Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 27, 1992 to September 14, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: Bone marrow erythrocyte micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dinaphtho[1,2,3-cd:1',2',3'-lm]perylene-9,18-dione, dodecyl derivs.

EC Number:

270-145-4

EC Name:

Dinaphtho[1,2,3-cd:1',2',3'-lm]perylene-9,18-dione, dodecyl derivs.

Cas Number:

68411-75-6

Molecular formula:

Unspecified

IUPAC Name:

Dinaphtho[1,2,3-cd:1',2',3'-lm]perylene-9,18-dione, dodecyl derivs.

Test material form:

liquid

Details on test material:

Lot No.: 7932-157

Specific details on test material used for the study:

Name: Fluorescent Yellow 131 SC (non-volatiles).
Lot No.: 7932-157.
Purity: Responsibility of the Sponsor.
Appearance and physical state at room temperature: Dark red liquid.
Stability: Responsibility of the Sponsor.
Storage conditions: Stored at room temperature in a plastic secondary container at the SRI Chemical Repository, Building M, Room 217.
Received: April 30, 1992.
Amount received at SRI: 100 grams.
Supplier: Morton International.
Expiration: Responsibility of the Sponsor.
Disposition: Remaining unused portion of the test article will be returned to the Sponsor unless otherwise stated. Dosing solutions were disposed at SRI.

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMAL SPECIFICATION:
Dose Range Finding Assay:
Swiss-Webster mice, 105 males and 105 females, born on approximately April 10, 1992 and March 14, 1992, respectively, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles Rivers Laboratories (P01) on May 19, 1992. Upon arrival, mice of the same sex were randomly placed in animal cages (10 per cage) and 21 male and 21 female mice were selected randomly to be weighed. The weights of the male mice ranged from 17.6 to 26.1 g, and those of the female mice ranged from 16.4 to 24.7 g at the time of receipt. The weighed mice constitute 20% of the total number of mice of each sex received.
Eighteen male and 18 female mice were used for the dose range finding assay initiated on May 26, 1992. Mice not used for this particular study were assigned to concurrent studies.

Definitive Assay:
Swiss-Webster mice, 234 males and 195 females, born on approximately April 7, 1992 and March 5, 1992, respectively, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles Rivers Laboratories (P01) on June 2, 1992. Upon arrival, mice of the same sex were randomly placed in animal cages (10 per cage) and 46 male and 40 female mice were selected randomly to be weighed. The weights of the male mice ranged from 20.1 to 27.5 g, and those of the female mice ranged from 21.2 to 28.3 g at the time of receipt. The weighed mice constitute 20% of the total number of mice of each sex received.
Fifty male and 50 female mice were used for the definitive assay initiated on June 16, 1992. Mice not used for this particular study were assigned to concurrent studies.

TEST SYSTEM IDENTIFICATION:
The animals were randomized and uniquely identified by ear punch. A card attached to the outside of each cage listed the project number, test article, dose level, time of sacrifice, sex, animal number, ear punch number, and tail marking.

SUPPLIER:
Charles River Laboratories
(Portage P01)
Shaver Road
Portage, Michigan 49081.

QUARANTINE:
Mice received on May 19, 1992 and June 2, 1992 were quarantined for 7 and 6 days, respectively, and released to the study on May 26, 1992 and June 8, 1992 by LAMD. Composite parasitology exams performed on May 26, 1992 and June 3, 1992 were negative, and no evidence of adverse clinical signs was observed during the two quarantine periods. No notable gross pathologies were found in any of the 8 male and 8 female mice sacrificed and necropsied during the quarantine periods for preliminary health evaluation.

ANIMAL ROOM ENVIRONMENTAL CONDITIONS:
Rooms: Building L, Room B134 for quarantines and Room W107 at SRI for dose range finding and definitive assays.
Temperature range: 68°-75°F.
Humidity range: 44-87%.
Light cycle: 12 hours light/12 hours dark.
Cage specification: Mice were housed no more than 10 to a cage during quarantine, 3 to a cage during the dose range finding assay, and 5 to a cage during the definitive assay. Polycarbonate cages with hardwood-chip bedding were used throughout the study.
There were no alterations in environmental conditions that could be expected to alter the results of this study.

