performed. This repetition test was performed with three groups, the negative and positive control groups containing each 12 animals per sex, and the test material group containing seven mice per sex and sampling time (48- and 72-hours) for each of the three doses used (total in the test material group: 84 animals). All animals were given a single intraperitoneal administration of 10.0 ml/kg body weight. The negative control group received physiological saline solution (0.9 %). The test material group (3 dose groups) received Nicotinamide at 681, 1000, and 1470 mg/kg body weight, respectively. The test material was given dissolved in aqua ad injectabilia. The positive control group received 51.1 mg/kg body weight cyclophosphamide dissolved in physiological saline solution (0.9 %). Nicotinamide related toxic symptoms were registered in test material group animals of all dose groups. The symptoms consisted in slight to severe hypokinesia, ptosis, lacrimation, slight to severe tremor, moderate clonic convulsions, decrease of muscle tone, cyanosis, and paralysis of hind leg. No test material group animal died. Forty-eight (48) and 72 hours after treatment, six mice per sex and control group and seven mice each per sex and dose of the test material group were sacrificed and bone marrow was removed from the femurs for examination. The first five animals each per sex and group, respectively per sex and dose in the test material group, were used for evaluation. One thousand polychromatic erythrocytes per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes was used to assess the toxicity of the test material by counting a total of 1000 erythrocytes. The incidences of polychromatic erythrocytes with micronuclei were statistically analyzed by means of a Poisson test. Estimation and testing were performed at the 48- and 72-hour sampling times for each dose and sex separately, as well as for both sexes combined per dose, as compared with the respective negative control group data. After single intraperitoneal administration of the test material in male and female animals at 681 and 1000 mg/kg body weight, respectively, no statistically significant test material-related increase of micronucleated polychromatic erythrocytes was observed, when compared with corresponding negative control group animals at the examined 48- and 72-hour sampling times. At 1470 mg/kg body weight no statistically significant increase in the number of micronucleated polychromatic erythrocytes was present in males at the 48-hour sampling time, as well as in both males and females at the 72-hour sampling time. In females of the 48-hour sampling time at 1470 mg/kg body weight a statistically significant increase in micronucleated polychromatic erythrocytes was present when compared to the respective negative controls. Due to this increase the same was true when analysis for animals administered 1470 mg/kg body weight was performed for both sexes combined at the 48-hour sampling time. Increased incidences of micronucleated polychromatic erythrocytes were found only in animals of the 1470 mg/kg body weight dose, in which a high cytotoxic effect on bone marrow could be evidenced from a clear reduction in the PCE/NCE ratio (animal No. 760, male, 0.009 % PCE/NCE = 0.70; animal No. 774, female, 0.007% PCE/NCE = 0.51; animal No. 775, female, 0.01 % PCE/NCE = 0.22). A reduction in PCE/NCE ratio was present in several test material treated mice compared to negative control group mice, indicating a toxic effect of the test material on bone marrow. The positive control group animals, which received cyclophosphamide, exhibited a statistically significant increase in the number of micronucleated polychromatic erythrocytes, except males at the 72-hour sampling time, and thus validated the test system.

EVALUATION

Under the experimental conditions reported, the positive results of the main test, i.e. statistically significant increase of micronucleated polychromatic erythrocytes in males at the 48-hour sampling time and in females at the 72-hour sampling time, were not reproducible during the repetition test using the same dose of Nicotinamide, i.e. 1470 mg/kg body weight, as in the main test. Neither there was a dose related response at any test point when additionally doses of 681 and 1000 mg/kg body weight were used. Nevertheless, during the repetition test a statistically significant increase of micronucleated polychromatic erythrocytes was present at a test point, where such a tendency was not observed during the main test, i.e. in females of the 48-hour sampling time at 1470 mg/kg body weight. In these females increased incidences of micronucleated polychromatic erythrocytes were found in animals with a clearly decreased ratio of PCE/NCE. The same phenomenon was present in one male of the 48-hour sampling time at 1470 mg/kg. These findings are to be considered as results of an exaggerated pharmacological action of the test material Nicotinamide. In so far, they are congruent with in vitro findings, where Nicotinamide increases the frequencies of SCEs when cytotoxic concentrations are used (Morris and Heflich, 1984; Darroudi and Natarajan, 1985). Nicotinamide, a vitamin, is part of the coenzyme NAD/NADP and in so far involved in many enzymatic reactions of the mammalian metabolism. So, for instance, high levels of Nicotinamide have an inhibitory effect on drug oxidation of type I substrates as N-nitrosomorpholine, the mutagenic activity of which is reduced in such a case (Braun et al., 1980). On the other hand, the mutagenic activity of N-nitrosomorpholine is increased by low levels of nicotinamide and NADP via inhibition of pyridine nucleotidase (Braun et al., 1980). Poly (ADP-ribose) synthetase is an enzyme which plays an important role in the DNA replication and repair. Nicotinamide as a precursor of the substrate of this enzyme, NAD, therefore is also needed in certain concentrations to guarantee sufficient DNA repair. On the other hand, in in vitro assays nicotinamide in high, cytotoxic concentrations proved to be an inhibitor of the poly (ADP-ribose) synthetase. As a result inhibitory effects on unscheduled DNA synthesis caused by nicotinamide could be seen (Barra et al., 1982). This inhibition of natural repair mechanisms at cytotoxic concentrations of nicotinamide is also considered the cause for increased SCE frequencies (Morris and Heflich, 1984). Inhibition of the poly (ADP-ribose) synthetase is also a result of a deficiency of nicotinamide leading to increased mutations in Chinese hamster V79 cells (Okada et al., 1987). Nicotinamide did not induce chromosomal aberrations in a Chinese hamster fibroblast cell line (CHL) and was negative in the AMES test (Ishidate et al., 1984). As the findings indicate Nicotinamide has no direct mutagenic potential, but at high, cytotoxic concentrations as well as in situations of a deficiency of Nicotinamide genotoxic effects may be observed, especially in in vitro assays. These effects are considered to be due to disturbances in NAD/NADP dependent enzyme systems, among which also enzymes responsible for DNA synthesis.

CONCLUSION

No statistically significant test material-related increase in the number of micronucleated polychromatic erythrocytes was present in either male or female mice at the 24-hour sampling time, in females at the 48-hour sampling time, in males at the 72-hour sampling time, and when analyzed both sexes combined at the 24- and 72-hour sampling times. The statistically significant positive response in males of the 48-hour sampling time and in females of the 72-hour sampling time of the main test could not be verified during the repetition test using two additional dose levels. Therefore, according to the criteria of assessment, Nicotinamide is non-mutagenic in the reported in vivo mouse micronucleus test.