Document GVQ18VznOqkRNb84664Dm2on
BUSHY RUN RESEARCH CENTER
R. 0. 4, Mellon Road, Export, Pennsylvania 16632
CONFIDENTIAL: Not to be released outside UCL without the written
consent of the UCC-sponsoring Pivision HS&EA Manager.
project Report 47-171 10 Pages January 16, 1985
BAXELITE* Cycloaliphatic Epoxy Resin ERI-4234 Salmonella/Mlcrosome (Ames) Bacterial Mutagenicity Assay
Sponsor: Union Carbide Corporation Specialty Polymers and Composites Division
Summary
BAKELITE* Cycloaliphatic Epoxy Resin IRL-4234 was tested for potential sutagenlc activity using the Salmonella/mlcrosome bacterial mutagenicity assay (Ames test). Epoxy Resin IRL-4234 was tested with and without metabolic activation in triplicate at five concentrations, ranging from 0.1 milligrams to 10 mllll- grams per plate. The highest concentration was cytotoxic in the autagenlclty tests and In a preliminary test to chooBe appropriate doses. Mutagenic activity was not observed with any of the five bacterial strains tested with or without metabolic activation. Epoxy Resin ERL-4234 was not sutagenlc in this screening test.
Sample
Chemical Name: BAKELITE* Cycloaliphatic Epoxy Resin IRL-4234 2.D. it T F3-98461 4193 WB BRRC Sample it 47-205 BRRC Account #: 84-18-18046-46 CAS #: 3388-03-02
Chemical Synonyms: ARALDITE CY175; UP612; 2-(3,4-epoxy)cyclohexylmethyl5,5-spiro(3,4-epoxy)cyclohexane-m-dioxane
Molecular Formula: C1&22P4
Submitted by: R. J. Cotter, Bound Brook, NJ
Sponsor: Division:
R. E. Plevan, Danbury, CT Specialty Polymers and Composites Bound Bro k, NJ
Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation
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BAKEL1TE* Cycloaliphatic Epoxy Resin ERX-4234 Salmonella/Mlcroeome (Ames) Bacterial Mutagenicity Assay
*****
I. Introduction and Theory
The Salmonella typhlmurlum/mlcroaome nutation assay is a microbial screening test which detects the ability of chemicals to cause genetic alterations In the histidine gene of selected indicator strains of Salmonella typhlaurlum. The Indicator strains used for this test are all histidinerequiring (his-) bacteria which carry a mutation In the histidine locus. The strains were obtained from Dr. Bruce Ames, University of California, Berkeley, CA. Histidine-independent (hls+) strains can arise either spontaneously or by the mutagenic action of a chemical or physical agent. Base pair mutagens cause a base change In the DNA molecule at the site of the original mutation or at a second site In the DNA which supresses the original mutation. Frameshlft mutagens cause the addition or deletion of single or multiple base pairs in the DNA molecule. The relative frequency of induced or spontaneous reversion to hls+ can be quantitltated by plating the Indicator strains on minimal agar and counting the number of revertant colonies, representing bacteria that were able to grow and form colonies in the absence of histidine.
To test the mutagenic potential of a test chemical, the bacterial strains are exposed to several concentrations of the chemical* Tests are done both with and without the addition of a mammalian liver homogenate (S9) b cause some chemicals require metabolic conversion for detection of their biological effects. Concurrent solvent and positive controls are also run with each assay to determine the responsiveness of the test system. After a suitable period of Incubation (48-72 hrs), direct revertant colony counts are made and evaluated. Test chemlcalB which produce at least a 2-fold and dose-related Increase In mutant colonies over the concurrent control value are considered to be bacterial mutagens and suspect mammalian mutagens.
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II. Salmonella typhlaurlun Mutagenicity Assay
A. Objective
The objective waa to assess the mutagenic potential of Epoxy Resin ERL-4234 as measured In the Salmonella typhlmuriuro/ mlcrosome mutagenicity assay. A more complete discussion of the theoretical basis of this test Is presented In Appendix 1.
B. Test Chemical (Available analytical and physical data is attached as Appendix 2)
1. Sample name; BAKELITE* Cycloaliphatic Epoxy Resin ERL-4234 2. BRRC Sample Number: 47-205 3. CAS Number: 3388-03-2 4. Purity; > 95X; IR spectra consistent with structure
(see Appendix 2) 5. Density: 1.1891 6. Date Received: July 25, 1984 7. Storage Conditions: Room Temperature 8. Storage Location: Chemical Hood/Genetic Toxicology Dept.
