Document Z4Ok3K6d1ygmyM9r3p2xBNKJV
I BUSHY RUN RESEARCH CENTER
R. D. 4, Mellon Road, Export, Pennsylvania 15632
CONFIDENTIAL: Not to be released outside uuTwithout the written
consent of the UCC-sponsoring Division HS&EA Manager.
Project Report 47-171 10 Pages January 16, 1985
BAKELITE* Cycloaliphatic Epoxy Resin ERL-4234 Salmonella/Mlcrosomc (Ames) Bacterial Mutagenicity Assay
Sponsor: Union Carbide Corporation Specialty PolymerB and Composl
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Summary
as '* 5
BAXELITE* Cycloaliphatic Epoxy Resin ERL-4234 was tested fo: sutagenlc activity using the Salmonella/mlcrosome bacterial mutag (Ames test). Epoxy Resin ERL-4234 was tested with and without me activation In triplicate at five concentrations, ranging from 0.1 to 10 mllll- grams per plate. The highest concentration was cyto' sutageniclty tests and in a preliminary test to choose approprlat Mutagenic activity was not observed with any of the five bacteria! tested with or without metabolic activation. Epoxy Resin ERL-423 sutagenlc In this screening test.
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Sample
Chemical Name: BAKELITE* Cycloaliphatic Epoxy Resin ERL-4234 I.D, #: T F3-98461 4193 WB BRRC Sample I: 47-205 BRRC Account #: 84-18-18046-46 CAS #: 3388-03-02 Chemical Synonyms: ARALDITE CY175; UP612; 2-(3,4-epoxy)cyclohexylmethyl-
5,5-spiro(3,4-epoxy)cyclohexane-m-dloxane Molecular Formula: C25H22O4
Submitted by: R. J. Cotter, Bound Brook, NJ
Sponsor: Division:
R. E. Pievan, Danbury, CT Specialty Polymers and Composites Bound Br k, NJ
Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation
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I BUSHY RUN RESEARCH CENTER
R. 0. 4. Mellon Road, Export, Pennsylvania 15632
Telephone M12) 733 5200
CONFIDENTIAL: Not to be released outside UCC without the written
consent of the UCC-sponsorlng Division HS&EA Manager.
.Project Report 47-171 10 Pages January 16, 1985
BAKELITE* Cycloaliphatic Epoxy Resin ERL-4234 Salmonella/Mlcrosome (Ames) Bacterial Mutagenicity Assay
Sponsor: Union Carbide Corporation Specialty Polymers and Composites Division
Summary
BAKELITE* Cycloaliphatic Epoxy Resin IR.L-4234 was tested for potential autagenlc activity using the Salmonella/mlcrosome bacterial mutagenicity assay (Ames test). Epoxy Resin HtL-4234 was tested with and vlthout metabolic activation in triplicate at five concentrations, ranging from 0.1 milligrams to 10 milll- grams per plate. The highest concentration was cytotoxic In the sutagenlclty tests and In a preliminary test to choose appropriate doses. Mutagenic activity was not observed with any of the five bacterial strains tested with or without metabolic activation. Epoxy Resin B&-4234 was not mutagenic In this screening test.
Sample
Chemical Name: BAKELITE* Cycloaliphatic Epoxy Resin ERL-4234 I.D. #: T F3-98461 4193 WB BRRC Sample #: 47-205 BRRC Account #: 84-18-18046-46 CAS #: 3388-03-02 Chemical Synonyms: ARALDITE CY175; UP612; 2-(3,4-epoxy)cyclohexylmethyl-
5,5-spiro(3,4-epoxy)cyclohexane-m-dioxane
Molecular Formula: 15^22^4
Submitted by: R. J. Cotter, Bound Brook, NJ
Sponsor: Division:
R. E. Pievan, Danbury, CT Specialty Polymers and Composites Bound Brook, NJ
Bushy Run Research Center A Joint Mellon Institute --Union Carbide Corporation Operation
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Report 47-171 Page 2
\
BAKELITE* Cycloaliphatic Epoxy Resin ERL-4234
Salmonella/Mlcrosome (Ames)
Bacterial Mutagenicity Assay
*****
I. Introduction and Theory
The Salmonella typhimurlua/mlcrosome mutation 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 typhimurium. 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 (his+) 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 supresseB 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 his+ can be quantititated 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 hiBtldine.
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) because 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 chemicals 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.
