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:
26 April 1989 - 30 January 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: M-3175; 1,3-propylenediamine-N,N,N',N'-tetra acetic acid, ammonium iron (+3) salt (mentioned in 28-day study report)
Appearance: yellow powder
Batch No.: VG-01
Purity: 99% (mentioned in 28-day study report)

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) limited, Margate, Kent, England
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 17.0 - 23.0 g
- Assigned to test groups randomly: yes, under following basis: computer-generated random numbers
- Fasting period before study: no data
- Housing: Male and female mice were housed in single-sex groups of two or five in high density polypropylene cages with stainless steel tops.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 26 April 1989 - 30 January 1990

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: saline, 0.9%
- Justification for choice of solvent/vehicle: The test material was found to form a doseable and apparently homogeneous suspension
- Concentration of test material in vehicle: 500 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing suspensions were freshly prepared in 0.9% saline on the day of dosing, each concentration being individually formulated, and mixed prior to use.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24-48-72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
120, 600, 3000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Positive control(s):
Chlorambucil
- Justification for choice of positive control(s): known postive
- Route of administration: oral gavage
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:
At least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1000 cells of each type were scored from each animal where possible, but where
there was an appreciable deviation from unity in the ratio of polychromatic to mature erythrocytes, scoring continued until a minimum of 2000 of the predominant cell type were counted.
Each erythrocyte scored was examined for the presence or absence of micronuclei. When examination had been completed, the slides were decoded. The resultant data were used to calculate the number of micronucleated cells per 1000 erythrocytes. The ratio of polychromatic to mature cells was also determined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study

DETAILS OF SLIDE PREPARATION: Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa)
staining technique. When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.
Evaluation criteria:
Statistics.
Statistics:
Using the frequency of micronucleated cells per 1000 polychromatic erythrocytes scored. the data were subjected to statistical
analysis-by the Mann-Whitney procedure. A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1. Data from males and females within each group were compared. As there was no significant difference within each group, the sexes were pooled for further analysis. For each sampllng time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The preliminary toxicity test was conducted using M-3175 dosages of 625, 1250, 2500 and 5000 mg/kg. All animals survived to scheduled termination. Evidence of toxicity of M-3175 was noted in all animals dosed with M-3175 at 5000 mg/kg; signs included watery faeces, ungroomed appearance at the base of the tail, severe piloerection, slow respiration and hunched posture. Piloerection was also observed in both male animals dosed with M-3175 at 2500 mg/kg. No adverse reactions to treatment were recorded for any other group of animals dosed with M-3175.
Incidences of weight loss were noted in all groups and these were marked in the animals dosed with M-3175 at 5000 mg/kg.
Severe toxicity (as evidenced by depression in bone marrow proliferation) was noted in animals dosed with M-3175 at 5000 mg/kg. There was no evidence of such toxicity in any other group of animals dosed with M-3175.

A supplementary toxicity test was conducted using M-3175 dosages of 3000 and 4000 mg/kg.
One animal dosed with M-3175 at 4000 mg/kg was found dead in its cage approximately 17 hours post-dose. Evidence of toxicity of M-3175 was noted in the three remaining animals dosed with M-3175 at 4000 mg/kg; signs included piloerection, watery faeces, ungroomed appearance at the base of the tail and hunched posture. Signs of toxicity including watery faeces, ungroomed appearance at the base of the tail and piloerection were also noted in both males dosed with M-3175 at 3000 mg/kg.
Large bodyweight losses were noted in the three surviving animals dosed with M-3175 at 4000 mg/kg. No such weight losses were noted in the animals dosed at 3000 mg/kg.
Severe toxicity (as evidenced by depression in bone marrow proliferation) was noted in animals dosed with M-3175 at 4000 mg/kg. There was no evidence of such toxicity in animals dosed at 3000 mg/kg.

