Document pE6ey92O5L5xDoj9xkRQOvXd
I BUSHY RUN RESEARCH CENTER
R. D. 4, Mellon Road, Export, Pennsylvania 15632
Tolophono (412) 733-5200
T3NFIDENTIAI: Not to be released iotfide UCC~without the written
:onecnt of the UCC-aponsoring Hvision HS&EA Manager.
Project Import 47-182
10 Pagea February 4, 1985
BAKSLITE* Cycloaliphatic Epoxy Reain ERL-4221
Salmonella/Microaome (Ames)
Bacterial Mutagenicity Assay
Sponsor: R. E. Plevan, HS&EA Manager Union Carbide Corporation Specialty Polymers & Composites Division
Summary
Epoxy Resin ERL-4221 was tested for potential autagenlc activity using the tlaonella/alcrosome bacterial mutagenicity assay (Aaes test). Epoxy Resin Hl-4221 was tested with and without aetabollc activation in triplicate at tve concentrations, ranging froa 0.1 to 10 milligrams per plate. The highest oncentratlon was cytotoxic In the mutagenicity tests and In a preliminary eat to choose appropriate doses. Positive dose-related Increases of the umbers of revertant colonies above the solvent control level were observed nly with metabolic activation with the two bacterial strains, TA100 and A1535, that detect base-pair substitution mutagens. Epoxy Resin ERL-4221 was a active mutagen in this In vitro screening test.
Sample
Chemical Name: BAXELITE* Cycloaliphatic Epoxy Resin ERL-4221 l.D. #: TF3-98171 4124 RCP BRRC Sample #: 47-204
BRRC Account #: 84-18-18049-4# CAS #: 2386-87-0 Chemical Synonyms: 7-Oxablcydo(4.1.0]heptane-3-carboxyllc add.
7-oxabl cydo [ 4.1.0 ]hept-3-ylmethyl ester j 3,4-Epoxycydohexyl methyl-3,4-epoxy-cydohexyl carboxylstc
Molecular Formula; C24H20O4
ibmltted by: R. J. Cotter, Bound Brook, NJ
7onsor: -vision:
R. E. Plevan, Danbury, CT Specialty Polymers & Composites Bound Brook, NJ
Bushy Rue Rssssrch Center A Joint Mellon Institute--Union Carbide Corporation Operation
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Report 47-182 Page 2
BAKELITE* Cycloaliphatic Epoxy Resin fRI-4221 Salmonella/Mlcrosome (Ames? Bacterial Mutagenicity Attay
1. Introduction and Theory
The Salmonella typhlmurlum/mlcrosome mutation attay it a microbial tcreenlng tett which detecta the ability of chemlcala to cauce genetic alterations In the hietldlne gene of selected indicator atrains of Salmonella typhlmurlum. The Indicator ttralnt 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. Frameshift 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 quantltltated 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) 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.
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II, Salmonella typhlmurlum Mutagenicity Assay
A. Objective
The objective was to assess the mutagenic potential of Epoxy Resin ERL-4221 as measured In the Salmonella typhlmurlum/ microtome 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-4221 2. BRRC Sample Number: 47-204 3. CAS Number: 2386-87-0 4. Purity: > 952; IR spectra consistent with structure
(see Appendix 2) 5. Density: 1.175 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 6 Jackson, Muskegon, MI; Lot IAD912; CAS #67-68-5
2. Positive Controls; a) 4-nltro-o-phenylenediamlne; 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-amlnoanthracene; BRRC #44-67; CAS #613-13-8
D. Metabolic Activation
S9 liver homogenate, prepared from Arodor 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 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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F. Test Dates
1. Test Initiated: October 29, 1984 2. Test Completed: November 2, 1984
G. Storage location of Materials
1. Rav Data: BRRC Archives 2. Final Report: BRRC Archives
H. Experimental Design (detailed procedures in Appendix 1)
1. Sample Preparation: The test substance was dissolved In dlmethylsulfoxlde (DMSO) to a concentration of 100 mg/ml for doses of 10 mg/plate and below. 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 wsb performed both with and without metabolic activation. Concurrent solvent and positive controls were run In each test.
Z. 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 la audited by the Independent Quality Assurance department at the Bushy Run Research Center.
