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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
An Ames test, in-vitro chromosome aberration study and in-vivo mouse micronucleus study were conducted on the substance. Ames test: The substance was not mutagenic to bacteria under the conditions of the test. Chromosome aberration study: The substance was considered positive for inducing chromosome aberrations in CHO cells with and without metabolic activation. Mouse micronucleus study: The substance is considered negative in the mouse bone marrow micronucleus assay under the conditions of this assay.
Link to relevant study records
Reference
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:
Experimental start date: July 22, 1998. Experimental termination date: December 3, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Young adult male and female mice of the Crl:CD-l®(ICR) BR strain were purchased from Charles River Laboratories, Raleigh, NC or Portage, MI.
- Age at study initiation: Approximately 9 weeks old.
- Weight at study initiation:
Micronucleus study 1: 29.1 - 37.1 grams.
Micronucleus study 2: 32.2 - 36.4 grams
- Assigned to test groups randomly: yes
- Housing: The animals were housed in sanitary polycarbonate cages containing Sani-Chips® Hardwood Chip Laboratory bedding. The animals were housed, separated by gender, up to seven animals per cage during acclimation, and full dose group after randomization.
- Diet (e.g. ad libitum): Commercial diet (Certified Rodent Diet) was available ad libitum
- Water (e.g. ad libitum): Tap water available ad libitum
- Acclimation period: At least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26°C
- Humidity (%): 30 - 70%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Because of difficulty passing the test article mixed with 0.5% methylcellulose through a 23-gauge needle, com oil was selected as the vehicle control article.
Details on exposure:
MICRONUCLEUS STUDY 1:
Dose selection:
Based on results from the dose range-finding study, dose levels of 225, 450 and 900 mg/kg were selected for testing in mice in this study. Only males were used in the micronucleus study because there were no substantial differences in clinical observations between the sexes in the dose range-finding study.

Dosing information:
A total of 52 male animals were used in this study.
6 males at dose level 225 mg/kg (harvest timepoint 24 hours).
6 males at dose level 450 mg/kg (harvest timepoint 24 hours).
6 males at dose level 900 mg/kg (harvest timepoint 24 hours).
6 males at dose level 900 mg/kg (harvest timepoint 48 hours).
6 males with vehicle control (harvest timepoint 24 hours).
6 males with vehicle control (harvest timepoint 48 hours).
6 males with positive control (harvest timepoint 24 hours).
10 satellite animals designated for blood sampling.

In order to ensure the availability of a minimum of five animals for analysis, the number of treated animals/group was six.

Blood sampling:
A satellite group consisting of 10 animals were included in the definitive assay. Five of the animals were dosed at 900 mg/kg dose level and the remaining five were dosed with the vehicle control article, corn oil. Approximately 4 hours post dose, these animals were anesthetized using CO2/O2 and blood samples were taken periorbitally. The blood samples were centrifuged and the serum was collected, transferred to labeled tubes, and frozen, prior to analysis.

Animal Observations:
All animals were examined immediately after dosing, about 1 hour after dosing, and at least daily for the duration of this study for toxic signs and/or mortalities.


MICRONUCLEUS STUDY 2:
Dose selection:
Due to unanticipated mortality in 900 mg/kg animals at the 48-hour harvest timepoint in the first micronucleus assay, dose levels of 0, 500 and 750 mg/kg were selected for testing in male mice.

Dosing Information:
A total of 34 animals were used in this study.
6 males at 500 mg/kg (harvest timepoint 48 hours)
6 males at 750 mg/kg (harvest timepoint 48 hours)
6 males with vehicle control (harvest timepoint 48 hours)
16 satellite animals designated for blood sampling (15 animals designated for blood sampling and one replacement animal).

Blood sampling:
A satellite group consisting of 15 animals were included in the definitive assay. Five of the animals/dose level were dosed at 500, and 900 mg/kg and the remaining five were dosed with the vehicle control article, corn oil. Approximately 4 hours post dose, these animals were anesthetized using CO2/O2 and blood samples were taken periorbitally. The blood samples were centrifuged and the serum was collected, transferred to labeled tubes, and frozen, prior to analysis.

