### Harcros Chemicals Inc. 5200Speaker Road Kansas City, KS 66106 Telephone : 913/621-7747 Fax : 913/621-7746 E-mail : OrganicsCS@Harcros.com Date 12/18/12 . ~{ELE 1 `~n

. 

## C ;3-i'T r-= ~ 

## 2012 DEC ! 9 00

Document Control Office (DCO) (7407M) Office of Pollution Control and Toxics (OPPT) U.S. Environmental Protection Agency (EPA) 1200 Pennsylvania Ave. NW Washington, D.C. 20460 (202-564-8930) Attn: TSCA Section 4 40 CFR § 799.5087 (Docket # EPA-HQ-OPPT-2007-0531) RE: Ames Test CASRN 31138-65-5 To Whom

It May Concern: 

# pe35d~(~~

In accordancewith EPA's final rule, Testing of Certain High Production Volume Chemicals; Second Group of Chemicals, published in the Federal Register on January 7, 2011 at 76 fed. Reg. 1067, please find enclosed the final report for the Ames Test on D-gluco-Heptonic acid, monosodium salt, (2.xi .)- [CAS# 31138-65-5] . This studywas conducted to fulfill the requirements of testing category E as set forth in Table 2 of 40 CFR 799.50870) . If anyfurther information is required, please do not hesitate to contact me at (913) 621-7802 or at asavini@harcros.com

. Regards,

Ann Marie Savini Harcros Chemicals Inc. Regulatory Specialist cc: Howard Stevenson James Grantham Don Meuschke John Miles File IIIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIII IIII 4 0 1 3 0 0 0 0 0 1 9



# -_ JAN 1 5 2012 CONTAINS NOCBIPAGE 1 OF 24 PAGES

SodiumGlucoheptonate: REVERSE MUTATION ASSAY °AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI PROJECT NUMBER

: 41104688 

# harlan®

AUTHOR

: PW

Thompson

STUDYSPONSOR

: TEST FACILITY: Harcros Chemicals Inc. Harlan Laboratories Ltd 3452 Corporate Drive ShardlowBusiness Park Dalton GA 30720 Shardlow UNITED STATES OF AMERICA Derbyshire DE72 2GD UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018

41104688

.doc/pPROJECT NUMBER: 41104688 PAGE 2 QUALITYASSURANCE REPORT This study type is classed as short-term . The General Study Plan for this study type was reviewed for compliance once only on initial production . Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases of this study type at least once every three months. From 16th May 2012 the frequency of these process based inspections was changed from a monthly to a three monthly cycle. This deviation is considered not to affect the purpose or integrity of the study. In addition, general facilities are inspected once at least once a year and the results are reported to management . This report has been audited by the Quality Assurance Unit, and is considered to be an accurate account of the data generated and of the procedures followed. In each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and inspections appropriate to the type and schedule of this study were as follows:

§ § § 06 January 2011 15 October 2012 10 October 2012 22 October 2012 15 October 2012 05 December 2012 Date of QASignature General Study Plan Compliance Audit Test ItemPreparation Test SystemPreparation Exposure Assessment of Response Draft Report Audit Final Report Audit Evaluation specific to this study



## .. . .. ....... .. ..... For the Qua 

### process



## .2012

. .... ............... .. .~ . ....... ..`. ......... . .......................... DATE: .. ... ...... .1.8. .DEC

. Assurance Uni

*Authorised QA Signatures: Senior Audit Staff: J G Riley BSc (Hons) MRQA, J M Crowther MIScT MRQA, GWren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQAPROJECT NUMBER: 41104688 PAGE 3 GLP COMPLIANCE STATEMENT With the exception noted belowthe work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)) . These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17) ; and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within four hours of it being applied to the test system . It is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study. This report fully and accurately reflects the procedures used and data generated.

P W Thompson HN Study Director 

## ...... .. ...... .................... DATE: ... . I

. .. ~ . .DEC . .201 .Z ................. ... .... ..