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HI. Results
Epoxy Resin DtL-4221 was tested In a preliminary toxicity screen at 117.5, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 milligrams per plate with strain TA100 only. The top three doses of 117.5, 30, and 10 milligrams per plate completely inhibited growth of the background lawn. Based on these results, mutagenicity testing was done with 5 doses of 10, 3, 1, 0.3, and 0.1 sllllgrams per plate in triplicate. The highest dose was expected to produce sooe 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 (HC Gene Muta-S. typhlmurlun, August 1982).
Gravimetric analysis of the Initial dilutions of the test substance for both tests Indicated no more than a 6.OX error from the stated concentration. All subsequent dilutions had no more than a 7.2Z 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 without metabolic activation, either by evidence of a dose-response relationship or a doubling of the number of colonies over the solvent control value. With metabolic activation, dose-related mutagenic activity was observed with strains TA100 and TA1535 which detect base-pair substitution mutagens. The ratios of the average numbers of revertant colonies to the average number observed in solvent control cultures increased with Increasing dose as follows: 1.0, 1.1, 1.4, and 2.5-fold Increases with TA100; and 1.6, 2.0, 5.7, and 15.3 with TA1535. No evidence of mutagenic activity was observed with strains TA98, TA1537, or TA1538.
All of the observed mutagenic effects of Epoxy Resin ERL-4221 appear to be base-pair substitutions following metabolic activation of the test agent* The mutagenic activity observed only with metabolic activation suggests the production of mutagenlcally active metabolites from the parent compound by the S9 activation system.
Pose 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, RBS, the test chemical and all solvents and controls were sterile.
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IV. Conclusion
Epoxy Reeln BLL-4221 produced a dose-dependent autagenlc response with tbe Salmonella typhimurlum strains IA100 and TA1535 ben tested with metabolic activation. No positive effects were observed In tests without a aetabollc activation aystea. Under tbe conditions of this assay* Epoxy Resin ERL-4221 was an active autagenlc In tbe Salnonella/microaome autagenldty assay.
Reviewed and Approved by:
w. Chrlstophey Hengle^, M*M.B.A. Study Director
/*. /er
Ronald S. Sleslnskl* Ph.D
Date
Manager* Genetic,Biochemical 4 Acute Toxicology
Fred R. Frank, Ph.D Director
Contributorsi Technical Assistance: Eileen R. Morablt* A.S
WPC/rkk/0619B-2 11-16-84
Date
UOC
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CHEMICAL* BRRC #47-204, Epoxy Resin ERL-4221 EXPERIMENT DATE: October 29 through Noveaber 1* 1984
WITHOUT ACTIVATION SOLVENT* DMSO
Test
dose
AGENT
FER PLATE
STRAIN TA98 SOLVENT
POS: 01 BRRC# 47-204
110 MG
0.01 MG 0.1 MG 0.3 MG 1 MG 3 MG 10 MG
PLATE COUNTS
12
3
22 29 819 953
28 35 27 18 27 22
18 18 TT
27 884
22 22 23
24 T
AVERAGE
26 885
28 22 24 20
T
STANDARD DEVIATION
3.6 67.0
6.5 4.5 2.6 3.5
STRAIN IA100 SOLVENT POS: 02
BRRC# 47-204
110 MG 0.01 MG
0.1 MG 0.3 MG
1 MG
3 MG 10 MG
134 2112
142
139 126
136 T
138 2340
131 128 143
121 T
129 2179
126 127 135 138
T
134 2210
133 131 135 132
T
4.5 117.2
8.2 6.7
8.5 9.3
w
STRAIN TA1535
SOLVENT
110 MG