Observations:
The satellite animals were examined immediately after dosing. All remaining animals were examined immediately after dosing, about 1 hour after dosing, and at least daily for the duration of this study for toxic signs and/or mortalities.





Duration of treatment / exposure:
All animals were dosed on an acute (one-time only) basis.
Frequency of treatment:
Animals dosed once only.
Post exposure period:
24 or 48 hours.
Remarks:
Doses / Concentrations:
Study 1: 225, 450 and 900 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Study 2: 500 and 750 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
6 males per dose level.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positve control: Cyclophosphamide
- Justification for choice of positive control(s): Known to induce a positive result.
- Route of administration: Cyclophosphamide was solubilized in sterile deionized water and dosed orally.
- Doses / concentrations: 80 mg/kg.
Tissues and cell types examined:
The objective of this study was to evaluate the test article for in vivo clastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in mouse bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Three dose range-finding studies were conducted to determine the dose levels for the main micronucleus study (see details in any other information on materials and methods section).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Extraction of Bone Marrow - At the appropriate harvest time points (24 or 48 hours following dosing), the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones (tibias or femurs) were removed for marrow extraction from the first five surviving animals (as determined by numerical order of the ear tags within a dose group). For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3-5 mL fetal bovine serum (one tube per animal). Animals not needed for bone marrow collection were euthanized at the completion of the study.


DETAILS OF SLIDE PREPARATION:
Following centrifiigatioh to pellet the tissue, the supematant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grunwald Solution followed by Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

METHOD OF ANALYSIS:
Slides prepared from the bone marrow collected from the five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The frequency of PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 200 erythrocytes on the slide.

Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 emd 1/5 the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.

The staining procedure permitted the differentiation by color of PCEs and NCEs
(bluish-grey and red, respectively).

OTHER:
Serum was collected in order to validate the high-performance liquid chromatographpy method developed for measuring levels of test article in mouse serum. The purpose of this was to validate a HPLC (High Performance Liquid Chromatography) method, and to quantitatively determine the test article concentration in mouse serum in support of toxicity studies.



Evaluation criteria:
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically
significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only
determinant of a positive response, the Study Director also considered the biological relevance of the results in the final evaluation.
Statistics:
Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on
untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance
was statistically significant (p ≤ 0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time. Additionally, parametric or nonparametric tests for trend may have been employed to identify any dose-related response.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
See results section for details.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
See any other information on results incl. tables section.

RESULTS OF DEFINITIVE STUDY
See any other information on results incl. tables section.

Dose Range-Finding Study 1:

The toxicity observations for the remaining animals are shown in the table: Animal Observations for Toxicity for Dose Range-Finding Study 1 (see attached background material). Effects were seen in animals at every observation timepoint and included; slightly hypoactive, rough haircoat, hypoactive, eyes sealed shut, distended abdomen, cold to touch, hunched posturem squinted eyes.

The mortality data for the animals that were not bled are summarized in the following table:

Treatment (mg/kg)

Number of Animals

Male

Female

2000

3/3

3/3*

Vehicle control

0/3

0/3

*One female died within 3 minutes of dosing and was replaced.

Conclusion:

Based on these results, the maximum tolerated dose could not be determined.

Dose Range-Finding Study 2:

The toxicity observations are shown the table: Animal Observations for Toxicity for Dose-Range-Finding 2 (see attached background material). Effects seen included; prostrate, dyspnea, squinted eyes, rough haircoat, slightly hypoactive, hypoactive, eyes sealed shut, polypnea, hunched posture, pale body.

Treatment (mg/kg)

Number of Animals

Male

Female

200

1/3

0/3

500

0/3

0/3

900

1/3

0/3

1200

2/3

2/3

1500

3/3

3/3

Conclusion:

Based on these results, the maximum tolerated dose could not be determined.