FOOD AND WATER SUPPLY:
Food: Purina Certified Rodent Chow #5002 ad libitum. Purina Mills, Inc., St. Louis, MO. Lot Nos. FEB12921B and MAR11921C.
Water: UV purified/deionized tap water ad libitum was provided via an automatic watering system for quarantine and the definitive assay and via water bottles for the dose range finding assay. Water purity is analyzed quarterly, and results are recorded in Building 205, Room 39 at SRI.
There were no known contaminants in the water or diet that could be expected to alter the results of this study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

VEHICLE CONTROL/DILUENT:
Name: Corn oil.
Lot No.: OCT0893A.
Purity: 100%, as per bottle label.
Appearance and physical state at room temperature: Yellow-gold liquid.
Storage conditions: Stored in original bottle at 4°C in Building L, Room 109.
Received: April 9, 1992.
Supplier: Best Foods CPC International, Inc. Englewood Cliffs, NJ 07632.
Expiration: April 9, 1993.
Disposition: Retained as vehicle control at SRI in Building L, Room 109.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose Range Finding Assay:
The test article was mixed well in corn oil immediately before dose administration. The volume of the test solution administered was 10 ml/kg of body weight.

Definitive Assay:
The test article was mixed well in corn oil immediately before dose administration. The volume of test suspension administered was 10 ml/kg body weight

Duration of treatment / exposure:

Dose Range Finding Assay:
The preliminary dose range finding assay was performed to determine appropriate dose levels for the definitive study and to define the effects of the test article on the development of erythrocytes in bone marrow. Male and female Swiss-Webster mice were given daily doses of the test article by gavage approximately 24 hours apart for 2 days. Three mice per sex per dose group were individually weighed and dosed with F. Yellow 131 SC in corn oil at dose levels of 300, 600, 1200, 2500, or 5000 mg/kg/day. A control group, consisting of 3 male and 3 female mice, received corn oil at a volume of 10 ml/kg/day. Approximately 48 hours after the final dose administration, all animals were anesthetized by i.p. injection of sodium pentobarbital (60 mg/kg), then euthanized by cervical dislocation.

Definitive Assay:
Mice were dosed by gavage with F. Yellow 131 SC at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. A corn oil control group, and a benzene positive control group were treated similarly and concurrently with the F. Yellow 131 SC test groups. Half the animals were sacrificed approximately 24 and 48 hours, respectively, after the final dose. Animals were anesthetized by i.p. injection with sodium pentobarbital (60 mg/kg), then euthanized by cervical dislocation.

Frequency of treatment:

Animals in both the range-finding and the definitive assay were dosed once per day.

Post exposure period:

Range-finding Assay: 48 hours
Definitive Assay: Half the animals were sacrificed approximately 24 and 48 hours, respectively, after the final dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day

Remarks:

Definitive Assay

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day

Remarks:

Definitive Assay

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day

Remarks:

Definitive Assay

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: Benzene.
Lot No.: 03825EV.
Purity: 99.9%, as per supplier.
Appearance and physical state at room temperature: Clear, colorless liquid.
Stability: Not available from supplier.
Received: October 10, 1988.
Expiration: October 10, 1998.
Storage conditions: Stored at room temperature in a plastic secondary container at the SRI Chemical Repository, Building M, Room 217.
Supplier: Aldrich Chemical Company P. 0. Box 14508 St. Louis, MO 63178.
Disposition: Retained as positive control at SRI Chemical Repository, Building M, Room 217.

Ten males were dosed with the the positive control at a dose of 500 mg/kg/day.

Examinations

Tissues and cell types examined:

PERIPHERAL BLOOD ANALYSIS:
Peripheral blood smears from the dose range finding assay were analyzed for the ratio of polychromatic erythrocytes (PCEs) to red blood cells (RBCs).

BONE MARROW ANALYSIS:
Bone marrow smears were analyzed in both the dose range finding and the definitive assays.