This report may be presented in final formas a digital (pdf) document . Such documents are prepared by scanning the paper original, and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the hand-signed paper original, held in secure archives, is the definitive document .PROJECT NUMBER: 41104688 PAGE 4 CONTENTS QUALITY ASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1 . INTRODUCTION 7 2 . TEST ITEM 8 3 . METHODS 8 3.1 Tester Strains 8 3.2 Preparation of Test and Reference Items 3.3 Microsomal Enzyme Fraction 10 3.4 S9-Mix and Agar 10 3.5 Test Procedure 11 3.6 Acceptance Criteria 13 3.7 Evaluation Criteria 13 4 . ARCHIVES 14 5. RESULTS 15 5.1 Preliminary Toxicity Test 15 5.2 Mutation Test 15 6. CONCLUSION 16 7. REFERENCES 17 Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls) 18 Table 2 Test Results: Experiment 1 - Without Metabolic Activation 19 Table 3 Test Results: Experiment 1 -With Metabolic Activation 20 Table 4 Test Results: Experiment 2 - Without Metabolic Activation 21 Table 5 Test Results: Experiment 2 - With Metabolic Activation 22 Appendix 1 History Profile ofVehicle and Positive Control Values 23 Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 24PROJECT NUMBER: 41104688 PAGE 5 SodiumGlucoheptonate: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI SUMMARY Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MET], MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, SodiumGlucoheptonate, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) . The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 to 5000 pg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. Results. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation . Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 Ng/plate. No test itemprecipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.PROJECT NUMBER: 41104688 PAGE 6

Conclusion. The test item, Sodium Glucoheptonate, was considered to be non-mutagenic under the conditions of this test .PROJECT NUMBER: 41104688 PAGE 7 SodiumGlucoheptonate: REVERSE MUTATION ASSAY '°AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 1 . INTRODUCTION The purpose of the study was to evaluate the test item, SodiumGlucoheptonate, for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria . The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLWand MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines . A copy of the Certificate of Compliance with GLP, issued by the UK Department of Health, is included . The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item . These strains have a deleted excision repair mechanism which makes themmore sensitive to various mutagens and they will not growon media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to growon a histidine deficient medium . Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976)) is used to complement the Salmonella strains. Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix)PROJECT NUMBER: 41104688 PAGE 8

prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes. The experimental phase of this study was performed between 22 February 2012 and 05 November 2012. 2 . TEST ITEM Sponsor's identification Description Purity Batch number Date received Expiry date Storage conditions SodiumGlucoheptonate Dark brown liquid 50.5% 921000100 09 February 2012 09 February 2015 Roomtemperature in the dark The integrity of supplied data relating to the identity, purity and stability of the test itemis the responsibility of the Sponsor. 3. METHODS 3.1 Tester Strains The five strains of bacteria used, and their mutations, are as follows: Salmonella typhimurium Strains Genotype TA1537 TA98 TA1535 TA100 Escherichia coli Strain WP2uvrA his C 3076; rfa; uvr8- :

his D 3052; rfa; uvr8' ;R-factor his G 46 ; rfa; uvr8": his G 46 ; rfa' ; uvr8" ;R-factor Genotype trp' ; uvrA" : Type of mutations indicated frame shift mutations base-pair substitutions Type of mutations indicated base-pair substitution All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived fromS . typhimuriumstrain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvr8-bio gene,PROJECT NUMBER: 41104688 PAGE 9

causes an inactivation of the excision repair systemand a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)) . In addition to a mutation in the tryptophan operon, the E . coli tester strain contains a uvrA" DNArepair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to showenhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel (1976)) . The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 1078364 08/16, 1119376 10/17 and 1014570 03/16) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. 3.2 Preparation of Test and Reference Items The test itemwas fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle. The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment . All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Asolvent treatment group was used as the vehicle control and the positive controls used in the series of plates without S9-mix were as follows:PROJECT NUMBER: 41104688

