SRC TR

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HPV4 CHEMICAL DATA A DEQUACY REVIEW

Hexachloroethane, CAS No. 67

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Prepared by:

Ashlee Aldridge, Chris Janson, Justine Von Runnen, and Joan D. Garey

Chemical, Biological and Environmental Center
SRC, Inc.

7502 Round Pon d Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027

Task FG007.0.009.002

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches, Alternate Project Officer

Me ena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

July 23 , 2010HPV4 Chemical Data Adequacy Review

Hexachloroethane, CAS No. 67

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- 1

J uly 2010

Submitted to: High Production Volume Chemicals Branch

Risk Assessment Division

Office of Pollution Prevention and Toxics Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks 1200 Pennsylvania Avenue, NW

Washington, DC 20460

- 0001

Prepared By:

SRC, Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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1 A. PREPARATION OF THE DATA ADEQUACY ASSESSMENTS

A

- 1. Source material used for assessment

For this unsponsored chemical, a comprehensive literature search was performed to identify available t oxicity data for the following SIDS endpoints:

Acute toxicity to fish Acute toxicity to Daphnia Toxicity to aquatic plants

Acute (oral, dermal, inhalation) toxicity Repeated

- dose toxicity Reproductive toxicity Developmental toxicity

Gene mutation

Chromoso mal aberrations

Studies relevant to non

- SIDS endpoints (e.g., eye and skin irritation; neurotoxicity) conducted via oral, inhalation, or dermal routes were also identified through literature searching.

The following sources were searched for toxicity dat a:

PubMed

- B ibliographic database that contains references to journal articles in the life

sciences with a concentration on biomedicine

T OXNET

- N ational L ibrary of M edicine databases on toxicology, hazardous chemicals, environmental health, and toxic re leases; includes:

**•** TOXLINE with BIOSIS and NTIS ;

**•** Development and Reproductive Toxicology File (DART) ;

**•** Chemical Carcinogenesis Research Information System (CCRIS)

- Carcinogenicity and mutagenicity test results for over 8000

**•** chemicals) ;

**•** Hazardous Subst ances Data Bank (HSDB)

- Comprehensive, peer

reviewed toxicology data for about 5000 chemicals ;

**•** Genetic Toxicology Data Bank (GENE

- TOX)

- Peer

- reviewed genetic toxicology test data for over 3000 chemicals.

TSCATS

- U npublished technical reports submitted by industry to the EPA under the Toxic Substances Control Act]

Registry of toxic effects of chemical substances (RTECs)

Environmental Fate Database (EFDB)

ECOTOX

- EPA’s peer

- reviewed ecotoxicology databaseDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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2 EPA’s Integrated Risk Information System (IRIS ) website

Agency for Toxic Substances and Disease Registry (ATSDR) website

National Toxicology Program (NTP) website

International Program on Chemical Safety Concise International Chemical Assessment Documents (IPCS INC HE M or CICADs)

European Chemical Substances Information System (ESIS)

- source of IUCLID Datasets

Any available IUCLID Datasets found through the ESIS website and robust summaries submitted to EPA by former sponsors on the assigned chemicals were retrieved. Original

sources were obtain ed for any relevant toxicity data found for the assigned chemicals.

While IUCLID D ataset s sometimes contained studies of interest, certain study descriptions contained insufficient detail to assess adequacy. In these cases, the primary source

of the st udy, if available and accessible, was obtain ed and review ed.

A

- 2. Evaluating data adequacy

Using standard OECD testing guidelines and professional judgment as guides, available toxicity studies for the unsponsored chemical were reviewed for adequacy. Summaries were prepared for all relevant data found, even if little or no study detail was available. Each study was summarized according to a standard format and assigned a Klimisch reliability code; all study summaries for the chemical were combined in to a single summary document. For studies already assigned Klimisch codes (e.g., studies summarized in IUCLID datasets), an explanation was provided if the reviewer’s assessment of data adequacy differed from the pre

- assigned

Klimisch code. A table summa rizing data availability/adequacy for each SIDS endpoint is

included at the end of the summary document.

B. SPECIFIC INFORMATION ON THE AVAILABLE TOXICITY DATA

The available data for each of the SIDS toxicity endpoints are evaluated on the following pages. Studies for a common endpoint are grouped together in the document and an endpoint adequacy assessment is provided near the top of the first page of the summaries for that endpoint. If the data available for the endpoint is determined to be inade quate, a basis for that conclusion is supplied.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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3 B

- 1. TOXICITY TO FISH

Summary of endpoint: A dequate

Study 1: Acute toxicity study in bluegill sunfish

Title: Bluegill (Lepomis macrochirus)

The study is: Adequate Not Adequate

This study is not adequate as it lacks important study details included information regarding study methodology.

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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Remarks: None

METHOD

Method/guideline followed: None state d

GLP: No t stated

Year study performed: 1978

Test type: Acute Fish Toxicity

Test species: Bluegill ( Lepomis macrochirus )

Analytical monitoring: No t stated

Exposure duration: 96 hours

Concentrations tested: Not stated

Test method/conditions rem arks:

None stated

RESULTS

Endpoint value(s):

24

- hour LC50 = 1.85 ppm (1.45

– 9.69)

48

- hour LC50 = 1.77 ppm (1.37

- 2.14)

72

- hour LC50 = 0.98 ppm (0.85

– 1.10)Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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4 96

- hour LC50 = 0.98 ppm (0.85

– 1.10) NOEC < 0.22 ppm

Results remarks: None stated

CONCLU SIONS

96

- hour LC50 = 0.98 ppm

STUDY RELIABILITY

[3 ] Not reliable

.

REFERENCE

TSCATS submission (OTS0511060, Doc ID 40

- 7848049): Syracuse Research Corporation. 1978. Results of Continuous Exposure of Fathead Minnow Embryo to 21 Priority Pollutants.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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5 Study 2: Acute toxicity study in Sheepshead minnow

Title: Sheepshead minnow ( Cyprinidon variegatus )

The study is: Adequate Not Adequate

This study is not adequate as it lacks important study details includ ing inf ormation regarding study methodology.

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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Remarks: None

METHOD

Method/guideline followed: None stated

GLP: Not stated

Year study performed: 1978

Test type: Acute Fish Toxicity

Test spe cies: Sheepshead minnow ( Cyprinidon variegatus )

Analytical monitoring: Not stated

Exposure duration: 96 hours

Concentrations tested: Not stated

Test method/conditions remarks: None stated

RESULTS

Endpoint value(s):

24

- hour LC50 = 3.06 ppm (2.44

– 4.09)

48

- hour LC50 = 2.76 ppm (2.20

– 3.60)

72

- hour LC50 = 2.40 ppm (1.89

– 3.05)

96

- hour LC50 = 2.50 ppm (1.89

– 3.05) NOEC = 1.0 ppm

Results remarks: None statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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6 CONCLUSIONS

96

- hour LC50 = 2.50 ppm

STUDY RELIABILITY

[3] Not reliable.

REFEREN CE

TSCATS submission (OTS0511060, Doc ID 40

- 7848049): Syracuse Research Corporation. 1978. Results of Continuous Exposure of Fathead Minnow Embryo to 21 Priority Pollutants.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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7 Study 3: Acute toxicity study in fathead minnow

Title: Acute Toxicity of Ten Chlorinated Aliphatic Hydrocarbons to the Fathead Minnow ( Pimephales promelas )

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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Remarks: None

METHOD

Method/guidel ine followed: None stated

GLP: Not stated

Year study performed: Not stated

Test type: Acute Fish Toxicity

– flow

- through

Test species: Fathead minnow ( Pimephales promelas )

Analytical monitoring: Not stated

Exposure duration: 96 hours

Concentrat ions tested: Not stated

Test method/conditions remarks: Exposure system

– Two 2

- L/cycle proportional diluters with dilution factors of 0.6 (Mount and Brungs 1967) were used to deliver five toxicant concentrations and a control, in duplicate, to randomly arranged exposure chambers. These chambers were all

- glass aquaria with a working volume of 41 L, 30 cm high by 30 cm by 60 cm, with a standpipe topped with a cylinder of

stainless

- steel screen to prevent loss of fish. Flows were greater than ten tank

- vol umes per day. Fluorescent lighting on a 16 hour photoperiod was used, and the light level was 48 lumens at the

water surface.

Physical and chemical conditions

– The laboratory lake water supply (Lake Superior) was the

source of the dilution water. This was heated to maintain a mean of 25 C with a standard deviation of 0.7 C in the test tanks. At least once during each 4

- day test, pH, dissolved oxygen (DO), hardness (as CaCO 3 ), and alkalinity (as CaCO 3 ) were determined for control, an intermediate, and t he high tanks. The pH ranged from 6.7 to 7.6. Means and ranges for DO, hardness, and alkalinity, were 8.0 mg/L (7.6

- 9.2), 45.1 mg/L as CaCO 3 (45.

- 45.5), and 41.8Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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8 mg/L as CaCO 3 (35.6

- 43.4), respectively, for all tests. Values obtained from high exposure concentrations did not deviate significantly from those observed in the controls. Water chemistry methods were those recommended by the American Public Health Association (APHA et al. 1980) and the U.S. Environmental Protection Agency (USEPA 1974).

Biolo gical methods

– Laboratory

- reared fathead minnows ( Pimephales promelas ), 30 to 35 days old, were used in these experiments. The rearing water was the same as the diluents water, with the temperature held at a temperature of 25 +/

- 2 C. Fish in the rearin g tanks were fed live brine shrimp nauplii in excess until 12 to 24 hr before testing, then not fed during the exposure period. At the beginning of a flow

- through test, 50 fish were randomly assigned to each of the 12 exposure tanks. Dead fish were counte d and removed at least twice during the first day, and twice daily after that. A saturator system was used to solubilize the chemicals (Phipps et al. 1982). This saturator uses an intermittent flow of water, delivered by a metering pump, through

one or m ore closed chambers (19 L stainless steel soda carbonation tanks). The water is continuously mixed with the toxicant, the latter was added in a single charge before the test began. Methods not discussed here followed those specified by the U.S. Environmen tal Protection Agency (1975).

Chemical methods

– Chemical analyses for hexachloroethane was performed with a Hewlett

Packard 5730A gas chromatograph equipped with a Model 3552A data system and a 63 Ni electric capture detector. The column was packed 100 /120 mesh Supelcoport coated with 1.5%

SP2250/1.95% SP

- 2401. The carrier gas was 5% methane in argon. Fifty ml hexane was added to 100 ml volumetric flasks, which were then brought up to the mark with the water sample. Samples were stirred vigorously o n magnetic stirrers for 1.5 hr and the organic layer was allowed to separate for 1 hr. Samples were diluted with hexane if necessary and analyzed by comparison to known hexane standards. Known amounts of the chemicals were added to water samples to deter mine the efficiency of this extraction procedure. The over

- all recovery for all compounds was 99 +/

- 4% (n = 69). Final results were not corrected for

recovery.

Statistical methods

– The LC50 concentrations were calculated by using the Trimmed Spearman

Karber method for estimating median lethal concentrations (Hamilton et al. 1977). This method is not subject to the deficiencies of the more common normal or logistic methods and it is not as sensitive to anomalous responses on the conventional Spearman

- Karber technique.

RESULTS

Endpoint value(s):

24

- hour LC50 = 1.80 mg/L (1.70

- 1.91)

48

- hour LC50 = 1.55 mg/L (1.47

- 1.63)

72

- hour LC50 = 1.55 mg/L (1.47

- 1.63)

96

- hour LC50 = 1.51 mg/L (1.43

- 1.58)

Results remarks: None statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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9 CONCLUSIONS

96

- hour LC50 = 1.51 mg/L

STUDY RELIABILITY

[1] Valid.

REFERENCE

ECOTOX Ref No: 11227. Walbridge, C. et al. (1983). Acute toxicity of ten chlorinated aliphatic hydrocarbons to the fathead minnow (Pimephales promelas). Archives of

Environmental Con tamination and To xicology 12: 661

- 666

.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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10 Study 4: Acute toxicity study in sheepshead minnow

Title: Evaluation of Hexachloroethane for Static Acute Toxicity Testing to the Sheepshead Minnow, Cyprinodon Variegatus with Cover Letter

This study is: Adequate Not Adequate

This study is considered inadequate because it lacked sufficient information to assess the toxicity, including stated test concentrations and LC50 values.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: The test compounds were purchased from the Aldrich Chemical Company, Inc.

METHOD

Method/guideline followed: Test methods described by the Committee on methods for Toxicity Tests with Aquatic Organisms (1975)

Test type: Acute Fish Saltwater Tox icity Study, static

GLP compliant?: Not specified

Year study was performed: Not stated

Test species: Cyprinodon variegatus (Sheepshead minnow), Surfside Beach salt marsh area. Average length and weight of the sheepshead minnow during the testing progra m was 3.4 cm and 2.1 g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Not specified

Test method/conditions remarks: Twenty fish per test concentration were exposed to the test article (concentrations not specified) in st ock solutions containing 5000 pppm; methanol was the carrier solvent. Five test concentrations were used for each compound. Test chambers consisted of all glass battery jars capable of holding 15 liters of seawater. A screening test was conducted

with e ach compound to establish a range of toxicity and a full scale static test was then run to

determine the 96

- hour LC 50. The control fish were exposed to the same amount of methanol as in the highest toxicant concentration. Filtered plant air was passed th rough air stones at the bottom of the test aquaria and control. Dissolved oxygen levels did not drop below 5.8 ppm during the 96

- hour testing period. Toxicant concentrations were renewed every 24 hours.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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11 Samples were collected and verified by Gas Chromog raphy using a 12’ x 1/8” glass column with

10% OV

- 101 on W

- HP for HCB.

The fish specimens collected for this study were kept in the laboratory for at least two weeks prior to testing where the water temperature was maintained at 80 oF. The photoperiod w as set at 18L:6D. The average length and weight of the sheepshead minnow during the testing program was 3.4 centimeters and 2.1 grams. Chemical characteristics of the water, analyzed according to Standard Methods (APHA, 1971) were: pH 7.9

- 8.0; 19, 525 pp m chloride (Cl

- ); and 5.8

- 6.1 ppm dissolved oxygen.