Details of tissue and slide preparation:

PERIPHERAL BLOOD ANALYSIS:
Peripheral blood smears from the dose range finding assay were analyzed for the ratio of polychromatic erythrocytes (PCEs) to red blood cells (RBCs). Blood samples were obtained by pricking the ventral tail vein with a 25-gauge needle and drawing 2-3 μ1 of blood into a capillary tube. The sample from each mouse was transferred to 3 clean, prelabeled microscope slides, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until stained. Slides from each test animal were visually examined, and the slide with the most uniform preparation was coded and stained with acridine orange (Hayashi et al., 1983). Unstained slides were filed for possible future use.

BONE MARROW ANALYSIS:
Bone marrow smears were analyzed in both the dose range finding and the definitive assays. One femur from each mouse was removed and flushed gently with 0.2 ml of fetal bovine serum (FBS) into 0.5 ml of FBS in a 1.5-m1 conical polycarbonate tube. Cells were concentrated by centrifugation, then resuspended in a volume of supernatant, approximately equal to the pellet volumes. The sample from each mouse was transferred to 3 clean, prelabeled microscope slides, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until stained. Slides from each test animal were visually examined, and the slide with the most uniform preparation was coded and stained with acridine orange (Hayashi et al., 1983). Unstained slides were filed for possible future use.

DATA COLLECTION:
Slides for micronucleus evaluation were coded by an individual not involved in the microscopic evaluation using random-letter codes generated by an SRI-developed software package on an IBM PC program. Slide labels were printed directly from the computer.

CYTOLOGICAL ANALYSIS:
Peripheral blood smears and bone marrow smears were evaluated by using epifluorescence microscopy at a magnification of 630X. In the dose range finding assay, peripheral blood smears and bone marrow smears were analyzed for the number of RNA-positive (polychromatic) erythrocytes (PCEs) in at least 1000 and 200 erythrocytes, respectively, per animal. In the definitive assay, two parameters were determined in the bone marrow smears: (1) the number of micronucleated PCEs in at least 1000 PCEs per animal, which provides an index of chromosomal damage; and (2) the number of PCEs in at least 200 erythrocytes per animal, which provides an index of cytotoxicity.
The criteria used for identifying micronuclei are those described by Schmid (1976), with the additional requirement that the micronuclei exhibit the bright yellow fluorescence characteristic of acridine orange stain. The data from a given slide were
registered directly to an IBM PC data file during scoring. After analysis, the slides were decoded and the data summarized by using a decoding program in an IBM PC.

Evaluation criteria:

DATA ANALYSIS AND INTERPRETATION:
Criteria for a Valid Assay:
The data from this assay were considered acceptable if (1) the frequency of micronucleated cells in the corn oil vehicle control group was within the normal historical range, (2) the administration of the positive control article resulted in a statistically significant elevation of micronucleated cells, and (3) at least 3 surviving animals of each sex in two or more dose groups showed a ratio of PCEs to total erythrocytes in bone marrow greater than or equal to 0.1.

Interpretation of Response:
Positive. The test article is considered positive if the incidence of micronucleated PCEs is significantly higher than that in the corn oil vehicle control group (p < 0.05) in (1) two different dose groups from one experiment, (2) at a single dose if confirmed by a separate experiment (e.g., an increased frequency of micronucleated cells in the dose range finding experiment), or (3) a positive, dose-related increase in the incidence of micronucleated cells.

Negative. The test article is considered negative if the criteria for a positive or inconclusive response are not met.

Inconclusive. The results are considered inconclusive if there is reason to believe that the concentrations of the test article selected for evaluation were inappropriate (e.g., excessive toxicity) or if a statistically significant elevation in the frequency of micronucleated PCEs is observed in only one treatment group and the dose-response trend is not significant.

Control of Bias:
Animals were randomized by using an IBM PC computer-generated, random-number form, then consecutively assigned to dose groups (i.e., animals numbered 1 through 8 were the first 8 animals used in the experiment). Before the microscope slides were scored, they were coded, using random-letter codes, by a person not assigned to score that experiment.

