5-ethyl-2-methylpyridine

Genetic toxicity: in vivo

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:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material: 5-ethyl-2-methyl-pyridine

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 4-5 weeks old
- Weight at study initiation: 17.9 - 25.8 g on arrival
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Male and female mice were housed in single-sex groups of two or five in high density polypropylene cages with stainless steel tops, in a limited-access animal holding room. Cages housing animals of different treatment groups were distributed so that the effects of any spatially-variable components were equalized between groups as far as possible.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least four days prior to treatment.

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

IN-LIFE DATES:
- From: 2 July 1990
- To: 20 July 1990

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used:
Test item: Corn oil
Positive control: Aqueous 10 % ethanol
- Concentration of test material in vehicle:
Preliminary toxicity test: 31.3, 62.5, 100, 125 mg/mL
Main micronucleus test: 15.6, 31.3, 62.5 mg/mL
- Amount of vehicle (if gavage or dermal): The volume-dosage was 10 ml/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was found to be immiscible with 0.9 % saline, but was miscible with corn oil at a concentration in excess of 500 mg/mL. Consequently, dosing formulations were freshly prepared in corn oil on the day of dosing, each concentration being individually formulated, and mixed prior to use (except for the formulation prepared at 31.1 mg/mL for the preliminary toxicity test which was prepared by dilution, in corn oil, from a higher concentration).
All concentrations and dosages cited in this report refer to the test item as received; no allowance has been made for purity or activity below 100 %. No determinations of homogeneity or concentration were performed on prepared formulations.
A suspension of chlorambucil (Sigma Chemical Company) in aqueous 10 % ethanol was prepared immediately prior to dosing, for use as a positive control.

Duration of treatment / exposure:

Preliminary toxicity test
Initially, the toxicity of test item to dividing bone marrow erythrocytes was examined: slide evaluation was restricted to determination of numbers of polychromatic and mature erythrocytes and calculation of the ratio of polychromatic to mature cells for each animal, and for each group. Dosing was by the oral route, on one occasion and at a volume-dosage of 10 ml/kg. All animals were killed 72 hours after treatment.
Main micronucleus Test
Preparations of the test compound or dosing vehicle (corn oil) were administered once, by the oral route, to groups of male and female mice. The volume-dosage was 10 mL/kg. The positive control agent, chlorambucil, was administered orally, at a dosage of 30 mg/kg bw in aqueous 10 % ethanol. Five male and five female mice per group were killed 24 hours after treatment; further lots of five males and five females from low and high dose groups were killed 48 and 72 hours after treatment.

Frequency of treatment:

Single dose

Post exposure period:

Preliminary toxicity test: 72 hours
Main micronucleus test: 24, 48 and 72 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Preliminary toxicity test: 313, 625, 1000, 1250 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Main micronucleus test: 0, 156.3, 312.5, 625 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Positive control: 30 mg/kg bw chlorambucil
Basis:
actual ingested

No. of animals per sex per dose:

Preliminary toxicity test (concentration: number of animals):
313 mg/kg bw: 2M /2F
625 mg/kg bw :2M /2F
1000 mg/kg bw: 2M /2F
1250 mg/kg bw : 2M /2F
Main micronucleus test:
Corn oil (vehicle control): 15M/15F
156.3 mg/kg bw: 5M/5F
312.5 mg/kg bw: 5M/5F
625.0 mg/kg bw: 15M/15F
30.0 mg/kg bw Chlorambucil (positive control): 5M/5F

Control animals:

yes, concurrent vehicle

Positive control(s):

Chlorambucil
- Route of administration: Oral
- Doses / concentrations: Single dose / 30 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Preliminary toxicity test: The levels initially selected for testing were 625 and 1250 mg/kg bw (selected on the basis of a rat oral LD50 value of 1737 mg/kg bw). However, as adverse reactions to treatment were observed in some of these animals, further mice were treated at 313 and 1000 mg/kg bw.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Single dose treatment with sampling at 24, 48 and 72 hours.

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.

METHOD OF ANALYSIS:
Preliminary toxicity test
The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1000, oil immersion) a total of 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.
Main micronucleus test
At least one slide from each animal was randomly coded by a person not subsequently involved in the scoring of the study. Care was taken to ensure that no unique slide identifications remained visible in order to eliminate bias.
Slides were examined as detailed for the preliminary toxicity test, but in addition 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; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.

Statistics:

Using the frequency of micronucleated cells per 1000 polychromatic erythrocytes scored, the data were subjected to statistical analysis by the Mann-Whitney procedure (Mann and Whitney, 1942). 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. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling 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
- Dose range: The test item dosages selected for use in the main micronucleus test were 156.3, 312.5 and 625 mg/kg bw.
- Solubility: Up to 500 mg/ml in corn oil
- Clinical signs of toxicity in test animals:
Initially, groups of two male and two female mice were dosed with test item at 625 and 1250 mg/kg bw. One female animal dosed at 1250 mg/kg bw showed reactions to treatment including lethargy, irregular breathing and prone posture, and was killed in extremis approximately 30 minutes after dosing. One male animal treated at this dosage showed lethargy, hunched posture and unstable gait immediately after dosing, but had recovered when observed approximately 5 hours later, and one male animal dosed at 625 mg/kg bw was seen to be lethargic immediately after dosing, but had also recovered 5 hours later. Further groups of two male and two female mice were then dosed with test item at 1000 and 313 mg/kg bw. All animals dosed at 1000 mg/kg bw showed irregular breathing and unstable gait immediately after dosing; in addition, one female animal showed prone posture and was killed in extremis approximately 30 minutes after dosing. Both males recovered and showed no reactions to treatment when observed approximately 2 hours later: the remaining female animal showed signs of lethargy and hunched posture at this time, but had recovered by the next morning. No animal dosed at 313 mg/kg bw showed adverse reactions to treatment.
Bodyweights were recorded at dosing and daily thereafter until termination; no marked or dose-related incidences of weight loss were recorded throughout the test.
- Evidence of cytotoxicity in tissue analyzed:
Slides were prepared and stained for all surviving animals. The ratios of polychromatic: mature erythrocytes were detailed. No evidence of bone marrow toxicity (i.e. reduced
cell proliferation) was apparent in animals treated at 313, 625 or 1250 mg/kg bw. However, bone marrow toxicity was apparent on slides prepared from the three surviving mice dosed at 1000 mg/kg bw (the polychromatic: mature cell ratio was reduced to 0.5).