Report 47-171 Page 3
II. Salmonella typhlmurlum Mutagenicity Assay
A. Objective
The objective was to assess the mutagenic potential of Epoxy Resin HtL-4234 as measured In the Salmonella typhimurlum/ 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: BAXELITE* Cycloaliphatic Epoxy Resin ERL-4234 2. BRRC Sample Number: 47-205 3. CAS Number: 3388-03-2 4. Purity: > 95%; 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: dimethylsulfoxide, Burdick & Jackson, Muskegon, Ml; Lot # AD912; CAS # 67-68-5
2. Positive Controls: a) 4-nitro-o-phenylenediamine; BRRC #44-71; CAS #99-56-9 b) 9-amlnoacridine; BRRC #44-233; CAS #90-45-9 c) sodium azide; BRRC #44-72; CAS #26628-22-8 d) 2-amlnoanthracene; BRRC #44-67; CAS #613-13-8
D. Metabolic Activation
S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0.5 ml of S9 mix containing 50 yl 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.
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Report 47-171 Page 4
Test Dates
1. Test Initiated: October 15. 1984 2. Test Completed: October 19, 1984
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 dimethylsulfoxlde (DMSO) to a concentration of 100 mg/ml for doses of 10 mg/plate and below. The mixture was heated to 50*C 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 gravlmetrlcally analyzed.
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 dose, 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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Report 47-171 Page 5
III. Results
Epoxy Resin ELL-4234 was tested In a preliminary toxicity screen at 30, 10, 3, I* 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 milligrams per plate with strain TA100 only. The test chemical was treated as a solid because of Its high viscosity. The top 2 doses of 30 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, sutagenlclty testing was done with 5 doses of 10, 3, 1, 0.3, and 0.1 allllgrams per plate In triplicate. The highest dose was expected to produce lome 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. typhlmurlum, August 1982).
Gravimetric analysis of the highest test substance dilutions for each sutagenlclty test indicated no more than a 1.32 error from the stated concentrations. Gravimetric analysis of the lowest two dilutions In the test vlthout metabolic activation Indicated errors > 102 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 9.42 error from the stated concentrations. All subsequent dilutions for the test with metabolic activation had no more than a 1.32 error from the stated concentrations.
All plate counts and the respective means and standard deviations are Bhown 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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Report 47-171 .3165
Page 7 TABLE 1 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY
' ' * T*
CHEMICAL: BRRC #47-205, Epoxy Resin ERL-4234
WITHOUT ACTIVATION
EXPDUMENT DATE: October 15 through October 18, 1984 SOLVENT : DMSO
TEST DOSE
AGENT
PER PLATE
SIRAIN TA98
SOLVENT
110 MG
POS: 01
0.01 MG
BRRC# 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
--
SIXAIN 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 SIXAIN 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 10 MG
46 T
53 T
39 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
5
1
7
4
3.1
I POS: 03
0.06 MG
67
88
93
83
13.8
BRRC#
0.09 MG
1
2
6
3
2.6
47-205
0.27 MG
5
2
3
3
1.5
1 MG
3
1
7
4
3.1
3 MG
3
5
3
4
1.2
10 MG
T
T
T
T
! ______
, SIXAIN TA1538
SOLVENT
110 MG
5
7
7
6
1.2
1 MS: 01
0.01 MG
666
983
872
840
160.9
1 RKC#
0.09 MG
5
4
9
6
2.6
47-205
0.27 MG
6
5
6
6
0.6
i
1 MG
5
3
7
5
2.0
3 MG
10
2
13
8
5.7
10 MG
T
T
T
T
4.
T: TOXIC
POS: POSITIVE CONTROLS
01: 4--NITRO-O-PHENYLENEDIAMINE
03: 9-AMINOACRIDINE
02: SODIUM AZIDE
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 POS: 04
110 MG 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
no
105
10.1
1 MG
105
117
96
106
10.5
3 MG 97 no
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
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 AZIDE
03: 9-AMINOACRIDINE 04: 2-AMINOANTHRACENE
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BUSHY RUN RESEARCH
R. 0. 4, Mellon Road, Export. Pennsylvania 16632
CENTER
Telephone <4i2) 733-5200
Good Laboratory Practices Compliance
This study was conducted In accordance with the U.S. Environmental Protection Agency's TSCA Good Laboratory Practices (GLF) Standards (40 CFR Part 792, effective December 29, 1983) with the following deviations:
1. The sponsor indicated that Epoxy Resin ER-L-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.
Prepared by:
VPC/rkk/0604B-l 11-01-84
Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation
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Report 47-171 Page 10
BUSHY RUN RESEARCH
R. 0. 4, Mellon Road. Export, Pennsylvania 15632
CENTER
Telephone (412) 733-5200
Quality Assurance Unit Study Inspection Summary
,, ,.
