SRC TR-12-004

HPV2 TEST RULE DATA ADEQUACY REVIEW

Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0

Prepared by:

Joan D. Garey, Teresa Manyin and William L. Richards

Chemical, Biological, and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.2.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

January 23, 2012
HPV2 TEST RULE DATA ADEQUACY REVIEW

Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0

January 2012

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.

Chemical, Biological, and Environmental Center 

2451 Crystal Drive, Suite 475 

Arlington, VA 22202
Table of Contents

A. INTRODUCTION .................................................................................................................... 1

B. INFORMATION ON THE TEST SUBSTANCE .................................................................... 1

C. EVALUATION OF TOXICITY DATA AVAILABLE FOR TEST RULE CHEMICAL ...... 1

D. DATA FOR REQUIRED ENDPOINTS .................................................................................. 3

D-1. Required Test: Toxicity to Fish ........................................................................................ 3

D-2. Required Test: Toxicity to Aquatic Invertebrates............................................................. 5

D-3. Required Test: Toxicity to Aquatic Plants...................................................................... 10

D-4. Required Test: Combined Repeated-Dose with Reproductive/Developmental 

Toxicity Screen ......................................................................................................................... 14

D-5. Required Test: Genetic Toxicity – Bacterial Reverse Mutation................................. 2121

D-6. Required Test: Genetic Toxicity – Chromosomal Aberrations .................................... 255

E. SUMMARY OF ENDPOINTS ............................................................................................. 299
A. INTRODUCTION

This document evaluates a submission by BrüggemannChemical U.S., Inc., received by EPA1 in 

response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second 

Group of Chemicals” (76 FR 1067, January 7, 2011; referred to here as HPV2) for 

Methanesulfinic acid, hydroxyl-, monosodium salt (CASRN 149-44-0). In its submission, 

BrüggemannChemical included data for the required test, Bacterial Reverse Mutation Test (in 

vitro). An evaluation of this submission is presented herein.

Data adequacy assessments of available data for CASRN 149-44-0 were performed previously 

on aquatic toxicity and mammalian endpoints required by the final test rule for “Testing of 

Certain High Production Volume Chemicals” (40 CFR Parts 9 and 799, March 16, 2006; referred 

to here as HPV1); these evaluations are also included in this document. The information for 

aquatic toxicity studies was obtained from robust summaries (dated 5/16/2008) submitted to EPA 

by the Sodium Formaldehyde Sulfoxylate Consortium and from the European Chemical Agency 

website (http://echa.europa.eu). Information for the chromosomal aberration and repeated dose 

with reproductive/developmental toxicity screen studies was obtained from test reports submitted 

by SOCMA, dated June 16, 2008 (EPA-HQ-OPPT-2005-0033-0309).

B. INFORMATION ON THE TEST SUBSTANCE

For certain studies described in this document that cover endpoints not in the test rule, it is noted 

that purity information is not provided. However, details provided in these study descriptions 

indicate that a commercial form of the test rule substance was used for testing. According to a 

letter from BrüggemannChemical US, Inc. to SOCMA Visions and supplied to EPA (letter dated 

5/12/2008), the composition of the commercial form of CASRN 149-44-0 (also known as 

sodium formaldehyde sulfinate, or SFS) theoretically contains approximately 76.6% CASRN 

149-44-0 and 23.4% water (by weight). The composition of one batch of the commercial 

formulation was reported as follows: SFS (76.9%); water (11.5%); sodium carbonate (1.9%); 

sodium hydroxymethanesulfonate (4.4%); sodium sulfite (1.9%); sodium sulfate (2.4%); iron 

(0.2 ppm) and zinc (0.2 ppm). 

EPA considers bacterial reverse mutation testing performed using the commercial formulation 

of CASRN 149-44-0 to be adequate for test rule purposes. 

In addition, EPA considers other testing (i.e., acute toxicity to fish and aquatic invertebrates, 

toxicity to aquatic plants, combined repeated-dose with reproductive/developmental toxicity 

screen, and chromosomal aberration tests) performed for HPV1 test rule purposes to be adequate.

 

1 Letter of Intent/Submission of Data in Response to TSCA section 4 Test rule; Methanesulfinic acid, hydroxyl-, 

monosodium salt (CASRN 149-44-0), along with Final Report, Determination of the mutagenic potential of NHS 

(Sodium hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B. 

13/14, received by EPA on March 8, 2011.
2

C. EVALUATION OF TOXICITY DATA AVAILABLE FOR TEST RULE CHEMICAL 

The available data for the test rule chemical toxicity endpoints are evaluated in Section D, 

including the data submitted for the test endpoint required by the HPV2 test rule. The sponsor 

submitted a full study report for this endpoint, i.e., bacterial reverse mutation testing. A 

summary was prepared for the submitted study, using a standard format and assigned a Klimisch 

reliability code. An adequacy determination was made using test rule-specified guidelines, as 

well as professional judgment. 

Studies for a common endpoint are grouped together in the document and an endpoint adequacy 

assessment is provided near the top of the first page of the summaries for that endpoint. If the 

data available for a given study are determined to be inadequate, a basis for that conclusion is 

supplied. A summary of the adequacy assessment for required endpoints is provided on the last 

page of this document (Section E).

This report includes evaluations of submitted toxicity data for the following endpoints:

Aquatic Toxicity (includes Acute Toxicity to Fish, Acute Toxicity to Daphnia, and

Toxicity to Aquatic Plants);

Mammalian Toxicity – Genotoxicity (Bacterial Reverse Mutation in vitro and 

Chromosomal Aberrations in vivo);

Mammalian Toxicity – Repeated dose/reproduction/developmental (Combined Repeated 

Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
3

D. DATA FOR REQUIRED ENDPOINTS

D-1. Required Test: Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in golden orfe (Nat, 1987) 

Title: Report on the Study of the Acute Toxicity: Rongalit C/Blankit C

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0

Remarks: The test material was a commercial formulation of methanesulfinic acid, hydroxyl-, 

monosodium salt (Rongalit C/Blankit C). No purity information was provided.

