SRC TR

- 1 2

- 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. 75 02 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027 Task FG007. 2 .009.00 1

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 , 201 2HPV2 TEST RULE DATA ADEQUACY REVIEW

Methanesulfinic acid, hydroxyl

- , monosodium salt, CASRN 149

- 44

- 0

January 201 2

Submitted to: High Prod uction Volume Chemicals Branch Risk Assessment Division Office of Pol lution 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 22202Table 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 REQ UIRED 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 Repr oductive/Developmental Toxicity Screen

................................ ................................ ................................ ......................... 14

D

- 5. Required Test: Genetic Toxicity

– Bacterial Reverse Mutation

................................ . 21 21

D

- 6. Required Test: Genetic Toxicity

– Chromosomal Aberrations

................................ .... 25 5

E. SUMMARY OF ENDPOINTS

................................ ................................ ............................. 29 9A. INTRODUCTION

This document evaluates a submission by Brü ggemann C hemical U.S., Inc. , received by EPA 1 in response to the final test rule, “Testing of Certai n 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,
Br ü ggemannChemical included data for the requi red 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 t he 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 obtaine d 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 aberr ation 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 t his 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%); sodiu m 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 Mutati on 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 endpoint s are evaluated in Section D

, including the data submitted for the test endpoint required by the HPV2 test rule. The sponsor submitted a f ull study report for this endpoint, i.e., bacterial reverse mutation testing

. A

s ummar y w as prepared for the submitted study, using a standard format and assigned a Klimisch reliability code. An adequacy determination was made using test rule

- specified g uidelines, 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 evaluat ions of submitted toxicity data for the following endpoints:

Aquatic Toxicity (includes Acute Toxicity to Fish

, Acute Toxicity to Daphnia , and Toxicity to A quatic P lants ); Mammalian Toxicity

– Genotoxicity ( Bacterial Reverse Mutation in vitro and Chromosomal Aberrations i n 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

I dentity: Met hanesulfinic acid, hydroxyl

- , m onosodium 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/guidel ine 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: 5 000 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 micro m ho. After resalting, the hardness, acid capacity, ratio of Ca/Mg i ons, ratio of Na/K ions and pH were 2.5 mm ol/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 d ay 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 te st 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 LC 50 > 1 0,0 00 mg/L

.

Results re marks: 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: Ron g alit 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: A dequate

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 a cid, hydroxyl

- , monosodium salt , CASRN 149

- 44

- 0 (referred to as

s odium 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 li mit 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/conditio ns remarks:

Each 2 ml vessel for testing contained 40 mL test solution and five daphnia, 2 replica t es for 1 and 10 mg/L; 4 replicates for con t rol and 100 mg/L. Testing was performed using deionized water, enriched with CaCl 2 * 2H 2 O ) at 293.8 mg/L; MgSO 4 * 7H 2 O ) at 123.3 mg/L; NaHCO 3 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 o C a nd hardness was 250 mg CaCO 3 /L. Potassium dichromate was used as a positive control. R enewal 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 4 8 hours was 103% of the start concentration.

RESULTS

Endpoint value(s): Based on nominal concentrations: 48

- hour EC 50 > 100 mg/L and 48

- hour NOEC = 10 mg/L.

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

r ange

- finding test was 10 mg/L.

CONCLUSIONS

The EC 50 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/ ", a nd 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 hydroxymethane 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, t he correct name for the sub stance 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 produ c t containing sodium hydroxymeth anesulfinate 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

s odium hydroxymethanesulphinat e ) , 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 )

Analy tical monitoring: No

Exposure duration: 48 hours

Concentrations tested: Nominal: 0, 1 0, 2 0, 40, 80 and 16 0 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

h ours. 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 m a cronu t rient materials, 60 trace8 elements, 8 vitamins and 3 amin o acids. Through the addition of CaCl 2 and NaHCO 3 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.

Twen t y daphnids were tested per concen tration (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 h ou rs

. The pH, oxygen content and temperature were determined at beginning and end of the 48

- h ou r test period. The medium was not aera ted. 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 h ang 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 EC 50 = 20

- 40 mg/L

Results remarks: Dissolved oxygen content ran ged 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 EC 50 of the test substance was 20

- 40 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. No measured concentra tions, 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 EC 50 of “Brueggolit E02” in the presence of Scenedesmus subspicatus

T his study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Br u ggolite E 02 (commercial methanesulfinic acid, hydroxyl

- , monosodium salt

, CASRN 149

- 44

- 0 , referred to as commercial s odium hydroxymethanesulphin ate in study description ), Batch 02112000 from Brü ggemann.