RESULTS OF DEFINITIVE STUDY
Reactions to treatment
Cases of bodyweight loss and stasis are clearly marked. In view of the relatively short timespan between weighings, no major significance can be attached to these results. Incidences of weight loss were recorded throughout the study and, although the majority of these were small, it should be noted that seven animals lost weight and one failed to gain weight in the group dosed with M-3175 at 3000 mg/kg and killed 24 hours post-dose. In the vehicle control group at this termination time only two animals were noted to have lost weight.
Evidence of toxicity of M-3175 was noted in all animals in the 24 hour period after dosing at 3000 mg/kg; signs included watery faeces, ungroomed appearance at the base of the tail and slight piloerection. No adverse reactions to treatment were recorded for any other group of animals dosed with M-3175. Of the ten mice given chlorambucil, the positive control agent, 9 lost weight during the 24 hour period before termination.

Incidence of micronucleated cells
No significant differences in the frequencies of micronucleated polychromatic cells were seen between sexes, in any group.
Among mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.3 for the vehicle control group, with a range of 0.0-1.0. Corresponding values for animals given M-3175 at 120, 600 or 3000 mg/kg were closely similar: 0.6, 0.6 or 0.7 with ranges of 0.0-2.0, 0.0-3.0 and 0.0-1.0 respectively.
Among mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in vehicle controls were 1.2 (range 0.0-3.9) and 0.3 (range 0.0-1.0) respectively. Corresponding values for animals given M-3175 at 3000 mg/kg were 0.4 (range 0.0-1.6) and 0.1 (range 0.0-1.0) respectively.
Thus, mean values for M-3175-treated groups were closely similar to mean control group values at all termination times.
Statistical analysis confirmed that there was no significant difference between the vehicle control group and M-3175-treated group at any termination time (p > 0.05).
Chlorambucil treatment produced a range of micronucleated cells per 1000 polychromatic erythrocytes from 48.7-92.7 with a mean of 64.0. Statistical analysis showed that animals treated with chlorambucil had significantly more micronucleated polychromatic cells than vehicle controls (p < 0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.

The recorded incidence of micronuclei per 1000 mature erythrocytes varied between 0.0 and 2.6 throughout all groups. These findings demonstrate the normal status of the animals used in the study: in particular, the low incidence in animals killed 24 hours after treatment shows
the absence of any pre-treatment abnormality in the bone marrow.
The ratio of polychromatic to mature erythrocytes was 0.8 in the vehicle control group at 24 hours. Ratios for groups given M-3175 at 120, 600 or 3000 mg/kg and terminated 24 hours post-dose were 0.9, 0.9 and 0.8 respectively. Forty eight hours after treatment, the ratio of the vehicle control group was 0.8, and in animals given M-3175 at 3000 mg/kg it was 0.2. Seventy two hours after treatment, the ratio of polychromatic to mature erythrocytes in the vehicle control group was 0.8, and in M-3175-treated animals it was 0.9. Ratios for all treatment groups were therefore closely similar to those of their respective vehicle control groups except in animals dosed with M-3175 at 3000 mg/kg and
terminated 48 hours later; this group showed severe bone marrow toxicity as evidenced by depression in bone marrow proliferation.
In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was reduced to 0.7.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of M-3175 even where there was evidence of severe bone marrow toxicity. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.
Executive summary:

The effect of M-3175 on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei. A preliminary toxicity test was first conducted, using dosages of 625, 1250, 2500 and 5000 mg/kg. A supplementary toxicity test was then conducted using M-3175 dosages of 3000 and 4000 mg/kg. Subsequently, male and female mice were given a single dose of M-3175 at 120, 600 or 3000 mg/kg. In all cases M-3175 was dosed orally, in 0.9% saline. Concurrent vehicle and positive control groups of mice were similarly dosed with 0.9% saline or chlorambucil (30 mg/kg) respectively. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and five females, given M-3175 at 3000 mg/kg or the vehicle control, were killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic: mature cells was also calculated for each animal, as an indicator of gross toxicity.

Severe bone marrow toxtcity, as evidenced by depression of bone marrow proliferation, was noted in animals dosed with M-3175 at 3000 mg/kg and killed 48 hours later. No evidence of such toxicity was noted for any other group of animals dosed with M-3175.

Evidence of toxicity of M-3175 was noted in all animals in the 24 hour period after dosing at 3000 mg/kg; signs included watery faeces, ungroomed appearance at the base of the tail and slight piloerection. No adverse reactions to treatment were recorded for any other group of animals dosed with M-3175.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of M-3175 were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant

increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p<0.01).

It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of M-3175 even where there was evidence of severe bone marrow toxicity. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.