BAKELITE Cycloaliphatic Epoxy
Test Substance: _Res.in 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 Study Director To Manaeene
2-2-84
2-3-84
10-2-84
10-5-84
10-16-84
11-28-84
1-14-85
1-16-85
('ILa : t, ijult?
L. 'J. Calisti, oup Leader
Date
Good Laboratory Practices/Quality Assurance
Bushy Run Research Center A Joint Mellon Institute--Union Carbide Corporation Operation
UC^ 062488
Report 47-171 Page 1 of 4
APPENDIX 1
Standard Procedures for the Salmonella/mlcroBome Mutagenicity Assay
A. Introduction
The strains used In this test are all Salmonella typhimurlum histidine iuxotroph6 developed by Dr. Bruce Ames of the University of California at Berkeley. The test Is designed to assay for reverse mutations In the histidine locus by plating the bacteria on minimal agar with only a trace of histidine. Bacteria that have undergone a reverse mutation In this locus can form colonies In minimal agar and are counted as mutants. The trace of histidine in the einimal agar allows the bacteria to undergo a small number of cell divisions which then form the background lawn, visible as a cloudiness In the agar. Observation of the lawn permits an estimate of toxicity. Testing with growing cultures also allows potential mutagens to act on replicating DNA which Is often a more sensitive test for several classes of mutagens.
B. Indicator Organisms
Salmonella typhlmurlum strains TA98, TA100, TA1535, TA1537, and TA1538 are used in this assay. They all lack the excision repair mechanism, which Increases their sensitivity to potential mutagens. They also have a mutation affecting their llpopolysaccharlde layer which allows large molecules to enter the bacteria more easily. Strains TA98 and TA100 were derived from strains 141538 and TA1535 by the addition of an R factor which Imparts amplcillln resistance and also Increases sensitivity to several classes of mutagens.
Indicator organisms are stored at -80*C. Working cultures are prepared losthly by Inoculating nutrient broth from the frozen cultures and Incubating vlth agitation overnight. Bacteria are then plated onto Vogel-Bonner Medium E igax plates (master plates) with an excess of histidine and biotin (required because of the llpopolysaccharlde deficiency). After Incubation for 24 hours, the strains are checked for their genetic markers to verify their Identity and Pwity (SOP #7.4.2A).
For testing, the broth cultures are prepared by Inoculating from the Mister plates into nutrient broth and Incubated overnight with agitation. The troth cultures are then kept on Ice during the day of testing. Fresh cultures ue sade each day of testing.
4 Toxicity Testing
Sterile tubes are prepared containing 2 ml soft agar (6 g/1 agar and 5 g/l kCl) with a final concentration of 0.05 mM L-hlstldlne and 0.05 mM D-biotln (the entire mixture Is called top agar). Dilutions of the test chemical are kde In an appropriate solvent so that the correct amount of chemical can be dded to each tube In 100 pi amounts. If less than 100 pi amounts of the fett chemical Is used, phosphate-buffered saline Is used to adjust the total *l<a>e to 100 pi.
Report 47-171 Appendix 1 Page 2 of 4
At least five doses are tested with maximum doses of 50 mg (for solids) or 100 yl (for liquids) per plate for nontoxic chemicals, unless limited by solubility. Generally, a chemical cannot be considered to be nonmutagenlc unless at least 5 mg/plate has been tested, unless limited by toxicity or solubility (deSerres and Shelby, 1979; HG-Gene Muta-S. typhimurlum, August 1982). A 100 yl aliquot of an overnight broth culture of strain TA100 Is added followed by the appropriate amount of test chemical. The mixture Is vortexed and poured onto the surface of a Vogel-Bonner Medium E agar plate (VB-E plate). The top agar Is allowed to harden and the plates are Incubated at 37*C for 24 to 48 hours. The plates are examined for the condition of their background lawns and growth Is recorded as either confluent, sparse, or absent. Confluency Is considered an Indication of nontoxlclty, sparse growth Indicates moderate toxicity, and lack of growth Is recorded as toxic.