Remarks: Purity not provided

METHOD

Method/guideline followed: OECD Guideline 201

Test type: Alga, Growth Inhibition Test

GLP compliant?: Yes

Year study was performed: 20 01

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 remar ks:

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 de mineralized 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/despo s itory of algae culture of the Un iversity 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

t he st ock c ulture (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 st ock solution of the dilution series was prepared and pipette d into the test vessel (4 ml each), such that a mixture of the added foreculture and nutrient conc entrate was achieved ( using 0.5 ml of each ). Each concentration was tested in triplicate. The test vessels were incubated open under stirring (magnet ic stirrers) at 8000 lux lighting at 21 ± 2 o C. 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 measure d values of the optical density were recorded and evaluated with a computer program.

RESULTS

Endpoint value(s): Based on nomina l concentrations reported: EC 50 (growth rate) = 370 mg/L; EC 100 (growth rate) >1000 mg/L; NOEC (growth rate) = 10 mg/L ; EC 50 (biomass) = 220 mg/L; EC 100 (biomass) >1000 mg/L.

Results remarks: The pH variation was 0.5 units. The nominal growth factor tha t 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 EC 50 s for growth rate and biomass were 370 and 220 mg/L, respective ly. 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 fo r th e 72 hour EC 50 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 l anguage 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: Al though 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 i ncorrectly 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 di hydrate

. 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

Expo sure 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 m onths. 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

sto ck culture and placed for 1

- 2 days in the light, and then incolated in about 100 mL sterile13 medium. Incubation then followed in a shaking machine (100

- 150 rpm) for about 72 hours at 23
± 1 o C, and 8000 l u x (without a dark phase).

Exponentially growing cu ltures of Scenedesmus subspicatus (10,000 cells/mL) in defined medium were exposed in triplicate to var y ing concentrations of test substance over a time period of 72 hours in a circular shaking machine (circular vibrator VKS 75 with standard lighting

attac hed (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: EC 50 (growth rat e) = 450 mg/L; EC 50 ( 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 EC 50 s for growth rate and cell density were 450 and 5 mg/L, respectively.

STUDY RELIABI LITY

[ 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. Unpu blished 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 reprodu ctive/developmental oral toxicity s creen 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

gav age

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), a nd 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: Comb ined 28

- day r epeated

- 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 Riv er, 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: O ral gavage

Vehicle: Water (Elix, Millipore S.A.S., Mo lsheim, 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, 10 0, 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 fir st 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 t o 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/s ex/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 cage

enrichment. During overnight activity monitoring, ani mals 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, pa per, 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 fo r 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 bo tt om, middle and top for the low

- and high

- dose groups were analyzed for homogeneity and stability at 0 hours after preparation. Duplicate sample s 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 Projec t 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). Base d on the results, it was concluded that the formulations were prepared correctly. The low

- and high

- dos e 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 pe rf ormed 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 prelimina ry range

- f inding 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 vagi nal 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 identifi ed 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 exa mined 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 duri ng 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

pa rameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total17 protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, and inorganic phosphate) were performed after appro priate 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, ma mmary 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 poss ible 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 ani mals 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 exsang uinated. 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 exa mined 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 ex amined. 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 al l 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 pu ps 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. Descriptions18 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 a minotransferase (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 th e 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 grou ps 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 mea n 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, bod y 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 a nd 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

- re sponse 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 c hanges. 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). Absolu te brain weight19 (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 observe d 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 ab solute 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 no rmal 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 ( 18 9 ± 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 bilirub in 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 conside red 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 fema les 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 r elated 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 10 0 100 100

Mean Precoital Time* 2.5 2.9 1.4 1.720 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

V iability 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

- b w/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 definit ive reproduction, breeding, and developmental NOAEL was established as being 1000 mg/kg

- bw/day.

STUDY RELIABILITY

[ 1 ] Reliable with out restriction

.

REFERENCES

Beekhuijzen, M.E.W. 2008. A combined 28

- day repeated dose toxicity study with the reproduc tion/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

Sum mary 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 ; Synonym s : Sodium hydroxymethanesulfinate; sodium formaldehyde sulfoxyla te (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 crystal lization ) ]; 3.6% sodium hydroxymethanesulfonate (impurity); 4.3% sodiu m 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 Br ü ggemann Chemical 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 TA 97a , TA 98 , TA 100 , TA 102 and T A1 535

Metabolic activation: With and without (using S

- 9 in the p late 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 ; s econd experiment: 0 (separate water and DMSO solvent controls), 1258, 2516 and 5031 μ g/plat e of commercial product test substance (equivalent to 957.3, 1914.7 and 3828.6 μ g SFS /plat e ); for both experiments, the test substance solutions were prepared usi ng deionized water as a solvent22 Statistical methods used: The colonies were counted visuall y, and the numbers were recorded. S preadsheet software (Microsoft Excel ® ) was used to calculate mean values and standard deviations as well as the increase factor of rev e rtant induction

.

