SRC TR-10-055

HPV1 TEST RULE – DATA ADEQUACY ASSESSMENT

Methanesulfinic acid, hydroxy-, mo nosodium salt, CAS No. 149-44-0

Genotoxicity - Chromosomal Aberrations

Prepared by:

Teresa Manyin, Chris Janson, and Joan D. Garey

Environmental Science Center
SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.004

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches, Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Wo rk Assignment Manager

March 5, 2010HPV1 Test Rule – Data Adequacy Assessment

Methanesulfinic acid, hydroxy-, mo nosodium salt, CAS No. 149-44-0

Genotoxicity – Chromosomal Aberrations

March 2010

Submitted to: High Production Volume Chemicals Branch

Risk Assessment Division

Office of Pollution Prevention and Toxics Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks 1200 Pennsylvania Avenue, NW

Washington, DC 20460-0001

Prepared By:

SRC, Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV1 Test Rule Study – Data Adequacy – Chromosomal Aberrations CAS No. 149-44-0

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city study conducted in response to the first

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test guideline and professional judgment as guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline is not always the test guideline required by the test rule, because some test sponsors requested and received approval from EPA to use a different guideline .) The study was summarized according to a standard format, assessed for adequacy, and assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w of TSCA Section 4 Final Study Report”. Any deviations from the applicable test guideline are described. The study is also evaluated for compliance with Standard Operating Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual animal data, calculations, and statistical analyses are described, and internal inconsistencies within the study report are identified. An overall assessment is provided for each of the following: study conduct, data reliability, and data utility.

1Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 149-44-0

GENETIC TOXICITY – CH ROMOSOMAL ABERRATIONS

Title: Micronucleus test in bone marrow cells of the mouse with methane sulfinic acid, hydroxy-, monosodium salt

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Methanesulfinic acid, hydroxy-, monosodi um salt, aka sodium hydroxymethane sulfinate (SFS), CAS No. 149-44-0

Remarks: 88.4% purity by weight (7 6.9% sodium hydroxymethane sulfinate, 11.5% water); 100.3% if calculated as dihydrate; impurities and stabi lizers include 1.9% alka linity (as sodium carbonate), 4.4% sodium hydroxymethanesulfonate, 1.9% sodium sulfite, 2.4% sodium sulfate, 0.2% iron (as Fe), and 0.2% zinc (as Zn); Batch Number 07041205 from Brüggemann; commercial substance; white powder of a crystall ine substance consisting of colorless crystals;

MW = 118.08

METHOD

Method/guideline followed: OPPTS 870.5375, OECD 474, or 40 CFR 799.9539; EEC Directive 2000/32/EC, Part B.12

Test type: Micronucleus assay

GLP compliant?: Yes

Year study performed: 2008

Test species: NMRI BR mouse; supplier: Charles River, Sluzfeld, Germany; age: 6 weeks old; mean initial body weight: males: 35 g, females: 27 g (individual body weights were within 20% of the mean by sex)

Number of animals/sex/dose: 5/sex/dose

Route of administration: Intraperitoneal

Vehicle: Saline

Exposure duration: 24 or 48 hours

Concentrations tested: 0, 500, 1000, or 2000 mg/kg-bw

Statistical methods used: The test was considered acceptable if: 1) the positive control

substance induced a statistic ally significant (Wilcoxon Rank Su m Test, one-sided, p < 0 .05)

2Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 149-44-0

increase in the frequency of micronucleated pol ychromatic erythrocytes; 2) the incidence of

micronucleated PCEs in the control animals was within the laboratory hi storical control data range (mean ± three times the standard devi ation): Males: 0.9‰ ± 3.2‰; Females: 1.0‰ ± 3.2‰. A test substance was considered positive in the micronucleus test if it induced a biologically as well as a stat istically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated PCEs (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately. A test substance was considered negative in the micronucle us test if none of the tested concentrations or sampling times showed a statistically si gnificant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated PCEs either in the combined data for both

sexes or in the data for male or female groups separately.

Test method/conditions remarks: Animals: The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of 21 ± 3°C ( actual range 21.4 - 22.9°C) and a relative humidity of 30 - 70% (actual range 41 - 78%). Due to cleaning procedures or performance of f unctional observations in the room, temporary deviations from the maximum level for humidity (with max. 8%) occu rred. Based on laboratory historical data,

these deviations were not considered to aff ect the study integrity. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day. The animals were grouphoused (5 per sex per cage) in labeled polycar bonate cages containing sterilized sawdust as bedding material. Paper bedding wa s provided as nest material. Certificates of analysis of bedding and paper were examined and then retained in the NOTOX archives. The acclimatization period was at least 5 days before start of treatment under laboratory conditions. The animals had free access to standard pellet ed laboratory animal diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany) and tap water. Results of analyses for

nutrients and contaminants of water a nd diet were examined and archived.

Study Design: Groups of 5 animals/sex received a single intraperitoneal injection of 0 (saline vehicle), 500, 1000 or 2000 mg/kg-bw methane sulfinic acid, hydroxy-, monosodium salt or positive control (50 mg/kg-bw cyclophosphamide). The dosing volume was 10 mL/kg-bw. The

doses of test material administered were based on results of a previous dose-range finding study. Bone marrow was sampled at 24 hours for all groups (with the exception of the positive control, which was sampled at 48 hours). An additional group of 5 animals/sex was injected with 2000 mg/kg-bw and sampled for bone marrow 48 hours late r. Animals were weighed just prior to dosing. Signs of systemic toxicity were recorded at least once per day. Animals were terminated by cervical dislocation 24 or 48 hours after dosing (depending on treatment) and bone marrow samples were collected.