D. Dose Selection
For the mutagenicity test, a minimum of five half-log doBes are tested within a range of moderately toxic to relatively nontoxic doses based on the results of the toxicity test. If all doses in the toxicity test were nontoxic, then a maximum dose of 50 mg or 100 yl is used In the mutagenicity test, unless limited by solubility.
E. Controls
Concurrent solvent and positive controls are run with each test. 3he solvent control Is the maximum amount of solvent added with the diluted test chemical. For chemicals tested without dilution (l.e., by direct addition), water is used as the solvent control. Both activation-dependent and activation-independent positive controls are used. The activation-independent controls are 4-nitro-o-phenylenediamine for TA98 and TA1538, sodium azide for TA100 and TA1535, and 9-amlnoacrldine for TA1537. The activation-dependent control In 2-aminoanthracene (2-anthramlne) for all strains. The concentrations are determined from prior dose-response experiments on these chemicals and are listed In the tables of results In this report.
F. Metabolic Activation
Each lot of Aroclor-1254 induced, rat-liver homogenate (S9) is prescreened for activity with 7,12-dimethylbenzanthracene to determine the appropriate amount to use for testing. The S9 Is added to the "S9 mix" which contains, per ml, S9 (50-250 yl), MgCl (8 imoles), KC1 (33 imoles), glucose-6phosphate (5 ymoles), NADP (4 ymoles), and sodium phosphate, pH7.4 (100 imoles). S9 mix Is prepared fresh each day of testing and kept at 0-4*C.
G. Test Chemicals and Solvents
The solvents of choice are water, DMSO or ethanol. A minimum of five concentrations of the test chemical are diluted in the appropriate solvent and tested. The chemical Is diluted so that 100 yl will deliver the required
11 nr 062^)0
Report 47-171 Appendix 1 Page 3 of 4
3171
dose* If smaller volumes are used, phosphate-buffered saline Is added to adjust the total volume to 100 yl. Test chemical solutions are prepared on the day of testing and are gravlmetrically analysed. If the analysis Indicates > 102 error from the Intended concentration, then either the dilution Is repeated or the actual concentration Is stated In the report.
B. Dosing
To a sterile tube containing 2 ml of top agar, 100 yl aliquot of the appropriate bacterial culture is added followed by the addition of 100 yl of the appropriate solvent, control, or test chemical solution. Either 0.5 ml of S9 mix or 0.5 ml of phosphate-buffered saline (PBS) is added for tests with and vlthout metabolic activation, respectively. The top agar mixture is then poured onto a VB-E plate. Each dose is tested in triplicate and with all five bacterial strains. Sterility checks are done on the PBS and/or S9 mix, all solvents, and the highest concentration of each test chemical. The plates are transferred to a darkened 37*C Incubator after hardening and Incubated for 48-72 hours.
I. Data Collection
An Artek Model #880 Colony Counter is used to count bacterial colonies or handcounts of the plates are done. The counter is calibrated for each test to check the counting accuracy (SOP I7.4.5A). The numbers of colonies per plate are counted and recorded. An examination is also made of the background lawn on each plate. If toxicity is observed as an inhibition of growth of the background lawn, the plate Is not counted, but Is recorded as toxic. If the background lawn Is sparse and the colony count is still recorded, that number la not used In the calculation of the mean number of colonies and its standard deviation.
J. Criteria for Test Validity and for Interpretation of Results
The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced In a second test, the test result will be considered negative. If there is no evidence of a dose-related Increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.
X. Data Storage and Retrieval
Copies of the final report, statistical analyses, analytical data and raw data are stored in the BRRC Archives.
3172
.Report 47-171 Appendix 1
Page 4 of 4
References
1. Ames, B.N., J. McCann and F. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammallan-mlcrosome mutagenicity test. Mutation Research, 31: 347-364 (1975).
2. Vogel, H.J., and D.M. Bonner, Acetylornithase of Escherichia coll: partial purification and some properties, Journal of Biological Chemistry, 218: 97-106 (1956).
3. deSerres, F.D., and M.D. Shelby, Recommendations on data production and analysis UBing the Salmonella/microBome mutagenicity assay, Mutation Research, 64: 159-165 (1979).
4. Environmental Protection Agency, Health Effects Test Guidelines, HG-Gene Muta-. typlmurlum, EPA Report No. 560/6-82-001, August, 1982.