RESULTS

Endpoint value(s): Values not specified

Results remarks: Results are reported in terms of the concentration necessary to produce death to 50% of the individuals (LC 50 ) within the 96

- hour expo sure period. Log probit paper was used to calculate percent mortality versus concentration. A standard LC 50 was determined according to the test methods described by the Committee on Methods of Toxicity Tests with Aquatic Organisms (1975).

Reviewer’s no te: An attached graph seems to indicate the LC50 is circa 2 mg/L

CONCLUSIONS

96h LC50 = ~ 2 mg/L

STUDY RELIABILITY

[4] Not assignable. The study lacked sufficient information to assess the toxicity, including

stated test concentrations and LC50 values

.

REFERENCE

TSCATS submission (OTS0206455, Document ID 878213724, received 06/29/83). The Dow Chemical Company 1982. Evaluation of Hexachloroethane for Static Acute Toxicity Testing to the Sheepshead Minnow, Cyprinodon Variegatus with Cover Letter 1982

.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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12 Study 5: Acute toxicity study in rainbow trout

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: 98% purity; Lot: ED0 80787

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: 1983

Test species: Rainbow trout ( Oncorhynchus mykiss ); Fingerlings used in the study were received from Lake Mills, WI National Fish Hatchery and from the Fattig Hatchery of Brady, NE. Fish used in this study had a mean standard length of 6.6 ± 1.0 cm and a mean standard weight of 4.3 ± 1.8 g (n = 20).

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrati ons tested: 0 (control), 0.35, 0.64, 0.92, 1.58 and 1.83 mg/L

Test method/conditions remarks: A proportional diluter system was used, in which there were five toxicant concentrations plus a control all in duplicate. Individual exposure chambers

measure d either 26 x 17 x 15 cm, or 20 x 35 x 15 cm and contained 3.1 L and 6.3 L of water. The diluter cycled every 10

– 16 minutes, providing from 6.8 to 10.6 volume additions per day. Water temperature was maintained at approximately 12C. Ten fish were test ed per chamber.

During the acclimatization period, fish were housed in Lake Superior water at 12C and maintained on a diet of Glencoe Mills trout chow until used in testing. Fish were not fed during testing. Observations were made at regular intervals though 96 hrs for mortalities and other gross behavioral effects. Death was defined as cessation of opercular movement.

Water quality parameters were routinely measured. Toxicant concentrations were measured daily. All chambers were analyzed at 0 an d 96 hours, with alternating replicated analyzed at 24,Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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13 48 and 72 hours.

Statistical Analysis: The LC50 value was determined by the trimmed Spearman

- Karber method.

RESULTS

Endpoint value(s): The 96

- hour LC50 value for bluegill fish was 0.94 mg/L.

Re sults remarks: WQCD/the CET '80 WQCD data point differs as it reports data from the text while encoded for ECOTOX is the value reported in the table.

Temperature: 10.9

– 12.7C Hardness: 42.5 mg/L CaCO3 pH: 7.2 Dissolved oxygen: 59

– 77.1% Alkalinity: 46. 6 mg/L CaCO3 Acidity: 2.4 mg/L CaCO3

CONCLUSIONS

96h LC50 = 0 .94 mg/L

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 10579. Toxicity and Metabolism Studies with EPA (Environmental Protection Agency) Priority Poll utants and Related Chemicals in Freshwater Organisms. Report

No.: EPA 600/3

- 83

- 095 (NTIS/PB83

- 263665). Authors: Call, D.J., L.T. Brooke, N. Ahmad, and J.E. Richter. September 1983. Performing Laboratory: Center for Lake Superior Environmental Studies, University of Wisconsin

- Superior.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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14 Study 6: Acute toxicity study in bluegill sunfish

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 6 7

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Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: Not stated

Test species: Bluegill sunfish ( Lepomis macrochirus ); Juveniles were obtained from Fa ttig Hatcheries, Brady, Nebraska; mean weight of 0.7g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 0 (control), 0.29, 0.41, 0.73, 1.28 and 2.10 mg/L

Vehicle: DMF

Test method/conditions remarks: Bluegills were held in 530

- litre fiberglass tanks at 17°C. And were fed frozen adult brine shrimp. Fish were not fed for 24 h before or during testing

A method was developed that entailed testing all species in separate screened compartments of the same exposure tank. To ve rify this method, tests with PCP were conducted in duplicate where each species was tested in individual exposure tanks (the traditional method) and then in different compartments of the same exposure tanks (the newly developed method) so comparisons could be made between methods. Tests were conducted at 17 oC, since this is one of the recommended test temperatures for warm water organisms.

Two modified nearly identical 2

- litre proportional diluters with dilution factors of 0

- 6 were used

for all tests. Du ring each diluter cycle, flow

- splitting chambers divided each of five exposure concentrations and a control into duplicate exposure tanks. Exposure tanks in were glass aquariaDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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15 with a volume of 22.5 liters. Each tank was divided into six compartments usin g stainless steel

screen dividers.

All test water was obtained from Lake Superior and heated to the appropriate test temperature. Means and standard deviations of test temperatures were 22.5± 0.4 oC for the PCP tests and 17.3

± 0.6 oC.

Death was the pr imary endpoint for all species, although effects on equilibrium, behavior,

coloration and deformities were noted.

Statistical Analysis: Determinations of LC50 values were made using a computerized Trimmed Spearman

- Karber method. Mortality data from the duplicate tanks were combined before LC50 determinations were conducted. Other procedures or methods not specified followed those recommended by the American Society for Testing and Materials (1980).

RESULTS

Endpoint value(s): The 96

- hour LC50 value for bluegill fish was 0.970 mg/L.

Results remarks: No mortality was observed in the DMF control group.

Temperature: 17.3 oC

Hardness: 40.7

– 46.6 mg/L CaCO3 pH: 7.1

– 7.8 Dissolved oxygen: 4

– 8.4 mg/L Alkalinity: 42.3

– 57 mg/L CaCO3 Photoperiod: 16h lig ht; 8h dark; 28 lumens

CONCLUSIONS

96h LC50 = 0.97 mg/L

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 10775. A Method for Aquatic Multiple Species Toxicant Testing: Acute Toxicity of 10 Chemicals to 5 Vertebrate s and 2 Invertebrates. Authors: Phipps, G.L. and Holcombe, G.W. (1985). Environmental Pollution (Series A) 38: 141

- 57.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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16 Study 7: Acute toxicity study in fathead minnow

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: Not stated

T est species: Fathead Minnow ( Pimephales promelas ); obtained from stock

- culture units of the Environmental Research Laboratory

- Duluth; mean weight of 0.3g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 0 (control), 0.29 , 0.41, 0.73, 1.28 and 2.10 mg/L

Vehicle: DMF

Test method/conditions remarks: Minnows were kept in a rearing system at 20 oC and were fed live brine shrimp until used for testing. Fish were not fed for 24 h before or during testing

A method was devel oped that entailed testing all species in separate screened compartments of the same exposure tank. To verify this method, tests with PCP were conducted in duplicate where each species was tested in individual exposure tanks (the traditional method) and th en in different compartments of the same exposure tanks (the newly developed method) so comparisons could be made between methods. Tests were conducted at 17 oC, since this is one of the recommended test temperatures for warm water organisms.

Two modified nearly identical 2

- litre proportional diluters with dilution factors of 0

- 6 were used for all tests. During each diluter cycle, flow

- splitting chambers divided each of five exposure concentrations and a control into duplicate exposure tanks. Exposure ta nks in were glass aquariaDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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17 with a volume of 22.5 liters. Each tank was divided into six compartments using stainless steel

screen dividers.

All test water was obtained from Lake Superior and heated to the appropriate test temperature.

Means and standard deviations of test temperatures were 22.5± 0.4 oC for the PCP tests and 17.3

± 0.6 oC.

Death was the primary endpoint for all species, although effects on equilibrium, behavior,

coloration and deformities were noted.

Statistical Analysis: Determination s of LC50 values were made using a computerized Trimmed Spearman

- Karber method. Mortality data from the duplicate tanks were combined before LC50 determinations were conducted. Other procedures or methods not specified followed those recommended by the Am erican Society for Testing and Materials (1980).

RESULTS

Endpoint value(s): The 96

- hour LC50 value for fathead minnow was 1.230 mg/L.

Results remarks: No mortality was observed in the DMF control group.

Temperature: 17.3 oC

Hardness: 40.7

– 46.6 mg/L CaCO3 pH: 7.1

– 7.8 Dissolved oxygen: 4

– 8.4 mg/L Alkalinity: 42.3

– 57 mg/L CaCO3 Photoperiod: 16h light; 8h dark; 28 lumens

CONCLUSIONS

96h LC50 = 1.23 mg/L

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 10775 . A Method for Aquatic Multiple Species Toxicant Testing: Acute Toxicity of 10 Chemicals to 5 Vertebrates and 2 Invertebrates. Authors: Phipps, G.L. and Holcombe, G.W. (1985). Environmental Pollution (Series A) 38: 141

- 57.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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18 Study 8: Acute toxicity stud y in rainbow trout

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed:

Test species: Rainbow Trout ( Oncorhynchus mykiss ); Juveniles were obtained from Fattig Hatcheries, Brady, Nebraska; mean weight of 1.8g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 0 (control), 0.29, 0.41, 0.73, 1.28 and 2.10 mg/L

Vehicle: DMF

Test method/conditions remarks: Trout were held in 530

- litre fiberglass holding tanks at 15 oC and fed a commercial dry food. Fish were not fed for 24 h before or during testing

A method was developed that entailed testing all species in separate screened compartments of the same exposure tank. To verify this method, tests with PCP were conducted in duplicate where each species was teste d in individual exposure tanks (the traditional method) and then in different compartments of the same exposure tanks (the newly developed method) so comparisons could be made between methods. Tests were conducted at 17 oC, since this is one of

the recomme nded test temperatures for warm water organisms.

Two modified nearly identical 2

- litre proportional diluters with dilution factors of 0

- 6 were used for all tests. During each diluter cycle, flow

- splitting chambers divided each of five exposure

concentrat ions and a control into duplicate exposure tanks. Exposure tanks in were glass aquariaDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

- 1

19 with a volume of 22.5 liters. Each tank was divided into six compartments using stainless steel

screen dividers.

All test water was obtained from Lake Superior and heated to the appropriate test temperature. Means and standard deviations of test temperatures were 22.5± 0.4 oC for the PCP tests and 17.3

± 0.6 oC.

Compartments were covered with a stainless

- steel screen to prevent the test organisms from escaping.

Dea th was the primary endpoint for all species, although effects on equilibrium, behavior,

coloration and deformities were noted.

Statistical Analysis: Determinations of LC50 values were made using a computerized Trimmed Spearman

- Karber method. Mortality d ata from the duplicate tanks were combined before LC50 determinations were conducted. Other procedures or methods not specified followed those recommended by the American Society for Testing and Materials (1980).

RESULTS

Endpoint value(s): The 96

- hour LC 50 value for rainbow trout was 0.970 mg/L.

Results remarks: No mortality was observed in the DMF control group.

Temperature: 17.3 oC

Hardness: 40.7

– 46.6 mg/L CaCO3 pH: 7.1

– 7.8 Dissolved oxygen: 4

– 8.4 mg/L Alkalinity: 42.3

– 57 mg/L CaCO3 Photope riod: 16h light; 8h dark; 28 lumens

CONCLUSIONS

96h LC50 = 0.97 mg/L

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 10775. A Method for Aquatic Multiple Species Toxicant Testing: Acute Toxicity of 10 Chemicals to 5 Vertebrates and 2 Invertebrates. Authors: Phipps, G.L. and Holcombe, G.W. (1985). Environmental Pollution (Series A) 38: 141

- 57.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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20 Study 9: Acute toxicity study in bluegill sunfish

Title: Acute toxicity to fish

This study is: Adequa te Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

- 72

- 1

Remarks: 98% purity

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: ~ 19 84

Test species: Bluegill sunfish ( Lepomis macrochirus ); obtained from stocks cultured and maintained at Fattig Fish Hatchery, Brady, Nebraska; mean weight of 0.68g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Not s tated

Test method/conditions remarks: Dilution water for all tests, and water in which all animals were cultured prior to each test, was obtained from a ground water spring source at FTC; the characteristics of this water were fairly constant during the test period with slight seasonal variations in temperature, pH, alkalinity and hardness. All test animals were acclimated to the ground spring water for a minimum of 2 weeks. The tanks were glass aquaria (20

– 60 L) and

dilution ratios between tanks were 50

– 75%.

Statistical Analysis: Median lethal concentration (LC50) values and their 95% confidence intervals were calculated using the Trimmed Spearman

- Karber method. Comparisons of toxicities among chemicals were made after converting the LC 50 values from mg/L to mol/L. Regression analyses were conducted using the BMDP program operating on a DEC PSP

- 11/70

computer.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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21 RESULTS

Endpoint value(s): The 96

- hour LC50 value for bluegill fish was 0.856 mg/L.

Results remarks: No mortality was observed in th e control group.

Temperature: 16.5

– 17.8 oC Hardness: 196 mg/L CaCO3 pH: 8.05

– 8.1 Dissolved oxygen: 6.01

– 7.01 mg/L Alkalinity: 172 mg/L CaCO3

CONCLUSIONS

Hexachloroethane is a non

- specific toxicant. Among all species tested, the LC50 range was fr om 0.86 mg/L for blueguills to 2.8 mg/L for tadpoles.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 12004. Comparative Toxicity of Ten Organic Chemicals to Ten Common Aquatic Species. Authors: Thurston et al

., Water Res 19(9): 1145

- 55 (1985).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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22 Study 10: Acute toxicity study in fathead minnow

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

- 72

- 1

Remarks: 98% purity

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: ~ 1984

Test species: Fathead minnow ( Pimephales promelas ); obtained from stocks cultured and

maintained at Fattig Fish Hatchery, Brady, Nebraska; mean weight of 0.44g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Not stated

Test method/conditions remarks: Dilution water for all tests, and water in which all animals

we re cultured prior to each test, was obtained from a ground water spring source at FTC; the characteristics of this water were fairly constant during the test period with slight seasonal variations in temperature, pH, alkalinity and hardness. All test anim als were acclimated to the ground spring water for a minimum of 2 weeks. The tanks were glass aquaria (20

– 60 L) and

dilution ratios between tanks were 50

– 75%.

