SRC TR-10-055 

HPV1 TEST RULE – DATA ADEQUACY ASSESSMENT 

Methanesulfinic acid, hydroxy-, monosodium 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 Work Assignment Manager 

March 5, 2010 
HPV1 Test Rule – Data Adequacy Assessment 

Methanesulfinic acid, hydroxy-, monosodium 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 22202 
Draft HPV1 Test Rule Study – Data Adequacy – Chromosomal Aberrations CAS No. 149-44-0

PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity 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 guides, the study was reviewed for adequacy and 

evaluated for compliance with the test guideline. (The applicable test guideline 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 assigned a Klimisch reliability code. If the study 

was determined to be inadequate, a basis for that conclusion is supplied. For studies already 

assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data 

adequacy differed from the pre-assigned Klimisch code. 

A detailed evaluation of the test report and study conduct is provided at the end of the 

summary document in a section entitled “Review 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 Procedures, Good Laboratory Practice Standards, and 

Quality Assurance procedures, as well as the use of appropriate controls and test substances. 

Any deficiencies in the descriptions of the study method, test system, animals studied, physical 

conditions, dosage information, individual 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. 

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

GENETIC TOXICITY – CHROMOSOMAL 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-, monosodium salt, aka sodium hydroxymethane 

sulfinate (SFS), CAS No. 149-44-0 

Remarks: 88.4% purity by weight (76.9% sodium hydroxymethane sulfinate, 11.5% water); 

100.3% if calculated as dihydrate; impurities and stabilizers include 1.9% alkalinity (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 crystalline 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 statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0 .05) 

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

increase in the frequency of micronucleated polychromatic erythrocytes; 2) the incidence of 

micronucleated PCEs in the control animals was within the laboratory historical control data 

range (mean ± three times the standard deviation): 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 statistically 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 micronucleus test if none of the tested concentrations 

or sampling times showed a statistically significant (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 functional observations in the room, temporary deviations from the 

maximum level for humidity (with max. 8%) occurred. Based on laboratory historical data, 

these deviations were not considered to affect 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 polycarbonate cages containing sterilized sawdust as 

bedding material. Paper bedding was 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 pelleted 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 and 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 later. 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 cell suspension was collected and 

centrifuged at 1000 rpm (approximately 100 g) for 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 

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Draft 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 angle of approximately 45° over the slide with the 

drop of bone marrow suspension. The preparations were air-dried, 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-procedure" 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 magnification of 1000x. The number of micronucleated polychromatic 

erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to 

normochromatic erythrocytes was determined by counting and differentiating the first 1000 

erythrocytes at the same time. Micronuclei were only counted in polychromatic 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 the corresponding solvent control 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 vehicle controls). The corresponding values in 

females were 13.2 ± 4.3, 18.0 ± 6.4, and 13.8 ± 7.3 (compared to 2.4 ± 1.1 in vehicle controls). 

The number of micronucleated PCEs in the bone marrow of all animals treated with test material 

and sampled after 24 hours was also above the historical control data range. Therefore, the 

increases are biologically relevant 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 corresponding 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 bone marrow of all negative control animals was 

within the historical solvent control data range. Cyclophosphamide, the positive control 

substance, induced a statistically significant increase in the number of micronucleated PCEs in 

both sexes (37.8 ± 5.5 and 35.8 ± 3.3 in males and females, respectively) compared to control 

values listed above. Hence, the acceptability 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 treated with cyclophosphamide showed an expected 

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

decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic 

effects on erythropoiesis. 

CONCLUSIONS

Methane sulfinic acid, hydroxy-, monosodium salt is clastogenic in the 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. 

5 
Review 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) inspections, their dates, and findings were missing or not signed. 

There is evidence that QA procedures 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 different 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 summary 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 risk 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/Branch/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 met, 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).