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) : N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) : N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) : 9-Aminoacridine (9AA) : 4-Nitroquinoline-1-oxide (4NQ0) : PAGE 10 2 pg/plate for WP2uvrA 3 lag/plate for TA100 5 Ng/plate for TA1535 80 Ng/plate for TAI 537 0 .2 Ng/plate for TA98 in addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix at the following concentrations: 2-Aminoanthracene (2AA) : 2-Aminoanthracene (2AA) : 2-Aminoanthracene (2AA) : Benzo(a)pyrene (BP) : 

### 1 Ng/plate for TA100 2 Ng/plate for TA1535 and TAI 537 10 Ng/plate for WP2uvrA 5 pg/plate for TA98 3.3 Microsomal Enzyme Fraction The S9 Microsomal fraction was prepared in-house (18 September 2011 (preliminary toxicity test), 01 July 2012 (Experiment 1) and 16 September 2012 (Experiment 2)) from rats induced with Phenobarbitone/p-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4 . The S9 homogenate was produced by homogenising the liver in a 0

.15M KCI solution (1g liver to 3 ml KCI) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/ml . Aliquots of the supernatant were frozen and stored at approximately -196°C . Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test . 3.4 S9-Mix and Agar The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.PROJECT NUMBER : 41104688 PAGE 11 S9 5.0 ml 1 .65 M KCI/0 .4 M MgC12 1 .0 ml 0.1 M Glucose-6-phosphate 2 .5 ml 0.1 M NADP 2 .0 ml 0.2 M Sodiumphosphate buffer (pH 7.4) 25.0 ml Sterile distilled water 14.5 ml A 0 .5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment . Top agar was prepared using 0.6% Bacto agar (lot numbers 0348877 10/15, 1297425 09/16 and 2129076 10/17) and 0 .5%sodiumchloride with 5 ml of 1 .0 mM histidine and 1 .0 mM biotin or 1 .0 mM

tryptophan solution added to each 100 ml of top agar. For the preliminary toxicity test, Vogel-Bonner Minimal agar plates were purchased from ILS Ltd (lot number 1193292 10/15) . For Experiments 1 and 2, Vogel-Bonner Minimal agar plates were purchased fromSGL Ltd (lot numbers 32918 10/12 and 33059 11/12) . 3.5 Test Procedure 3.5.1 Preliminary Toxicity Test In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item . The concentrations tested were 0, 0.15, 0.5, 1 .5, 5, 15, 50, 150, 500, 1500 and 5000 Ng/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test item formulation and 0 .5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate) . Ten concentrations of the test itemformulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item . After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.PROJECT NUMBER: 41104688 PAGE 12

3.5.2 Mutation Test - Experiment 1 Five concentrations of the test item (50, 150, 500, 1500 and 5000 Ng/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0 .1 ml of the vehicle, test itemformulation or positive control and either 0 .5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate) . This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test itemboth with and without S9-mix. All of the plates were incubated at 37°Cfor approximately 48 hours and the frequency of revertant colonies assessed using an automated colony counter. 3.5.3 Mutation Test - Experiment 2 The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was the same as Experiment 1 (50 to 5000 Ng/plate) . As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation . The test item formulations and vehicle control were therefore dosed as follows: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test itemformulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate) . This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described in Section 3.5.2.PROJECT NUMBER: 41104688 PAGE 13

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using an automated colony counter. 3.6 Acceptance Criteria The reverse mutation assay may be considered valid if the following criteria are met: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000) . All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 4 (negative controls) . Combined historical negative and solvent control ranges for 2010 and 2011 are presented in Appendix 1 . All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per ml . Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive controls for 2010 and 2011 are presented in Appendix 1 . There should be a minimumof four non-toxic test itemdose levels. There should be no evidence of excessive contamination. 3.7 Evaluation Criteria There are several criteria for determining a positive result . Any, one, or all of the following can be used to determine the overall result of the study: 1 . A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)) .PROJECT NUMBER: 41104688 PAGE 14

