Protocol for the Genotoxicity Assay of Ammonium Perchlorate (Revised)

I. Overall Objectives:

Research will be conducted to determine the potential genotoxicity associated with the exposure to ammonium perchlorate (AP), a chemical that is being considered for potential military and space application.

Three short-term genotoxic assays will be used to examine the genotoxicity of ammonium perchlorate, which include:

1. Salmonella/Mammalian microsome reverse mutation assay

- per EPA (TSCA) Health Efect Testing Guidelines

(40 CFR 798.5265)

2. Mouse lymphoma assay

- per EPA (TSCA) Health Efect Testing Guidelines

(40 CFR 798.5300)

3. In vivo mouse bone marrow micronucleus test

- per EPA (TSCA) Health Efect Testing Guidelines

(40 CFR 798.5395)

II. Salmonella/Mammalian Microsome Reverse Mutation Assay

II-A. Purpose

The Salmonella/Mammalian microsome reverse mutation system is a microbial assay which measures the reversion from his^-- (histidine dependent) to his+ (histidine independent) induced by chemicals which cause base changes or frameshift mutations in the genome of this organism.

II-B. Background

A reverse mutation assay using Salmonella typhimurium detects mutations in a gene of a histidine requiring strains to produce a histidine independent strain of this organism. A reverse mutation can be achieved by base pair changes, which may occur at the site of the original mutation or at a second site in the chromosome; or by frameshift mutations resulted from the addition or deletion of single or multiple base pairs in the DNA molecule.

In this assay, bacteria are exposed to the test agent with and without a metabolic activation system and plated onto minimum agar medium which is deficient in histidine. After a suitable period of incubation, revertant colonies are counted and compared with the number of spontaneous revertants in an untreated and / or vehicle control culture. The mutagenicity of the test agents is evident by the increased number of revertants.

II-C. Test Methods

1. Tester strains:

Four tester strains will be used in this assay, which include TA1535 and TA100 for the detection of base pair mutagens, and TA1537 and TA98 for the detection of frameshift mutagens. The tester strains will be obtained from Dr. Bruce N. Ames in University of California, CA.

2. Confirmation of the genotypes of the tester strains:

Following genotypes should be confirmed in each tester strain based on the methods described by Maron and Ames (1983) prior to the mutagenesis study:

1. Requirement of histidine for growth (His^--)
2. Sensitivity to Crystal violet (rfa mutation)
3. Sensitivity to U.V. light (uvrB mutation)
4. Resistance to ampicillin (R factor)
5. Spontaneous revertant

3. Bacteria growth:

Fresh culture of the tester strains should be used for each assay.

The bacteria are cultured in nutrient broth at 37^oC in an environmental shaker incubator for 10-15 hours to reach the late exponential or early stationary phase of growth (10⁸-10⁹ cells per mL).

4. Metabolic activation:

The test compound should be examined both in the presence and absence of an appropriate metabolic activation system. The most commonly used activation system in this assay is S9 mixture, a cofactor supplemented postmitochondrial fraction prepared from the liver of rats treated with enzyme inducers such as Aroclor-1254.

5. Test agent:

Ammonium Perchlorate will be freshly dissolved in sterile distilled water to the required concentrations. A prescreening test including 5 log doses (with 5 mg/plate as the top dose) will be conducted in TA100 for the dose selection. Toxicity will be evident by a reduction in the spontaneous revertants per plate, and /or a clearing of the background lawn. Five concentrations with adequate intervals will be selected and tested in the mutagenesis.

6. Controls:

In each assay, the following concurrent positive and negative controls will be set up:

a. Negative controls (untreated and/or solvent):

Untreated cultures with and without S9 mixture are set up as negative control. They are used for the measurement of spontaneous revertants, which will serve as the background level of reverse mutation. Appropriate solvent controls will also be included in each assay.

b. Positive control:

Positive controls with known mutagens shall ensure the responsiveness of the tester strains as well as the efficacy of the activation system. Sodium azide (without S9) will be the positive control for TA1535 and 9-aminoacridine (without S9) for TA1537. The positive controls for TA98 and TA100 will be 2-aminofluorene (with S9). The above positive control agents will be dissolved in DMSO.