Statistical Analysis: Median lethal concentration (LC50) values and their 95% confidence

i ntervals were calculated using the Trimmed Spearman

- Karber method. Comparisons of toxicities among chemicals were made after converting the LC50 values from mg/L to mol/L. Regression analyses were conducted using the BMDP program operating on a DEC PSP

- 1 1/70

computer.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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23 RESULTS

Endpoint value(s): The 96

- hour LC50 value for fathead minnow was 1.1 mg/L.

Results remarks: No mortality was observed in the control group.

Temperature: 16.6

– 18 oC Hardness: 196 mg/L CaCO3 pH: not measured Dissolved oxygen : 5.27

– 6.01 mg/L Alkalinity: 172 mg/L CaCO3

CONCLUSIONS

Hexachloroethane is a non

- specific toxicant. Among all species tested, the LC50 range was from 0.86 mg/L for blueguills to 2.8 mg/L for tadpoles.

STUDY RELIABILITY

[1] Reliable without restri ctions.

REFERENCE

ECOTOX Reference No. 12004. Comparative Toxicity of Ten Organic Chemicals to Ten Common Aquatic Species. Authors: Thurston et al ., Water Res 19(9): 1145

- 55 (1985).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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24 Study 11: Acute toxicity study in fathead minnow

Title: Acute toxic ity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: 98% purity

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP co mpliant?: Not stated

Year study was performed: ~ 1984

Test species: Fathead minnow ( Pimephales promelas ); obtained from stocks cultured and maintained at Fattig Fish Hatchery, Brady, Nebraska; mean weight of 0.56g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Not stated

Test method/conditions remarks: Dilution water for all tests, and water in which all animals were cultured prior to each test, was obtained from a ground water spring source at FTC; the

characterist ics of this water were fairly constant during the test period with slight seasonal variations in temperature, pH, alkalinity and hardness. All test animals were acclimated to the ground spring water for a minimum of 2 weeks. The tanks were glass aquaria (20

– 60 L) and

dilution ratios between tanks were 50

– 75%.

Statistical Analysis: Median lethal concentration (LC50) values and their 95% confidence intervals were calculated using the Trimmed Spearman

- Karber method. Comparisons of toxicities among che micals were made after converting the LC50 values from mg/L to mol/L. Regression analyses were conducted using the BMDP program operating on a DEC PSP

- 11/70

computer.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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25 RESULTS

Endpoint value(s): The 96

- hour LC50 value for fathead minnow was 1.39 mg/L.

Results remarks: No mortality was observed in the control group.

Temperature: 18.8

– 20.2 oC Hardness: 196 mg/L CaCO3 pH: 8.04

– 8.11 Dissolved oxygen: 6.31

– 7.44 mg/L Alkalinity: 172 mg/L CaCO3

CONCLUSIONS

Hexachloroethane is a non

- specific toxica nt. Among all species tested, the LC50 range was from 0.86 mg/L for blueguills to 2.8 mg/L for tadpoles.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 12004. Comparative Toxicity of Ten Organic Chemicals to Ten Common Aquatic Species. Authors: Thurston et al ., Water Res 19(9): 1145

- 55 (1985).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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26 Study 12: Acute toxicity study in rainbow trout

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

- 72

- 1

Remarks: 98% purity

METHOD

Method/guideline followed: Not stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: ~ 1984

Test species: Rainbow trout ( Oncorhynchus myki ss ); obtained from both the U.S. Fish and Wildlife Service Fish Technology Center (FTC), Bozeman, Montana and Spring Creek Trout Hatchery, Lewistown, Montana; mean weight of 1.93g.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations t ested: Not stated

Test method/conditions remarks: Dilution water for all tests, and water in which all animals were cultured prior to each test, was obtained from a ground water spring source at FTC; the characteristics of this water were fairly constan t during the test period with slight seasonal variations in temperature, pH, alkalinity and hardness. All test animals were acclimated to the ground spring water for a minimum of 2 weeks. The tanks were glass aquaria (20

– 60 L) and

dilution ratios betwe en tanks were 50

– 75%.

Statistical Analysis: Median lethal concentration (LC50) values and their 95% confidence intervals were calculated using the Trimmed Spearman

- Karber method. Comparisons of toxicities among chemicals were made after converting the LC50 values from mg/L to mol/L. Regression analyses were conducted using the BMDP program operating on a DEC PSP

- 11/70

computer.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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27 RESULTS

Endpoint value(s): The 96

- hour LC50 value for rainbow trout was 1.18 mg/L.

Results remarks: No mortality was ob served in the control group.

Temperature: 12.7

– 14.8 oC Hardness: 196 mg/L CaCO3 pH: 7.86

– 7.99 Dissolved oxygen: 6.15

– 6.49 mg/L Alkalinity: 172 mg/L CaCO3

CONCLUSIONS

Hexachloroethane is a non

- specific toxicant. Among all species tested, the LC50 range was from 0.86 mg/L for blueguills to 2.8 mg/L for tadpoles.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 12004. Comparative Toxicity of Ten Organic Chemicals to Ten Common Aquatic Species. Authors: Thur ston et al ., Water Res 19(9): 1145

- 55 (1985).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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28 Study 13: Acute toxicity study in rainbow trout

Title: Acute toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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- 1

Remarks: None stated

METHOD

Method/guideline followed: In general, all methods followed closely those of the committee for toxicity tests with aquatic organisms (1975).

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: 1984

Test species: Rainbow trout ( Oncorhynchus mykiss ); The fish used in this study were laboratory

- reared to an age of 30

- 35 days and had a mean standard length of 66.4 ± 9.9 cm and a mean weight of 4.3 ± 1.8 g.

Analytical monitoring: Yes

E xposure duration: 96 hours

Concentrations tested: 0 (control), 0.34, 0.67, 0.97, 1.58 and 1.83 mg/L

Test method/conditions remarks: The dilution water was unfiltered Lake Superior water. The water temperature in the test tanks was held at a mean of 25 ± 0.7 oC. The rearing was done in a system similar to the exposure system. The rearing water was the same as the diluent water, with the temperature held at 25 ± 2 oC. Fish in the rearing tanks were fed live brine shrimp nauplii in

excess until 12 to 2 4 h before testing. The fish were not fed during the exposure period.

When starting a test, 10

- 50 fish were randomly assigned to each of the 12 exposure tanks. Methods not discussed here followed those specified by the U.S. Environmental Protection Agen cy (1975). Fish were exposed in a flow

- through diluter with room air temperature maintained at 12oC, and a controlled photoperiod of 16 hr light. Test chamber water exchange rates ranged from 3.2 to 9.3 times per 24 hours. Ten fish were exposed per cham ber resulting in chamber loadings ranging from 1.3 to 4.1 g/L. Five exposure concentrations and a control were

used.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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29 Statistical Analysis: The LC50 concentrations were calculated by using the Trimmed Spearman

Karber method for estimating median lethal co ncentrations.

RESULTS

Endpoint value(s): The 96

- hour LC50 value for rainbow trout was 0.84 mg/L.

Results remarks: Fish that lost equilibrium or were motionless on the chamber bottom were

considered affected. The 96

- hour EC50 concentration was also 0.8 4 mg/L.

Temperature: 11.6

– 12.7 oC

Hardness: 50.6

– 56.8 mg/L CaCO3 pH: 6.8

– 7.5 Dissolved oxygen: 8

– 9.6 mg/L Alkalinity: 44.6

– 53.1 mg/L CaCO3 Acidity: 1.97

– 4.1 mg/L CaCO3

CONCLUSIONS

96

- h EC50 and LC50 = 0.84 mg/L

STUDY RELIABILITY

[1] Rel iable without restrictions.

REFERENCE

ECOTOX Reference No: 4433. Aquatic Toxicity Tests to Characterize the Hazard of Volatile Organic Chemicals in Water: A Toxicity Data Summary

-- Parts I and II. Authors: Ahmad, N., D. Benoit, L. Brooke, D. Call, A. Ca rlson, D. Defoe, J. Huot, A. Moriarity, J. Richter, P. Shubat, G. Veith (1984). Source: EPA 600/3

- 84

- 009, U.S.EPA, Duluth, MN; 103p. (Author Communication Used) (Publ in Part As 11227, 15981, 10448, 12124, 12123, 15301).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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30 B

- 2. TOXICITY TO AQUATIC INVERTE BRATES

Summary of endpoint: A dequate

Study 1: Acute toxicity study in Daphnia magna

Title: Acute toxicity to daphnids

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 6 7

- 72

- 1

Remarks: 98% purity; Lot: ED080787

METHOD

Method/guideline followed: ASTM (1980); Proposed Standard Practice of Conducting Basic Acute Tests with Fishes, Macroinvertebrates and Amphibians

- Draft No. 8

Test type: Static

GLP compliant?: Not sta ted

Year study was performed: 1983

Test species: Water flea ( Daphnia magna ); Brood stock obtained from the Environmental Research Laboratory

- Duluth, MN, U.S. EPA. Daphnids used in this study were in the first instar ( ≤ 24 hours old).

Analytical monitor ing: Yes

Exposure duration: 48 hours

Concentrations tested:

Unfed Test: 0 (control), 0.805, 1.08, 1.54, 2.55, 4.65 and 5.44 mg/L Fed Test: 0 (control), 0.631, 0.844, 1.12, 2.04, 3.81 and 3.87 mg/L

Test method/conditions remarks: Daphnids were mainta ined at a temperature of approximately 20C on a diet consisting of a mixture of finely ground trout chow and baker’s

yeast.

A combination of Gro

- Lux and Duro

- Test (Optima FS) fluorescent bulbs provide 32 ft

- candles of light at the air

- water interface, a nd were set for a 16L:8D photoperiod coupled with a 15 minute transition period between light and dark phases. All culturing and testing was done withDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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31 Lake Superior water which was filtered (5 um) and aerated. All chemical stock solutions were prepared b y saturating lake water with the test chemical on a stirring plate. Test containers were 200 mL Erlenmeyer flasks filled to 200 or 160 mL for tests which the daphnids were unfed or fed, respectively. The flasks were tightly stoppered with foil wrapped ne oprene stoppers. Food concentration was 20 mg/L. The measure of acute toxicity was the 48

- hour EC50 based upon complete immobilization and the 48

- hour LC50 based upon death as determined by cessation of heart beat and gut movement. Both were determined using a 30X dissection scope. Toxicant concentrations were measured during the test.

RESULTS

Endpoint value(s): The 48

- hour LC50 values for daphnids in the unfed and fed tests were 2.90 and 2.35 mg/L, respectively. The 48

- hour EC50 values for daphnid s in the unfed and fed tests were 2.10 and 1.81 mg/L, respectively.

Results remarks: The 48h LC50 and EC50 values in the unfed test were calculated by the probit method. The 48h LC50 and EC50 values of 2.35 and 1.81 mg/L in the fed test were calculated by the binomial method and the moving average method, respectively.

Generally, little difference was noted between endpoints (LC50 or EC50) whether test animals were fed or not fed during the exposures.

Temperature: 20 ± 1 oC Hardness: 45 mg/L CaCO3

p H of new solution (fed or unfed): 7.4 ± 0.1 pH of old solution: 7.0 ± 0.1 (fed) and 7.6 ± 0.1 (unfed) Dissolved oxygen in new solutions (fed or unfed): 86.1

– 87.9% Dissolved oxygen in old solutions: 59.0

– 74.5% (fed) and 80.2

– 89.4% (unfed) Alkalinity: 42 mg/L CaCO3

CONCLUSIONS

48h LC50 = 2.90 mg/L (unfed) and 2.35 mg/L (fed) 48h EC50 = 2.10 mg/L (unfed) and 1.81 mg/L (fed)

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 10579. Toxicity and Metabolism Studies w ith EPA (Environmental Protection Agency) Priority Pollutants and Related Chemicals in Freshwater Organisms. Report

No.: EPA 600/3

- 83

- 095 (NTIS/PB83

- 263665). Authors: Call, D.J., L.T. Brooke, N. Ahmad, and J.E. Richter. September 1983. Performing Labor atory: Center for Lake Superior EnvironmentalDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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32 Studies, University of Wisconsin

- Superior.

ECOTOX Reference No. 15981. Acute and Chronic Toxicity of some Chlorinated Benzenes, Chlorinated Ethanes, and Tetrachloroethylene to Daphnia magna. Authors: Rich ter, J.E., S.F. Peterson, and C.F. Kleiner. 1983. Source: Arch.Environ.Contam.Toxicol. 12(6):679

- 684 (OECDG Data File).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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33 Study 2: Acute toxicity study in Daphnia magna

Title: Acute toxicity to daphnids

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

- 72

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Remarks: 98% purity

METHOD

Method/guideline followed: Not stated

Test type: Static

GLP compliant?: Not stated

Year study was performed: ~ 1984

Test species: Water flea ( Daphnia magna ); Daphnia were reared at the Fisheries Bioassay Laboratory located at the U.S. Fish and Wildlife Service Fish Technology Center, Bozeman, Montana; parent stocks for came from the U .S. EPA Environmental Research Laboratory

Dulu th, Minnesota; Daphnids used in this study were < 24 hours old.