METHOD

Method/guideline followed: DIN 38412

Test type: Acute, static

GLP compliant?: No data

Year study was performed: 1987

Test species: Golden orfe (Leuciscus idus) 

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: Nominal: 5000 and 10,000 mg/L 

Test method/conditions remarks: Test water was reconstituted freshwater, prepared from 

fully demineralized tap water according to the guideline. The maximum conductivity was 10 

micromho. After resalting, the hardness, acid capacity, ratio of Ca/Mg ions, ratio of Na/K ions 

and pH were 2.5 mmol/L, 0.8 mmol/L, 4:1, 10:1 and approximately 7.7. Fish were acclimated to 

test water for 3 days prior to exposure. The photoperiod was 16 hours light and 8 hours dark. 

Food (type not specified) was withdrawn 1 day prior to testing. Test material was added to the 

test water (10 liters/vessel) without pretreatment. The temperature, pH and hardness of the water 

were 20 o

C, approximately 7.7 and 2.5 mmol/L, respectively. Fish (10/concentration, 2.4 g 

fish/liter test water) were added to the aquaria (30 cm x 22 cm x 24 cm all-glass aquaria). The 

medium was continuously aerated with oil-free air throughout the test. 
4

RESULTS

Endpoint value(s): Based on nominal concentrations: 96-hour LC50 > 10,000 mg/L.

Results remarks: None of the animals died and no symptoms of toxicity were reported.

CONCLUSIONS

No toxicity was observed at the highest nominal concentration of 10,000 mg/L.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

Nat, R. 1987. Report on the Study of the Acute Toxicity: Rongalit C/Blankit C, BASF 

Aktiengesellschaft, Department of Toxicology, Report No. 10F0335/875145, dated 1987-09-17.
5

D-2. Required Test: Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (From ECHA website, Exp Key short-term 

toxicity to aquatic invertebrates.001) 

Title: Not available

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0 (referred to as 

sodium hydroxymethanesulphinate on ECHA website)

Remarks: Information on purity not provided

METHOD

Method/guideline followed: OECD TG 202, EU Method C.2

Test type: Acute static renewal limit test

GLP compliant?: Yes

Year study was performed: 2010

Test species: Water flea (Daphnia magna Straus), ≤ 24 hours old

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: Nominal: 0, 1, 10, and 100 mg/L

Test method/conditions remarks: 

Each 2 ml vessel for testing contained 40 mL test solution and five daphnia, 2 replicates for 1 

and 10 mg/L; 4 replicates for control and 100 mg/L. Testing was performed using deionized 

water, enriched with CaCl2*2H2O) at 293.8 mg/L; MgSO4*7H2O) at 123.3 mg/L; NaHCO3 at 

64.8 mg/L and KCl at 5.8 mg/L. The pH was 7.6-7.7 and the photoperiod was 16 hrs light, 8 

hours dark. Test temperature was 20 ± 2 oC and hardness was 250 mg CaCO3/L. Potassium 

dichromate was used as a positive control. Renewal of test solution (frequency/flow rate) was 

twice a week. A first main study was performed as a limit test using 100 mg/L; 95% 

immobilization was observed. On this basis, the second study was performed using 1, 10 and 

100 mg/L. In the second study, 100 mg/L showed only 10% immobilization. The concentration 

of dissolved oxygen was above 8.8 mg/L during the entire test.
6

Analytic testing was indicated as demonstrating good correlation between nominal and measured 

concentrations. The recovery after 48 hours was 103% of the start concentration.

RESULTS

Endpoint value(s): Based on nominal concentrations: 48-hour EC50 > 100 mg/L and 48-hour 

NOEC = 10 mg/L. 

Results remarks: The highest concentration causing no immobilization within the period of the 

range-finding test was 10 mg/L.

CONCLUSIONS

The EC50 of the test substance was > 100 mg/L (highest concentration tested).

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Study available from ECHA website, http://echa.europa.eu, for CASRN 144-49-0. Study 

indicated as Exp Key Short-term toxicity to aquatic invertebrates.001. The data source is 

referred to as a “study report”, with a report date of 2010-02-14. No other reference information 

is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
7

Study 2: Acute toxicity study in Daphnia magna (Jaeger, 1999) 

Title: Acute toxicity of Daphnia according to OECD 202 Part 1 for sodium 

hydroxymethanesulfinate

This study is: Adequate Not Adequate

NOTE: Although the study itself is adequate, there is a question regarding the test substance 

used for the study. According to the dossier, the correct name for the substance was used in the 

study report, but the incorrect CASRN was used. The test material was incorrectly identified as 

CASRN 497-19-8 (sodium carbonate). The dossier indicates that the test substance was a 

commercial product containing sodium hydroxymethanesulfinate and 1-3% CASRN 497-19-8. 

The exact purity of the test substance was stated in the dossier as unknown.

TEST SUBSTANCE 

Identity: Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0 (referred to as 

sodium hydroxymethanesulphinate), with 1-3% sodium carbonate.

Remarks: Information on purity not provided

METHOD

Method/guideline followed: OECD TG 202, Part 1

Test type: Acute static

GLP compliant?: Yes

Year study was performed: 1999

Test species: Water flea (Daphnia magna) 

Analytical monitoring: No

Exposure duration: 48 hours

Concentrations tested: Nominal: 0, 10, 20, 40, 80 and 160 mg/L

Test method/conditions remarks: 

The storage of the animals was at a constant temperature of 23 1 o

C. The photoperiod was 14 

hours. The food was a pure culture of the green algae Scenedesmus suspicatus (3x daily via a 

tube pump). The animals were transferred 3x per week to a new medium via a sieve. 