WPC/rkk/0604B-l 10-24-84
APPENDIX 2
REOOESTFOh'STANbXkb'tOxicOLOGY'iitubjSj
[ ; W: At BUSH'y;rUN RESEARCH CENTEBffifK*'j
U*tO* I
jCARBiDC
Report 47-171 Page 1 of 9
f. BUSINESS na , confident!*?-1- y3
Mr. R.C. Myers Bushy Run Research Center RD 4 Mellon Road Export, PA t5632
tram: Name Sponsoring niui.xinnSP^GjiHsTY
Dept -------------------------------------------- ----------------location (city) Jklt//%IK .
Charge to Account No. 3-T'~' 5~9& ^tVVO---------------------
Technology Manager ijhoriyation; 71 j.
S*i-g--n-a- .tu--re
1
i/9lpy Dt
COMPLETE COPIES OF THIS FORM MUST BE SENT (BY THE SUBMITTERVTO:
A. Dr. Bryan Ballantyne Technical Center (3005) South Charleston, WV 25303
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B. Dr. W.C. Kuryla Technical Center (3005) Soulh Charleston, WV 25303
C. Dr. E.R. Homan
Bushy Run Research Center
RD 4 Mellon Road Export, PA 15632
'` J
D. Sponsoring Division HS A EA Manager
Name.
loc.:
STS REQUESTED (Standard procedures; other tests require specific protocols. Please contact the Corporate
Applied Toxicology Dept (or advice and guidance.)
R ECEIVED
' RANGE FINDING;
3 IN VITRO:
D Acute Oral P Acute Percutaneous D Acute Inhalation 0 Skin Irritancy D Eye Irritancy
O Other.
(tp*c*X)
IKNDWN PROCEDURAL REQUIREMENT (circle)
^ Ames (Salmonella) Test
AUG 2 4 i9d4
0 Gene Mutation (CHO)
w. C HLNIGI",
Chromosome Exchange (SCE)
Chromosome Damage (Micronucleus) DNA Damage (UDS)
u
0 Macrophage (RAM) test
:=CD, FMSA, DOT, FIFRA. TSCA. RCRA, other
0 Other
ADDUCT/COMPOSITION NAME: PfUr- VtS*/
;CS NOMENCLATURE.-^^
~f/ 3 ~/7)
(W-O)
J _rj.
^ 3S86-03-A
'cMICAL SYNONYMS/ALTERNATE/TRADE NAMES:
_
-------- ~cY~n?j a/'Vtz.y-fiTiX t-WtfyKtotte&y&TML.
~RUCTURAL FORMULA OR COMPOSITION OF MIXTURES:
Utu-0 UhL-O
O"
_____ ,, ^ -W-- QA/pvt- .
.STRlBUTlQN OF REPORT: A standard divisional distribution list will be used unless otherwise requested
(see page 4).
rec
MMBCUSE) teceiv rt*
ACKNOWLEDGEMENT OF SAMPLE RECEIPT
7-hfk*/ "
ay
^RCSamnla Nn v ? "
______________
v
3174 ? y>
| CHEMICAL INFORMATION J
Report 47-17 Appendix 2 Page 2 of 9
1. SAMPLE PURITY/COMPOSITION: Analytical test method employed* "
'(Send copy of available data, source and location of analyses.)
Percent Composition Amounts and identities of known impurities: Amount of unidentified impurities: -----------Test material:. %
------- AJxtZc/
3E -- - --
-E Ar
2. REQUIRED SAMPLE QUANTITY:
Acute Tests:
In vitro Tests: 4 ounces
1. 32 ounces for full series of tests
2. 16 ounces for chemicals with known high toxicity
3. 2 ounces for irritancy tests only
NOTE; To.reduce problems involving disposal of unused amounts of the sample, supply only the amount specified above.
3. PHYSICAL CHARACTERISTICS: (Give all which are known):
Gas
Liquid
_ SolidPowder. Granular
Molecular Wt. _
AU______________
Density (liquids) _____LJ_ Itrtir)______
pH (aqueous sol.) __ ______ _______________
Freezing point
O------------------------ C
Boiling point
^ 2. S> ~C__C
Vapor Pressure (@ 3 temp, between 15* and 1S0*C);
1. *~Q' 0 ly 'ifltn rnrn 0
.