POS: 02
0.01 MG
BRRC#
0.1 MG
47-204
0.3 MG
1 MG
3 MG
10 MG
46
2095 57
40 25
46 T
31 2383
43 38
50 35
T
28 2290
36 43 43
36 T
35 2256
45 40 39
39 T
9.6 147.0
10.7 2.5
12.9 6.1
STRAIN TA1537
SOLVENT
110 MG
4
7
4
5
1.7
POS: 03 0.06 MG 78 102
93
91
12.1
BRRC#
0:1 MG
7
5
4
5
1.5
47-204
0.3 MG
5
6
3
5
1.5
1 MG 6 10
4
7
3.1
3 MG
9
1
7
6
4.2
10 MG
T
T
T
T
**
STRAIN TA1538
SOLVENT POS: 01
110 MG 0.01 MG
BRRC#
0.1 MG
47-204
0.3 MG 1 MG
3 MG 10 MG
10 1016
9
8 5
9 T
7 1023
17
6 4
5 T
9 1058
10
8 6
6 T
9 1032
12 7
5
7 T
1.5 22.5
4.4 1.2
1.0
2.1
UCC 063005
T: TOXIC POS* POSITIVE CONTROLS
01* 4-NITRQ-O-PHENYLENEDIAMINE
02: SODIUM AZIDE
03: 9-AMINOACRIDINE 04: 2-AMINOANTHRACENE
3330
TABLE 2 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY
Report 47-182 Peg 8
CHEMICAL: BRRC #47-204, Epoxy Resin ERL-4221 EXPERIMENT BATE: October 30 through November 2, 1984
WITH ACTIVATION SOLVENT: DMSO
TEST AGENT
BOSE PER PLATE
STRAIN TA98
SOLVENT
110 MG
POS: 04 0.01 MG
BRRC#
0.1 MG
47-204
0.3 MG
1 KG
3 MG
10 MG
PLATE COUNTS
12
3
20 1314
20 20 28 21
T
22 1342
21 16 29 39
T
22 1614
18 17 31 22
T
AVERAGE
21 1490
20 18 29 27
T
STANDARD DEVIATION
1.2 280.9
1.5 2.1 1.5 10.1
--
STRAIN TA100 SOLVENT POS: 04 BRRC# 47-204
110 MG 0.01 MG
0.1 MG 0.3 MG
1 MG
3 MG 10 MG
113 1268
97 108
138
248 T
100 1217
109
108 134
248 T
87 1183
93
126
148
263 T
100 1223
100 114
140
253 T
13.0 42.8
8.3 10.4
7.2 8.7
mm
STRAIN TA1535
SOLVENT
110 MG
POS: 04
0.01 MG
BRRC#
0.1 MG
47-204
0.3 MG
1 MG
3 MG
10 MG
11 40 10 12 55 145
T
10 95
19 15 53
121 T
7 89 12 28 46
149 T
9 75 14 18
51 138
T
2.1 10.2
4.7 8.5 4.7
15.1
STRAIN TA1537
SOLVENT
110 MG
5
8
6
6
1.5
POS: 04
0.01 MG
79
32
79
63
27.1
BRRC#
0.1 MG
7
5
2
5
2.5
47-204
0.3 MG
3
3
2
3
0.6
1 MG
1
5
3
3
2.0
3 MG
9
5
10
8
2.6
10 MG
T
T
T
T
*
STRAIN TA1538
SOLVENT
110 MG
POS: 04
0.01 MG
BRRC#
0.1 MG
47-204
0.3 MG
1 MG
3 MG 10 MG
17 184
18 8
13 13
T
7 209
15 4 15
15 T
20 226
9 8 19 14 T
15 206
14
7 16 14
T
6.8 21.1
4.6 2.3
3.1 1.0
UCC 063006
T: TOXIC POS: POSITIVE CONTROLS
01: 4-NITRO-O-PHENYLENEDIAMINE
02; SODIUM AZIDE
03: 9-AMINOACRIDINE 04: 2-AMINOANTHRACENE
Rep rt 47-182 Page 9
-33<Jl
BUSHY RUN RESEARCH
R. 0. 4, Mellon Road, Eapon, Pennsylvania 15632
CENTER
Telephone (412) 733 5200
Good laboratory Practices Compliance
this study wee conducted In sccordence with the U.S. Environmental Protection Agency's Good Laboratory Practices (CLP) Standards (40 CFR part 792, effective December 29, 1983) with the following deviations:
1. The sponsor Indicated that Epoxy Resin ERL-4221 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 analysed by weight or by volume, and no additional analyses were performed.
In the opinion of the study director, these deviations do not affect the validity or reliability of this study.
Prepared by;
rV. Christoph^ Hengler,
Study Director
^
M.B.A.
WC/rkk/0619B-l 11-08-84
Rushy Run Rssurch Center A Joint Mellon Institute--Union Carbide Corporation Operation
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Report 47-182 Page 10
BUSHY RUN RESEARCH CENTER
R. 0. , Mton Head. Esport. Pennsylvania 1M32
Telephone taw 733-6300
-- Quality Aipurance Unit Study Inspection Summery Test Substance: BAKELITE Cycloaliphatic Epoxy Resin ERL-4221
Study: Anas Bacterial Mutagenicity Assay
Study Director: V. 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 nanageaent 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 aanageaent.