Approximately every 4-6 weeks the old animals were replaced with young animals on the basis 

of ongoing reproduction.

The medium was aerated ahead of time to the saturation point of oxygen and adjusted to a pH 

value of 7.8 ± 0.2. The synthetic seawater salt contained 7 macronutrient materials, 60 trace 
8

elements, 8 vitamins and 3 amino acids. Through the addition of CaCl2 and NaHCO3 the total 

hardness (2.5 mmol/L), the Ca/Mg –ratio (4:1), and the alkalinity (0.8 mmol/L) was adjusted 

according to the medium recommended by the OECD Guideline 202. 

Twenty daphnids were tested per concentration (4 x 5 in each 20 or 50 mL glass beaker). 

Temperature of test chambers was 20 ± 1 o

C and the photoperiod was 14 hours. The pH, oxygen 

content and temperature were determined at beginning and end of the 48-hour test period. The 

medium was not aerated. The measurements at time 0 were conducted in an extra run without 

animals.

The following parameters were observed at 0, 24, and 48 hours: Immobilized animals;

immobilized animals that float on the water’s surface; animals not immobilized, but that hang on 

the surface of the water; and animals not immobilized, but that are weak and gravitate to the 

bottom of the test container.

RESULTS

Immobilization and Number of Animals Hanging on Water Surface

Concentration (mg/L)

Control 10 20 40 80 160

24 hour (%) 0 0 5 0 0 5

48 hour (%) 0 0 15 65 75 90

Animals on

Water Surface (%) 0 0 0 0 0 0

Endpoint value(s): Based on nominal concentrations: 48-hour EC50 = 20-40 mg/L 

Results remarks: Dissolved oxygen content ranged from 96% - 98% for each concentration at 

0 and 48 hours. The pH values ranged from 7.75 to 7.92.

CONCLUSIONS

The 48-hr EC50 of the test substance was 20-40 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. No measured concentrations, purity of test substance not known.
9

REFERENCE

Jaeger, W. 1999. Acute toxicity of Daphnia according to OECD 202 Part 1 for sodium 

hydroxymethanesulfinate. Analytik GmbH study dated 1999-05-12 (English translation of an 

unpublished German language study).
10

D-3. Required Test: Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Scenedesmus subspicatus (Paulus, 2001)

Title: Determination of the 72 hour EC50 of “Brueggolit E02” in the presence of Scenedesmus 

subspicatus

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Bruggolite E 02 (commercial methanesulfinic acid, hydroxyl-, monosodium salt, 

CASRN 149-44-0, referred to as commercial sodium hydroxymethanesulphinate in study 

description), Batch 02112000 from Brüggemann.

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD Guideline 201

Test type: Alga, Growth Inhibition Test

GLP compliant?: Yes

Year study was performed: 2001

Test species: Freshwater green algae (Desmodesmus subspicatus, formerly Scenedesmus 

subspicatus) 

Analytical monitoring: No

Exposure duration: 72 hours

Concentrations tested: Nominal: 0, 10, 20, 40, 80, 160, 320, 640 and 1000 mg/L

Test method/conditions remarks: 

Two stock solutions of the concentrations 1250 mg/L and 800 mg/L were prepared for the main 

study. The dose solutions were then prepared out of these through dilution. The dilution series 

was prepared through dilution of the stock solution with demineralized water. Concentrations 

tested (10-1000 mg/L) were based on the results of a preliminary study.
11

Scenedesmus subspicatus were procured on agar for the LAUS GmbH from the Institut fuer 

Pflanzenwissenschaften/despository of algae culture of the University of Goettingen as the strain 

culture. The last procurement took place in June 2000. The strain culture was stored in a 

refrigerator. 

Every 3 days before the start of the preliminary and main tests, a batch of algae was taken out of 

the stock culture (Ch.B. 210600), the algae were apportioned in a culture flask with 50 ml 

foreculture medium (Ch.B. 11122000). This was incubated for 72 hours with stirring and 

lighting. After adjusting the cell number at about 100,000/ml by means of a photometric

measurement and the addition of foreculture medium, the culture was ready for the test. 

The stock solution of the dilution series was prepared and pipetted into the test vessel (4 ml 

each), such that a mixture of the added foreculture and nutrient concentrate was achieved (using 

0.5 ml of each). Each concentration was tested in triplicate. The test vessels were incubated 

open under stirring (magnetic stirrers) at 8000 lux lighting at 21 ± 2 oC. Every 24 hours, the cell 

number was determined by means of a photometric measurement at 440 nm. Before and after 

the test, the pH values of the control were measured. The measured values of the optical density 

were recorded and evaluated with a computer program. 

RESULTS

Endpoint value(s): Based on nominal concentrations reported: EC50 (growth rate) = 370 mg/L; 

EC100 (growth rate) >1000 mg/L; NOEC (growth rate) = 10 mg/L; EC50 (biomass) = 220 mg/L; 

EC100 (biomass) >1000 mg/L.

Results remarks: The pH variation was 0.5 units. The nominal growth factor that is expected 

for a control run is 0.9. The control for the preliminary test had a growth rate of 1.07 and the one 

for the main test had a growth rate of 1.04. 

CONCLUSIONS

The 72-hour EC50s for growth rate and biomass were 370 and 220 mg/L, respectively. The 72-

hour NOEC for growth rate was 10 mg/L. 

STUDY RELIABILITY 

[2] Reliable with restrictions. No information was provided on test substance purity and no 

analytical monitoring was performed.