7. Mutagenesis assay (plate incorporation method):

All dose points (with and without S9 mixture) will be set up in triplicates. 0.1 mL of the culture is added to 2 mL of top agar which is melt and held at 45^oC heating block, along with 0.1 mL of the test agent, and 0.5 mL of S9 mixture (in S9+ plates only). The contents are mixed and then poured onto the surface of a minimum glucose agar plate and spread out evenly. The top agar is allowed to solidify and the plates are inverted and incubated at 37^oC for 48 hours. The number of revertants per dish is counted manually or by an automatic colony counter.

II-D. Data collection and reporting

The number of revertants per dish will be determined by automatic colony counter, and the results are stored and processed by computer (Excel spreadsheets). Following specific information will be reported for the Salmonella mutagenesis assay: (1) Tester strains used (results of genotypic confirmation), (2) Metabolic activation system used (source, amount, cofactors, method for preparation), (3) Dose levels and the rationale for their selection, (4) Positive and negative controls, (5) Individual plate counts, means, and standard deviation, and (6) Dose response relationship if applicable.

II-E. Result analysis and interpretation

1. Criteria for acceptability:

The data generated will be considered acceptable if:

1. The spontaneous revertant frequency is in the normal range as reported in the literature or within the laboratory's historical range.
2. A sufficient number of nontoxic concentrations have been tested.
3. The strain-specific positive mutagens significantly increase the revertant in the corresponding strains.

2. Criteria for interpretation:

a. Positive result:

A compound will be considered positive in this assay if a dose-dependent increase in the number of revertants is observed in three concentrations, and the highest increase in TA1535 and TA1537 is equal to three times the spontaneous control value or the highest increase in TA98 and TA100 is equal to two times the spontaneous level (Brusick and Hayes, 1989). Sometimes the precise fold increase will not be necessary if a clear dose-dependent pattern is noted over several concentrations.

A positive result in Salmonella/microsome mutagenesis indicates that under the experimental conditions, the test compound induces point mutation by base changes or frameshift in the genome of this organism.

b. Negative result:

A test agent will be considered negative in this assay if the criteria for positive response are not met, and the tester strains are sensitive to the positive mutagens.

A negative result indicates that under the experimental conditions, the test compound is not mutagenic in Salmonella typhimurium.

III. Mouse Lymphoma Assay

III-A. Purpose

Mammalian cell culture systems can be used to detect mutations induced by chemical substances. One of the most commonly used mammalian cell mutagenesis system, the L5178Y mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base pair changes, frameshift and small deletions. Mutant cells deficient in TK due to the forward mutation in the TK locus (from TK+ to TK-) are resistant to the cytotoxic effect of pyrimidine analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or trifluorothymidine (TFT). The mutagenicity of the test agents is indicated by the increase in the number of mutants after treatment.

III-B. Background

Thymidine monophosphate (TMP) occupies a unique position in DNA replication. Of the four principle deoxyribonucleotide monophosphates, TMP alone does not undergo significant conversion to other nucleotides. This conservation makes the TMP pool size quite small and constant under normal growth condition, which serves as a regulator for DNA synthesis. If the TME is replaced by other lethal TMP analogues, the cell will be killed. The phosphorylation of these analogues is mediated by the "salvage" enzyme thymidine kinase (TK), which normally phosphorylates thymidine to TMP in most mammalian cells. TK-deficient cells lack this enzyme activity and therefore are resistant to the cytotoxic effect of the lethal analogues. In the mouse lymphoma cell forward mutation assay, the TK-conpetent L5178Y (TK+/+ or TK+/-) cells are treated with the test agents. After certain period of expression, the cells are shifted to a selective medium containing the lethal analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or trifluorothymidine (TFT). Only the mutant cells (TK-/-) can survive under the selection condition, and the mutagenicity of the test compound is evident by the increase in the number of mutants.

III-C. Test Methods

1. Cells and culture maintenance:

The L5178Y TK^+/- mouse lymphoma cells, clone 3.7.2C are used throughout the study, which were originally obtained from Dr. Donald Clive of former Burroughs Wellcome Co. (Research Triangle Park, NC). The cells used in the mutagenesis assay should have a high cloning efficiency and low spontaneous mutation frequency. The cells are maintained as suspension culture in F_10p media in culture flasks equilibrated with 5% CO₂.95% air and incubated at 37^oC in a rotary shaker.

The cells have a doubling time as 10-11 hours. Each week the cells will be grown in the F_10p media containing THMG (thymidine, hypoxanthine, methotrexate and glycine) to select against newly arising TK^-/^- mutants, and then placed in in the F_10p media containing THG (thymidine, hypoxanthine, and glycine) for 1-3 days prior to use in mutagenesis study.

