Harcros Chemicals Inc. 

5200 Speaker Road 

Kansas City, KS 66106 

Telephone : 913/621-7747 

Fax : 913/621-7746 

E-mail : OrganicsCS@Harcros.com 

Date 12/18/12 

~{E LE . 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 accordance with 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 study was conducted to fulfill the requirements of testing category E as set forth in Table 2 of 40 

CFR 799.50870) . 

If any further 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 NO CBI 
PAGE 1 OF 24 PAGES 

Sodium Glucoheptonate: 

REVERSE MUTATION ASSAY °AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND 

ESCHERICHIA COLI 

PROJECT NUMBER: 41104688 

harlan® 

AUTHOR: P W Thompson 

STUDY SPONSOR: TEST FACILITY: 

Harcros Chemicals Inc. Harlan Laboratories Ltd 

3452 Corporate Drive Shardlow Business 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/p 
PROJECT NUMBER: 41104688 PAGE 2 

QUALITY ASSURANCE 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 QA Signature 

General Study Plan Compliance Audit 

Test Item Preparation 

Test System Preparation 

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, 

G Wren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQA 
PROJECT NUMBER: 41104688 PAGE 3 

GLP COMPLIANCE STATEMENT 

With the exception noted below the 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 form as 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 of Vehicle and Positive Control Values 23 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 24 
PROJECT NUMBER: 41104688 PAGE 5 

Sodium Glucoheptonate : 

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, Sodium Glucoheptonate, 

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 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. 
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 

Sodium Glucoheptonate : 

REVERSE MUTATION ASSAY '°AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND 

ESCHERICHIA COLI 

1 . INTRODUCTION 

The purpose of the study was to evaluate the test item, Sodium Glucoheptonate, 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, 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 . 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 them more sensitive to various mutagens and they will not grow on 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 grow on 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 

Sodium Glucoheptonate 

Dark brown liquid 

50 .5% 

921000100 

09 February 2012 

09 February 2015 

Room temperature in the dark 

The integrity of supplied data relating to the identity, purity and stability of the test item is 

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 from S. typhimurium strain 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 system and 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" DNA repair deficiency which enhances its sensitivity 

to some mutagenic compounds . This deficiency allows the strain to show enhanced 

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 item was 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. 

A solvent 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 Sodium phosphate 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% sodium chloride 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 from SGL 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 item formulation 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 item formulation 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 item both with and without S9-mix. 

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. 

3.5 .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 item formulation 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 minimum of four non-toxic test item dose 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 . A reproducible 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 item will 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, UK archives 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 item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The 

test item formulation 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 

+ TAI 00 110 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 5 for 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 dose 
PROJECT 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, Sodium Glucoheptonate, 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, M H 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 M D (1979), Recommendations on data production and 

analysis using the Salmonella/microsome mutagenicity assay, Environmental 

Mutagenesis, 1, 87-92 . 

Mahon G A T 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 D J Ed .), Cambridge University Press Report, 26-65. 
PROJECT NUMBER: 41104688 PAGE 18 

Sodium Glucoheptonate: 

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) 

18 
PROJECT NUMBER: 47104688 

Sodium Glucoheptonate : 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Table 2 Test Results : Experiment 1 - Without Metabolic Activation 

PAGE 19 

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

Number ofrevertants mean +/-SD 

Dose Level Base- air substitution strains Framesh ift 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 

6 9 (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 150 jig 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 0 6 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 2 1 15 1 0 25 5.7 30 3.5 0 .0 

87 . 16 . 14 24 8 

71 (77) 15 (17) 27 (23) 28 (27) 

5000 Ng 75 3 (8) 

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 

D L l 3 u 5 N 2 N 0.2 ug 80 con ro s ose eve 

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 deviation 
PROJECT NUMBER: 41104688 PAGE 20 

Sodium Glucoheptonate : 

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 st rains 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) 

(+) 150 Ng 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) 

500 p9 88 

9 7 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 6 8 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 

Dose eve 

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 deviation 
PROJECT NUMBER: 41104688 

Sodium Glucoheptonate : 

REVERSE MUTATION ASSAY "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 150 tlg 

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 12 9 21 2 14 0 6 29 1 2 20 4.2 1 .2 

95 . 14 . 29 . 1 g 5 _ 

ENNG ENNG ENNG 4NQ0 9AA 

Positive 

t l 

Name 

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

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 deviation 
PROJECT NUMBER: 41104688 PAGE 22 

Sodium Glucoheptonate : 

REVERSE MUTATION ASSAY "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 I c 

Number ofrevertants mean +/-SD 

Dose Level Bas e- air substitution strains Framesh ifl 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 

150 Ng 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) 

500 tlg 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) 

1500 P9 97 3 2 14 0 6 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 deviation 
PROJECT NUMBER: 41104688 PAGE23 

Sodium Glucoheptonate : 

REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 

Appendix 1 History Profile of Vehicle and Positive Control Values 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 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 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 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= Maximum value 
PROJECT 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 

Shardlow Business 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 Authority 
ORIGIN 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 

TD (DCO)(7407M)(OPPT) 

US EPA TSCA SECTION 4 

1200 PENNSYLVANIA AVE NW 

WASHINGTON DC 20460 

REF-POSTAGE 

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