REFERENCE

Paulus, J. 2001. Determination for the 72 hour EC50 of “Brueggolit E02” in the presence of 

Scenedesmus subspicatus. LAUS-Libertaere Arbeitsgemeinschaft Environmental & Hazard 

Substance Consultation GmbH Report No. 00110605J01, dated 2001-02-21. English translation 

of an unpublished German language study.
12

Study 2: Toxicity to Scenedesmus subspicatus (Jaeger, 1999)

Title: Algae inhibition test according to OECD 201 with sodium hydroxy methane sulfinate

This study is: Adequate Not Adequate

NOTE: Although the study itself is adequate, there is a question regarding the test substance 

used for the study. According to the dossier, the correct name for the substance was used in the 

study report, but the incorrect CASRN was used. The test material was incorrectly identified as 

CASRN 497-19-8 (sodium carbonate). The chemical is a commercial product containing sodium 

hydroxymethanesulfinate and 1-3% CASRN 497-19-8. The exact purity is unknown.

TEST SUBSTANCE

Identity: Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0, referred to as 

Sodium hydroxymethanesulfinate in study description, Batch Number 07041205 from 

Bruggemann.

Remarks: Analytical purity: 100.3% calculated as dihydrate. The data came from an English 

translation of a German language study. 

METHOD

Method/guideline followed: OECD Guideline 201

Test type: Alga, Growth Inhibition Test

GLP compliant?: Yes

Year study was performed: 1999

Test species: Scenedesmus subspicatus (SAG 86.81)

Analytical monitoring: No

Exposure duration: 72 hours

Concentrations tested: Nominal: 0, 0.1, 1, 10, 100, 500 mg/L

Test method/conditions remarks: 

The stock culture was inoculated with a 0.8% agar-agar consolidated, sterile nutrient medium in 

an inclined agar tubule every two months. Between incoculations, the culture was kept in a 

cooling cabinet at about 8 o

C. Before the inoculation, the inclined agar tube was placed 1-2 days 

in light. For the preparation of the foreculture, a transverse agar tublet was charged with the 

stock culture and placed for 1-2 days in the light, and then incolated in about 100 mL sterile 
13

medium. Incubation then followed in a shaking machine (100-150 rpm) for about 72 hours at 23 

± 1 oC, and 8000 lux (without a dark phase).

Exponentially growing cultures of Scenedesmus subspicatus (10,000 cells/mL) in defined 

medium were exposed in triplicate to varying concentrations of test substance over a time period 

of 72 hours in a circular shaking machine (circular vibrator VKS 75 with standard lighting 

attached (120-150 rpm). The pH for each concentration was determined at 0 and 72 hours. The 

cell count was determined by measuring the extinction at 520 nm in each run every 24 hours. 

The mean value of the cell number was plotted for every test concentration versus the time, in 

order to obtain the growth curves. The inhibition of the cell growth rate and the specific growth 

rate was determined in comparison to a control.

RESULTS

Endpoint value(s): Based on nominal concentrations reported: EC50 (growth rate) = 450 mg/L; 

EC50 (cell density) = 5 mg/L.

Results remarks: The test was valid, as algae increased by a factor of 100 in 72 hours. 

CONCLUSIONS

The 72-hour EC50s for growth rate and cell density were 450 and 5 mg/L, respectively. 

STUDY RELIABILITY 

[2] Reliable with restrictions. No information was provided on test substance purity and no 

analytical monitoring was performed.

REFERENCE

Jaeger, W. 1999. Algae inhibition test according to OECD 201 with Sodium Hydroxy Methane 

Sulfinate. Unpublished report dated 1999-05-12. English translation of a German language 

study.
14

D-4. Required Test: Combined Repeated-Dose with Reproductive/Developmental Toxicity 

Screen

Summary of endpoint: Adequate

Study 1: Combined repeated-dose with reproductive/developmental oral toxicity screen in rats 

(Beekhuijzen, 2008)

Title: A combined 28-day repeated dose toxicity study with the reproduction/developmental 

toxicity screening test of methane sulfinic acid, hydroxy-, monosodium salt in rats by oral 

gavage

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Methanesulfinic acid, hydroxy-, monosodium salt, aka sodium hydroxymethane 

sulfinate (SFS), CASRN 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: OECD 422 and EPA, 40 CFR 799.9365

Test type: Combined 28-day repeated-dose toxicity study with reproductive/developmental 

toxicity screening test

GLP compliant?: Yes

Year study performed: 2007

Test species: Male and virgin female Wistar (Crl:WI(Han); outbred, SPF-Quality) rats; 

supplier: Charles River, Deutschland, Sulzfeld, Germany; age at initiation: approximately 10 

weeks old; mean initial body weights: males: 292 g, females: 190 g

Number of animals/sex/dose: 10

Route of administration: Oral gavage

Vehicle: Water (Elix, Millipore S.A.S., Molsheim, France)
15

Exposure duration: Males were exposed for 30 days (during 2 weeks prior to mating, during 

mating [up to 14 days], and to termination). Females were exposed for 42 - 53 days (during 2 

weeks prior to mating, during mating and gestation, and up to Day 4 of lactation).

Doses/concentrations tested: 0, 100, 300 and 1000 mg/kg/day 

Statistical methods used: Normally distributed data were analyzed using the Dunnett test, 

based on a pooled variance estimate. The Steel test was applied if data were not normally 

distributed. The Fisher Exact test was applied to frequency data. The following were calculated 

for each group: percentage mating, fertility index, conception rate, gestation index, duration of 

gestation, percentage of live males at first litter check, percentage of live females at first litter 

check, percentage of postnatal loss from Days 0-4 post partum, and viability index. All tests 

were two-sided, with p < 0.05 accepted as the lowest level of significance. 