Inspection
Date
Type
1-31-84
Standard Protocol
10-3-84
Standard Protocol Fora
10-30-84
Event-Preparing Agar
1-15 to 1-18-85
Final Data and Final Report
Date QAU Report Issued To Study Director To Management
2-2-84
2-3-84
10-3-84
10-3-84
11-2-84
11-28-84
1-18-85
2-1-85
,<^r ,( n
{' '
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-J-
Linda J. Calisfi, Croup Leader
Date
Good Laboratory Practices/Quality Assurance
Bushy Bun Rssssrch Canter A Joint Mellon Institute--Union Carbide Corporation Operation
Report 47-182 Page 1 of 4
33* 333
APPENDIX 1
Standard Procedures for the Salmonella/nlcrosome Mutagenicity Assay
A. Introduction
The strains used In this test are all Salmonella typhimurlum histidine
auxotrophs 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 ainimal 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 typhimurium 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 effecting their llpopolysaccharide layer which allows large molecules to enter the bacteria more easily. Strains TA98 and TA100 were derived from strains TA1538 and TA1535 by the addition of an R factor which Imparts ampldllln resistance and also Increases sensitivity to several classes of mutagens.
Indicator organisms are stored at -80*0. Working cultures are prepared sonthly by Inoculating nutrient broth from the frozen cultures and Incubating vlth agitation overnight. Bacteria are then plated onto Vogel-Bonner Medium E gar plates (master plates) with an excess of histidine and biotin (required because of the llpopolysaccharide deficiency). After Incubation for 24 hours, the strains are checked for their genetic markers to verify their Identity and purity (SOP #7.4.2A).
For testing, the broth cultures are prepared by Inoculating from the setter plates Into nutrient broth and Incubated overnight with agitation. The broth cultures are then kept on Ice during the day of testing. Fresh cultures re made each day of testing.
C. Toxicity Testing
Sterile tubes are prepared containing 2 ml soft agar (6 g/1 agar and 5 g/1 NaQ) with a final concentration of 0.03 mM L-hlatldlne and 0.05 mM D-biotin (the entire mixture Is called top agar). Dilutions of the test chemical are ade In an appropriate solvent so that the correct amount of chemical can be added to each tube In 100 pi amounts. If less than 100 pi amounts of the test chemical Is used, phosphate-buffered saline Is used to adjust the total volume to 100 pi.
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Report *7-182 Appendix 1 Page 2 of 4
At least five doses are tested vltb maximum doses of 50 ag (for solids) or 100 id (for liquids) per plate for nontoxic chemicals, unless limited by solubility. Generally, a chemical cannot be considered to be nonautagenlc unless at least 5 ag/plate has been tested, unless limited by toxicity or solubility (deSerres and Shelby, 1979; HG-Gene Muta-S. typhlmurlum, August 1982). A 100 pi aliquot of an overnight broth culture of strain TA100 Is added followed by the appropriate amount of test chemical. The alxtur Is
vortexed and poured onto the surface of a Vogel-Bonner Medium E agar plate (VB-E plate). The top agar la allowed to harden and the plates are Incubated
at 37*C for 2* to 48 hours. The plates are examined for the condition of their background lawns and growth Is recorded as either confluent, sparse, or absent. Conflueacy Is considered an Indication of nontoxlclty, sparse growth
Indicates moderate toxicity, and lack of growth la recorded as toxic.
D. Dose Selection
For the mutagenicity test, a minimum of five half-log doses are t sted 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 pi Is used In the mutagenicity test, unless limited by solubility.
E. Controls
Concurrent solvent and positive controls are run with each test. The 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-nltro-o-phenylenediamlne for TA98 and TA1536, sodium azide for TA100 and TA1535, and 9-amlnoacrldlne for TA1537. The activation-dependent control In 2-amlnoanthracene (2-anthramlne) for all strains. The concentrations are determined from prior dose-response experiments on these chemicals and are Hated In the tables of results In this report.
F. Metabolic Activation
Each lot of Arodor-1254 Induced, rat-liver homogenate (S9) Is prescreened for activity with 7,12<dlmethylbenzanthracene to determine the appropriate amount to uae for testing. The S9 Is added to the "S9 mix" which contains, per ml, S9 (50-250 pi), MgCl2 (8 moles), KC1 (33 moles), glucose-6phosphate (5 moles), NADF (4 moles), and sodium phosphate, pH7.4 (100 moles). 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, DMS0 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
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Report 47-182 Appendix 1 Page 3 of 4
dose. If smaller volume* are u*ed, phosphate-buffered saline Is added to adjust the total volume to 100 v>I* Test chemical solutions are prepared on the day of testing and are gravimetrically analyzed. If the analysis indicates > lOZ error from the Intended concentration, then either the dilution Is repeated or the actual concentration Is stated In the report.