2. Metabolic activation system:

Cells will be exposed to the test agent both in the presence and absence of an appropriate metabolic activation system. Cofactor- supplemented liver S9 from Aroclor-induced rats will be used in each assay.

3. Test agent:

Ammonium perchlorate will be freshly dissolved with distilled water prior to each use. A preliminary range finding experiment will be conducted using 10 doses over a 3-4 log range with 5000 ug/mL as the top concentration. The procedures for range finding are identical to that used for mutagenesis except that the cultures are terminated after 24-48 hours without further cloning. The toxicity is indicated by the decrease of cell number in the suspension culture compared with that in untreated control. Four to five concentrations will be selected based on the result and used in the mutagenesis assay. The highest dose should produce a low level of survival (approximately 10-15%), and the survival in the lowest dose should be the same as the negative control.

4. Controls:

Negative control without treatment and positive control with known mutagens should be included in each assay. Ethyl methanesulfonate (EMS, without S9 mixture) and 3-methylcholanthrene (3-MCA with S9 mixture) will be used as the positive controls. Both mutagens are dissolved in DMSO, and corresponding solvent control will also be included.

5. Mutagenesis assay:

a. Exposure:

Cells (6 x 10⁶ cells in 10 mL medium for each culture) are treated with test agents with and without S9 mixture, and incubated at 37^oC with rotation for 4 hours. Chemicals are removed and cells are washed twice by centrifugation then resuspended in non-selective medium at a density of 3 x 10⁵ cells/mL, and maintained in roller drum for 2 days at 37^oC.

b. Expression:

The 2 day maintenance after exposure is the expression period for mutation. During this period, cell density is checked daily and adjusted to 3 x 10⁵ cells/mL.

c. Cloning:

On the second day of expression, cells are seeded onto soft agar medium to determine the survival and the mutation frequency. For each dose group, 3 cultures containing 200 cells/dish in non-selective medium are set up for viability measurement, another set of 3 cultures with 1 x 10⁶ cells/dish in selective medium containing TFT are used for mutant counting. Dishes are incubated at 37^oC in an atmosphere of 5% CO₂ .95% air.

d. Colony counting:

Colonies are counted 11-12 days after cloning using an automatic colony counter. The mutant frequency is calculated and adjusted based on the survival percentage.

III-D. Data collection and reporting

All the original records about cell maintenance, medium and chemical preparation, cell counts, S9 preparation, details for experimental set-up of range finding and mutagenesis assay will be kept in standard forms. Results will be expressed in tabular form which include colony forming efficiency (CFE %), relative CFE (RCFE), number of mutants, mutation frequency (MF) and relative mutation frequency (RMF) for each culture. Specifically for the mouse lymphoma cell mutagenesis assay , following information will be included in the report: 1. Cells (type, number of cultures, methods for maintenance), 2. test agents (dose selection and rationale). 3. Experimental conditions (incubation temperature, CO₂ concentration, treatment schedule, cell density, metabolic activation system and its preparation, positive and negative controls, length of expression, selective agent and concentration, etc.).

III-E. Result analysis and interpretation

1. Criteria for acceptability:

The data generated will be considered acceptable if:

a. normal range as reported in the literature or within The spontaneous mutation frequency is in the the laboratory's historical range.

b. The test system is sensitive to the known mutagen as judged by the results in the concurrent positive control cultures.

2. Criteria for interpretation:

a. Positive result:

A test agent will be considered to be positive in the mouse lymphoma cell mutagenesis assay if it induces a statistically significant dose-related increase in the mutant frequency, or generates a reproducible and statistically significant increase in the mutant frequency for at least one concentration.

A positive result in mouse lymphoma cell mutagenesis assay indicates that under the experimental conditions, the test compound induces gene mutation in the cells used.

b. Negative result:

A test agent which does not produce either a statistically significant dose-related increase, or a reproducible and statistically significant increase of the mutant frequency in any one of the concentrations tested will be considered nonmutagenic in this system.

A negative result indicates that under the experimental conditions, the test compound does not produce gene mutation in the cells used.