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: Not stated

Test method/conditions remarks: Dilution water for all tests, and water in which all animals

were cultured prior to each test, was obtained from a ground water spring source at FTC; the characteristics of this water were fairly constant during the test period with slight seasonal variations in temperature, pH, alkalinity and hardness. All test animal s were acclimated to the ground spring water for a minimum of 2 weeks. The test was conducted in 250

- mL hard

- glass beakers which were covered with watch glasses during the test.

Tests on Daphnia were conducted on 0 to 24

- hour old neonates obtained from g ravid females which were isolated from the stock colony 20 to 24 hours prior to the start of each test. Twenty Daphnia were used for controls and tested at each toxicant concentration, five each in four beakers or jars.

At the end of the 48

- h test, live and dead Daphnia were counted. Immobilized animals thatDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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34 showed respiratory or appendage movement were counted as alive.

Statistical Analysis: Median lethal concentration (LC50) values and their 95% confidence intervals were calculated using the Trimmed Spearman

- Karber method. Comparisons of toxicities among chemicals were made after converting the LC50 values from mg/L to mol/L. Regression analyses were conducted using the BMDP program operating on a DEC PSP

- 11/70

computer.

RESULTS

Endpoint value(s) : The 48

- hour LC50 values for daphnids were 1.36 mg/L.

Results remarks: No mortality was observed in the control group.

Temperature: 22.0 oC Hardness: 196 mg/L CaCO3 pH: 8.16

– 8.29 Dissolved oxygen: 6.45

– 6.78 mg/L Alkalinity: 172 mg/L CaCO3

CONCL USIONS

Hexachloroethane is a non

- specific toxicant. Among all species tested, the LC50 range was from 0.86 mg/L for blueguills to 2.8 mg/L for tadpoles.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

ECOTOX Reference No. 12004. Com parative Toxicity of Ten Organic Chemicals to Ten Common Aquatic Species. Authors: Thurston et al ., Water Res 19(9): 1145

- 55 (1985).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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35 Study 3: Acute toxicity study in mysid shrimp

Title: Mysid shrimp (Mysidopsis bahia)

The study is: Adequate Not Adequate

This study is not adequate as it lacks important study details including information regarding study methodology.

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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Remarks: None

METHOD

Method/gu ideline followed: Not stated

GLP: Not stated

Year study performed: 1978

Test type: Acute toxicity

Species/strain/supplier: Mysid shrimp ( Mysidopsis bahia )

Analytical monitoring: Not stated

Exposure period: 96 hours

Number of animals/sex/dose: Not s tated

Statistical methods used: Not stated

Method remarks: Not stated

RESULTS

Nominal concentrations: Not stated

Measured concentrations: Not stated

Element value: 24

- hour LC50 = 1.48 ppm (0.71

– 2.80)

48

- hour LC50 = 0.99 ppm (0.74

– 1.28)

72

- ho ur LC50 = 0.99 ppm (0.74

– 1.28)Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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36 96

- hour LC50 = 0.94 ppm (0.69

– 1.48)

NOEC = 0.1 ppm

Remarks: None stated

CONCLUSIONS

96

- hour LC50 = 0.94 ppm

RELIABILITY CODE

[ 3 ] Not reliable

.

REFERENCES

TSCATS submission (OTS0511060, Doc ID 40

- 7848049): Syra cuse Research Corporation. 1978. Results of Continuous Exposure of Fathead Minnow Embryo to 21 Priority Pollutants.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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37 B

- 3. TOXICITY TO AQUATIC PLANTS

Summary of endpoint: Inadequate

Study 1: Acute toxicity in Selenastrum capricornutum

Title: Algae

– Selenastrum capricornutum (freshwater species)

The study is: Adequate Not Adequate

This study was considered inadequate due to an absence of information on testing methodology.

TEST SUBSTANCE

Identity: Ethane, he xachloro

- , CAS No. 67

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Remarks:

METHOD

Method/guideline followed: Not stated

Test type: Acute

GLP: Not stated

Year: 1978

Species/strain/supplier: Selenastrum capricornutum

Element Basis: Not stated

Exposure Period: 96 hours

Analytical M onitoring: Not stated

Statistical Methods: Not stated

Method remarks: None stated

RESULTS

Nominal concentrations: Not stated

Measured concentrations: Not statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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38 Element value:

24

- hour EC 50 = 99.2 ppm

48

- hour EC 50 = 67.3 ppm

72

- hour EC 50 = 91.7 p pm 96

- hour EC 50 (chlorophyll a) = 87.0 ppm

96

- hour EC 50 (cell count) = 93.2 ppm

Remarks: None

CONCLUSIONS

96

- hour EC 50 (chlorophyll a) = 87.0 ppm

96

- hour EC 50 (cell count) = 93.2 ppm

RELIABILITY CODE

[ 3 ] Not reliable

.

REFERENCE

TSCATS submission (O TS0511060, Doc ID 40

- 7848049): Syracuse Research Corporation. 1978. Results of Continuous Exposure of Fathead Minnow Embryo to 21 Priority Pol lutants

.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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39 Study 2: Acute toxicity in the saltwater algae

Title: Algae

– saltwater species ( Skeletonema co statum )

The study is: Adequate Not Adequate

This study was considered inadequate due to an absence of information on testing methodology.

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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- 1

Remarks:

M ETHOD

Method/guideline followed: Not stated

Test type: Acute

GLP: Not stated

Year: 1978

Species/strain/supplier: Saltwater alga e ( Skeletonema costatum )

Element Basis: Not stated

Exposure Period: 96 hours

Analytical Monitoring: Not stated

Stat istical Methods: Not stated

Method remarks: None stated

RESULTS

Nominal concentrations: Not stated

Measured concentrations: Not stated

Element value:

24

- hour EC 50 > 18 ppm 48

- hour EC 50 = 8.47 ppm (6.20

– 10.5)

72

- hour EC 50 = 8.34 ppm (5.47

– 14.3)Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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40 96

- hour EC 50 (chlorophyll a) = 8.57 ppm (5.48

– 15.9)

96

- hour EC 50 (cell count) = 7.75 ppm (3.85

– 17.1) NOEC = 1.0 ppm

Remarks: None

CONCLUSIONS

96

- hour EC 50 (chlorophyll a) = 8.57 ppm

96

- hour EC 50 (cell count) = 7.75 ppm

RELIABILITY CODE

[3] Not reliable

.

REFERENCE

TSCATS submission (OTS0511060, Doc ID 40

- 7848049): Syracuse Research Corporation. 1978. Results of Continuous Exposure of Fathead Minnow Embryo to 21 Priority Pollutants.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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41 Study 3: Acute toxicity in Selenastrum capricornutum

Title : Algal toxicity

This study is: Adequate Not Adequate

This study is considered to be inadequate due to lack of important study details; primary source has not been made available to the public.

TEST SUBSTANCE

I dentity: Hexachloroethane, CAS No. 67

- 72

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Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: Static

GLP compliant?: Not stated

Year study was performed: 1978

Test species: Green algae ( Pseudokirchneriella subcapitata/S elenastrum capricornutum )

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: Not stated

Test method/conditions remarks: Not stated

RESULTS

Endpoint value(s): 96h EC50 (chlorophyll) = 87 mg/L 96h EC50 (general population changes) = 93.2 mg/L NOEC (chlorophyll) = 5.6 mg/L

Results remarks: Not statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

- 72

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42 CONCLUSIONS

The 96h EC50 values for Pseudokirchneriella subcapitata were 87 mg/L based on chlorophyll content and 93.2 mg/L based on population.

STUDY RELIABILITY

[3] N ot Reliable.

REFERENCE

ECOTOX Reference No. 9607. "U.S. EPA. 1978. In

- depth studies on health and environmental impacts of selected water pollutants. (Table of data available from Charles E. Stephan, U.S. EPA, Duluth, MN.)"Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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43 B

- 4. ACUTE TOXICITY

S ummary of endpoint: Adequate, based on a weight

- of

- evidence approach using several oral toxicity studies that are each missing many details, but all demonstrate low oral toxicity of

hexachloroethane.

Study 1: Acute oral toxicity study in rats

Title : T he toxicity of hexachloroethane in laboratory animals

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to a lack of information on testing methodology, including numbe r of animals tested and mortality data.

TEST SUBSTANCE

Identity : Ethane, hexachloro

- , (HCE), CAS No. 67

- 72

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Remarks : Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluf f Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Plainfield, NJ, under the specifications of

MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not s tated

Type: Acute oral toxicity

GLP : Not stated

Year : Not stated

Species/Strain: Sprague

- Dawley rats

Sex: M ale and female

No. of animals per sex per dose: Not stated

Vehicle : Corn oil (50% w/v) or methylcellulose (5% w/v)

Route of administra tion: GavageDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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44 Method remarks: Solutions were administered by stomach tube. Test substance was dissolved in either corn oil or methylcelluose

.

RESULTS

LD50 value: Rat, female (corn oil) = 4450 mg/kg (3900

- 5110) Rat, female (methylcellulose) = 7080 mg/k g (6240

- 8040) Rat, male (corn oil) = 5160 mg/kg (4250

- 6270) Rat, male (methylcellulose) = 7690 (6380

- 9250)

Number of deaths at each dose level: Not stated

Remarks: None stated

CONCLUSIONS

LD50 = 4450

– 5160 mg/kg (corn oil); 7080

– 7690 mg/kg (methylce llulose)

RELIABILITY CODE

3

– Not reliable

REFERENCES

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

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45 Study 2: Acute oral toxicity stu dy in rabbits

Title : The toxicity of hexachloroethane in laboratory animals

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to a lack of information on testing

meth odology, including number of animals tested, concentrations tested and mortality data.

TEST SUBSTANCE

Identity : Ethane, hexachloro

- , (HCE), CAS No. 67

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Remarks : Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Plainfield, NJ, under the specifications of

MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not stated

Type: Acute oral toxicity

GLP : Not stated

Year : Not stated

Species/Strain: New Zealand albino rabbits

Sex: M ale

No. of animals per sex per dose: Not stated

Vehicle : methylcellulose (5% w/v )

Route of administration: Gavage

Method remarks: Solutions were administered by stomach tube. Test substance was dissolved in methylcelluose

.

RESULTS

LD50 value: An LD50 value was not reported

.

Number of deaths at each dose level: Not statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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46 Rema rks: Approximate lethal dose > 1000 mg/kg

CONCLUSIONS

ALD > 1000 mg/kg

RELIABILITY CODE

3

– Not reliable

REFERENCES

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

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47 Study 3: Acute intraperitoneal toxicity study in rats

Title : The toxicity of hexachloroethane in laboratory animals

The study is: Adequate Not Adequate

This study is not adequate for t he acute toxicity endpoint due to a lack of information on testing methodology, including number of animals tested, concentrations tested and mortality data. Furthermore, intraperitoneal administration is not a preferred dosing method.

TEST SUBSTANCE

Id entity : Ethane, hexachloro

- , (HCE), CAS No. 67

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Remarks : Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and sup plied by Hummel Chemical Company, Plainfield, NJ, under the specifications of

MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not stated

Type: Acute toxicity

GLP : Not stated

Year : Not stated

Species/Strain: Sprague Dawley rat

Sex: M ale

No. of animals per sex per dose: Not stated

Vehicle : Corn oil

Route of administration: Intraperitoneal

Method remarks:

None stated

RESULTS

LD50 value: An LD50 value was not reported

Number o f deaths at each dose level: Not statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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48 Remarks: Approximate lethal dose = 2900 mg/kg

CONCLUSIONS

ALD = 2900 mg/kg

RELIABILITY CODE

3

– Not reliable

REFERENCES

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of

hexachlor oethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

- 199.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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49 Study 4: Acute oral toxicity study in guinea pigs

Title : The toxicity of hexachloroethane in laboratory animals

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to a lack of information on testing methodology, including number of animals tested, concentrations tested and mortality data.

TEST SUBSTANCE

Identity : Ethane, hexachloro

- , (HCE), C AS No. 67

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Remarks : Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Pla infield, NJ, under the specifications of

MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not stated

Type: Acute oral toxicity

GLP : Not stated

Year : Not stated

Species/Strain: Hart ley guinea pigs

Sex: M ale

No. of animals per sex per dose: Not stated

Vehicle : corn oil

Route of administration: Gavage

Method remarks: Solutions were administered by stomach tube. Test substance was dissolved in corn oil.

RESULTS

LD50 value: 49 70 mg/kg (4030

- 6150)

Number of deaths at each dose level: Not statedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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50 Remarks: None stated

CONCLUSIONS

LD50 = 4970 mg/kg

RELIABILITY CODE

3

– Not reliable

REFERENCES

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of

he xachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

- 199.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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51 Study 5: Acute dermal toxicity study in rabbits

Title : The toxicity of hexachloroethane in laboratory animals

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to a lack of information on testing methodology, including number of animals tested, concentrations tested and mortality data.

TEST SUBSTANCE

Identity : Ethane, hexachloro

- , (HCE ), CAS No. 67

- 72

- 1

Remarks : Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Plainfield, NJ, under the specifications of

MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not stated

Type: Acute dermal toxicity

GLP : Not stated

Year : Not stated

Species/Strain : New Zealand albino rabbits

Sex: Male

No. of animals per sex per dose: Not stated

Vehicle : Water paste

Route of administration: Dermal

Method remarks: The test compound was mixed into a paste with equal quantities of distilled water. The paste

was applied to the shaved skin of rabbits and covered with a plastic wrap for a 24

- hour period.

RESULTS

LD50 value: ≥ 32,000 mg/kgDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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52 Number of deaths at each dose level: Not stated

Remarks: Signs in rats at lethal dosages were ataxia, tremors and convulsions, at nonlethal dosages, red discharge from the eyes. No primary skin irritation was observed in dermal LD50

rabbi ts.