H. Dosing
To a sterile tube containing 2 ml of top agar, 100 pi aliquot of the appropriate bacterial culture Is added followed by the addition of 100 pi of the appropriate solvent, control, or test chemical solution. Either 0.5 ml of 59 mix or 0.5 ml of phosphate-buffered saline (PBS) Is added for tests with and without 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 la also made of the background lawn on each plate. If toxicity Is observed as an Inhibition of growth of the background lawn, the plat* Is not counted, but Is recorded as toxic. If the background lawn Is sparse and the colony count Is still recorded, that number Is 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 produce* 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.
E. Data Storage and Retrieval
Copies of the final report, statistical analyses, analytical date and raw data are stored In the BRRC Archives.
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Report 47-182 Appendix 1 Fige 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, Acetylornlthase of Escherichia colli 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 using the Salmonella/mlcrosome 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/0619B-l 11-08-84
UCC
063012
request for standard toxicology studies
I AT SUSHY RUN RESEARCH CENTER
IVC^UCC.
ofJ*Agg 1
9
3337
BUSINESS CONFIDENTIAL
To Mr. R.C. Myers Bushy Run Research C nter RD4M lion Road Export, PA 15632
Name
Sponsoring r>wiian<ft%S>/fc-7y
&i*t4`fs7Ef
Dept -jLjJi------------------------------------- ------- --------------------
Location (city)
Charge to Account No,
Technology Manager & / /CvJ/A'TZ&JSa /
Study Authorization:
Signature
tZ Wkn.
Date
COMPLETE COPIES OF this Fpbm MUST tE SENT (BY THE SUBMITTER, TO
A. Dr. Bryan Balfantyne Technical Center (3005) South Charleston, WV 25303
B. Dr. W C. Kuryla Technical Center (3005) South Charleston, WV 25303
C. Dr. E.R. Homan Bushy Run Research Center RD 4 Mellon Road Export, PA 15632
D. Sponsoring Division H$ 6 EA Manager
Name:
Loc . ,a
TESTS REQUESTED (Standard procedures; other tests require specific protocols. Please contact the Corporate
Applied Toxicology Dept, for advice and guidance.)
RECEIVED
1 RANGE FINDING:
D Acute Oral
D Acute Percutaneous
Other.
***!i
3.IN VITRO:
_
& Ames (Salmonella) Test
Gene Mutation (CHO)
AUG 24 1984
W- C HENGl rP
P Acute Inhalation
p O Skin Irritancy
O Eye Irritancy
2 KNOWN PROCEDURAL REQUIREMENT (circle):
O Chromosome Exchange (SCE)
O Chromosome Damage (Mtcronueleus)
DNA Damage (UDS) Macrophage (RAM) test
OECD, FHSA. DOT, FlFRA. TSCA. RCRA, Other.
Other
PLEASE PROVIDE THE FOLLOWING INFORMATION. IT IS NEEDED IN ORDER TO EXPEDITE WORK ON THE REQUESTED STUDIES.
MQDUCT/COMPOSITION NAME: &C
i I *CS N0MENCLATURE:7t^^C.^///. `hm: &3 6-87-0
`J-Qfr&ryc 'C, Mj
(V/.-) ftJ*C
I CHEMICAL SYNONYMS/ALTERNATE/TRADE NAMES:
, nexrQH&m.
STRUCTURAL FORMULA OR COMPOSITION OF MIXTURES:
li 9
.
] DISTRIBUTION of REPORT: A standard divisional distribution list will be used unless otherwise requested (see page 4).
ha Mac ust)
ACKNOWLEDGEMENT OF SAMPLE RECEIPT
Received: ' **C Samp), Niff
-By
S
UCO 063013
CHEMICAL INFORMATION
Report 47-182 Appendix 2 Fege 2 of 9
#
i i i :
1 SAMPLE PURITY/COMPOSITION:
It
Analytical test method employed* *(Snd copy of available data. source and location o< anaiyaet.)