2. Areproducible increase at one or more concentrations. 3. Biological relevance against in-house historical control ranges . 4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)) . 5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response) . A test itemwill be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal . 4 . ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd., Shardlow, UKarchives for five years, after which instructions will be sought as to further retention or disposal .PROJECT NUMBER: 41104688 PAGE 15

5 . RESULTS 5.1 Preliminary Toxicity Test The test itemwas non-toxic to the strains of bacteria used (TA100 and WP2uvrA) . The test itemformulation and S9-mix used in this experiment were both shown to be sterile. The numbers of revertant colonies for the toxicity assay were: With (+) or Dose (Ng/plate) without (-) S9-mix Strain 0 0 .15 0 .5 1 .5 5 15 50 150 500 1500 5000

- TA100 104 104 95 115 98 95 121 99 103 107 126 + TAI00 110 90 109 91 108 109 102 102 92 82 98

- WP2uvrA 34 31 40 35 26 33 28 30 29 33 34 + WP2uvrA 37 37 26 19 40 32 34 43 42 34 35 5.2 Mutation Test Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory) . The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report . Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test . The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5for Experiment 2. A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Appendix 1 . The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dosePROJECT NUMBER: 41104688 PAGE 16

level of 5000 Ng/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. 6 . CONCLUSION The test item, SodiumGlucoheptonate, was considered to be non-mutagenic under the conditions of this test .PROJECT NUMBER: 41104688 PAGE 17

7. REFERENCES Ames B N, McCann J, and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364. Maron D M

and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173- 215. Mortelmans K and Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation Research, 455, 29-60 . Green, MH L. and Muriel W J (1976), Mutagen Testing Using TRP' Reversion in Escherichia coli, Mutation Research, 38, 3- 32. De Serres F J and Shelby MD (1979), Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92. Mahon GAT et al (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland DJ Ed.), Cambridge University Press Report, 26-65 .PROJECT NUMBER: 41104688 PAGE 18 SodiumGlucoheptonate: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1 Number of revertants (mean number of colonies per plate)

Base-pair substitution type Frameshift type TA100 TA1535 WP2uvrA TA98 TA1537 89 89 (87) 82 18

20 (16) 10 21

18 (21) 25 24

21 (21) 18 9

13 (9) 5
EXPERIMENT 2 Number of revertants (mean number of colonies per plate)

Base-pair substitution type Frameshift type TA100 TA1535 WP2uvrA TA98 TA1537 107 112 (110) 112 17 11 (17) 22 30

26 (31) 36 19

26 (25) 29 13

13 (15) 18PROJECT NUMBER: 47104688 SodiumGlucoheptonate: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM ANDESCHERICHIA COLI Table 2 Test Results: Experiment 1 - Without Metabolic Activation PAGE 19

Test Period t"rom : ut5 ucrooer zui[ io: i i vciuuei IV ic

Number ofrevertants mean +/-SD Dose Level Base- air substitution strains Frameshift strains Per Plate TA100 TA1535 WP2uvrA TA98 TAI 537 Solvent Control 82 (85) 20 (18) 28 (22) 2~ (30) 1q (10) (Water) 91 5 .5# 16 2

.1 22 4.5 28 4.4 7 3.8 9 6 (89) 25 (22) (20) (28) 13 (11) 50 ply 0 21 1 26 5 5 25 4.0 25 3.1 10 2.1

91 . 16 . 18 31 9 S9-Mix 150jig 99 91 (93) 21 (16) 18 20 (19) 24

24 (25) 11

13 (12) (-) 88 5 .7 9 6

.4 19 1 .0 28 2.3 11 1 .2 77 (91) 18 (18) 18 (24) 37 (31) 12 (12) 500 Ng O1 12 3 19 06 29 5.5 34 8.5 12 0.6

94 . 18 . 24 21 11 0 (89) 14 (15) 17 (19) 30 (28) 8 (8) 1500 Ng 91 1 2 15 0

1 25 5.7 30 3.5 0.0 87 . 16 . 14 24 8 71 (77) 15 (17) 27 (23) 28 (27) 3 (8) 5000 Ng 75 6 7 21 3 8 15 7.2