At termination, both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 mL of fetal calf serum. The ce ll suspension was collected and centrifuged at 1000 rpm (approximately 100 g) fo r 5 minutes. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned with a 1:1 mixture of

3Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 149-44-0

96% (v/v) ethanol/ether, dried with a tissue, and labeled. The drop was spread by moving a clean slide with round-whetted sides at an angl e of approximately 45° over the slide with the drop of bone marrow suspension. Th e preparations were air-drie d, fixed for 5 minutes in 100% methanol, and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-pro cedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

Slides were scored at a ma gnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. Th e ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei we re only counted in polyc hromatic erythrocytes.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not applicable

Results remarks: In both sexes, all doses of test material induced statistically significant increases in the mean number of micronucleated PCEs compared to th e corresponding solvent c ontrol group at the 24- hour sampling time. At 24 hours, the numbers (mean +/- SD) of micronucleated PCEs/2000 PCEs in males treated with 500, 1000, and 2000 mg/kg-bw were 11.0 ± 3.2, 16.6 ± 5.7, and 16.8 ± 6.2, respectively (compared to 1.6 ± 0.9 in vehi cle controls). The corresponding values in females were 13.2 ± 4.3, 18.0 ± 6.4, and 13.8 ± 7.3 (com pared to 2.4 ± 1.1 in vehicle controls).

The number of micronucleated PCEs in the bone marro w of all animals treated with test material and sampled after 24 hours was also above the hi storical control data range. Therefore, the increases are biologically re levant and methane sulfinic acid, hydroxy-, monosodium salt is considered clastogenic.

The test material did not induce a statistically significant increase in the mean number of micronucleated PCEs compared to the correspondi ng solvent control group in male and female animals dosed with 2000 mg/kg-bw at the 48-hour sampling time. Values in treated males and females at 48 hours were 1.6 ± 0.9 and 3.4 ± 1.1 micronucleated PCEs/2000 PCEs (compared to 1.6 ± 0.9 and 2.4 ± 1.1 in vehicle-treated males and females).

The incidence of micronucleated PCEs in the bo ne marrow of all negative control animals was

within the historical solven t control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the numb er of micronucleated PCEs in both sexes (37.8 ± 5.5 and 35.8 ± 3.3 in males and fe males, respectively) compared to control values listed above. Hence, the acceptabi lity criteria of the test were met.

Animals treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on erythropoiesis. The animals of the groups treat ed with cyclophosphamide showed an expected

4Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 149-44-0

decrease in the ratio of polychromatic to nor mochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

CONCLUSIONS

Methane sulfinic acid, hydroxy-, monosodium salt is clastogenic in th e micronucleus test.

STUDY RELIABILITY

[1] = Valid without restriction.

REFERENCE

Buskens, C.A.F. 2008. Micronucleus test in bone marrow cells of the mouse with Methane Sulfinic Acid, Hydroxy-, Monosodium Salt. NOTOX B.V. Project No. 485472, dated 22 May 2008, NOTOX Substance 173421/A.

5Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: Methanesulfinic acid, hydroxy-, monosodium salt

CAS Registry Number: 149-44-0

Report Title: Micronucleus test in bone marrow cells of the mouse with Methane Sulfinic Acid, Hydroxy-,

Monosodium Salt

Test Sponsor: The SFS Consortium, consisting of BrüggemannChemical US, Inc. and Royce Associates

Testing Lab: NOTOX B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands

Test Standard: 40 CFR 799.9539

Date Study Received by EPA: June 2008 Date Study Due to EPA: May 17, 2007

Special Instructions (e.g., regarding approved protocol modifications, etc.):

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [ A ], Disagree [ D ], Not Applicable [ NA ], or Cannot Determine [ C ], and add comment, below, as needed

[D] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from the EPA test standard requirements, including “shall,” “must,” or “will” violations. List significant test standard deviations and “shall,” “must,” or “will” violations in comments below.

[D] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol that may have a bearing on the study conclusions. List these in comments below.

[D] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not followed or are inadequately documented.

[D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed.

[D] 5. The Statement of Quality Assurance (QA) inspecti ons, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures may be inadequate.

[D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed.

[D] 7. The test, control, or reference substances are di fferent from that specified in the section 4 test rule or

Enforceable Consent Agreement (ECA).

[D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations from temperature, lighting, dosing) are apparent.

[D] 9. The final study report inadequately describes the study method.

[D] 10. The final study report inadequately describes the test system.

[D] 11. The final study report inadequately describes the dosage information.[D] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete observations pertinent to the type of study conducted), or individual data were not provided.

[D] 13. The final study report inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used.

Part II. Conclusions

Place a check mark beside the summar y statement(s) that apply to this review and add comments below as appropriate

Part A. To be filled out by Reviewer

Study Conduct 1. The study was well-conducted. [X] 2. The study was generally well-conducted, with some minor [ ] deficiencies (list in notes below). 3. The study conduct was significantly deficient or questionable, (give [ ] reasons in notes below).

Data Reliability 4. The study results are precise and accurate. [X] 5. Appropriate methods were used to determine [X] statistical significance. 6. If repeated, the test results would be expected to be the [X]

same.

Data Utility 7. The data are useful for ri sk assessment for the endpoint tested. [X] 8. The data are useful for some other purpose (e.g., dose-setting). [ ] 9. The data are not useful for any purpose (describe in notes below). [ ]

Reviewer Comments (continue on separate page, if needed):

Reviewer Name/Title/Bran ch/Division/Office: Teresa Manyin/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was me t, additional testing is indicated. [ ] (when applicable) 2. The trigger for additional (tiered) testing was not met. [ ]

Study Acceptability 3. The study is acceptable. [ ] 4. The study may not be acceptable [ ] (provide rationale in memo to CITB chief).