"C
2.
mm @_ _ _ _ _ _ _
- "C
3.
mm @ _ -
*C
Flash point _(& C
(Tag, closed cup)
4. SOLUBILITY: Solvent
Water _ Acetone Ethanol __ Dimethyisulfoxide-- Vegetable Oil Other (specify)
Percent Solution
5. STABILITY: (Check appropriately):
Shelf Life Light sensitive Temp sensitiue Oxidize* _ Polymerizes^
< 6 mo; . Z - >6 mo. ves: i/ ^ no
ves; .no
ves: J -- no
i/ ves:
.no
Known solvent Incompatibility (specify)
* &L r*ii
IJCC 062494
47-itj*
2 of 9 -
pANDUNG/STORAGE/D<SPOSAUNFORMATtON:|
Report Appendix 2 * *' Page 3 of 9
3175
rtrOMMENDED PRECAUTIONS FOR HANDLING*:
fcJM f&rt S/U/J AfJ2>
^netpM UCC (or other company) Material Safety Data Sheet H available.
Any unusual fire or explosion hazards: ___ LQL_____________
My chemical incompatibilities (materials to avoid):
Ac/pf &*Jj)
': SPECIAL PERSONNEL PROTECTIVE EQUIPMENT NEEDED: *JAf6&L
^COMMENDED INACTIVATION/DECONTAMINATION PROCEDURE(S): MAfti GCfilfO /fcMf
'C ! MA H&-
.MATSrt
;ECOMVIENDED STORAGE CONDITIONS:
Tenperature (Check appropriately);
torn Temp
3. Wrigerate
0fueze
0 lm
1/
c ?nc <r nc
j*WPLE DISPOSAL: (Check appropriately):
0 Return unused sample to:
(attach DOT shipping instructions)
Special Conditions Reauired (Check appropriately)
Under Nr gas Deaairatnr Other
^Disposal by BRRC is authorized in compliance with applicable regulations.
UCC 062495
j)THER PERTINENT INFORMATION]
Report 47-17J Appendix "J Page 4 of 9
I
The following information will be useful in determining or confirming the necessity of specific toxicity tests:
1. Proposed use of material (circle):
Qndustriap Intermediate
Home Use
Experimental
Industrial Product
Food
Drug
Cosmetic Packaging (specify Food, Drug, Cosmetic)
Other use (specify): ------------------- ------------- ------------------------------------------------------------------------------
2. Potential hazard route(s) (circle): Other (specify): -----------------------
Inhalation
3. Form shipped in commerce (circle): Solvent or Vehicle composition: --
Solid
iquidy Gas
Suspension or Slurry
ATTACHMENTS (TO BE PROVIDED BY SUBMITTER): * - Analytical data for sample. * - Material Safety Data Sheet of sample material (if available). * - DOT shipping information (if applicable).
DISTRIBUTION LIST: Unless indicated below, the standard distribution list for the sponsoring division will be used.
D Please use the following divisional distribution list lor the final study report:
i
WCK/Mar. S3
IJCC 062496
MATERIAL SAFETY DATA SHEET
EFFECTIVE DATE: Oecember 1.1881
Report 47-171 ^
ZiTJ, air?
PRODUCT NAME: BAKELITE Cydoaliphatie Epoxy Resin ERL4234
SS5U.NAM6:
Formula:
CiHhQ4
W,yl CHEMICAL FAMILY:
~ MOLECULAR WEIGHT:
266.33
SYNONYMS
3-(3,4-epoxycydohexyl)-8, 9-Eposy-2,4-Dioxaspire (6, 6] undecane
DEPARTMENT of TRANSPORTATION
HAZARD CLASSIFICATION SHIPPING NAME
None None
CAS | 3388-03-2
CAS NAME Spiro[1,3>dioxane-5,3*-[7]oxabicydo[4.1.0] heptane], 2-(7-oxabicydo[4.1.0]hept-3-yl)-
' r
SOILING POINT, 760 mm Hg
>250*CO482,F)
POUR POINT
15 *C
SPECIFIC GRAVITY (H.0 1)
VAPOR DENSITY (air - 1)
PER CENT VOLATILES BY VOLUME APPEARANCE AND ODOR
1.1891 at 20/20 C
VAPOR PRESSURE at 20*C
< 0.01 mm Hg
9.2
SOLUBILITY IN WATER, % by wt. at 25 #C
4.2
Nil
EVAPORATION RATE
<0.01
(Butyl Acetate 1)
High viscosity liquid; faint, burnt odor. '**-- .-- . .-.z- -TewexraweyMwr^r^v - -
BAKELITE Cycloaliphatic Epoxy Resin ERL4234
100
TLV (Units)
Not established
HAZARD
Carcinogenic in laboratory animals. Irritant.