CONCLUSIONS

LD50 ≥ 32,000 mg/kg

RELIABILITY CODE

3

– Not reliable

REFERENCES

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

- 199.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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53 Study 6: Acute inhalation toxicity study in rats

Title: Acute range

- finding inhalation toxicity study in rats

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to lack of i mportant study details.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Purity: 99.8%

METHOD

Method/guideline followed : N ot specified

Test type: Inhalation

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Sprague

- Dawley

Number of animals/sex/dose: 6 males

Route of administration: Vapors (Inhalation)

Vehicle: Air

Doses/concentrations tested: 2.5 mg/L (260 ppm) or 57 mg/L (5900 ppm)

Statistical methods used: Not specified

Test method/condition s remarks: Groups of six male rats were exposed to test substance vapors generated by passing dried air over the solid material contained in a long heated tube, for

6

- 8 hour periods. Surviving animals were necropsied 14 days after exposure and selected ti ssues and organs were examined.

RESULTS

Endpoint value(s): Not specifiedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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54 Results remarks: Rats exposed to a nominal concentration of 2.5 mg/L (260 ppm) for 8 hours showed no toxic signs during exposure or for 14 days thereafter. Body weight gain and organ

to

- body weight rations of liver, spleen, kidney, lung and testes from exposed rats were not significantly different from chamber controls. No exposure

- related gross or histopathologic changes were noted in tissues and organs. Rats exposed to a nomi nal concentration of 57 mg/L (5900 ppm) for 8 hours showed severe toxic signs including death. At 6 hours, one exposed rat showed a staggered gait, 2 out of 6 were dead at eight hours. Surviving rats showed a reduced body weight gain over the 14 day obse rvation period as compared with controls. No gross lesions were observed although two of the four remaining exposed animals showed exposure

related microscopic changes. The lesions in both animals were a subacute diffuse interstitial pneumonitis of minim al to moderate severity. The vapor concentrations in this study were derived from a heated container of hexachloroethane and when this supersaturated air passed into the cooler chamber, as expected, crystals of the chemical formed throughout the exposure

chamber.

CONCLUSIONS

LD50 > 57 mg/L (5900 ppm)

NOEC = 2.5 mg/L (260 ppm)

STUDY RELIABILITY

[4] Not assignable. The study lacked sufficient information to assess the toxicity.

REFERENCE

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

- 199.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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55 Study 7: Acute oral toxicity study in rats

Title: Acute Oral Toxicity in Rats

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to a lack of information on testing methodology and non

- standard study duration.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Colorless, crystalline material th at sublimes at 350 oF

METHOD

Method/guideline followed: Not specified

Test type: Acute oral

GLP compliant?: Not specified

Year study performed: ~ 1962

Test species: Rat, strain n ot specified

Number of animals/sex/dose: Groups of 5

Route of administrati on: Gavage

Vehicle: Aqueous methyl cellulose solution

Doses/concentrations tested: 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16 g/kg body weight

Statistical methods used: Not specified

Test method/conditions remarks: Groups of 5 rats were dosed orally by stom ach tube, with suspensions of the chemical in aqueous methyl cellulose solution.

RESULTS

Endpoint value(s): LD 50 > 3.16 g/kg

Results remarks: The rat oral LD 50 > 3.16 g/kg indicative of a low order of acute toxicity by this route. Signs of intoxicatio n were observed in animals receiving the highest dosage level,Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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56 3.16 g/kg, and were referable chiefly to the central nervous system. There were no pathological organ changes grossly visible at autopsy.

CONCLUSIONS

Hexachloroethane has a low degree of acu te toxicity by ingestion, as indicated by an oral LD 50 greater than 3 g/kg for the rat.

STUDY RELIABILITY

[3] Not reliable. The study did not follow an OECD guideline and study duration was not stated.

REFERENCE

TSCATS submission (OTS0206402, Documen t ID 878213533, received 04/18/82). Exxon Chemical Americas 1982. Initial Submission: Environmental Affairs Department Acute Oral Toxicity Study in Rats with Cover Letter Dated 1983.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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57 Study 8: Acute dermal toxicity study in rabbits

Title: Acute Dermal Toxicity in Rabbits

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to a lack of information on testing methodology, missing important study details and insuf ficient number of animals tested.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Colorless, crystalline material that sublimes at 350 oF

METHOD

Method/guideline followed: Not specified

Test type: Acute dermal

GLP compliant?: Not specified

Year study performed: ~ 1962

Test species: Rabbits, sex and strain not specified

Number of animals/sex/dose: 4/dose level

Route of administration: Dermal

Vehicle: Aqueous methyl cellulose solution

Doses/concentrations tested: 0.1, 0.316 , 1.0, or 3.16 g/kg

Statistical methods used: Not specified

Test method/conditions remarks: A paste of hexachloroethane moistened with aqueous methyl cellulose solution was applied to rabbit skin and allowed to remain in contact with the skin for 2

- hour s.

RESULTS

Endpoint value(s): LD 50 > 3.16 g/kg

Results remarks: The LD 50 value is considered to be a low degree of toxicity of skin absorption.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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58 The animals were normal in appearance, behavior, and weight gain during the 14

- day post

treatment observat ion period, and there were no visible signs of pathological organ changes at autopsy. The paste was very poorly absorbed, if at all, at the 3 higher dosage levels, but apparently well absorbed at the lowest level. There were no signs of skin irritation a t any time during the 14

- day observation period.

CONCLUSIONS

The hazard of direct skin contact is low because the chemical is poorly absorbed through the skin

and is non

- irritant. Furthermore, no toxic effects were observed in animals after 24 hours of continuous skin exposure to the chemical in concentration up to 3g/kg.

STUDY RELIABILITY

[3] Not reliable. Limited study details were provided , the number of animals was insufficient and

sex and strain of animals were not noted.

REFERENCE

TSCATS subm ission (OTS0206402, Document ID 878213533, received 04/18/82). Exxon Chemical Americas 1982. Initial Submission: Environmental Affairs Department Acute Dermal Toxicity Study in Rats with Cover Letter Dated 1983.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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59 Study 9: Acute inhalation toxicity st udy in mice, rats and guinea pigs

Title: Acute Inhalation Toxicity in mice, rats, and guinea pigs

The study is: Adequate Not Adequate

This study is not adequate for the acute toxicity endpoint due to missing importan t study details

.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Colorless, crystalline material that sublimes at 350 oF

METHOD

Method/guideline followed: Not specified

Test type: Inhalation

GLP compliant?: Not specified

Year study performed: ~ 1962

Test species: Mice, rats and guinea pigs (sex and strain not specified)

Number of animals/sex/dose: Not specified

Route of administration: Inhalation

Vehicle: Not specified

Doses/concentrations tested: 850 ppm

Statistical meth ods used: Not specified

Test method/conditions remarks: Mice, rats and guinea pigs were exposed to hexachloroethane

for 6 hours.

RESULTS

Endpoint value(s): Not specified

Results remarks: During the exposure period, the signs of toxic systemic absorpti on were chiefly those of central nervous system involvement, and the guinea pigs appeared moreDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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60 susceptible to the toxic effects of the vapor than did the two other species. One guinea pig died after 1⁄2 hours of exposure and another died within 2 hours afte r termination of exposure. There were no deaths among the mice or rats. At autopsy pulmonary hemorrhage was observed in all animals and was most severe in the guinea pigs and least in mice.

CONCLUSIONS

A 6

- hour exposure to approx 850 ppm, generated by heating the chemical was fatal to guinea pigs and caused pulmonary hemorrhage in this species and also in rats and mice.

STUDY RELIABILITY

[4] Not assignable. The study lacked sufficient information to assess the toxicity.

REFERENCE

TSCATS submissi on (OTS0206402, Document ID 878213533, received 04/18/82). Exxon Chemical Americas 1982. Initial Submission: Environmental Affairs Department Acute Inhalation Toxicity Study in Rats with Cover Letter Dated 1983.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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61 B

- 5. REPEATED

- DOSE TOXICITY

Summary of endpoint: Adequate

Study 1: Repeated

- dose toxicity study in rats

Title: NTP Technical Report on the Toxicology and Carcinogenesis Studies of

Hexachloroethane in F344/N Rats

The study is: Adequate Not Adequate

This study is not adequate for the repeated

- dose toxicity endpoint as i t is not of the minimum (28

- day ) duration.

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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- 1

Remarks: molecular weight

– 236.7. > 99% pure

METHOD

Method/guideline fo llowed: Not stated

Test type: Sixteen

- day studies

GLP compliant?: Not stated

Year study was performed: ~ 1981

Test species: Fischer 344 Rat. F344/N rats were obtained from Harlan Industries, Inc. (males) or Charles River Breeding Laboratories (fema les) and held for 20 days (males) or 21 days (females) before the studies began. The rats were approximately 7

- 8 weeks old when placed on the study.

Number of animals/sex/dose: 5/sex/dose

Route of administration: Gavage

Vehicle: corn oil

Doses/conce ntrations tested: 0, 187, 375, 750, 1500 or 3000 mg/kg

Exposure duration: 12 doses over 16 days

Statistical methods:Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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62 Not stated

Test method/conditions remarks: Groups of five rats of each sex were administered 0, 187, 375, 750, 1500 or 3000 mg/kg hex achloroethane in corn oil by gavage for 12 doses over 16 days. Animals were housed five per cage. Water and feed were available ad libitum. The rats were observed two times per day and weighed on days 0, 7, 14 and at the end of the studies. A

necropsy was performed on all animals.

RESULTS

Endpoint value(s): NOEC not established

Results remarks: All rats that received 1500 or 3000 mg/kg and 1/5 males and 2/5 females that received 750 mg/kg died before the end of the studies. The final mean body wei ghts of rats that received 750 mg/kg were 25% lower than that of vehicle controls for males and 37% lower for females. Compound

- related clinical signs seen at 750 mg/kg or more included dyspnea, ataxia, prostration, and excessive lacrimation. Compound

- re lated effects observed microscopically included hyaline droplet formation in the cytoplasm of the renal tubular epithelium in all dosed males and tubular cell regeneration and eosinophilic granular casts of cell debris in the tubule

lumina at the corticome dullary junction in the kidney at 187 and 375 mg/kg.

CONCLUSIONS

LOEC = 187 mg/kg

STUDY RELIABILITY

3

– Not reliable.

REFERENCE

National Toxicology Program (1989). NTP Technical Report on the Toxicology and Carcinogenesis Studies of Hexachloroethan e (CAS No. 67

- 72

- 1) in F344/N Rats (Gavage Studies). NIH Publication No. 89

- 2816Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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63 Study 2: Repeated

- dose toxicity study in rats

Title: NTP Technical Report on the Toxicology and Carcinogenesis Studies of

Hexachloroethane in F344/N Rats

The study i s: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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- 1

Remarks: molecular weight

– 236.7. > 99% pure

METHOD

Method/guideline followed: Not stated

Test type: Thirteen

- week stud y

GLP compliant?: Not stated

Year study was performed: ~1981

Test species: Fischer 344 Rat. 4

- 5 week old male and female F344/N rats were obtained from Charles River Breeding Laboratories, observed for 14 days, and then assigned to dose groups so

that the average cage weights were approximately equal for all animals of the same sex. Rats were approximately 6

- 7 weeks old when placed on study.

Number of animals/sex/dose: 5/sex/dose

Route of administration: Gavage

Vehicle: corn oil

Doses/concentrat ions tested: 0, 47, 94, 188, 375, 750 mg/kg

Exposure duration: 5 days/week for 13 weeks

Statistical methods:

Not stated

Test method/conditions remarks:

Groups of 10 rats of each sex were administered 0, 47, 94, 188, 375 or 750 mg/kg

hexachloroethane in corn oil by gavage, 5 days per week for 13 weeks. Rats were housed fiveDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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64 per cage. Feed and water were available ad libitum.

Animals were observed two times per day; moribund animals were killed. Individual animal weights were recorded one time pe r week. At the end of the 13

- week studies, survivors were killed. A necropsy was performed on all animals except those excessively autolyzed or

cannibalized. At the end of the 13

- week studies, urine from each animal was collected over a cold pack (1

- 3 C ) for 18

- 224 hours.

RESULTS

Endpoint value(s): No data

Results remarks: Five of 10 male rats and 2/10 female rats that received 750 mg/kg died before the end of the studies. The final mean body weights of rats that received 750 mg/kg were 19%

lower th an that of vehicle controls for males and 4% for females. Compound

– related clinical signs included post gavage hyperactivity for both sexes at doses of 94 mg/kg or more and convulsions at 375 or 750 mg/kg; the week of first appearance decreased with incr eased dose. Increased relative organ weights at 750 mg/kg included liver, heart, kidney and brain for males and females. Kidney changes were seen in all dosed male groups (9/10 at the lowest dose), and the severity increased with dose. At the highest do se, only the five male rats that died before the end of the study were examined microscopically. All five had renal papillary necrosis and degeneration and necrosis of the renal tubular epithelium. Other changes seen in the kidney in

male rats at this do se and all lower doses were identical to those observed in the 16

- day study and consisted of hyaline droplet formation, tubular regeneration, and tubular casts. The rounded to crystalloid

- shaped hyaline droplets were present in tubular cells throughout th e cortex of the kidney and were characterized by epithelial cells with an increased basophilic staining of the cytoplasm. Tubule lumina at the corticomedullary junction were slightly distended by the eosinophilic granular casts. Hepatocellular necrosis w as observed in 2/5 males and 8/10 females at 750 mg/kg, 1/10 males and 4/10 females at 375 mg/kg, and 2/10 females at 188 mg/kg. These focal areas of necrosis were centrilobular and contained a minimal inflammatory cell infiltrate. Hemorrhagic necrosis o f the urinary bladder was seen in 5/5 males that received 750 mg/kg. Urinalysis indicated findings consistent with the histopathologic changes in the kidney of dosed male rats (fine and coarse granular casts accompanied by cellular casts and epithelial ce lls). These same changes were not seen in female rats.

CONCLUSIONS

Hexachloroethane caused increased incidences of hepatocellular carcinomas in dosed mice of

each sex

.