Test method/conditions remarks :

Preincubation period of culture in nutrient broth : 12 hours at 3 7 °C

.
PLATE INCORPORATION METHOD (First Experiment): Per strain and dose, four plates with and four plates without S9 mix were use d. 10 mL of the test solution of the appropriate concentration were membrane filtrate d 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 bottl e 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 pho sphate buffer (only for

treatment s without S9) or 0. 5 mL S9 mix, 2 ml. Top

- Agar was added. The mixture was gently

v ortexed, then poured on a minimal glucose plate and distributed ev enly using a Drigalski

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

t hen inverted and placed in the dark incubator at 3 7 °C

.

PRE

- INCUBATION METHOD (S econd experiment):
Per st r ain 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 f il t rated into sterile vessels. Top agar basis was m elt ed in a microwave oven; after melting, 10 m L . of h istidinebiotin

- solution

, 0.5 mMol per 100 mL basis

, was added and the bottle was pl aced in the water bath at 45 °C

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

m L 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

a t 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 v ortexed gently, then poured on a minimal gl ucose plate and distributed evenl y, using a Drig alski spatula. The plates w ere cl osed, 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

- phenylen e 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

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

NUMBER OF REPLICATIONS : Four replicates, with and wi thout S9, for each solvent were used in the test

.23 EVALUATION CRITERIA: A tes t item is considered to have mutagenic potential if a significan t, reproducible increase of revertant colonies per plate ( i ncrease factor ≥ 2) in at least one strain can be observed. A concentration

- related increase over the range tested can also be ta ken as a sign of mutagenic activity

.

RESULTS

FIRST EXPERIMENT: Th e 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 mu t agens) showed

mu t agenic effects with and without metabolic activation. No sig ns of toxicity of the test item towards the tested strains w ere observed; t he background lawn was visible and the number of revertant colonies was not significantly reduced. No significant increase of the number of

revert ant colonies in the treatments wit h and without metabolic activation c ould be observed. No

concentr ation

- related increase over the tested range was found.

SECOND EXPERIMENT: The treatments for the confi rmation of the genotype, the ste rility control and the determination of the titer did no t show any inconsistencies. All positive controls showed mut agenic effects with and without metabolic activation. No signs of toxicity of the test item towards the tested strains were observed; t he background law n was visible and the number of rev ertant colonies was not significantly reduced. No significant increase of the number of revertant colonies in the treatments with and without metabolic acti vation was observed. No concentration

- related increase over the tested range was found.

Genotoxicity re sult: 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 determine d val ues for the spontaneous revertant s 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 c onsidered to be non

- mut agenic in the bacterial reverse mutation test

.

STUDY RELIABILITY

[ 1 ] Reliable with out restrictions.24 REFERENCES

Paulus, J. 2008. Determination of the mutagenic potential of NHS (Sodium hydroxymethanesulfinate) with the Bacter ial Mutation Test following OEC D 471 and EU B. 13/14 . Report no. A B07120702G803. 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: A dequate

Stu dy 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 SUBSTAN CE

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 powd er 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

- bw26 Statistical methods used: The test was considered acceptable if: 1) the positive control

s ubstance 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 his torical 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 (Wilcox on 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 pe rf ormance of functional observations in the room, tempora ry 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 a rt ificial 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. Ce rt ificates of analysis of bedding and paper were examined and then retained in the NOTOX archives. The acclimatization period was at le ast 5 days before sta rt of treatment under laborato ry conditions. The animals had free access to standard pelleted laborato ry animal diet (SM R/M

- Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany) and tap water. Results of analyses for nutrients and cont aminants 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 positi ve 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 onc e 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 sh o rt ened 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 re moved 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 previou sly cleaned with a 1:1 mixture of27 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, fi xed 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 N ederland B.V.). The dry slides were dipped in xylene ( Klinipath, Duiven, The Netherlands) before they were embedded in Pe rt ex ( Klinipath) and mounted with a coverslip. Slides were scored at a magnification of 1000x. The number of micronucleated polychro matic 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 pol ychromatic 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 t o 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 (c ompared 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 s tatistically 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 hou rs 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 so lvent 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 restriction s

.

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

Endpoin t 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 Mutation 1 Yes Yes No

Chromosomal Aberrati ons 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)