Test method/conditions remarks: Animals were acclimated at least 5 days prior to start of 

treatment. They were housed in rooms maintained at 19.0-23.5 °C, with a relative humidity of 

26-95% and a 12-hour light/ 12 hour dark cycle. There were 15 air changes per hour. Prior to 

mating, animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, 

height 18 cm). Females were caged with males on a one-to-one basis during mating. After 

mating, males were housed in their home cage, with a maximum of 5 animals/sex/cage. Females 

were individually housed after mating and were kept with their offspring until termination.

Cages were generally supplied with sterilized sawdust as bedding material and paper as cageenrichment. During overnight activity monitoring, animals were housed individually with 

sterilized sawdust bedding material without paper cage-enrichment. Animals were allowed free 

access to tap water and pelleted rodent diet (SMR/M-Z from SSNIFF Spezialdiaten GmbH, 

Soest, Germany). Analyses of bedding, paper, diet and water did not reveal any findings that 

were considered to affect study integrity.

Test material: Formulations (w/w) were prepared daily, within 4 hours of dosing, and were 

homogenized to a visually acceptable level. No adjustment was made for specific gravity, 

vehicle and/or formulation. The pH of the solutions was determined weekly. The pH of a 10% 

solution was approximately 10. Sampling and chemical analysis of formulations were performed 

on one day during the treatment period; duplicate samples from each group were analyzed for 

accuracy. Duplicate samples of dosing solutions taken from the bottom, middle and top for the 

low- and high- dose groups were analyzed for homogeneity and stability at 0 hours after 

preparation. Duplicate samples of these solutions (taken from the middle) were also analyzed for 

stability at 5 hours after preparation. The analytical method used was based on the results of a 

separate project for the development and validation of the analytical method (NOTOX Project 

485209).

The pHs of the formulations were between 8.31-8.68 (control), 10.12-10.17 (low dose), 10.10-

10.17 (mid dose) and 9.94-10.02 (high dose). The accuracy of the formulations was 102-109% 

(low dose), 98-103% (mid dose) and 87-105% (high dose). Based on the results, it was 

concluded that the formulations were prepared correctly. The low- and high-dose formulations 

were homogeneous (≤ 10% coefficient of variation). Formulations at the entire range were 

stable for at least 5 hours when stored at room temperature.
16

A health examination was performed prior to treatment to ensure that the animals were in a good 

state of health. A total of 40 animals/sex were randomized into four groups of 10/sex according 

to body weight, with all animals within 20% of the sex mean. They were identified by earmark 

and tattoo.

Groups of animals were given 0 (water vehicle), 100, 300 or 1000 mg/kg-bw test material (at 5 

mL/kg-bw) by oral gavage, once daily, 7 days/week up to the day of scheduled termination. A 

limit dose of 1000 mg/kg-bw/day was selected as the high dose level based on data obtained in a 

preliminary range-finding study. Actual dose volumes were calculated according to the latest 

body weight. Following 14 days of exposure, males and females were co-housed (1:1). Sibling 

mating was avoided. Detection of mating was confirmed by presence of sperm in the vaginal 

lavage or by the appearance of an intravaginal copulatory plug. This day was designated as Day 

0 postcoitum. Once mating had occurred, the males and females were separated. A maximum of 

14 days was allowed for mating. There were at least 8 pregnant females per dose level. Females 

were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was 

found completed. Females that were littering were left undisturbed. On Day 1 of lactation, all 

pups were individually identified by a tattoo. Examination of maternal care revealed no 

deficiencies. 

Parental animals were observed for mortality and viability at least twice daily. At least once 

daily, detailed clinical exams were conducted for all animals. This was also performed outside 

the home cage once before treatment and at weekly intervals. These observations were not 

performed when the animals were mating or housed individually. The time of onset, degree and 

duration was recorded. Cage debris of pregnant females was examined for evidence of abortion 

or premature birth. No signs of abortion, premature birth or difficult or prolonged parturition 

were recorded. 

Functional observations (hearing ability, pupillary reflex, static righting reflex and grip strength 

and a motor activity test) were recorded for the first five mated males/group and the first five 

females/group with live offspring. During the motor activity test, males were caged individually 

and females were caged with their offspring. The males were tested during week 4 of treatment 

and the assigned females were tested during lactation (all before blood sampling). 

Males and females were weighed on the first day of exposure and weekly thereafter. Mated 

females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on 

Days 1 and 4. Food consumption was recorded weekly (with the exception that it was not 

recorded during breeding). Food consumption of mated females was measured on Days 0, 4, 7, 

11, 14, 17 and 20 post-coitum and after delivery on Days 1 and 4 postpartum. Water 

consumption was not quantified, but was monitored subjectively. Identities of animals paired, 

mating date, confirmation of pregnancy, and delivery day were recorded.

After an overnight fast, blood was collected from the retro-orbital sinuses of the first five mated 

males/group and the first five females/group with live offspring, under isoflurane anesthesia. 

Standard hematological (white blood cells, differential leukocyte count, red blood cells, 

reticulocytes, red blood cell distribution width, hemoglobin, hematocrit, mean corpuscular

volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, 

prothrombin time, and activated partial thromboplastin time) and clinical biochemistry 

parameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total 
17

protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, 

chloride, calcium, and inorganic phosphate) were performed after appropriate blood processing. 