STUDY RELIABILITY

1

– Reliable without restrictions

.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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65 REFERENCE

National Toxico logy Program (1989). NTP Technical Report on the Toxicology and Carcinogenesis Studies of Hexachloroethane (CAS No. 67

- 72

- 1) in F344/N Rats (Gavage Studies). NIH Publication No. 89

- 2816Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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66 Study 3: Repeated

- dose toxicity study in rabbits

Title : The tox icity of hexachloroethane in laboratory animals

The study is: Adequate Not Adequate

This study is not adequate for the repeated

- dose endpoint as it is not of a sufficient duration.

TEST SUBSTANCE

Identity : Ethane, h exachloro

- , (HCE), CAS No. 67

- 72

- 1

Remarks : Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel C hemical Company, Plainfield, NJ, under the specifications of

MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not stated

Test type: Repeated

- dose oral

GLP compliant?: Not stated

Year stu dy was performed: Not stated

Test species: New Zealand white rabbits

Number of animals/sex/dose: 5 males/dose

Route of administration: Oral

Vehicle: 5% aqueous methylcellulose

Doses/concentrations tested: 100, 320, 1000 mg/kg

Exposure duration: 12 days

Statistical methods: Not stated

Test method/conditions remarks: Daily oral dosages of hexachloroethane suspended in 5% aqueous methylcellulose (1000, 320, 100 mg/kg and control 3 mL/kg) were given for 12 days to groups of five male rabbits each to monitor the developmental toxic signs and changes in blood chemistry values.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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67 Blood was taken from the central ear artery of rabbits on treatment days 1, 4, 8, 12 and on day 4 following termination of dosing. The following parameters were measured in s erum: glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT), blood urea nitrogen (BUN), alkaline phosphatase, bilirubin, total protein, potassium and sodium. Animals were necropsided on day 4 following the last dosing and samples of the following tissues and organs were examined for microscopic lesions: eye, brain, lung, kidney, liver, spleen, heart, stomach, pancreas, large intestine, skeletal muscle, bone, urinary bladder, small intestine and testes. Daily body weights were reco rded and at necropsy organs were weighed (lungs, liver, kidneys, spleen and testes) in order to compute relative organ

- to

- body weight ratios for each dosage level.

RESULTS

Endpoint value(s): NOEL = 100 mg/kg/day; LOEL = 320 mg/kg/day

Results remarks : Daily oral dosages of 1000 mg/kg caused a significant (p < 0.05) reduction in body weight beginning at day 7 of dosing and relative organ weight increases in liver and kidneys. The 320 mg/kg dosages caused a significant (p < 0.05) reduction in body weig ht gain beginning on day 10. No other toxic signs were noted. A dosage of 100 mg/kg/day produced no changes in the criteria studied.

Liver degeneration and necrosis were observed in the two highest dosed groups (320 and 1000 mg/kg). These changes inclu ded fatty degeneration, coagulation necrosis, hemorrhage, ballooning degeneration, eosinophilic change and hemosiderin

- laden macrophages and giant cells. Similar liver degeneration as described above was not seen in controls or in rabbits dosed at 100 mg/kg. The liver lesions in the 1000 mg/kg rabbits were more severe than those observed in the 320 mg/kg group and thus followed a dose response pattern.

An exposure

- related toxic tubular nephrosis of the convoluted tubules in the corticomedullary region of the kidney was present in the 320 mg/kg and 1000 mg/kg dosed rabbits. This change was not present in the controls or 100 mg/kg dosed rabbits. A dose

- response relationship was not observed between the 320 mg/kg and 1000 mg/kg dosed rabbits.

Tubular neph rocalcinosis of a minimal degree was observed in the 320 and 1000 mg/kg rabbits. This change was not seen in the controls or 100 mg/kg dosed rabbits.

Blood chemical parameters were affected only in that the potassium and glucose values were decreased sig nificantly at the 1000 and 320 mg/kg dosage levels. A trend toward increase in alkaline phosphatase, SGOT and bilirubin levels was seen at the 1000 mg/kg dosage but was not statistically signifi cant when compared to controls.

CONCLUSIONS

NOEL = 100 mg/k g/day LOEL = 320 mg/kg/day

STUDY RELIABILITYDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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68 3

– Not reliable

REFERENCE

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

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69 Study 4: Repeated

- dose toxicity study in rats, guinea pigs, quail and dogs

Title: Subchronic toxicity study in rats, guinea pigs, quail and dogs

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Plainf ield, NJ, under the specifications of MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not specified

Test type: Subchronic repeated

- dose toxicity

GLP compliant?: Not stated

Year study perfor med: Not stated

Test species: Sprague

- Dawley rats (male and female), Hartley guinea pigs (male), Coturnix Japonica quail (male and female), and Beagle dogs (male), Marland Breeding Farms, Hewitt, New Jersey (rats), Hazelton Laboratories, Vienna Virginia ( dogs), and rats, guinea pigs and quail were selected from ‘our breeding colonies.’

Number of animals/sex/dose: Rats

– 25/sex/dose; Male dogs

– 4/dose; Rats in behavior test

– 15/sex/dose; Male/female quail

– 20/dose; Male guinea pigs

– 10/dose

Route of a dministration: Inhalation

Vehicle: Air

Exposure duration: 6 hours/day, 5 days/week for 6 weeks

Doses/concentrations tested: 15 ± 4, 48 ± 15, 260 ± 48 ppm

Statistical methods used: Not specified

Test method/conditions remarks:

Subchronic inhalation s tudies were conducted using 2000

- liter glass and stainless steel dynamicDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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70 flow chambers operated at 750 liters of air per minute and under negative pressure of 0.2 cm of red oil. The hexachloroethane was vaporized into the chamber by passing dried air over reservoirs of the compound and sweeping the vapor into the entering air at the cylindrical top. Four species of laboratory animals were exposed to analytically determined concentrations of hexachloroethane in air, 6 hours daily, 5 days per week for 6 week s. Male and female rats, male guinea pigs, quail and dogs were observed for development of signs and histopathologic changes during the 6

- week exposure and for 12 weeks following exposure. Blood from dogs was examined for development of changes in chemis tries and hematology. An evaluation of pulmonary function in dogs was made before and following exposure as well as changes in oxygen consumption rates in rats. Guinea pigs were studied for sensitization potential following inhalation exposure.

The subc hronic inhalation toxicity for hexachloroethane vapor was tested by exposing animals to control air and 3 concentrations of HCE for six hours a day, five days a week for 6 weeks. All animals used in these experiments were observed during a preliminary per iod and control groups exposed to chamber air were matched with each treatment group in respect to number, age, sex, and body weight. Animals were weighed weekly and observed daily for general appearance and attitude. Periodic clinical and hematological examinations were made to establish baselines on dogs for pre

- exposure control values and during the course of the experimental period. Half of all animals were sacrificed and necropsied at the end of the seix week exposure while the remained were necrops ied 12 weeks after termination exposure.

Behavior

– Behavioral tests were performed in order to measure possible changes in both learned and unlearned responses after hexachloroethane exposure. The avoidance testing apparatus

consisted of a small transl uscent plexiglass start box with a grid bar floor which opened into a

cardboard

- lined safe compartment. The animals were trained to avoid shock 1

- 3 days prior to hexachloroethane exposure, tested for avoidance just before being placed in the chambers on d ay 1, and again tested immediately after removal from the chambers after 1 day, 3 weeks and 6 weeks in the high and middle dose levels or just 6 weeks in the low dose level of exposure. Training consisted of four brief exposures to unavoidable foot shock through the grid floor of the start compartment followed by two avoidance trials. During these trials individual animals were placed in the start box and allowed 20 seconds to avoid shock by running into the safe compartment before shock onset. The depen dent measure of avoidance performance was the animal’s latency to escape into the safe compartment after being placed in the start box. Spontaneous motor activity was tested following 3 weeks exposure (high and middle dose levels) and 6 weeks exposure (hig h, middle and low dose levels). During this test, individual rats were placed inside an actophotometer, a darkened cylindrical drum with a metal grid floor. Activity counts were comprised of the total number of photo beam interruptions accumulated during individual 5 minute test sessions. The activity testing immediately followed avoidance testing (approximately 1 hour after removal from the exposure chambers) and required approximately 4 1⁄2 hours for the animals in the high and middle dose levels and 3 h ours for those in the low dose levels. During these tests, representatives of each group were run in random sequence so that there were no systematic differences between the groups in order or time of testing.

Histopathologic techniques

– A complete necr opsy was performed on all animals that diedDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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71 during the study and on all sacrificed animals. The brain, lungs, heart, liver, kidneys, spleen, eyes, bone marrow, trachea, nasal turbinates, thymus, stomach, small intestine, large intestine, pancreas, adrenal glands, urinary bladder, testes, skin, skeletal muscle and bone were taken from each animal and fixed in 10% neutral buffered formalin. The lungs were infused via the trachea and submerged in formalin for completion of fixation. Sections from organs and tissues taken at necropsy were prepared for histologic examination with hematoxylin and eosin stain and examined by light microscopy for evidence of

abnormalities. Upper respiratory evaluation was accomplished microscopically by examination of longitudinal H&E stained sections of nasal turbinates. Upper respiratory inflammation was judged on the presence of polymorphonuclear leukocytes in close association with excess mucus within the lumens of the nasal passages.

Pulmonary function studies

– Changes in c ompliance and resistance functions of the lungs due to inhalation of hexachloroethane vapor were determined in all groups of dogs. Dogs were anesthetized with Surital, 4 percent, (thiamylal sodium) by intravenous injection through a pediatric catheter sec ured in the cephalic vein. A cuffed endotracheal tube was inserted in the trachea and the distal end connected to a Fleisch pneumotach for the measurement of airflow. Intrapleural pressure was measured by means of an esophageal balloon. Transpulmonary

p ressure (the difference between esophageal pressure and airway pressure derived from a lateral top at the distal end of the endotracheal tube) was used for all calculations. Both flow and transpulmonary pressures were recorded with Statham Model PM 15 dif ferential pressure transducers. Flow and pressure signals were processed in a Buxco Electronics Inc., Pulmonary Function Computer and the following parameters were recorded on a Honeywell Model 1858 Fiber Optic recorder: flow, tidal volume, transpulmonary pressure, compliance and resistance. These parameters were measured or computed dynamically on a breath to breath basis and averaged every fifth breath.

RESULTS

Endpoint value(s): Not stated

Results remarks:

Body weight and survival

– 260 ppm group : Dogs exposed to this concentration developed tremors, were ataxic, hypersalivated, showed severe head bobbing, facial muscular fasciculations and held their eyelids closed during the exposure. One dog convulsed during the first exposure and died after 5 hours of exposure. Cause of death for the animal was not grossly evident. All surviving dogs showed similar signs intermittently throughout the remaining exposures with signs disappearing overnight. Evaluation of body weights of the dogs revealed no ef fects related to the exposures. No clinically significant changes were found in any of the blood parameters measured in dogs.

Guinea pigs showed a reduction in body weight gain beginning at the second week of exposure. Two guinea pigs died during each o f the fourth and fifth week. Challenging these animals with an interadermal injection of 0.1 suspension of hexachloroethane in saline produced noDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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72 sensitization response. The liver

- to

- body weight ratio was significantly higher than control.

Body weight g ain of the male, but not he nonpregnant female rats, was reduced starting with the third week of exposure. All rats showed tremors, ruffled pelt and red exudates around the eyes following exposure during the fourth week with one male and one female rat fo und dead. During the post exposure period all signs disappeared and the body weight gain reflected that of the controls. No exposure

- related gross changes were evident at necropsy. The kidney

- , spleen

and testes

- to

- body ratios in the male rat and the l iver ratio in the female rats were significantly larger than controls. The quail showed no signs, no effects in body weight and no gross organ or tissue changes at necropsy. All animals sacrificed 12 weeks after termination of exposure showed no gross ex posure

- related changes in tissues and organs. All sings had disappeared and body weight changes of all animals were comparable to the control.

The older male rats (12

- 14 weeks, 300

- 350 g) in the behavior group showed no toxic signs during exposure. The body weight gain was reduced compared to controls and at necropsy the relative weights of lung, liver, kidney and testes were increased over the controls.

15 ppm

– 48 ppm groups: Evaluation of body weights, gross necropsy changes and organ weights of all groups of male and nonpregnant female rats, quail, guinea pigs and dogs revealed no effects related to hexachloroethane exposure. No signs were seen in any of the above. No sensitization response was found following challenge doses of 0.1 percent in guin ea pigs.

Behavior

– None of the rats tested displayed obvious signs of toxicity. The avoidance latencies of the exposed group were compared with those of controls tested on the same day using the

Mann

- Whitney U test. Similar comparisons were conducted o n the motor activity scores using Student’s “t” test. No significant differences were found. Thus is appears that these hexachloroethane exposures produced no measurable effects on either avoidance performance or spontaneous motor activity in rats.

Pu lmonary function studies

– The mean values in all groups of dogs were not different from pre

test control values and were within reported normal limits.

Histopathologic findings

– No exposure related lesions were observed in dogs or guinea pigs.

Excess m ucus without inflammatory cells was present within sections of nasal turbinates in 2 of 10 (20%) quail in the 260 ppm exposure group. The change was not observed in tissue sections of nasal turbinates taken from 10 control quail.

Male and nonpregnant fem ale rats sacrificed immediately after 6 week’s exposure to 260 ppm had a higher incidence of severity of mycoplasma related lesions in the nasal turbinates, trachea and lung when compared to nonexposed controls. The lesions in the exposed rats were

subcli nical and consisted of mucopurulent exudates in the nasal cavities, lymphoid hyperplasia in the lamina propria of the trachea and pneumonitis rarely associated with bronchiectasis. No exposure influenced lesions were observed in male or female rats expose d at 48 and 15 ppm

HCE.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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73 No exposure

- related lesions were observed in quail or dogs sacrificed 12 weeks post exposure.

No histopathologic difference was observed between control and exposed rats sacrificed 12 weeks post

- exposure. No inflammatory exudat e was present in the nasal turbinates. The degree of subclinical tracheal lymphoid hyperplasia and pneumonitis was similar for both exposed and

control animals.