Smi AND"feXPLOSlON HAZARD DAIVr^ -; - ' ' ;
FLASH POINT (lest melhod(s)]
368 F, Pensky-Martens dosed cup 410 F, Cleveland open cup
FLAMMABLE LIMITS IN AIR, % by volume
LOWER
jN^^fiiXttri.lJ
UPPER
(NomMaKdSrial)
extinguishing
MEDIA
Use water sprey, carbon dioxide, dry chemical, alcohol-type or universal-type foams applied by manufacturer's recommended technique.
SPECIAL FIRE FIGHTING
procedures
Don't spray pool fires directly; a solid stream of water directed Into hot burning liquid can cause frothing. Use supplied breathing air and full protective dothing.
unusual fire AND
explosion hazards
None
304/744-3487
Thu number is twldblt davt* nsflhts, weekends, bndhoiidtvS-
Union CdfbidB Corporotion btUdvdB tnw tho dot* contained horom * foelwci and tf* opmiont diprdiMd #ft these of Qu*(t*o oiportc rooordmg tht rtsuiis of ft* tests
Mdeeted. the dta ere not to be token m 0 wertenty or rogrOBontotion tor which Union Carbide Corporet ion essumss 1*901 roopontib'Hty They er offered soteiy for your considereiion
k~t ------ -- - ***-- a.. -- .k*.***.. kU
bs deiermmed by the ueer tobemoccordonc* withoppticeb^e^eoerei.&iete.end ioc*i tewsenoieguieuons
0^97
3*78
' .4 >
Report 47-171 Appendix 2 Page 6 of 9
TLV AND SOURCE:
None established by ACGIH or OSHA.
ACUTE EFFECTS OF OVEREXPOSURE SWALLOWING
May cause nausea, vomiting, and diarrhea.
SKIN ABSORPTION
None currently known.
INHALATION
SKIN CONTACT
EYE CONTACT
CHRONIC EFFECTS OF OVEREXPOSURE
None currently known.
May cause slight redness for a short period of time.
Causes irritation. Causes skin cancer in mice in lifetime skin painting studies. Skin contact and breathing of vapor should be avoided.
OTHER HEALTH HAZARDS
None currently known.
EMERGENCY AND FIRST AID PROCEDURES: SWALLOWING SKIN INHALATION EYES
Give two glasses of water and induce vomiting by putting finger down throat.
Remove contaminated clothing and wash skin with plenty of soap and water. Launder clothing before reuse.
Remove to fresh air.
Immediately flush eyes with plenty of water for at least 15 minutes. Call a physician if discomfort persists.
NOTES TO PHYSICIAN
There is no specific antidote. Toxicology studies have shown the material to be of low acute toxicity. Treatment of overexposure should, therefore, be directed at the control of symptoms and the clinical condition.
171 31Tb Report 47-171*' Appendix 2 Page 7 of 9
1 ) E3S2SS^E22ESSeacgyny PaSZS^
stability
UNSTABLE
STABLE
CONDITIONS TO AVOID
None
incompatibility
fniterials to avoid)
HAZARDOUS COMBUSTION OR decomposition PRODUCTS
HAZARDOUS POLYMERIZATION
May Occur
Will not Occur
Aminas, alkalies, acids, and alcohols. Burning can produce CO and/or C02.
CONDITIONS TO AVOID
Contamination by amines, alkalies, and acids.
STEPS TO BE TAKEN IP MATERIAL IS RELEASED OR SPILLED
VhTSPILLDR LEAK PROCEDURES
. .. -- - ^ i v '
Wear suitable protective equipment; avoid skin and eye contact. Collect for disposal.
WASTE DISPOSAL METHOD
Incinerate in a furnace where permitted under appropriate Federal, State, and local regulations.
w* Try.' _* *'
. SPEC|AL PROTECpON INFORMATION
RESPIRATORY PROTECTION (specify type)
Air-supplied mask as noted below.
Central (mechanical) room ventilation is satisfactory with embitnt temperature conditions. To minimise exposure to vapors
VENTILATION
during processing at elevated temperatures, additional precautions should be taken, such as the use of a local ventilation system or air-supplied mask. Sine* this product has been shown to be a weak animal carcinogen in skin painting studies, exposure to skin
-------------------------- ----AbPvJdJ&iyaideEUhrough tbs us* ofgloves and other protective clothing, as appropriate.