On lactation Day 5 (or shortly thereafter), parental females were anesthetized with isoflurane and 

exsanguinated. Animals were subjected to a gross examination, organ weights (adrenal glands, 

brain, heart, kidneys, liver, spleen, and thymus) of the first 5 females with live offspring per 

group were collected and terminal body weights were recorded. Tissues (adrenal glands, aorta, 

brain, cecum, cervix, clitoral gland, colon, duodenum, eyes with optic nerve and Harderian 

gland, mammary gland area, femur, heart, ileum, jejunum, kidneys, larynx, exorbital lacrimal 

gland, liver, lung, mandibular and mesenteric lymph nodes, nasopharynx, esophagus, ovaries, 

pancreas, Peyer's patches, pituitary gland, preputial gland, rectum, mandibular and sublingual 

salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, sternum with bone 

marrow, stomach, thymus, thyroid with parathyroid, tongue, trachea, and urinary bladder) and 

gross lesions of these animals were preserved for possible future histopathological examination. 

The cervix, clitoral gland, ovaries, preputial gland, uterus, vagina and all gross lesions from 

remaining animals also were preserved. Slides of preserved organs and tissues of selected 

control and high dose animals and gross lesions of all animals were prepared and examined by a 

pathologist. The numbers of former implantation sites and corpora lutea were counted and 

recorded.

On post-mating Days 24-26, females that had not delivered were anesthetized and exsanguinated. 

No organs were collected for weighing. Animals were subjected to a gross examination and 

tissues described above for parental females (and any gross lesions) were preserved. The 

reproductive organs of all females that failed to conceive were examined by a pathologist. 

Additionally, nongravid uteri were stained using the Salewski technique to identify former 

implantation sites.

After completion of the mating period, parental males were anesthetized with isoflurane and 

exsanguinated and grossly examined. Organ weights (adrenal glands, brain, heart, kidneys, liver, 

spleen, and thymus) of the first 5 mated males per group were collected and terminal body 

weights were recorded. Weights of epididymides and testes of all males were also recorded. 

The same tissues preserved for selected females (see above) were preserved for the first 5 mated 

males per group (with the exception of female reproductive organs and the addition of the 

epididymides, prostate gland, seminal vesicles, testes, and coagulation gland). The coagulation 

gland, epididymides, preputial gland, prostate gland, seminal vesicles, testes and all gross lesions 

from remaining animals also were preserved. Slides of preserved organs and tissues of selected 

control and high-dose animals and gross lesions of all animals were prepared and examined by a 

pathologist. Of the selected 5 males/group of the control and high-dose group, additional slides 

of the testes were prepared to examine staging of spermatogenesis. The reproductive organs of 

all males that failed to sire were examined by a pathologist.

The numbers of live and dead pups at the first litter check (Day 1 of lactation) and daily 

thereafter were determined. Detailed clinical observations of pups were made at least once daily. 

Live pups were weighed during lactation Days 1 and 4. Sex of all pups was determined on Days 

1 and 4 of lactation (by measuring the anogenital distance).

Pups were killed by decapitation on Day 5 of lactation (or shortly thereafter). All offspring were 

sexed and externally examined. Stomachs were examined for the presence of milk. Descriptions 
18

of all external abnormalities were recorded. If possible, defects or causes of death were 

evaluated. Any abnormal pup, organ or tissue was preserved for possible future examination.

RESULTS

Endpoint value(s): The parental LOAEL was 1000 mg/kg-bw/day, based on reductions in mean 

body weight, body weight gain, and food consumption, as well as increases in the number of 

reticulocytes (males only) and the level of alanine aminotransferase (males only). The parental 

NOAEL was 300 mg/kg-bw/day. The reproductive and developmental NOAEL was 1000 

mg/kg-bw/day, based on no adverse effects observed at the highest dose tested. 

Results remarks: Formulation analysis showed that the formulations were prepared accurately, 

were homogeneous (i.e., coefficient of variation ≤ 10%) and were stable for at least 5 hours at 

room temperature. The accuracy of formulation (determined by chemical analysis) for the low-, 

mid-, and high-dose groups were 102-109%, 98-103%, and 87-105%, respectively.

Parental animals (effects by dose level):

1000 mg/kg-bw/day: None of the animals died and functional observations were not affected. 

One female showed hunched posture and piloerection from Day 20 post-coitum until Day 3 of 

lactation. Piloerection was noted in another female on Day 4 of lactation. Slight salivation noted 

incidentally in both sexes was attributed to the taste of the formulation and was not considered 

toxicologically relevant. Lower mean body weights were recorded for males and females at 

1000 mg/kg-bw during the complete treatment period (not always statistically significant). Body 

weight gains of males were also lower than the control throughout the treatment period. For 

females, body weight gain was decreased on Day 8 during the pre-mating period and on Day 1 of 

mating. Food consumption before or after allowance for body weight was reduced in males and 

females during the whole observation period (not always statistically significant). A slightly 

higher white blood cell count was noted in males (not statistically significant) and females. 

Relative numbers of reticulocytes were increased in males (3.6 ± 0.8% in treated vs. 2.3 ± 0.1 % 

in control). Increased mean corpuscular volume and decreased mean corpuscular hemoglobin 

concentration in females were not considered to be related to treatment as they were either within 

the range of normal or showed no dose-response relationship. Increased alanine 

aminotransferase was found in males (59.2 ± 20.4 U/I in treated vs. 38.0 ± 3.6 U/I in control). 

One male had increased activity of aspartate aminotransferase. Increased total bilirubin and 

decreased sodium in males were not considered to be related to treatment due to the lack of a 

dose-response relationship. One male showed exophthalmus of the eye, which was considered to 

be caused by blood sampling. Increases in organ/body weight ratios for the liver (2.57 ± 0.14% 

in treated vs. 2.33 ± 0.12% in control), spleen (0.225 ± 0.032 in treated vs. 0.162 ± 0.033 in 

control), testes (1.16 ± 0.15% in treated vs. 0.94 ± 0.16% in control) and epididymides (0.388 ± 

0.071 % in treated vs. 0.301 ± 0.050 in control) of males and liver (3.64 ± 0.22% in treated vs. 