CONCLUSIONS

Concentrations of 260 ppm HCE vapor caused severe systemic effects in dogs. mode rate effects in guinea pigs and rats and almost no responses in quail. No differences were found between exposed and control animals at 3 months after termination of these exposures.

STUDY RELIABILITY

[2 ] Reliable with restrictions. The study appears g enerally acceptable, although it is missing information including guideline followed and GLP compliance.

REFERENCE

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc . J. 40 (3) : 187

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74 Study 5: Repeated

- dose toxicity study in rats

Title: 2

- week Tolerance Study in the Diet of CF Fischer 344 Rats

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroeth ane

Remarks: Purity: 98.3%. Molecular weight: 236.7. Physical state: Rhombic crystals, sublimes

at 187 o F. The sample used for this study was obtained from Eastman Kodak Company and identified as No. 114, Lot E5C.

METHOD

Method/guideline followed: Not specified

Test type: 2

- week Tolerance Study in the Diet

GLP compliant?: Yes

Year study performed: 1977

Test species: CDF Fischer 344 rats, 6

- 7 weeks old, 125

- 128 g (females), 167

- 179 g (males) Charles River Breeding Laboratories Wilmington, MA

Number of animals/sex/dose: 5/sex/dose

Route of administration: Oral

Vehicle: Not specified

Exposure duration: 16 days

Doses/concentrations tested: 0, 10, 50, 200 or 500 mg/kg/day

Statistical methods used: Data on body weights, food consumption, clinical chemistry determinations, absolute (g) and relative (g/100g) organ weights were evaluated by a one

- way analysis of variance. Differences between experimental groups and their corresponding controls were examined using Dunnett’s test (Steel and Torrie, 196 0). The level of significance chosen for all cases was p < 0.05.

Test method/conditions remarks: A tolerance study on hexachloroethane (HCE) was conducted in rats to obtain data that would aid in the selection of dose levels for a subsequent 90

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75 day feedi ng study. Diets formulated to provide dose levels of 0, 10, 50, 200 or 500 mg/kg/day were given to male and female CDF Fischer 344 rats for 16 days. Parameters evaluated were general appearance and demeanor, body weights, food consumption, clinical chemist ry determinations, terminal absolute (g) and relative (g/100g) organ weights, and gross examination at necropsy. Histopathological examination of tissues was not conducted.

Clinical chemistry determinations of serum urea nitrogen (BUN), serum alkaline pho sphatase activity (AP) and serum glutamic pyruvic transaminase activity (SGPT) were made on blood samples obtained from all rats at the termination of the study.

Necropsy and Pathology

– After 16 days on the test diets, the rats were fasted overnight, wei ghed and killed by decapitation prior to gross examination at necropsy. The sample of blood was obtained from the severed cervical blood vessels following decapitation. Each rat was examined externally and internally for gross pathologic alterations. We ights of brain, heart, liver, kidney and testes were recorded, and a portion of the liver and kidney of each rat was preserved in phosphate

- buffered 10% formalin for possible histopathological examination. Samples of liver, kidney and fat were submitted t o the Analytical Section of the Toxicology Research Laboratory for development of an analytical method to determine HCE in tissues (Karbowski, 1978).

RESULTS

Endpoint value(s): Not specified

Results remarks: All rats survived the 16

- day test period. No overt signs of toxicity or changes in demeanor were detected during the routine examination of the animals. Palatability did not appear to be a factor in ingestion of the test diets because there were no indications of excessive wasting of food. There w ere no statistically significant differences between experimental and control body weights. However, the average body weight gain of the males and females receiving 500 mg/kg/day was lower than that of the controls. The value for control males on day

14 is unusually low and resulted in statistically significantly increased values for all the experimental groups on that day. The reason for this discrepant control value is not known. None of the statistically significant differences between treated and co ntrol means indicate a

treatment

- related effect on food consumption. Although the statistically significant increase in BUN of the 500 mg/kg/day male rats is within the range of values obtained from 145 control male rats of this age and strain in this labo ratory, this increase may be related to treatment in view of the gross pathological alterations observed in the kidney. There were no other statistically significant differences between treated and control means for the clinical chemistry parameters.

A tr end toward increased absolute and relative liver weights was evident in both sexes at the 200 and 500 mg/kg/day doses, with a statistically significant increase in the relative weight of this organ for females receiving 500 mg/kg/day. Statistically signifi cant increases in absolute and relative kidney weights occurred in male rats at the 200 and 500 mg/kg/day doses. There were no other statistically significant differences between treated and control means, nor any trends to suggest an effect of HCE ingesti on on organ weights. The kidneys of all the male rats receiving dose levels of 200 or 500 mg HCE/kg/day were observed to be diffusely pale. Equivocal swelling was observed in the kidneys of 4 of 5 maleDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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76 rats receiving 500 mg/kg/day. These gross observatio ns are considered to be related to ingestion of HCE. One male rat given 50 mg/kg/day showed equivocal diffuse paleness of the kidneys which may have been related to treatment. The livers from 2 of the 5 male rats receiving the 500 mg/kg/day revealed colo r and size alterations that are also probably associated with treatment. Gross examination of female rats revealed only a few alterations, none of which indicate an effect of ingestion of HCE. Due to the preliminary nature of the study, histopathologic

e xamination was not conducted.

CONCLUSIONS

Results of this study indicate that the kidney is the major target organ. Male rats appeared to be more sensitive than females to the renal toxic properties of HCE; examinations of gross

appearance at necropsy and organ weight data did not reveal an effect on the kidneys of female rats. The liver also appears to be affected by ingestion of HCE, but the nature and severity of the hepatic alterations observed in this study (trend toward increased weight, low inc idence of grossly observed alteration) indicate that this organ may be less sensitive than the kidney to

HCE.

Sublimation of HCE from the surface of the feed limits the feasibility of administration via the diet. However, preparation of the diets at fr equent intervals and control of sublimation by reducing the surface area of the feed exposed to the environment and by covering the feed jars for approximately 18 hrs of a 24

- hr day can minimize this limitation. The effects observed in the kidneys of male rats receiving doses of 200 or 500 mg/kg/day indicate that the highest dose

selected for a 90

- day feeding study should be less than 200 mg/kg/day.

STUDY RELIABILITY

[2] Reliable with restrictions. The study appears generally acceptable, although it is missing information including guideline followed and GLP compliance.

REFERENCE

TSCATS submission (OTS0215326, Document ID 878221265, received 06/29/83). The Dow Chemical Company 1983. Initial Submission: Repeated

- dose Toxicity Study in Rats with Cover

Letter Dated 1983.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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77 Study 6: Repeated

- dose toxicity study in rats

Title: A 16

- week toxicity study in the diet of CDF Fischer 344 Rats

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroe thane, CAS No. 67

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Remarks: Purity: 98.3%, Physical state: Rhombic crystals. Molecular weight: 236.7. Sample used was obtained from Eastman Kodak Company and identified as No. 144, Lot E5C

METHOD

Method/guideline followed: Not specified

Test type: 16

- week toxicity study

GLP compliant?: Yes

Year study performed: ~ 1978

Test species: CDF Fischer 344, 13 weeks of age Charles River Breeding Laboratories, Inc., Wilmington, MA

Number of animals/sex/dose: 10/sex/dose

Route of administration: Oral

V ehicle: Not specified

Exposure duration: 109

- 110 days or 16 weeks

Doses/concentrations tested: nominal: 0, 3, 30 or 100 mg/kg/day; actual: 0 (control), 1.3, 20 or 82 mg/kg/day

Statistical methods used: Dunnett’s test and a one

- way analysis of variance were used.

Test method/conditions remarks: The rats were exposed to the test substance for approximately 17 hours/day. The rats were observed at least twice weekly for overt signs of toxicity or changes in demeanor. Parameters evaluated were general app earance and demeanor, body weights, food consumption, serum biochemical determinations, hematologic values, urinalyses, urinary excretion of creatinine, coproporphyrin, uroporphyrin and delta aminolevulinic acid (delta

- ALA), gross examination at necropsy, terminal organ weight data and histopathologic examination of a full complement of tissues from control and high dose rats andDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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78 of liver, kidney (both sexes) and adrenals (males) from lower dose rats.

RESULTS

Endpoint value(s): NOAEL = 1.3 mg/kgbw/day

Re sults remarks: All rats survived the test period. No overt signs of toxicity or changes in demeanor were detected during the regular observation of the animals. There was no evidence of wasting of food to indicate unpalatability of the test diets. Body weight gains of all treated groups were comparable to control values. The statistically significant differences that occurred between treated and control mean body weights at some of the weekly weighings were random

and showed no relation to dose. Mean f ood consumption values, showed only random statistically significant differences between treated and control means that were not correlated

with dose. Gross observations that appeared to be related to treatment were confined to the kidneys of all

male rats and one female receiving 100 mg/kg/day. Male rats exhibited a pale and mottled appearance of the kidneys whereas only an equivocal mottled appearance was noted in one female rat. Increased absolute and relative liver and kidney weights of male rats at t he highest dose, and an increase in relative liver weight of female rats at the highest dose was observed. Histopathologic effects were observed in the kidneys and livers of the male rats at the 30 and 100 mg/kg/day doses and in the kidneys of females at the highest dose only. The kidneys of male rats at the 100 mg/kg/day dose showed increased slight to moderate renal tubular atrophy and degeneration with or without peritubular fibrosis, increased slight to moderate renal tubular cytoplasmic clumping and droplet formation, and scattered or isolated renal tubules showing slight hypertrophy and/or dilation of the proximal convoluted tubules. Lesions observed in the kidneys of the 30 mg/kg/day males were similar to those seen at the highest dose but were les s severe. Kidneys of female rats receiving 100 mg/kg/day showed only an increase incident of very slight renal tubular atrophy and degeneration. Livers of male rats at the 100 mg/kg/day dose showed an increased incidence of slight swelling of the hypetoc ytes when compared to controls; this increase was equivocal in males at the 30 mg/kg/day dose.

CONCLUSIONS

Results of this study indicate that the kidney is the primary target organ in rats ingesting HCE for 16 weeks, and male rats are more sensitive to the nephrotoxicity of HCE than are female rats. A dose of approximately 1.3 mg HCE/kg body weight/day was judged to be the no untoward effect

dose for both sexes.

STUDY RELIABILITY

[2 ] Reliable with restrictions. The study appears generally acceptable

, although it is missing information including guideline followed.

REFERENCE

TSCATS submission (OTS0206709, Document ID 878214911, received 06/29/84). The DowDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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79 Chemical Company 1984. Hexachloroethane: Results of a 16

- week toxicity study in the diet of

C DF Fischer 344 rats with cover letter.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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80 Study 7: Repeated

- dose toxicity study in rats

Title: Subchronic Oral Toxicity, Tissue Distribution and Clearance of Hexachloroethane in the

Rat

The study is: Adequate Not Ade quate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , (HCE), CAS No. 67

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Remarks: white crystalline solid. The HCE sample was obtained from Eastman Kodak Co., Rochester, NY. Gas chromatographic analysis determined the purity to be 99.4%.

METHOD

Method/guideline followed: Not stated

Test type: 16

- week in vivo study

GLP compliant?: Not stated

Year study was performed: Not stated

Test species: CDF Fisher 344 rats (Charles River Breeding Laboratories, Inc., Wilmington, MA). Males (260

- 300 g rams); Females (150

- 175 grams).

Number of animals/sex/dose: 10/sex/dose

Route of administration: Diet

Vehicle: Not stated

Doses/concentrations tested: 0 (control), 1, 15, 62 mg/kg

- bw/day

Exposure duration: 16 weeks

Statistical methods: Data from the subchronic toxicity study were examined stati stically by a

one

- way analysis of variance; differences between treated and control groups were identified using Dunnett’s test. The level of significance is reported as α = 0.05 for all cases, while recognizing that the repetitive use of ANOVA on several test parameters from the same group of rats causes an increased but undefined probability of false positive results. Thus, these statistical analyses are descriptive in nature and should be carefully interpreted.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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81 Test method/conditions remarks:

Test material and diet preparation

- Test diets were prepared every 2 to 3 days using Purina Laboratory Chow (Ralston Purina Co., St. Louis, MO). A feed concentrate of HCE was serially diluted to prepare the lower dose levels. To minimize postmixing loss (due to sublimation) of HCE from the feed, test diets were stored in covered glass jars following preparation. Feed crocks (o.d., 7 cm; h, 6 cm) were filled daily in the afternoon (about 4 pm) with fresh diets from the stock jars. The crocks were removed the following morning (about 7 am), at which time the feed in the crocks was discarded. Thus, the rats had access to the treated food for approximately 15 hours per day.

Diurnal eating pattern

– Since HCE is volatile and could also act as an aversive adulte rant of food, there was a possibility that the rats would delay eating until the HCE levels had declines to a “tolerable” level. To test for this possibility, four Fischer 344 female rats were placed in cages (one per cage) and eating behavior was monitor ed by utilization of a timer that was controlled by an infrared photocell. The photobeam crossed the top of the food crock. Concentration of HCE in food was approximately 1.2 mg/g when put into the crocks (time = 0). This HCE content was

the same as for the high dose group in the main portion of the dietary study. The daily distribution of eating time was estimated by logging the cumulative amount of time the photobeam was broken during consecutive 2

- hour intervals. There were 2

- hour intervals a day,

an d the timing ‘day’ began at 4pm. Lights were turned off at 6 pm and turned on at 6 am. Data were collected for four days. Untreated food was presented at 5 pm on the first and fourth days, and HCE treated food was presented at 5 pm on the second and thi rd days. To test for effects of removing the food during the daytime, food crocks were left in the cages during the first and second day, and were removed at 7 am on the third and fourth days.