Percent Composition Amounts and identities of known impurities: Amount of unidentified impurities: -------------Test material: %
%
i^fUiyRb'
(njMl ixjtu. Aulk,
d~o+}L* (U*J
Am*/jLa-Z^v wt f
%
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 problem* involving ditposat of unused amounts of the sample, supply only the amount specified above
3. PHYSICAL CHARACTERISTICS: (Give all which are known):
fine
1 iquirt
Solid
Molecular Wt Density (liquids) .
l.iTf izflvs4^
pH (aqueous sol.) Freezing point
~Ze
Bailing point
TIpO/sm--
Fla$h point
__________ uz_____________
(Tag, dosed cup)
c c c
Powder.
Granular.
Vapor Pressure (@ 3 temp, between is* and 150*0
1.
<_______ mm@
Zp(-y
C
2. mm@ C
3.
mm (B)
*C
4. SOLUBILITY:
Solvent
Water
u if*
Acetone Ethanol. Dimethylsulfoxide Vegetable Oil____ Other (specify) --
Percent Solution
> o$
5. STABILITY: (Check appropriately):
Shelf Life _ _________< 6 mo;
Light sensitive
yes;
Temp, sensitive
yes;
Oxidizes _____
Polymerizes*1
y
yes; yes;
l/ > 6 mo.
X _ no Jj- no
no no
Known solvent Incompatibility (specify) ________________ --
UCC
063014
Report 47-182 Appendix 2 Page 3 of 9
|^ANDUNG/STORA(5E/PlSPOSAUNFOftMATON:l
< BPCOMMENDED PRECAUTIONS FOR HANDLING*: tW AM* f&M S&AJ AJJ\ &*
`Enclose UCC (or other company) Material Safety Data Sheet if available.
MlAny unusual fire or explosion hazards:
Any chemical incompatibilities (materials to avoid):
faj)S
f
2. SPECIAL PERSONNEL PROTECTIVE EQUIPMENT NEEDED: Ul^fA
/kZ&H
uirm kuA* A%i2 uiAv^T.___________________________________________ __
3 RECOMMENDED INACTIVATION/DECONTAMINATION PROCEDURE(S):
* RECOMMENDED STORAGE CONDITIONS:
Temperature (Cheek appropriately): Room Temp. _____ ______________________ Refrigerate;< 20*C freeze < 0*C
Special Conditions ReQuired (Check appropriately)' Under N2 gas Dessicator Other;
j 5 SAMPLE DISPOSAL: (Check appropriately)
0 Return unused sample to:
(attach DOT shipping instructions)
UCC 063015
Disposal by BRRC is authorized in compliance with applicable regulations.
. 3340
B
Report 47-182 Appendix 2 Page 4 of 9
The following information will be useful in determining or contii mine the necessity of specific toxicity tests:
1. Proposed use of material (circle):
Industrial Product
Food
Drug
Other use (specify):
Intermediate
Home Use
Experimental
Cosmetic Packaging (specify Food, Drug. Cosmetic)
2. Potential hazard route(s) (circle):
Inhalation
Other (specify): _________________________________________________
3. Form shipped in commerce (circle): Solvent or Vehicle composition:
Solid
Gas
Suspension or Slurry
ATTACHMENTS (TO BE PROVIDED BY SUBMITTER): * Analytical data (or 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.
Please use the following divisional distribution list for the final study report
WCK/Mar S3
ucc
063016
MATERIAL SAFETY DATA SHEET
EFFECTIVE DATE: -tonmry 1,1ei
3341
Report 47-182 ,i'. Appendix 2 Page 5 of 9
pflOOUCT NAME: 'CHEMICAL NAME: FORMULA:
SAKELITE Cycioaliphstic Epoxy Resin ERL-4221, ERL-4221G
1.4- Epoxycyclohexyl methyl1.4-Epoxyeyclohexvl carboxylat*
CHEMICAL FAMILY:
C1Ht0O4
MOLECULAR WEIGHT:
SYNONYMS:
--
Diepoxide 2S2.30
department OF
transportation
HAZARD CLASSIFICATION SHIPPING NAME
None None
CAS# 2386-87 0
CAS NAME 7-Oxabicyc!o[4.1.0]heptane-3<arboxylic acid, 7-oxabicyclo(4.1.0] hept-3-ylmethyl attar
,11 PHYSICAL OATA.^
BOILING POINT. 780 mmHg 101.325 kP
SPECIFIC GRAVITY (H0 1)
364 *C (869 *F) Extrapoleted FREEZING POINT
1.175 at 25/25 *C
VAPOR PRESSURE at 20 *C
Sets to glass below -20*C(-4*F)
<0.001 kPs <0.01 mm Hg
VAPOR DENSITY (air * 1)
8.7
SOLUBILITY IN WATER, % by wt. at 20
0.05
PER CENT VOLATILES BY VOLUME
Nil
EVAPORATION RATE (Butyl Acetate * 1)
<0.01
APPEARANCE AND ODOR
Low viscosity liquid; charactaristie odor.