Statistics:

Statistical Tests:
The data were analyzed separately for each sex. The ratio of micronucleated PCEs to PCEs and the ratio of PCEs to total erythrocytes in percentages were calculated for each animal.
The statistical significance of differences in the percentage of PCEs to total erythrocytes among F. Yellow 131 SC-treated and corn oil control groups was evaluated by using the Kruskal-Wallis analysis of variance on ranks (calculated by using an SRI-developed software program in an IBM PC).
In experiments where the frequencies of micronucleated PCEs were determined by scoring 1000 PCEs per animal, data are not expected to be distributed normally. Such data were analyzed by the Cochran-Armitage test for a trend in binomial proportions to determine a significant dose-response relationship (using an SRI-developed software package), and a test for equality of binomial proportions to determine whether values for individual dose groups were statistically different from those for controls (Kastenbaum and Bowman; using an SRI-developed software package). These tests and their rationale are discussed in the ATSM Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes and other publications (ATSM Committee, 1988; Margolin et al., 1983).
Animal mean body weights were analyzed by the Student's t-test (in an SRIdeveloped software package) run in an IBM PC.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

At doses up to and including 5000 mg/kg/day, F. Yellow 131 SC

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING ASSAY:
For this assay, 3 mice per sex per dose group were individually weighed and dosed orally with F. Yellow 131 SC at 300, 600, 1200, 2500, or 5000 mg/kg/day for 2 consecutive days. A control group, consisting of 3 male and 3 female mice, received corn oil only. Mice were observed daily for four days from the start of dosing until sacrifice.
At 600, 1200, 2500 and 5000 mg/kg/day F. Yellow 131 SC, 1 male mouse, 1 female mouse, 1 male mouse and 1 female mouse, and 2 male mice and 1 female mouse, respectively, had discolored fur due to excretion of the test article. All other mice appeared normal throughout the assay. All mice were sacrificed approximately 48 hours after the last dose administration and cytotoxicity was evaluated by the frequency of RNA-positive erythrocytes (PCEs) among total red blood cells (RBCs).
The number of PCEs among total RBCs was counted in both peripheral blood and bone marrow smears to estimate the frequency of PCEs among erythrocytes. In the peripheral blood, treatment with 300, 600, 1200, 2500, or 5000 mg/kg/day F. Yellow 131 SC produced percentages of PCEs among total RBCs (PCE/RBC ratios) of 1.8, 1.7, 2.3, 2.1, and 1.9% in male mice and 1.5, 1.8, 2.1, 1.6, and 2.6% in female mice, respectively. Treatment with corn oil produced PCE/RBC ratios of 1.9% in male mice and 2.0% in female mice. In bone marrow, treatment with the above doses of F. Yellow 131 SC produced percentages of PCEs among total RBCs (PCE/RBC ratios) of 45, 50, 51, 52, and 50% in male mice and 47, 51, 46, 52, and 51% in female mice, respectively. Treatment with corn oil produced PCE/RBC ratios of 54% in male mice and 47% in female mice. No statistical PCE suppression was observed in either sex in any of the dose groups. No statistical evaluations of the dose range finding data were deemed necessary. The doses selected, in consultation with the Sponsor, for the definitive assay were 1250, 2500, and 5000 mg/kg/day of F. Yellow 131 SC.