Dose Range-Finding Study 3:

All animals were normal at both observation intervals.

Conclusion:

Based on these results, the maximum tolerated dose was estimated to be 900 mg/kg.

Micronucleus Study 1:

All animals positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoint. The vehicle control animals appeared normal immediately after dosing but were noted with a rough haircoat at the 1-hour post dose observation interval. These animals retumed to normal by Day 1 and remained healthy until the appropriate harvest timepoints. The satellite animals dosed with the vehicle control material appeared normal immediately postdose and at the 1-hour postdose observation interval.

The toxicity observations for the test-article treated animals are shown in the table: Animal Observations for Toxicity for Micronucleus Study 1 - males (see attached background material).Effects seen included; rough haircoat, slightly hypoactive, squinted eyes and tremors.

5 animals were found dead at the 900 mg/kg dose group at 48 hours.

Micronucleus Study 2:

All animals in the vehicle control group appeared normal after dosing and remained healthy until the appropriate harvest timepoints. The toxicity observation for the test-article treated animals are shown in the table: Animal Observations for Toxicity for Micronucleus Study 2 - males (see attached background material).

Effects seen included; rough haircoat, slightly hypoactive, squinted eyes

Results and Interpretation:

The test article induced signs of clinical toxicity in the treated animals and was cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) at 225 and 450 mg/kg at the 24-hour harvest timepoint.

The test article induced no statistically significant increases in micronucleated PCEs over the levels observed in the vehicle controls at any of the harvest timepoints.

The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs as compared to the vehicle controls, with a mean and standard error of 2.63% ± 0.80%.

Result data on PCE:NCE ratio's and PCE numbers are summarised by dose groups for the different timepoints in Table 1 (Micronucleus data summary table). Individual animal data are presented in Tables 2 and 3 (micronucleus test - individual animal data). Historical control data is presented in Table 4.

Please see attached background materila for result tables 1 - 4.

Conclusions of Sample Analysis of Mouse Serum:

The test article is stable in the mouse serum for 23 days when stored at approximately -20°C and one cycle of freeze/thaw has no impact on the stability of test substance in mouse serumm.

The sample analyses results indicated that all the samples contained detectable amounts of test article, except all the controls, in which no test article can be detected. The concentrations of test article in dosed mouse serum increased with the increasing of

dose levels, and leveled off after 750 mg/kg.

Conclusions:
Interpretation of results (migrated information): negative
The test article induced signs of clinical toxicity in the treated animals. There was a statistically significant decrease in the PCE:NCE ratio at
225 and 450 mg/kg at the 24-hour harvest timepoint, demonstrating that the test article was cytotoxic to the bone marrow. The test article did not induce a
statistically significant increase in the frequency of micronucleated PCEs and is considered negative in the mouse bone marrow micronucleus assay under the
conditions of this assay.
Executive summary:

The objective of this study was to evaluate the test article for in vivo clastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-l (ICR) BR mouse bone marrow. In addition, serum was collected in order to validate the high-performance liquid chromatography method developed for measuring the levels of the test article in mouse serum.

In the first dose range-finding study, the test article was dissolved in corn oil and dosed by intraperitoneal injection to 12 animals/sex at 2000 mg/kg (30 animals); three animals/sex received the vehicle control article, com oil, only. The animals were anesthetized using CO2/O2 and blood samples were taken periorbitally at approximately 0.5, 2 and 4 hours post dose from three animals/sex/sampling time. At the 0.5-hour time point, blood samples were also taken from three animals/sex that received only the vehicle. The blood samples were centrifuged and the serum was transferred to labeled tubes and frozen. The animals were observed after dosing for toxic signs and/or mortality. The remaining three animals/sex were not bled; however, clinical observations for toxic signs and/or mortality were performed for 2 days (immediately and at 1 hour post dose and on Days 1 and 2).

In the second dose range-finding study, the test article was dissolved in corn oil and dosed by intraperitoneal injection to three animals/sex/dose level (30 animals). The animals were dosed at 200, 500, 900, 1200, and 1500 mg/kg and observed for 2 days after dosing for toxic signs and/or mortality.