*** W*. \-it ,
*.,**
MATERIAL BAKELITE Epoxy Resin ERL-4221. ERL-4221G
% 100
TLV (Units) Not established
HAZARD SlitPit skin end eye irritant
V'- IV. FIRE AND EXPLOSION HAZARD DATA
flash point
l*st method(s))
245* F, Pensky-Martens Clotad Cup 400 F, Cleveland Open Cup
flammable limits in air. % by volume
Not determined
UPPER I Not determined
Extinguishing
media
Use water spray, carbon dioxids, dry chemical, alcohol-type or universaltype Foams applied by manufacturer's recommended technique.
fecial 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 protective clothing.
WUSUAL FIRE AND
explosion hazards
Hazardous polymerization may occur, due to heat from exposure to a fire.
SZ. EMERGENCY PHONE NUMBER
304/744 3487 Thi* nufntoer it avi<b< dv. mftt. w**#d. and ttoiidevt
ucc
06301?
*"< v--oft Cwm* ca'M'tiw' <*** uw w* Mi
umma m* iac<uw me in* Mmim iimim at* mu < mmmw) IIM'1 laewa-c in* ' o' m* iti t
""S nitoauart cai toaa laaafi aa auananir e MMtMMww <0* uak* U*n Cariiaa Carwwon aaavmaa wav laaaanainiin, Tn*. a>t FVH I 'O' raw ce"a*>a' o-
--Miifaiian.aiM raniicaiioa A0y waa o' '"aa# aataana rfawiiaii moat aa aa'anvinaa Or tna raw to Da--acco-avet wimaae'icaa* roea?a Si* wo 'oca< I**, O-O'rfl.'i'.cri
UNION CARBIDE CORPORATION SPECIALTY CHEMICALS AND PLASTICS DIVISION OLD RIDGEBURY ROAD. DANBURY, CT 06817
f.U
3342
Report 47-182
Appendix 2 Page 6 of 9
V. HEALTH HAZARQ DAflC
TLV AND SOURCE. None established by ACGIH or OSHA.
~ -------------
M*1
ACUTE EFFECTS OF OVEREXPOSURE
_1
i SWALLOWING
Moderetely toxic. May cause nausea, vomiting, and diarrhea.
SKIN ABSORPTION
t
INHALATION
i
( SKIN CONTACT
\
EYE CONTACT
\ CHRONIC EFFECTS
OF OVEREXPOSURE
None currently known. None currently known. May cause irritation. Mey cause irritation. None currently known.
OTHER HEALTH HAZARDS
None currently known.
|
EMERGENCY AND FIRST AID PROCEDURES:
1 1
I SWALLOWING
Give two glasses of water and induce vomiting by putting finger down throat.
SKIN
I I INHALATION
Wash with soap and water. No emergency cart anticipated.
EYES
Flush with water.
NOTES TO PHYSICIAN
There is no specific antidote. Treatment of overexposure should be directed at the control of symptoms and the clinical condition.
UCC
063018
Report 47-182
Appendix 2 Page 7 of 9
334
I lf* UWSTA8LE
MWe>
STABILITY
| STABLE
REACTIVITY BATA __
CONDITIONS TO AVOID
None
^COMPATIBILITY fMtritis to avoid)
Acids, strong bases, amines.
hazardous combustion or decomposition products
Burning can produce CO and/or C03.
hazardous polymerization
May Occur
Will not Occur
CONDITIONS TO AVOID
Acids, strong bases, amines.
-- " -.`T
r J _ VII. SPILL OR LEAK PROCEDURES
STEPS TO BE TAKEN f MATERIAL IS RELEASED OR SPILLED
Collect for disposal. Avoid discharge to waterways; see Section IX.
WASTE DISPOSAL METHOD
Dilute with an inert solvent end incinerate in a furnace where permitted under appropriate Federal, State, and local regulations.
ill VIII. SPECIAL PROTECTION INFORMATION
RESPIRATORY PROTECTION
None required in normal use.
(specify type)
VENTILATION
General (mechanical) room ventilation is expected to be satisfactory.
PROTECTIVE GLOVES
Plastic
t *- "
I EYE
! PROTECTION
OTHER PROTECTIVE EQUIPMENT
Eye bath and/or safety shower.