DEFINITIVE ASSAY:
For this assay, 10 mice per sex per dose group were individually weighed and dosed orally with F. Yellow 131 SC at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. A corn oil vehicle control group and a benzene positive control group were treated similarly and evaluated concurrently with the test groups. Five mice per sex per dose group were sacrificed at approximately 24 hours and the rest at approximately 48 hours after the final dose. Mice were observed daily for four days from the start of dosing until death or sacrifice.
Discolored fur due to excretion of F. Yellow 131 SC was observed in 3 male and 1 female mice, 1 male and 2 female mice, and 5 male and 2 female mice at 1250, 2500 and 5000 mg/kg/day, respectively. Rough fur was observed in 1 male mouse treated with benzene, 1 female mouse treated with F. Yellow 131 SC at 2500 mg/kg/day. One male mouse had a humped back and 1 female mouse had discolored fur and rough fur at 5000 mg/kg/day F. Yellow 131 SC. All other mice appeared normal throughout the assay. All mice were evaluated for toxicity and micronucleus formation in bone marrow erythrocytes.
Erythrocytes in bone marrow from treated mice were examined, and the frequency of PCEs among total RBCs and of micronucleated PCEs among PCEs were determined.
At the 24-hour harvest, male and female mice treated with corn oil alone averaged background micronucleus incidences of 0.2 and 0.4%, respectively. F. Yellow 131 SC treated groups had micronucleus counts not statistically different from that of the corn oil vehicle control group.
At the 48-hour harvest, male and female mice treated with corn oil alone averaged background micronucleus incidences of 0.3 and 0.2%, respectively. Again, F. Yellow 131 SC treated groups had micronucleus counts not statistically different from that of the corn oil vehicle control group.
Male mice treated with benzene had micronucleus frequencies approximately 23- and 8-fold greater than that of the corn oil control group at the 24- and 48-hour harvests, respectively. Positive control data were compared with data from the corn oil control group by using binomial proportions, which found statistically significant differences between the treated and control groups at p < 0.01.
It was concluded that F. Yellow 131 SC at doses up to 5000 mg/kg/day did not induce increased incidences of micronuclei in the bone marrow erythrocytes of male and female Swiss-Webster mice. Therefore, F. Yellow 131 SC was considered nongenotoxic under these test conditions.

Applicant's summary and conclusion

Conclusions:

In the definitive assay, 10 mice per sex per dose group were dosed orally with F. Yellow 131 SC in corn oil at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. Five mice per sex per dose group were sacrificed 24 hours after the final dose and the same number 48 hours after the final dose; all mice were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A corn oil control group and a benzene positive control group were treated similarly and evaluated concurrently with the test groups.
At doses up to and including 5000 mg/kg/day, F. Yellow 131 SC did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice. Therefore, F. Yellow 131 SC was considered nongenotoxic under these test conditions.

Executive summary:

The genotoxic potential of orally administered Fluorescent Yellow 131 SC (non-volatiles) (F. Yellow 131 SC) to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice.

A dose range finding assay was performed to determine a 2-day maximum tolerated dose (MTD) that would be used to select doses for the definitive study. In this assay, 3 mice per sex received F. Yellow 131 SC in corn oil by oral intubation (gavage) at dose levels of 300, 600, 1200, 2500, or 5000 mg/kg body weight/day (milligrams per kilogram per day). A control group, consisting of 3 male and 3 female mice, received corn oil only. All mice were dosed for 2 consecutive days and were observed daily from the start of dosing until sacrifice. Bone marrow and peripheral blood samples were

obtained approximately 48 hours after the administration of the last dose from all surviving mice. These samples were evaluated for evidence of cytotoxicity reflected in the frequency of RNA-positive (polychromatic) erythrocytes (PCEs) among total erythrocytes (RBCs).

Erythrocytes in both bone marrow and peripheral blood from all mice were examined, and the frequency of PCEs among RBCs was determined. Significant PCE suppression was not observed in either sex in any of the dose groups. No other statistical evaluations were deemed necessary. Therefore, the doses of F. Yellow 131 SC selected for the definitive assay were 1250, 2500, and 5000 mg/kg/day.

In the definitive assay, 10 mice per sex per dose group were dosed orally with F. Yellow 131 SC in corn oil at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. Five mice per sex per dose group were sacrificed 24 hours after the final dose and the same number 48 hours after the final dose; all mice were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A corn oil control group and a benzene positive control group were treated similarly and evaluated concurrently with the test groups.

F. Yellow 131 SC-treated groups had micronucleus counts similar to their respective corn oil control groups both for male and female mice at the 24-and 48-hour harvests.

Male mice treated with benzene had micronucleus frequencies approximately 23-fold greater than those of the corn oil control group at the 24-hour harvest, and approximately 8-fold greater at the 48-hour harvest.

At doses up to and including 5000 mg/kg/day, F. Yellow 131 SC did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice. Therefore, F. Yellow 131 SC was considered nongenotoxic under these test conditions.