3. Statistical analysis:

The toxicity of the test agent will be indicated by a decrease in CFE (or relative CFE, RCFE), which will be determined as follows:

CFE (%) = number of colonies/number of cells plated x 100%

RCFE = CFE in treated culture/CFE in negative control

The mutagenicity of the test agent will be evident by the increase in mutation frequency (MF, or expressed as relative mutation frequency, RMF) based on the number of mutants and adjusted by the survival fraction of cells:

MF= No. of mutants/ No. of clonable cells x 10⁶

RMF = MF in treated culture/MF in negative control

The differences in CFE and MF between control and treated cultures are evaluated by a two-tail Student's t-test. The dose-dependent response is examined by the linear regression.

IV. In Vivo Mouse Bone Marrow Micronucleus Test

IV-A. Purpose

The in vivo mammalian micronucleus test detects the damage of chromosome or mitotic apparatus caused by chemicals. Polychromatic erythrocytes (PCE) in bone marrow of rodents are used in this assay. When the erythroblast develops into an erythrocyte, the main nucleus is extruded and may leave a micronucleus in the cytoplasm. The visualization of the micronucleus is facilitated in the PCEs because they lack the main nucleus. Micronuclei form under normal conditions. The assay is based on an increase in the frequency of micronucleated PCEs in bone marrow of the treated animals.

IV-B. Background

Micronuclei are small particles consisting of acentric fragments of chromosome or entire chromosomes, which lag behind at anaphase of cell division. After telophase these fragments may not be included in the nuclei of the daughter cells and form single or multiple micronuclei in the cytoplasm. The clastogenic effect or mitotic apparatus damaging effect of the test agent will be evident by the increased frequency of micronucleated PCEs in the bone marrow.

IV-C. Test Methods

1. Experimental animals:

Swiss CD-1 mice, both sex, 8-10 week old will be used in the study. Animals will be procured from Charles River Laboratory. Five males and five females will be included for each test group. Animals are quarantined for 1 week, and then randomized and assigned to treatment and control groups.

2. Test agent:

Ammonium perchlorate will be dissolved in distilled water prior to each use and administered by gavage for three consecutive days. In the initial assessment of cytotoxicity, at least three doses (up to a maximum of 5000 mg/kg) will be used. The cytotoxicity will be judged by a decrease in the ratio of PCEs/NCEs (normochromatic erythrocytes) in the bone marrow. Five doses will be used in the dose-response study.

3. Controls:

Concurrent negative (saline) control (without treatment) and positive control (cyclophosphamide, a known micronucleus inducer dissolved in physical saline) will be included. Animals in the negative control group are used for the measurement of background frequency of micronucleated cells, and the positive control is used to verify the responsiveness of the test system.

4. Dosing and sampling:

Since the in vivo pharmacokinetic information of the test agent is unavailable, the experiment will be conducted by multiple dosing (one dose per day for 3 days), one of the most commonly used schedule for this assay. Briefly, the test compounds will be dissolved in distilled water and administered by gavage dosing. Twenty-four hours after the injection, mice are sacrificed and bone marrow cells are collected.

5. Preparation of bone marrow smears:

The bone marrow cells will be collected and suspended in 2 mL of a mixture of 1% sodium citrate and fetal bovine serum (70:30). After centrifugation at 1000 rpm for 5 minutes, the pellet will be resuspended with a few drops of the mixture, and smears prepared on standard microscopic slides.

6. Staining of the slides:

The slides will be stained the next day of preparation by May-Gruenwald and Giemsa solution as described by Schmid et al (1975). The slides are treated with xylene for 5 minutes and then embedded with coverslips.

7. Micronuclei observation:

The frequency of micronucleated cells are observed in 1000 polychromatic erythrocytes (PCE) per animal by using blind-coded slides. The PCEs/NCEs ratio is determined by counting 1000 erythrocytes, and used as the indicator of toxicity. Micronuclei are some round bodies in cytoplasm with a diameter of 1/20 to 1/5 of an erythrocyte. They stain intensively, similar to the staining of the main nuclei in the nucleated cells.

IV-D. Data collection and reporting

All the original observation for micronucleated cell frequency and PCEs/NCEs ratio will be recorded in standard scoring forms. Criteria for scoring of micronuclei should be given. Individual data will be presented in a tabular form which includes positive control, negative control, solvent control and treatment groups. The number of PCEs scored, the number of micronucleated PCEs, the percentage of micronucleated PCEs, and the ratio between PCEs and NCEs will be listed separately.