# 33

6.6 1 4.5

84 . 14 . 28 20 8

_ ENNG ENNG ENNG 4N40 9AA Positive t l Name

L l D 3 u 5 N 2 N 0 .2 ug 80 con ro s eve

ose 536 275 755 161 724 59-Mix No, of 572 (569) 218 (309) 722 (713) 159 (150) 578 (624) (-) Revertants 600 32.1 432 110.7 663 46.6 131 16.8 570 86 .7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 4NQ0 4-Nitroquinoline-1-oxide 9AA 9-Aminoacridine # Standard deviationPROJECT NUMBER: 41104688 PAGE 20 SodiumGlucoheptonate: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIACOLI Table 3 Test Results: Experiment 1 - With Metabolic Activation Test Period From : 08 October 2012 To : 11 October 2012 Number of revertants mean +/-SD Dose Level Base- air subshtution strains Frames ft strains Per Plate TA100 TA1535 WP2uvrA TA98 TA1537 SolventControl 99 (110) ~3 (12) zs (32) zz (22) ~Q (13) 

# F (Water) 116 13.7# 13 1 .2 33 4 .6 19 2

.5 13 2

.5 118 (111) 10 (12) 24 (27) 20 18 (19) 13

7 (9) 50 pg 106 6 4 11 2.1 26 4.2 1 .2 7 3.5

108 . 14 32 20 S9-Mix 104 (92) 10 (10) 25 (24) 128 (19) ~~ (13) (+) 150Ng 8 10.8 1 .5 4.2 21 2.9 11 3.8 7 11 27 107 (96) 10 (it) 22 (23) 16 16 (19) 10

19 (13) 500p9 88 7 9 10 1 .2 18 5.0 4.6 11 4.9 94 . 12 28 24 100 (105) 11 (11) 31 (27) 17 17 (16) 12

15 (12) 1500 N9 113 68 13 1 5 30 5.5 2.3 3.5

103 . 10 . 21 13 8 69 01 (96) 8 (13) 25 (30) 20 20 (21) 15 (12) 5000 N9 1 24 .9 4.6 8 .1 22 1 .2 10 2.6 118 14 35 2AA 2AA 2AA BP 2AA Positive Name l

L 1 N 2 N 10 5 N 2 Ir controls S9-Mix eve

Dose No. of 904 ( ) 219 218) ( 254 (333) 200 (178) 155 139 ( (+) Revertants 847 366 227 207 10.1 380

364 68.6 168

165 19

.4

# 139

9.2 833

2AA 2-Aminoanthracene BP Benzo(a)pyrene # Standard deviationPROJECT NUMBER: 41104688 SodiumGlucoheptonate: REVERSE MUTATIONASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI Table 4 Test Results: Experiment 2 - Without Metabolic Activation PAGE 21

Test Period From : 01 November 2012 To: 04 November 2012

Number of revertants mean +/- SD Dose Level gase- air substitution strains Frameshift strains Per Plate TA100 TA1535 WP2uvrA TA98 TA1537 Solvent Control 26 (109) 2~ (18) ZO (31) ~3 (25) ~6 (14) (Water) 128 30.6# 20 3.8 37 9.3 28 10.4 12 2.1

82 (94) 22 (22) 21 (27) 25 (22) 15 (11) 50 pig 109 13 7 24 2 0 32 5.5 25 5.2 12 4.0

92 . 2~ . 27 16 7 S9-Mix 150tlg 122 123 (122) 21

15 (20) 31

33 (28) 17

14 (19) 10

13 (10) (-) 120 1 .5 24 4.6 20 7 .0 27 6.8 6 2.5 95 (104) 9

13 (18) 37

34 (35) 31

28 (24) 11

18 (14) 500 Ng 93 17 3 1 4 2 1 .7 10.2 3.5

124 . 21 . 34 12 14 ill (920) 19 (17) 33 (30) 17 (19) 8 (6) 1500 Ng 123 T9 15 2 1 27 3.1 19 2.0 0.6