PROTECTIVE GLOVES
P,Mtic
I eye
J PROTECTION
Safety goggles
OTHER PROTECTIVE EQUIPMENT
Eye bath and safety shower
m,m a *
' ' W. ,, . . > ' -a... ....
1/V- AJf ^ ... ^
W
| ,,
PRECAUTIONS TO BE TAKEN IN HANDLING AND STORING
WARNING:
Suspect Cancer Agent. Do not get in eyes, on skin, or on clothing. Avoid breathing vapor.
Keep container closed. Wash thoroughly after handling.
-------
T
I OTHER PRECAUTIONS
FOR INDUSTRY USE ONLY
This product is resistant to rapid biodegradation; void discharge to natural waterways.
uai-sm
Printed in U-* A.
ucc
062499
3180
UNION CARBIDE CORPORATION
specialty pcxvmeps & composites aveoY
RECEIVED
SEP 121984
W-O HENGLER
Report 47 Appendix .
Page 8 of
P.D. BOX 670, BOUND BROOK. Nj near telephone ffioij 356-eoc
September 6, 1984
Mr. W. C. Hengler Bushy Run Technical Center RD4, Mellon Road Export, Pennsylvania 15632
Dear Mr. Hengler:
Infrared spectra have been run on the samples of cycloaliphatic epoxide that you returned to me recently. The spectra are enclosed for your files. The spectra that were obtained on these samples were compared with standard spectre for these products. In two cases, 4205 and 4299, comparisons were made with ot samples of the these products that are being utilised in current laboratory progrt
Comparison of these various spectra by the infrared experts in our Analytical Group (Dr. Clark and Mr. Lewis) led to the conclusion that the sample that you have are authentic samples of the UCC cycloaliphatic epoxides. Thus, th< mutagenicity testing will be performed on valid samples of 4221, 4299, 4234, 4206 and 4205.
ends.
Very truly yours.
RJCtmjk cc:Dr. E. M. Clark, Mr. J. Lewis, Dr. R. E. Plevan - DB
ttica.ttiaM ilu
o
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go oO
*rs *>o 5(Tdf
09 *0 T3
aft
ft
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h
v CL rt
O X sj VO to r
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3182
Report 47-171 Page 1 of 1
APPENDIX 3
Salmonella typhlmurlum
A. Historical Data on Spontaneous Reversion Rates at BRRC As of June 30, 1984
Strain TA98
+/-S9
-S9 +S9
N
98 98
Mean
IO 25.6
Standard Deviation
5.02 7.40
Median --------- jy-
26
Range
7. 33 14, 44
TA100
-S9 98 123.1 +S9 98 101.5
26.89 17.86
120 61, 187 100 65, 137
TA1535 -S9 98 32.5 +S9 98 13.4
TA1537 -S9 98 4.3 +S9 98 5.0
TA1538 -S9 98 7.2 +S9 98 15.0
13.61 7.34
1.90 2.61
3.72 5.41
32 7, 60 14 2, 35
4 1, 9 5 1, 13
7 2, 19 16 4, 29
Strain
B. Historical Data on Postlve Control Values at BRRC As of January 26, 1984
Positive Control
+/-S9
N
Mean
Standard Deviation Median
Range
TA98 NPD,10 pg -S9 84 877.1 166.73
870
2AA.10 pg +S9 84 1434.2 327.54 1457
532, 1301 604, 2037
TA100 NZ, 10 pg -S9 84 1621.8 277.37 1696 2AA.10 pg +S9 84 1181.7 323.54 1201
869, 2069 475, 2225
TA1535 NZ, 10 pg -S9 84 1622.0 253.42 1666
2AA.10 pg +S9 64
99.3
30.84
98
837, 2050 37, 182
TA1537 9AA,60 pg -S9 84 140.6 130.70 2AA,10 pg +S9 84 122.5 45.52
82 120
28, 489 36, 230
TA1538 NPD.10 pg -S9 84 1124.2 196.66 1168
2AA.10 pg +S9 64 532.0 230.61
466
573, 1582 146, 1249
$9- rat liver homog;enate; NPD- 4-ndLtrophenyl.enedlamine; .2AA- 2-am:Inoanthracene; NZ- sodium aside; 9AA- 9-amlnoacrldlne.
WPC/rkk/0604B-l 10-24-84