p.1 

Harcros Chemicals Inc. 

5200 Speaker Road 

Kansas C'rty, KS 66106 

Telephone: 913/621-7802 

Fax: 913/621-7876 

E-mail: asavini@harcros.com 

Date 05/22/13 

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) 

354347 

RE: Ames Test Amendment for CASRN 31138-65-5 

To Whom It May Concern: 

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, 

Harcros Chemicals Inc. submitted a final report for the Ames test for D-gluco-Heptonic acid, 

monosodium salt, (2.xi.)- [CAS# 31138-65-5] on December 18, 2012. In response to a request from 

EPA to provide information on the dilution of the sample used in the study, please find attached an 

amendment to the Ames test containing the required information. 

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. 

Ann Marie Savini 

Harcros Chemicals Inc. 

Regulatory Specialist 

cc: Peter Radford 

John Miles 

Regards, 

James Grantham 

Randy Buckman 

File iiiiiiiiiigii) 

4 0 1 3000003 9 

III 

CONTAINS NO CBI 
harlan 

HARLAN LABORATORIES LTD 

AUTHENTICATION OF AMENDMENT TO FINAL REPORT 

Sodium Glucoheptonate: 

REVERSE MUTATION ASSAY "AMES TEST" USING 

SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

PROJECT NUMBER: 41104688 

REASON FOR AMENDMENT 

A test item purity correction for 49.5% water present in the sample was made in 

Experiments 1 and 2. However, this information was not recorded in the appropriate section 

of the final report. 

DETAILS OF AMENDMENT 

Section 3.2 Preparation of Test and Reference Items: The second paragraph has been 

amended with an additional sentence to confirm that an appropriate purity allowance was 

made in Experiments 1 and 2. The additional sentence forced the final paragraph on page 

9 onto page 10; therefore 2 pages have been amended. 

Pages 9 and 10 are hereby amended. 

This amendment does not affect the validity or interpretation of the data. 

PW Thompson HNC 

Study Director 

This amendment has been audited by Harlan Laboratories Ltd, Shardlow, UK Quality 

Assurance Unit and is considered to be an accurate account of the project. 

DATE: 

DATE: 1 6 MAY 2013 

For the Quality Assurance Unit* 

"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 9 (Amendment) 

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. 

Formulated concentrations were adjusted in Experiments 1 and 2 to allow for the stated 

water content (49.5%) of the test item. 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. 
PROJECT NUMBER: 41104688 PAGE 10 (Amendment) 

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: 

N-ethyl-N'-nitro-N-nitrosoguanidine 

N-ethyl-N'-nitro-N-nitrosoguanidine 

N-ethyl-N'-nitro-N-nitrosoguanidine 

9-Aminoacridine (9AA): 

4-Nitroquinoline-1 -oxide (4NQO): 

(ENNG): 2 ug/plate for \NP2uvrA 

(ENNG): 3 ug/plate for TA100 

(ENNG): 5 ug/plate for TA1535 

- 80 ug/plate for TA1537 

0.2 ug/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 ug/plate forTA100 

2 ug/plate for TA1535 and TA1537 

10 ug/plate for \NP2uvrA 

5 ug/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. 
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ORIGIN ID: KCKA <913) 621-7733 

JAKE BQOREM 

HARCROS CHEMICALS INC 

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