C. Control Substances
1. Solvent: dlmethylsulfoxide, Burdick 4 Jackson, Muskegon, MI; Lot # AD912; CAS # 67-68-5
2. Positive Controls: a) 4-nitro-o-phenylenedlamlne; BRRC #44-71; CAS #99-56-9 b) 9-amlnoacrldlne; BRRC #44-233; CAS #90-45-9 c) sodium azide; BRRC #44-72; CAS #26628-22-8 d) 2-aminoanthracene; BRRC #44-67; CAS #613-13-8
D. Metabolic Activation
S9 liver homogenate, prepared from Aroclor 1254-lnduced, Sprague-Davley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0.5 ml of S9 mix containing 50 pi of S9 was added per plate,
E. Test Protocol
The assay was performed according to BRRC Standard Operating Procedures 7.4.1A through 7.4.7A, 7.4.12A, and 7.4.13. A general description of the test methods is attached to this report as Appendix 1.
, * #, 4i >
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Test Dates
Test Initiated: October 15, 1984 Test Completed: October 19, 1984
III-
G. Storage Location of Materials
1, Raw Data: BRRC Archives 2. Final Report: BRRC Archives
Experimental Design (detailed procedures In Appendix 1)
1. Sample Preparation: The test substance was dissolved In dlmetbylsulfoxide (DMSO) to a concentration of 100 mg/ml for doses of 10 mg/plate and below. The mixture was heated to 50C to hasten dissolution. All subsequent dilutions were made in the same solvent. Dilutions of the test substance were made fresh each day of testing. All dilutions for the mutagenicity tests were gravlmetrically analysed.
2. Dose Selection: A preliminary toxicity test was performed using strain TA100 to determine the level of toxicity of the test substance. Ten doses were tested for toxicity with a plate assay performed In the manner used for mutagenicity determinations. Toxicity was assessed at 24 to 48 hours after treatment by either growth inhibition of the background lawn or a reduction in the number of spontaneous mutants.
3. Testing: The test chemical was tested in triplicate at five doses chosen to span a range which Included moderately toxic to relatively nontoxic concentrations. If the substance was nontoxic in the preliminary toxicity test, 100 pi of liquid or 50 mg of solid was used as the maximum d se, unless limited by solubility. Testing was performed both with and without metabolic activation. Concurrent solvent and positive controls were run in each test.
Quality Control
Data from each test Is checked for accuracy and integrity by a second Investigator not Involved with the study. Compliance of testing procedures with Good Laboratory Practices is audited by the Independent Quality Assurance department at the Bushy Run Research Center.
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III. Results
Epoxy Resin ERL-4234 was tested In a preliminary toxicity screen at 50, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 milligrams per plate with (train TA100 only. The teat chemical was treated as a solid because of its high viscosity. The top 2 doses of 50 and 10 mg per plate precipitated in the top agar and a slight precipitate formed at 3 mg/plate. The top dose of 50 mg per plate inhibited all growth of the background lawn and 10 mg per plate permitted only sparse growth of the background lawn. Based on these results, utagenlclty testing was done with 5 doses of 10, 3, 1, 0.3, and 0.1 illigrams per plate in triplicate. The highest dose was expected to produce some degree of cytotoxicity based on preliminary toxicity test results, observed as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn. The range of doses that was tested followed the exposure concentration recommendations of the U.S. E.P.A. Health Effects Test guidelines (HG Gene Muta-S. typhlmurium, August 1982).
Gravimetric analysis of the highest test substance dilutions for each sutageniclty test indicated no more than a 1.3Z error from the stated concentrations. Gravimetric analysis of the lowest two dilutions in the test without metabolic activation indicated errors > 10Z from the Intended concentrations. Therefore, the Intended doses of 0.1 and 0.3 mg/plate are restated in Table 1 as the actual doses of 0.09 and 0.27 mg/plate. The remaining dilutions for the test without metabolic activation had no more than a 9.4Z error from the stated concentrations. All subsequent dilutions for the test with metabolic activation had no more than a 1.5Z error from the stated concentrations.