126 16 . 29 21 9 90 (105) 15 (14) 27 (28) 26 (21) ~ (6) 5000 pg 129 21 2 14 6 0 29 2

1 20 4.2 1 .2 95 . 14 . 29 . 1 g 5

_ ENNG ENNG ENNG 4NQ0 9AA Positive t l Name

L l D 3 N 5 N 2 N 0.2 N 80 Ng con ro s eve

ose 549 982 953 114 1188 S9-Mix No. of 491 (534) 573 (746) 985 (915)

# 136

(132)

# 1722

(1237) (-) Revertants 563 38.2 

## 683 211 .7 806 95.5 147 16.8 

# 801 462.5

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 4NQ0 4-Nitroquinoline-1 -oxide 9AA 9-Aminoacridine # Standard deviationPROJECT NUMBER: 41104688 PAGE 22 SodiumGlucoheptonate: REVERSE MUTATIONASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI Table 5 Test Results: Experiment 2- With Metabolic Activation Test Period I-rom : 01 Novemoer zuu i o: uv noverrluef cu Ic

Number ofrevertants mean +/-SD Dose Level Base- air substitution strains Frameshifl strains Per Plate TA100 TA1 535 WP2uvrA TA98 TA1537 Solvent Control 6

2 (94) 13

10 (12) 41

42 (42) 24

34 (30) s (11) (Water) 108 27.2# 14 2.1 42 0.6 31 5.1 16 5.0

116 (110) 12 (14) 34 (41) 30 25 (27) 19 (11) 50 p9 116 11 .6 14 1 .5 44 6.1 2.5 12 1 .5

97 15 45 27 S9-Mix 150Ng g5 (107) 11 11 (13) 43

37 (40) 25

29 (27) 11 (10) (+) 106 12.5 16 2.9 39 3.1 28 2.1 11 2.3 103 (102) 11 (13) 31 (34) 25 (30) 11 9 (10) 500tlg 97 5 0 16 2 6 37 3 1 34 4.7 1 .2

107 . 12 . 33 . 32 9 102 (101) 14 (14) 43 (38) 27 (24) 11 (12) 1500P9 97 3 2 14 06 39 6.1 26 3.8 12 1 .0

103 . 15 . 31 20 13 99 (99) 15 (14) 39 (36) 28 (26) (12) 5000 N9 99 0 6 10 3 2 32 3.6 26 1 .5 10 2.1

98 . 16 . 37 25 11 2AA 2AA 2AA BP 2AA Positive l Name D L l 1 2 10 5p2 2 N contro s ose eve 945 253 341 124 511 S9-Mix No. of 897 (781) 265 (255) 329 (357) 

# 131 (137) 

## 566 (498) (+) Revertants

# 500

244 .2 248 8

.7 401 38.6

# 157

17.4 417 75.3

2AA 2-Aminoanthracene BP Benzo(a)pyrene # Standard deviationPROJECT NUMBER : 41104688 PAGE23 SodiumGlucoheptonate: REVERSE MUTATIONASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM ANDESCHERICHIA COLI Appendix 1 History Profile of Vehicle and Positive Control Values COMBINEDVEHICLE ANDUNTREATED CONTROLVALUES 2010 Strain S9-Mix TA100

-Se +S9 TA1535

-S9 +59 TA102

-S9 +S9 WP2uvrA

-S9 +S9 TA98

-59 +89 TA1537

-S9 +S9 WP2uvrA

pKM101 -S9 +S9 WP2pKM101

-S9 +S9 Mean 104 98 19 15 266 318 29 33 21 25 12 13 141 169 90 117 SD 14

.7 13 .8 4.7 3.3 35.4 31 .8 9 .1 9.2 3.9 4

.3 2 .4 2.4 47.1 44,3 21 .9 30 .5 Min 71 62 8 7 206 234 14 16 11 14 6 7 75 101 46 48 Max 171 145 34 28 359 346 54 55 42 42 26 23 235 274 130 186 Values 604 441 504 345 31 13 512 369 590 388 566 335 40 32 29 20 POSITIVE CONTROL VALUES 2010