RESULTS OF DEFINITIVE STUDY
- Reactions to treatment
Bodyweights were recorded at the times of dosing and termination. Incidences of weight loss were noted throughout the study but these were small and are not dose related. Two male mice dosed with test item at 625 mg/kg bw showed reactions to treatment, including unstable gait and prone/hunched posture, immediately after dosing, but had recovered approximately 4 hours later. All animals survived to termination.
Of the ten mice given chlorambucil, the positive control agent, six lost weight during the 24 hour period before termination.
- Incidence of micronucleated cells
Values of micronucleated cells per 1000 erythrocytes scored and group means and ranges were recorded. Results were broken down according to sex and statistically analyzed.
There was 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 1.4 for the vehicle control group, with a range of 0.0-3.0. Corresponding values for animals given test item at 156.3, 312.5 or 625.0 mg/kg bw were closely similar: 2.4, 1.4 or 1.6 with ranges of 0.9-3.8, 0.0-2.7 and 0.0-2.9 respectively.
Among mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in vehicle controls were 2.0 (range 0.0-3.9) and 2.7 (range 0.9-4.9) respectively. Corresponding values for animals given test item at 625 mg/kg bw were 2.6 (range 0.0-4.9) and 1.9 (range 0.0-3.9) respectively.
Thus, mean values for test item 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 any test item treated group, at any termination time (p>0.05).
Chlorambucil treatment produced a range of micronucleated cells per 1000 polychromatic erythrocytes from 57.2-122.7 with a mean of 81.1. 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 4.5 throughout all groups. These findings demonstrate the normal status of the animals used in the study.
- Ratio of PCE/NCE (for Micronucleus assay):
The ratio of polychromatic to mature erythrocytes was 0.9 in the vehicle control group at 24 hours. Ratios for groups given test item at 156.3, 312.5 or 625.0 mg/kg bw and terminated 24 hours later were 0.9, 0.8 or 0.9, respectively. Forty eight hours after treatment, the ratio of the vehicle control group was 1.0, and in animals given test item at 625 mg/kg bw it was 0.8. Seventy two hours after treatment, the ratio of polychromatic to mature erythrocytes in both the vehicle control and test item treated animals was 0.7.
Ratios for all treatment groups were therefore closely similar to those of their respective vehicle control groups, and no clear indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was apparent.
In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was 0.8.

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 the test item.
The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

Executive summary:

In this study carried out according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) equivalent or similar to EU Method B.12, the effect of test item 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 test was first conducted in order to assess the toxicity of the test item. Initially, groups of two male and two female mice were dosed with test item at 625 and 1250 mg/kg bw. One female animal dosed at 1250 mg/kg bw showed adverse reactions to treatment (including disturbed breathing and prone posture) and was killed in extremis approximately 30 minutes after dosing. One male animal treated at this dosage, and one male animal dosed at 625 mg/kg bw also showed adverse reactions to treatment but had recovered 5 hours later. Further groups of two male and two female mice were dosed with test item at 1000 and 313 mg/kg bw. All animals dosed at 1000 mg/kg bw showed similar adverse reactions to treatment, and one female was killed in extremis approximately 30 minutes after dosing: the remaining mice recovered and survived to termination. No animal dosed at 313 mg/kg bw showed adverse reactions to treatment.

No marked or dose-related incidences of weight loss were recorded throughout the test. Inspection of slide preparations showed no evidence of bone marrow toxicity in animals treated at 313, 625 or 1250 mg/kg bw. However, bone marrow toxicity was apparent in slides prepared from the three surviving mice dosed at 1000 mg/kg bw (the polychromatic: mature cell ratio was reduced, indicating reduced cell proliferation).

Subsequently, male and female mice were given a single dose of test item at 156.3, 312.5 or 625.0 mg/kg bw. In all cases test item was dosed orally, dissolved in corn oil. Concurrent vehicle and positive control groups of mice were similarly dosed with corn oil or chlorambucil (30 mg/kg bw) respectively. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and five females, given test item at 625.0 mg/kg bw or the vehicle, 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 analyzed 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.

Two male mice, dosed with test item at 625 mg/kg bw showed reactions to treatment, including unstable gait and prone/hunched posture, immediately after dosing, but had recovered approximately 4 hours later. All animals survived to termination.

No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with test item. Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of test item were similar to those in concurrent controls. This lack of

treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis (p > 0.05). Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg bw (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 test item. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.