All plate counts and the respective means and standard deviations are shown in Table 1 (without activation) and in Table 2 (with activation). No indication of mutagenicity was observed at any of the tested doses, either by evidence of a dose-response relationship or a doubling of the number of colonies over the solvent control.
Dose selection appeared to be in a suitable range in the mutagenicity tests because some toxicity was evident in both tests. In both tests, toxicity was observed at 10 mg/plate with all strains.
All strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain, and the spontaneous reversion rates were within the historical ranges at this laboratory (see Appendix 3). All positive and negative controls were run concurrently with the test chemical* Concurrently run sterility checks showed that the S9 mix, PBS, the test chemical and all solvents and controls were sterile.
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IV. Conclusion
Epoxy Resin ERL-4234 did not produce a dose-dependent uutagenlc response in any of the Salmonella typhimurium strains. Under the conditions f this assay. Epoxy Resin ERL-4234 was not mutagenic in the Salmonella/mlcrosome mutagenicity assay.
Reviewed and Approved by:
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M.B.A. 'Date
/} A. i*s
Ronald S. Sleslnskl, Ph.D.
Date
Manager, Genetic, Biochemical, & Acute Toxicology
Fred R. Frank, Ph.D Director
Contributors: Technical Assistance: Eileen R. Morabit, A.S. Senior Technologist
WPC/rkk/0604B-2 11-01-84
Date
TABLE 1 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY
Report 47-171 V.lt>5
Page 7
` I f '.
CHEMICAL: BRRC #47-205, Epoxy Resin ERL-4234
WITHOUT ACTIVATION
EXPERIMENT DATE: October 15 through October 18, 1984 SOLVENT: DMSO
TEST
agent
DOSE PER PLATE
SIRAIN TA98 SOLVENT POS: 01
110 MG 0.01 MG
BSRC# 47-205
0.09 MG 0.27 MG
1 MG
3 MG 10 MG
PLATE COUNTS
12
3
14 19 715 743
14 16 13 13 15 14
22 16 TT
21 734
13 18 13
20 T
AVERAGE
18 731
14 15 14 19
T
STANDARD DEVIATION
3.6 14.3
1.5 2.9 1.0 3.1
"
SIRAIN TA100
SOLVENT
110 MG
138
POS: 02
0.01 MG 1888
BRRC# t 47-205
0.09 MG 0.27 MG
131 119
1 MG
150
3 MG
143
10 MG
T
137 1950
123 121 164 132
T
119 1771
132 120 139 132
54 (T)
131 1870
129 120 151 136
T
10.7 90.9
4.9 1.0
12.5 6.4
i SIRAIN TA1535
SOLVENT
110 MG
43
39
44
POS: 02
0.01 MG
963
951
1057
BRRC#
0.09 MG
39
41
33
47-205
0.27 MG
35
48
46
1 MG
29
33
47
3 MG
46
53
39
10 MG
T
T
T
42 990
38 43
36
46 T
2.6 58.0
4.2 7.0
9.5 7.0
--
SIRAIN TA1537
SOLVENT
110 MG
1 POS: 03
0.06 MG
BRRC#
0.09 MG
1 47-205
0.27 MG
J 1 MG
I 3 MG
10 MG
!