3.04 ± 0.33% in control), spleen (0.287 ± 0.023% in treated vs. 0.225 ± 0.0303% in control), 

kidneys (0.83 ± 0.05% in treated vs. 0 .73 ± 0.06% in control) and adrenals (0.043 ± 0.004% in 

treated vs. 0.034 ± 0.004% in control) of females were observed. There were no corresponding 

histological changes. An increase in the organ/body weight ratio of the brain in males (0.66 ± 

0.05% in treated vs. 0.60 ± 0 .01 % in control) was considered to be due to the relatively low 

terminal body weight (300 ± 14 g in treated vs. 336 ± 19 g in control). Absolute brain weight 
19

(but not relative) was slightly decreased in females (1.77 ± 0.05 g in treated vs. 1.87 ± 0.05 in 

control). Reproductive and breeding parameters were not affected by treatment at any dose 

tested. No treatment-related findings were observed for mating performance, fertility parameters 

(fertility index, conception rate, and gestation index), and duration of gestation.

300 mg/kg-bw/day: None of the animals died and functional observations were not affected. 

Females had slightly lower mean absolute body weights on Day 8 of premating (189 ± 6.6 g in 

treated vs. 197 ± 7.2 g in control). As this had recovered on Day 1 of mating (198 ± 11.8 g in 

treated vs. 203 ± 8.2 g in control), it was not considered to be toxicologically relevant. Increased 

basophils in males, increased mean corpuscular volume and mean corpuscular hemoglobin in 

females, decreased total bilirubin in males, and decreased albumin in females were not 

considered to be related to treatment as they were either within the range of normal or showed no 

dose-response relationship. No other abnormal findings were noted.

100 mg/kg-bw/day: None of the animals died and functional observations were not affected. 

Females had slightly lower mean absolute body weights on Day 8 of premating (189 ± 6.6 g in 

treated vs. 197 ± 7.2 g in control). As this had recovered on Day 1 of mating (196 ± 6.3 g in 

treated vs. 203 ± 8.2 g in control), it was not considered to be toxicologically relevant. Increased 

basophils in males and decreased total bilirubin in males were not considered to be related to 

treatment as they were either within the range of normal or showed no dose-response 

relationship. No other abnormal findings were noted.

Control: One female showed exophthalmus of the eye, which was considered to be caused by 

blood sampling.

Offspring:

The numbers of dead and living pups at first litter check were not affected by exposure to the test 

substance. Postnatal loss and viability index were similar for the control and treated groups. 

Development of pups was not affected by treatment with up to 1000 mg/kg-bw/day. Mean pup 

body weights were similar for the control and treated groups (means ranged from 5.6-5.8 g and 

5.3-5.6 g in males and females on Day 1, and 8.3-8.8 and 8.0-8.6 g in males and females on Day 

4, with none of the low values in the listed ranges being in high-dose animals). None of the 

clinical or macroscopic findings (scabs at the neck, dark red discoloration of tail apex, blue spot 

on neck, wound at the head) were considered to be related to treatment as they were considered 

to be within normal biological variation.

Summary Table:

Parameter Control 100 mg/kg 300 mg/kg 1000 mg/kg

Percentage Mating 100 100 100 100

Fertility Index 80 100 90 100

Gestation Index 100 100 100 100

Mean Precoital Time*2.5 2.9 1.4 1.7
20

Duration of 

Gestation (days) 21.1 ± 0.4 21.1 ± 0.3 21.0 ± 0.0 21.1 ± 0.3

Dead Pups at 

First Litter Check 0 0 0 0

Postnatal Loss (%) 0.0 2.6 2.9 1.0

Litters Affected 0 3 2 1

Viability Index 100.0 97.4 97.1 99.0

*units not listed, but presumed to be days

CONCLUSIONS

Treatment with methane sulfinic acid, hydroxy-, monosodium salt by oral gavage in male and 

female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg-bw/day revealed parental 

toxicity at 1000 mg/kg-bw/day. No reproduction, breeding, and developmental toxicity was 

observed for treatment up to 1000 mg/kg-bw/day. Based on these findings, the parental NOAEL 

was established at 300 mg/kg-bw/day. The definitive reproduction, breeding, and developmental 

NOAEL was established as being 1000 mg/kg-bw/day.

STUDY RELIABILITY

[1] Reliable without restriction. 

REFERENCES

Beekhuijzen, M.E.W. 2008. A combined 28-day repeated dose toxicity study with the 

reproduction/developmental toxicity screening test of methane sulfinic acid, hydroxy-, 

monosodium salt in rats by oral gavage. NOTOX B.V. Project No. 485218 (and 485615), dated

12 June 2008.
21

D-5. Required Test: Genetic Toxicity – Bacterial Reverse Mutation

Summary of endpoint: Adequate 

Study 1: Mutagenicity studies in Salmonella typhimurium (Paulus, 2008)

Title: Determination of the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) 

with the Bacterial Reverse Mutation Test following OECD 471 and EU B. 13/14

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Methanesulfinic acid, hydroxyl-, monosodium salt, CASRN 149-44-0; Synonyms: 

Sodium hydroxymethanesulfinate; sodium formaldehyde sulfoxylate (SFS)

Remarks: A commercial product was used having composition as follows: 88.3% SFS 

(hydrated) [contains 76.1% SFS (no water of crystallization) and 12.2% water (water of 

crystallization)]; 3.6% sodium hydroxymethanesulfonate (impurity); 4.3% sodium sulphite 

(impurity); 2.3% sodium sulphate (impurity); 1.5% sodium carbonate (stabilizer). This 

information was provided to EPA via an email to P. Campanella from BrüggemannChemical 

dated 12/2/11. 