In the third dose range-finding study, the test article was dissolved in com oil and dosed by intraperitoneal injection to three animals at 900 mg/kg. In addition, two males were dosed with the vehicle control article, corn oil. The animals were observed after dosing for toxic signs and/or mortality. The animals were anesthetized using CO2/O2 and blood samples were taken periorbitally at 4 hours post dose. The blood samples were centrifuged and the serum was transferred to labeled tubes and frozen.

Based on the results of the dose range-finding studies, the maximum tolerated dose was estimated to be 900 mg/kg. In the micronucleus assay, the test article was dissolved in corn oil and dosed by intraperitoneal injection to six males per dose level for each harvest time (24 and 48 hours). The animals were dosed at 225, 450, and 900 mg/kg. Five animals dosed at the 225 and 450 mg/kg dose levels and with the positive control were euthanized approximately 24 hours after dosing for extraction of the bone marrow. Five animals dosed at the 900 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal. In addition, a satellite group, consisting of 10 males, was designated for collection of blood serum for analyses. Five animals were dosed with the test article at 900 mg/kg and the remaining five animals were dosed with the vehicle control article, com oil. Approximately 4 hours post dose, the animals were anesthetized using CO2/O2 and blood samples were taken periorbitally. The blood samples were centrifuged and the serum was transferred to labeled tubes and frozen.

Due to unanticipated mortality at the 900 mg/kg dose level in the animals designated for the 48-hour harvest, a repeat of the 48-hour portion of the study was performed using dose levels of 500 and 750 mg/kg. In addition, a satellite group consisting of 15 animals were included for blood collection. Five animals were dosed at 500 and five at 750 mg/kg; the remaining five animals were dosed with the vehicle control article, corn oil. Approximately 4 hours post dose, these animals were anesthetized using CO2/O2 and blood samples were taken periorbitally. The blood samples were centrifuged and the serum was transferred to labeled tubes and frozen.

The test article induced signs of clinical toxicity in the treated animals that appeared dose dependent. There was a statistically significant decrease in the PCE:NCE ratio at 225 and 450 mg/kg at the 24-hour harvest time point, demonstrating that the test article was cytotoxic to the bone marrow.

The test article did not induce a statistically significant increase in the frequency of micronucleated PCEs and is considered negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Ames test:

The test substances mutagenic activity was investigated in a Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay.

The test substance did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor 1254 induced rat liver (S9).

Chromosome Aberration:

The objective of this in vitro assay was to evaluate the ability of the test material to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation.

The top concentration tested in this assay was limited by toxicity.

The test material was considered positive for inducing chromosome aberrations in CHO cells with and without metabolic activation.

Mouse Micronucleus:

The objective of this study was to evaluate the test article for in vivo clastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-l (ICR) BR mouse bone marrow.

The test article induced signs of clinical toxicity in the treated animals that appeared dose dependent. There was a statistically significant decrease in the PCE:NCE ratio at 225 and 450 mg/kg at the 24-hour harvest time point, demonstrating that the test article was cytotoxic to the bone marrow.

The test article did not induce a statistically significant increase in the frequency of micronucleated PCEs and is considered negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Justification for classification or non-classification

The substance is not classified as mutagenic based on results from two in-vitro studies (Ames test and chromosome aberration study) and on in-vivo study (mouse micronucleus study).

The Ames test showed the substance was not mutagenic to bacteria under the conditions of the test.

The chromosome aberration study showed the substance was considered positive for inducing chromosome aberrations in CHO cells with and without metabolic activation.

The mouse micronucleus study showed the substance is considered to be non-genetoxic under the conditions of this study.

The substance is not classified as the positive result from the in-vitro chromosome aberration study is not sufficient to classify the substance individually. The subsequent in-vivo mouse micronucleus study conducted gave a negative result.

It is therefore considered that the substance is not classified.