, STRAIN TA1538
SOLVENT
110 MG
1 POS: 01
0.01 MG
1 BRRC#
0.09 MG
47-205
0.27 MG
i 1 MG
3 MG
10 MG
5 67
1 5 3 3 T
5 666
5 6 5 10 T
1 88
2 2 1 5 T
7 983
4 5 3 2 T
7 93
6 3 7 3 T
7 872
9 6 7 13 T
T: TOXIC 1i POS: POSITIVE CONTROLS [ 01: 4-NITRO-O-PHENYLENEDIAMINE
' 02: SODIUM AZIDE
4 3.1 83 13.8
3 2.6 3 1.5 4 3.1 4 1.2 T
6 1.2 840 160.9
6 2.6 6 0.6 5 2.0 8 5.7 T
03: 9-AMINOACRIDINE 04: 2-AMINOANTHRACENE
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TABLE 2 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY
Report 47-171 Page 8
CHEMICAL: BRRC #47-205, Epoxy Resin ERL-4234
WITH ACTIVATION
EXPERIMENT DATE: October 16 through October 19, 1984 SOLVENT: DMSO
TEST
DOSE
AGENT
PER
PLATE
STRAIN TA98
SOLVENT
110 MG
POS: 04
0.01 MG
BRRC#
0.1 MG
47-205
0.3 MG
1 MG
3 MG 10 MG
PLATE COUNTS
12
3
15 1106
. 17 26 26
30 T
28 1160
29 27 19
27 T
27 1478
15 22
17
21 T
AVERAGE
23 1248
20 25 21 26
T
STANDARD
deviation
7.2 201.0
7.6 2.6 4.7 4.6
--
STRAIN TA100
SOLVENT
110 MG
106
80
113
100
17.4
POS: 04
0.01 MG
812
824
1009
882
110.4
BRRC#
0.1 MG
108
110
79
99
17.3
47-205
0.3 MG 93 111
110
105
10.1
1 MG
105
117
96
106
10.5
3 MG 97 110
86
98
12.0
10 MG
T
T
T
T
*
STRAIN TA1535
SOLVENT
110 MG
14
13
21
16
4.4
POS: 04
0.01 MG
82
84
58
75
14.5
BRRC#
0.1 MG
10
6
9
8
2.1
47-205
0.3 MG
12
13
12
12
0.6
1 MG 9 11
15
12
3.1
3 MG 9 14
17
13
4.0
10 MG
T
T
T
T
*
STRAIN TA1537
SOLVENT
110 MG
9
5
9
8
2.3
POS: 04
0.01 MG
105
104
86
98
10.7
BRRC#
0.1 MG
5
4
8
6
2.1
47-205
0.3 MG
5
6
6
6
0.6
1 MG
5
5
8
6
1.7
3 MG
6
6
2
5
2.3
10 MG
T
T
T
T
mm
STRAIN TA1538
SOLVENT
110 MG
POS: 04
0.01 MG
BRRC#
0.1 MG
47-205
0.3 MG
1 MG
3 MG
10 MG
17 423
9 21 19
13 T
21 403
19
8 13
19 T
17 250
8
9 17
7 T
18 359
12 13 17
13 T
2.3 94.6
6.1 7.2
3.5 6.0
T: TOXIC POS: POSITIVE CONTROLS
01: 4-NITRO-O-PHENYLENEDIAMINE
02: SODIUM A2IDE
03: 9-AMINOACRIDINE 04: 2-AMINOANTHRACENE
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BUSHY RUN RESEARCH CENTER
R. D. 4, Mellon Read. Export, Pennsylvania 19632
Telephone (412) 733-5200
Good Laboratory Practices Compliance
This study was conducted In accordance with the D.S. Environmental Protection Agency's TSCA Good Laboratory Practices (GLP) Standards (40 CFR Part 792, effective December 29, 1983) with the following deviations;
1. The sponsor Indicated that Epoxy Resin BRL-4234 was soluble and stable in dlmethylsulfoxlde and no additional analyses of stability In the test system were performed*
2. The content of the test substance and control mixtures were documented and analyzed by weight or by volume, and no additional chemical analyses were performed.
In the opinion of the study director, these deviations do not affect the validity or reliability of this study.
4 Prepared by;
W. Christophe, Study Direct
WPC/rkk /0604B-1 11-01-84
Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation
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BUSHY RUN RESEARCH
R. D. 4, Mellon Road. Export, Pennsylvania 15632
CENTER
Telephone (412) 73X5200
Quality Assurance Unit Study Inspection Summary BAKELITE Cycloaliphatic Epoxy
Test Substance: Regln ERL_4234
Study: Ames Bacterial Mutagenicity Assay
Study Director: W. C. Hengler, M.S., M.B.A.
The Quality Assurance Unit of BRRC conducted the inspections listed below and reported the results to the study director and to management on the dates indicated. It is the practice of this Quality Assurance Unit to report the results of each inspection to both the study director and management.
Inspection
Date
Type
1-31-84 Standard Protocol
*
10-1-84 Standard Protocol Form
10-16-84 Event-Preparing Agar
1-10-85 Final Data and Final Report
Date QAU Report Issued To Studv Director To Manager
2-2-84
2-3-84
10-2-84
10-5-84
10-16-84
11-28-84
1-14-85
1-16-85
Buehy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation
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