IX. SPECIAL PRECAUTIONS
PRECAUTIONS TO BE TAKEN IN HANDLING AND STORING
Safety glasses
Avoid contact with eyes, skin, and clothing. Wash thoroughly after handling.
Do not take internally.
i OTHER PRECAUTIONS
l
rH7*a i/ss-sm
FOR INDUSTRY USE ONLY
This product is essentially insoluble in water. It is resistant to rapid biodegradation and may be toxic to aquatic life. Avoid discharge to natural watars.
ucc
063019
Printed w U.S.A
3344
Report 47-182 Appendix 2
Page 8 of 9
UNION CARBIDE CORPORATION
ikdm.tv POt.wow a coMPosnes oveoN
RECEIVED SEP 131384 W.CHENGLtR
W.Q BOX TO, MOUND BTIPOK MJOBSOS
Tiupoe poii 3.aooo
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 epoxides 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 spectra for these products. In two cases, 4205 and 4299, comparisons were made with other samples of the these products that are being utilized in current laboratory programs.
Comparison of these various spectra by the infrared experts in our Analytical Group (Dr. Clark and Mr. Lewis) led to the conclusion that the samples
that you have are authentic samples of the UCC cycloaliphatic epoxides. Thus, the
mutagenicity testing will be performed on valid samples of 4221, 4299, 4234, 4206 and 4205.
ends.
Very truly yours.
RJCsmjk cc:Dr. . M. Clark, Mr. J. Lewis, Dr. R. E. Plevan - DB
UCC
063020
40<JO
JWO
3000 KM t 2V0U
:jrw
iooo
ABSCISSA
EXPANSION
.
owjiM* rt
EXPANSION
. ..
% T AOS . . _
SAMPI.E * fit-
<
remai*xs
OMC4N Aul>& ft*- /}**L_
______JUMt^ /JijtL-
mho
upo
moo
Sf AN TtMf MUITIPUUt . MH PPOGItAM.......................
SOIVEHT CONCENIP A TION
iooo km i **
too 400 700
HEP SCAN TIME OPT/E OPERATOR
SINGIE BEAM___________
__ OATE^MfV
CEll PATH . f IEPENCE ___ --
Ucc 063021
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t It
gIo* t3!J* *o33
tO & rt O M *>
Ml M ~JI tO MMa>
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334k
APPENDIX 3 Salmonella typhlnurlua
Report 47-182 Page 1 of 1
A. Hlatorleal Data on Spontaneoua Reversion Ratea at BRRC At of June 30, 1984
Strain IA98 TA100 TA1535 TA1537 TA1538
+/-S9 N Mean
-S9 98 18.2 +S9 98 23.6
-S9 98 123.1 +S9 98 101.5
-S9 98 32.5 +59 98 13.4
-S9 98 4.3 +S9 98 5.0
-S9 98 7.2 +S9 98 15.0
Standard Deviation
5.02 7.40
26.89 17.86
13.61 7.34
1.90 2.61
3.72 5.41
Median
18 26
120 100
32 14
4 5
7 16
Range
7, 33 14. 44
61. 187 65. 137
7. 60 2. 35
1, 9 1. 13
2. 19 4, 29
Strain
B. Hlatorleal Data on Poatlvc Control Values at BRRC As of January 26, 1984
Positive Control +/-S9 N
Standard Mean Deviation Median
Range
TA98 NPD,10 yg -S9 84 877.1 166.73
870
2AA.10 yg +S9 84 1434.2 327.54 1457
532, 1301 604, 2037
TA100 NZ. 10 yg -S9 84 1621.8 277.37 1696 2AA.10 yg +S9 84 1181.7 323.54 1201
869, 2069 475, 2225
TA1535 NZ, 10 yg -S9 64 1622.0 253.42 1666
2AA.10 yg +S9 84
99.3
30.84
98
837, 2050 37, 182
TA1537 9AA.60 yg -S9 84 140.6 130.70 2AA.10 yg +S9 84 122.5 45.52
82 120
28, 469 36, 230
TA1538 NPD.10 yg -S9 84 1124.2 196.66 1168
2AA.10 yg 459 84 532.0 230.61
486
573, 1582 146, 1249
S9- rat liver homogenate; NPD- 4-nltrophenylenedlamine; 2AA- 2-amlnoanthracene; NZ- sodium azide; 9AA- 9-amlnoacrldlne.
WPC/rkk/0619B-l 11-08-84
UCC
083022