The test report for micronucleus assay will also include the following specific information: 1. Experimental animals (species, age, body weight, sex, number), 2. Test agent (vehicle, doses and rationale for dose selection), 3. Treatment and sampling schedule, 4. Toxicity data, 5. Positive and negative controls, 6. Procedures for slide preparation and staining, and 7. Criteria for micronuclei identification.

IV-E. Result analysis and interpretation

1. Criteria for acceptability:

The data generated will be considered acceptable if:

a. The background frequency of micronucleated cells is in the normal range as reported in the literature or within the laboratory's historical range.

b. The test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.

2. Criteria for interpretation:

a. Positive result:

There are several criteria for determining a positive response in the micronucleus assay. One of which is a statistically significant dose-related increase in the number of micronucleated PCEs. Another criterion is that a reproducible and statistically significant increase in the micronucleated PCE frequency is detected for at least one concentration.

A positive result in micronucleus test indicates that under the experimental conditions, the test compound induces micronuclei by the damages of either chromosome or mitotic apparatus.

b. Negative result:

A test agent which does not produce either a statistically significant dose-related increase in the number of micronucleated PCEs, or a reproducible and statistically significant increase in the micronucleated PCE frequency in any one of the concentrations tested will be considered nonmutagenic in this system.

A negative result indicates that under the experimental conditions, the test compound does not produce micronuclei in the bone marrow of the test species.

3. Statistical analysis:

The differences in the micronucleated PCE frequency and the ratio of PCEs/NCEs among treated and control animals are statistically evaluated by Chi-square analysis, and the dose-dependent response is examined by linear regression.

V. Identification, Handling and Storage and of the Test Agent

The test agent, ammonium perchlorate will be provided by the sponsor in solid powder form. All the chemical and physical identifications will be verified and information will be provided by the manufacturer. The compound is highly soluble in water (500 g/liter) and stable below 50^oC. It will be kept in sealed dark or opaque glass container and stored at room temperature, avoiding direct sunlight and sudden temperature rise. The primary routes of exposure include skin absorption, ingestion and inhalation. Safety glasses, rubber gloves, and protective clothing is needed for handling.

VI. Good Laboratory Practice and Quality Assurance

All assays will be conducted in accordance with the provisions of the United States Environmental Protection Agency/Toxic Substances Control Acts (EPA/TSCA) Good Laboratory Practice (GLP) Standards as defined in the Federal Register (40 CFR, Part 792, 1992) and the TSCA Test Guidelines (40 CFR 798.5265, 40 CFR 798.5300, and 40 CFR 5395, 1995). All the procedures are performed in accordance with the Standard Operating Procedures (SOPs) of ManTech Environmental for the Salmonella/microsome mutagenesis assay, L5178Y mouse lymphoma cell mutagenesis and mouse bone marrow cell micronucleus test.

The Quality Assurance Auditors of ManTech Environmental Inc. will document inspections on all procedures used in this study. After the initiation of the study, modifications of the protocol will be in the form of Protocol Amendments, which will state the specific modifications and the reasons for the modifications.

VII. Schedule

In the starting phase (about 4 weeks), all the test agents, media, equipments, cells, tester bacteria and animals will be ordered. The genotypes of the tester strains will be confirmed, and cells are maintained. The prescreening studies for dose selection will also be conducted in this period. The 3 genotoxicity assays will be completed within 3 months (12 weeks). Independent confirmatory experiments will be conducted for the mouse lymphoma cell mutagenesis assay and Salmonella/microsome mutagenesis assay depending upon the decision of the sponsor. Another month will be contributed to data analysis and report preparation.

VIII. Reports and Deliverables

An interim technical progress report will be prepared and submitted to the Project Officer, indicating the state of completion of the requested genotoxic assays on March 10, 1998. The final report will be submitted by May 26, 1998. The study is to be completed by April 24, 1998. The final report constitutes the study's deliverable and ManTech considers acceptance of the deliverable to occur when it is received by TERA.

IX. References:

Brusick D. (1989) In: Principles and Methods of Toxicology, Second Edition, A.W. Hayes Ed., Raven Press, NY, p 424.

D.M. Maron and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113, 173-215.

A.D. Mitchell, B.C. Myhr, C.J. Ruddd, W.J. Caspary and V.C. Dunkel (1988) Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: Methods used and chemicals evaluated. Environ. Mol. Mutagen., 12 (suppl.13), 1-18.

W. Schmid (1975) The micronucleus test. Mutation Res., 31, 9-15.