TA100 TA1535 TA102 WP2uvrA TA98 TA1537 PP2uvrA WP2pKM101 Strain KM701 S9-Mix _S9 +S9 -S9 +S9 -59 +59 -S9 +S9 -S9 +59 -S9 +S9 -59 +S9 -Se +S9 Mean 459 1091 353 218 1163 1031 395 282 155 249 1285 202 811 1833 1443 537 SD 131 .9 452.8 389.4 77.4 385 .1 423.0 200

.1 81 .0 53.5 134

.7 833.7 72.4 335,6 695.6 603 .8 207.0 Min 228 302 87 103 717 575 129 130 89 84 128 83 446 799 476 321 Max 1218 3777 2003 726 1836 2392 978 661 488 1146 4743 530 1660 3255 2323 1028 Values 159 187 156 180 18 18 144 144 236 169 229 158 11 17 12 18 COMBINEDVEHICLE AND UNTREATEDCONTROLVALUES 2011 Strain S9-Mix TA100

-S9 +S9 TA1535

-59 +S9 TA102

-S9 +S9 WP2uvrA

-S9 +59 TA98

-S9 +S9 TA1537

-S9 +S9 WP2uvrA

pKM101 -S9 +S9 WP2pKM101

-S9 +S9 Mean 108 101 22 15 256 298 30 35 23 25 11 12 121 163 93 115 SD 3

.7 5.1 0.8 1 .0 5 .7 3.2 0.9 0.8 0 .7 1 .5 0.5 1 .4 5 .3 10.7 2

.2 25.9 Min 58 80 10 8 198 209 11 10 11 10 4 5 85 108 69 81 Max 171 18B 34 35 337 385 59 55 43 43 23 25 168 223 125 154 Values 1090 716 810 425 165 86 851 495 1167 482 1124 409 37 19 27 11 POSITIVE CONTROL VALUES 2011

TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA WP2pKM101 Strain pKM101 S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S8 -S9 +S9 Mean 528 888 307 304 986 875 843 283 194 249 946 235 1034 1583 1384 628 SD 223

.9 398.3 254.4 122.9 284.1 209.5 213.2 83 .1 115.2 186.7 840.7 72 .9 298

.8 881 .5 1054.4 198.4 Min 249 348 87 126 622 828 198 121 69 81 103 86 692 331 233 432 Max 2282 3072 1773 1150 2151 1780 1719 504 1631 2115 5224 491 1600 2916 2798 1150 Values 191 202 192 196 51 51 177 177 255 204 254 193 7 12 5 12

SD = Standard deviation Min = Minimum

value Max= MaximumvaluePROJECT NUMBER : 41104688 PAGE 24 Appendix 2 Statement of GLP Compliance in Accordance with Directive 200419/EC

Of D~ Department of Health THE DEPARTMENT OF HEALTH OF THE GOVERNMENT

OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC TEST FACILITY Hartan Laboratories Ltd ShardlowBusiness Park London Road Shardlow Derby DE72 2GD

DATE OF INSPECTION 10 July 2012

Dr. Andrew J. Gray An inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practlce Compliance Monitoring Programme
. This statement confirms that, on the date of issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance with the OECD Principles of Good Laboratory Practice. This statement constitutes a Good Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999) . TEST TYPE(S) Analytical/Clinical

Chemistry Environmental Toxicity Environmental Fate Mutagenicity Phys/Chem

. Tests Toxicology

30 A I tZ Head, UK GLP Monitoring AuthorityORIGIN ID: KCKR <913> 621-7733 ISHIP DATE : 18DEC12 JRKE BOOREM HCTWGT : 1 .0 LB MAN HRRCROS CHEMICALS INC CAD : 0978415/CRFE2606 5200 SPEAKER RD KANSAS CITY, KS 66106 I BILL THIRD PARTY UNITED STATES US 

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