METHOD

Method/guideline followed: OECD 471 and EU B. 13/14

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 2008

Test species: Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation: With and without (using S-9 in the plate incorporation method)

Concentrations tested: First experiment: 0 (separate water and DMSO solvent controls), 51, 

151, 505, 1514 and 5047 μg/plate of commercial product test substance (equivalent to 38.8, 

114.9, 384.3, 1152.2 and 3840.8 μg SFS/plate; second experiment: 0 (separate water and DMSO 

solvent controls), 1258, 2516 and 5031 μg/plate of commercial product test substance 

(equivalent to 957.3, 1914.7 and 3828.6 μg SFS/plate); for both experiments, the test substance 

solutions were prepared using deionized water as a solvent
22

Statistical methods used: The colonies were counted visually, and the numbers were recorded. 

Spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard 

deviations as well as the increase factor of revertant induction.

Test method/conditions remarks: 

Preincubation period of culture in nutrient broth: 12 hours at 37 °C.

PLATE INCORPORATION METHOD (First Experiment):

Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test 

solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar 

basis was melted in a microwave oven; after melting, 10 mL of histidinebiotin-solution, 0.5 

mMol per 100 mL basis, was added and the bottle was placed into the water bath at 45 °C. 0.1 

mL of the appropriate solution of the test item were given into a sterile tube. After mixing with 

0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for 

treatments without S9) or 0.5 mL S9 mix, 2 ml. Top-Agar was added. The mixture was gently 

vortexed, then poured on a minimal glucose plate and distributed evenly using a Drigalski 

spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, 

then inverted and placed in the dark incubator at 37 °C.

PRE-INCUBATION METHOD (Second experiment):

Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test 

solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar 

basis was melted in a microwave oven; after melting, 10 mL. of histidinebiotin-solution, 0.5 

mMol per 100 mL basis, was added and the bottle was placed in the water bath at 45 °C. 0.1 mL 

of the appropriate solution of the test item was placed into a sterile tube. After mixing with 0.1 

mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments 

without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber 

at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 

2 mL top agar were added. The mixture was vortexed gently, then poured on a minimal glucose 

plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with 

brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator 

at 37 °C.

POSITIVE CONTROLS:

Without metabolic activation, the following positive control substances were used: 4-nitro-1,2-

phenylene diamine (80 μg/plate) in DMSO with TA97a, TA98, and TA102; sodium azide (6 

μg/plate) in deionized water with TA100 and TA1535. With metabolic activation, positive 

controls were as follows: benzo(a)pyrene (40 μg/plate) in DMSO with TA98; 2-aminoanthracene 

(3 μg/plate) in DMSO with TA97a, TA100, TA102, and TA1535.

NUMBER OF REPLICATIONS:

Four replicates, with and without S9, for each solvent were used in the test.
23

EVALUATION CRITERIA:

A test item is considered to have mutagenic potential if a significant, reproducible increase of 

revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A 

concentration-related increase over the range tested can also be taken as a sign of mutagenic 

activity.

RESULTS

FIRST EXPERIMENT:

The treatments for the confirmation of the genotype, the sterility control and the determination of 

the titer did not show any inconsistencies. All positive controls (diagnostic mutagens) showed 

mutagenic effects with and without metabolic activation. No signs of toxicity of the test item 

towards the tested strains were observed; the background lawn was visible and the number of 

revertant colonies was not significantly reduced. No significant increase of the number of 

revertant colonies in the treatments with and without metabolic activation could be observed. No 

concentration-related increase over the tested range was found.

SECOND EXPERIMENT:

The treatments for the confirmation of the genotype, the sterility control and the determination of 

the titer did not show any inconsistencies. All positive controls showed mutagenic effects with 

and without metabolic activation. No signs of toxicity of the test item towards the tested strains 

were observed; the background lawn was visible and the number of revertant colonies was not 

significantly reduced. No significant increase of the number of revertant colonies in the 

treatments with and without metabolic activation was observed. No concentration-related 

increase over the tested range was found.

Genotoxicity result: First and second experiments: Negative with and without metabolic 

activation

Cytotoxic concentration (if applicable): First and second experiments: No signs of toxicity 

towards the tested strains could be observed.

Results remarks: The determined values for the spontaneous revertants of the negative controls 

were in the normal range of the test laboratory historical data of the laboratory (in the first and in 

the second experiment).

CONCLUSIONS

The test item is considered to be non-mutagenic in the bacterial reverse mutation test.

STUDY RELIABILITY

[1] Reliable without restrictions. 
24

REFERENCES

Paulus, J. 2008. Determination of the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) with the Bacterial Mutation Test following OECD 471 and EU B. 

13/14. Report no. AB07120702G803. BrüggemannChemical U.S., Inc., subsidiary of L. 

Brüggemann KG Germany. Report date: May 2, 2008.
25

D-6. Required Test: Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate 

Study 1: Micronucleus study in mice (Buskens, 2008)

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), CASRN 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 (in vivo)

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

Doses tested: 0, 500, 1000, or 2000 mg/kg-bw
26

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) 

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 group-housed (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 
27

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 dose: 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 

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

effects on erythropoiesis.
28

CONCLUSIONS

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

STUDY RELIABILITY

[1] Reliable without restrictions.

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.

 
29

E. SUMMARY OF ENDPOINTS 

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Daphnia Yes Yes No

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Bacterial Reverse Mutation1 Yes Yes No

Chromosomal Aberrations Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No

1

Required by HPV2 test rule (40 CFR Parts 9 and 799, Federal Register, Vol. 76, No. 5, January 

7, 2011)