Experimental design

– The animals were randomly divided into dose groups of 10 per sex and were fed 0 (control), 1, 15 or 62 mg HCE/kg body weight/day. (Targeted dose levels were 3, 30 and 100 mg/kg/day, however, loss of HCE during diet preparation resulted in the 1, 15 and 62 mg/kg/day dose levels). An additional group of 20 male rats was fed the highest dose to obtain data on tissue distribution and clearance. The rats were housed singly in stainless steel cages in a room designed for controlled humidity, temperature, 12

- hour photocycle, and approximately 10

air exchanges per hour. Body weights were recorded weekly, and food consumption was measured at least once and usually twice a week (nonconsecutive days). Routine urinalyses and hematologic determinations were conducted during the 13 th week of the study. He matologic parameters measured (coulter Counter ZBI and Hemoglobinometer, Coulter Electronics, Hialeah, FL) were packed cell volume (PCV) (Microhematocrit Centrifuge, Clay

- Adams Co., NY), erythrocyte count (RBC), hemoglobin concentration (Hgb) and total and differential leukocyte counts (WBC). Routine urinalyses included measurement of specific gravity (T. S. Meter, American Optical Co., Keene, NH) and semiquantitative estimates (Multistix

- Ames Company, Elkhart, IN) of urinary pH, glucose, protein, ketones, occult blood and urobilinogen. Clinical biochemical determinations (CentrifiChem System 400, Union Carbide Corp., Rye, NY) of serum urea nitrogen (BUN), creatinine and activities of glutamic

- pyruvic transaminase (SGPT) and alkaline phosphatase (AP)

were made on serum obtained at necropsy after 16 weeks on test. At necropsy, each rat was examined grossly and the brain, heart, liver, kidneys and testes wereDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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82 excised and weighed. Representative portions of the following organs and tissues form each rat

were preserved in phosphate

- buffered 10% formalin: esophagus, salivary gland, stomach, small intestine, large intestine, cecum, pancreas, liver, kidneys, urinary bladder, prostate, seminal vesicles, coagulating gland, testes, epididymis, ovaries, uterus, mammar y gland, brain (cerebellum, cerebrum, brain stem), spinal cord, pituitary gland, peripheral nerve (sciatic), trachea (larynx), lungs (bronchi), sternum, spleen, thymus, lymph nodes (thoracic, mesenteric), heart, aorta, skeletal muscle, adrenal glands, thyr oid gland, parathyroid glands, mesenteric adipose tissue, skin, tongue, any gross lesion or mass. The eyes of 5 rats/sex/dose were preserved in Zenker’s fixative and those of the remaining 5 rats/sex/dose were preserved in formalin. Hematoxylin and eosin

- stained sections were processed and examined from the tissues listed above, except sternum, of all rats in the control and highest dose group. Sections of liver and kidneys from both sexes and adrenals from males were similarly processed and examined

fro m rats in the lover two dose groups.

Tissue clearance and distribution

– A group of 20 male rats was given the high dose diet for 8 weeks, and then placed on untreated control diets. Three to four of these rats were sacrificed after 3, 6, 13, 22 and 31 d ays on control feed to obtain samples of fat, liver, kidney and while blood for analysis of HCE. Samples of the above tissues were also taken for HCE analysis from 4 rats/sex/dose that were necropsied at termination of the 16

- week subchronic toxicity stud y. Analyses were conducted on hexane extracts of the tissues by GLC using a Varian (varian Assoc., Walnut Creek, CA) 3700 gas chromatograph (GC) equipped with an electron campture detector, and a glass column (7 ft x 2 mm ID) packed with 10% OV

- 11 on 100/ 120 mesh Supelcoport (Supelco Inc., Bellefonte, PA). Area responses for the GC peaks were obtained using a Varian CDS 111 integrator. The extraction efficiencies for HCE from blood, liver, kidney and fat into hexane were determined to be 98.6%, 82.9%, 10 0% and 71%, respectively. The limit of detection was 0.001 μg/g. Half

- lives for tissue clearance were obtained by linear

least

- square analysis.

RESULTS

Endpoint value(s): NOEL

– 1 mg/kg

- bw/day (based on renal histologic changes)

Results remarks:

Di et Analysis

– Assay of the top 0.5 cm of meal revealed an HCE loss of about 10% in 2.5 hours and about 40% in 12 hours. Aliquots below the 0.5 cm level were assayed up to 48 hours and found to be comparable to initial concentration. Since rats eat deeper into the meal than 0.5 cm,

the actual amount of HCE lost would have been somewhat less than 40% for the total amount of food consumed. The rate of food consumption during the night was shown to be reasonably linear so that estimates of daily dose were ca lculated from measurements of food consumption and average HCE content of food. As a result, treatment doses were conservatively estimated to be 0, 1, 15 or 62 mg/kg/day rather than the targeted 0, 3, 30 or 100 mg/kg/day dose levels.

Diurnal eating patte rn

– The rats ate the HCE treated food at the same time intervals as untreated food. About 14% of eating activity occurred just after fresh crocks were placed in the cages, during the 4 to 6 pm interval (visual observation indicated near zero activity unt il the rats wereDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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83 disturbed by the caretakers). Most of the eating activity (78%) occurred during the 12 dark hours, and approximately 4% occurred soon after the lights came on at 6 am. Even when food was available between 8 am and 4 pm, minimal eating ac tivity was recorded during these daytime hours. Thus, there was no practical difference in feeding behavior when food was withheld or available during the daytime. Because the feeding pattern was linear, the HCE daily

doses were estimated from routine fo od consumption measurements and HCE analyses of food.

Subchronic toxicity evaluation

– All rats survived the 16

- week test period with no overt manifestations of toxicity or changes in behavior. Body

- weight gains, food consumption, hematologic parameters, urinalysis and clinical biochemical determinations showed no differences between treated and control groups. Male rats were slightly more sensitive than female rats to the nephrotoxic properties of HCE. Kidney effects in male rats were manifested by incr eased kidney weights and gross pathological alterations at the highest dose. Tubular atrophy, degeneration, hypertrophy and/or dilation were seen at the highest two doses. Evidence of renal toxicity in females consisted of very slight renal tubular atrop hy and degeneration observed histopathologically at the highest dose level. Liver weights were increased in male rats given the highest dose. The liver exhibited a slight swelling of the hepatocytes in males given the highest two doses. Female rats given the high dose had increased relative liver weight ratio unaccompanied by microscopic alterations. All other histopathologic observations in treated rats were similar in type and severity to those seen in control rats and were considered to be spontaneous in nature and typical of those commonly observed in rats of the age and strain used in this study.

Tissue distribution and clearance

– The highest concentration of HCE was consistently found in

the fat. Concentrations in fat were between 2.5 to 5 times t hat found in the kidney, and over 100

times the concentrations found in the liver or blood. The concentration of HCE in the fat

decreased from 303 +/

- 50 μ/g in a first

- order manner (t 1⁄2 = 2.7 days). The concentration of HCE in the liver, kidney and bloo d also decreased in an apparent first

- order manner with half

lives very similar to the fat. The concentration of HCE in the kidneys of male rats was higher than that of females at all dose levels. The concentration of HCE in the kidneys of the males were generally proportional to dose, while in females there was a disproportionately smaller increase in HCE levels in the kidney relative to dose. HCE content in fat was similar for male and female rats. There was a disproportionately small increase in the c oncentration of HCE in the liver and blood as the dose increased; this pattern was similar for both sexes.

CONCLUSIONS

Based on renal histologic changes, the no

- observed

- effect

- level (NOEL) for toxicity for male and female rats in this study was 1 mg HC E/kg

- bw/day. The apparent first

- order elimination suggests that HCE metabolism and excretion were saturated not saturated in rats receiving 62 mg/kg/day.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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84 STUDY RELIABILITY

[ 1 ] Reliable without restrictions.

REFERENCE

Gorzinski SJ, Nolan RJ, McColli ster SB, Kociba RJ, Mattsson JL., Drug Chem Toxicol. 1985;8(3):155

- 69.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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85 B

- 6. GENE MUTATION STUDIES

Summary of endpoint: Adequate

Study 1: Bacteria reverse mutation assay

Title: Ames test

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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- 1

Remarks:

METHOD

Method/guideline followed: Not stated

Test type: in vitro Ames test

GLP compliant?: Not stated

Year study was performed: ~ 1983

Test species: Salmonell a typhimurium TA1535, TA100, TA1537, TA98

Metabolic activation: With and without

Statistical methods used: No statistical analysis is performed. Results are a tabulation of the number of colonies appearing on the plates. A positive response is indica ted by a reproducible,

dose

- related increase in the number of histidine

- independent colonies per plate.

Concentrations tested: Not stated

Test method/conditions remarks: Prior to testing, the test article is serially diluted from an

initial stock. A s uitable dose range is determined in a preliminary range

- finding experiment. The article is usually tested over a minimum of six dose levels, the highest nontoxic dose level being 10 mg/plate unless solubility, mutagenicity, or toxicity dictates a lower up per limit. When extracts are made, various undiluted aliquots are tested usually over a dose range of 5 to 100, or 200 μl/plate. Liquids are occasionally also tested without diluting the sample using various aliquots. All assays are repeated at least on ce on a separate day.

The plate incorporation assay is performed in the following way. To a strile 13 x 100 mm test tube placed in a 43 C heating block we add:Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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86 1) 2.00 ml of 0.6% agar containing 0.6% NaCl, 0.05 mM biotin and 0.05 mM histidine 2) 0.05 ml of indicator organisms (about 10 8 bacteria) 3) 0.05 ml of a solution of the test article 4) 0.05 ml of metabolic activation mixture (if appropriate)

This mixture is stirred gently and then pour e d on plates containing about 25 ml of minimal glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37 C. The number of his+ revertant colonies is counted using a BioTran II automated colony counter when possible. When accurate counts cannot be obtained (e.g., because of precipitate), the plates are counted manually using a electric probe colony counter.

Concurrent sterility , negative (solvent), and positive controls are run with every experiment. Sterility controls include plating out separately steps (3) and (4). For negative cont rols, we use steps (1), (2), (3), and (4) with the following mutagens:

- Sodium azide for the base

- pair substitution mutants TA1535 and TA100.

- 9

- Aminoacridine for the frameshift mutant TA1537

- 2

- Nitrofluorene for the frameshift mutants TA1538 and TA98

- 2

- Anthramine for all tester strains, in the presence of metabolic activation

Assays in Desiccators for Volatile Compounds: The standard Ames plate test is not entirely suitable for the testing of highly volatile chemicals, so we have modified the procedur e to conduct such testing. The Salmonella plates are prepared as described for the assays in agar, but no test chemical is added. The plates without their lids are placed side by side on a perforated

shelf in a 9

- liter desiccators. A known volume of the test chemical is added to a glass petri plate that is placed in the center of and attached to the bottom of the shelf. A control chemical is tested similarly in each experiment. The desiccators is sealed and placed on a magnetic stir plate

in a room mai ntained at 37 C. A magnetic stirrer with vanes, placed in the base of each desiccators, ensures adequate dispersion of the chemical. After incubation for 8 hours, the plates are removed from the desiccators, their lids are replaced, and they are incubate d at 37 C for an

additional 42 hours. The number of his+ revertants is counted and recorded.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration: None specified

Results remarks: No reproducible, dose

- related increase in the number of histidi ne

- independent

revertants was observed with hexachloroethane.

CONCLUSIONS

No mutagenicity was seen with bacterial test strains specific for base substitution and frame shift

mutation in the Ames test both with and without metabolic activationDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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87 STUDY R ELIABILITY

(2) valid with restrictions; Only 2 plates/dose and strain were used and only one experiment was performed; No GLP, not guideline study

REFERENCE

TSCATS submission (OTS0509408, Doc ID 40

- 8424225): Stanford Research Institute

International. 1 984. Investigations of the Species Sensitivity and Mechanism of Carcinogenicity of Halogenated Hydrocarbons Final Report.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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88 Study 2: Bacteria reverse mutation assay

Title: Toxicology and Carcinogenesis Studies of Hexachloroethane (CAS No. 67

- 72

- 1) in

F 344/N Rats (Gavage Studies)

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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Remarks: molecular weight

– 236.7.

METHOD

Method/guideline followed: Test was perf ormed as reported by Ames et al. (1975) with modifications by Haworth et al. (1983)

Test type: in vitro Ames test

GLP compliant?: Not stated

Year study was performed: 1979, 1980

Test species: Salmonella typhimurium TA1535, TA100, TA1537, TA98

Metabo lic activation: With and without

Statistical methods used: Not stated

Concentrations tested: 0, 10, 33, 100, 333.3, 1000, 3333.3, 10,000 μg/plate

Test method/conditions remarks: Testing was performed as reported by Ames et al. (1975) with modificat ions listed below and described in greater detail by Haworth et al. (1983). Hexachloroethane was sent to two testing laboratories as coded aliquots from Radian Corporation (Austin, TX). The study chemical was

incubated with the Salmonella typhimurium tes ter strains (TA98, TA100, TA1535 and TA1537) either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254

- induced male Sprague Dawley rat or Syrian hamster liver) for 20 minutes at 37 C before the addition of soft agar supplemen ted with L

- histidine and D

- biotin and subsequent plating on minimal glucose agar plates. Incubation was continued for an additional 48 hours.

Hexachloroethane was tested in duplicate in all four strains at each laboratory. Each test consisted of triplic ate plates of concurrent positive and negative controls and of at least five doses of the study chemical. The high dose was limited by toxicity or solubility but did not exceed 10 mg/plate.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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89 A positive response was defined as a reproducible, dose

- related increase in histidine

- independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants which was not dose related, not reproducible, or of insufficient magnitude to support a determi nation of mutagenicity. A response was considered negative when no increase in revertant colonies was observed after chemical treatment.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration: None specified

Results remarks: Hexachloroethane, tested with a preincubation protocol at concentrations up to 10 mg/plate, was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535 or TA1537; all strains were tested with or without Aroclor 1254

- induced male Sprague Dawley rat or Syrian hams ter liver S9.

CONCLUSIONS

The test substance was n ot mutagenic.

STUDY RELIABILITY

(1 ) Valid without restrictions

REFERENCE

National Toxicology Program (1989). NTP Technical Report on the Toxicology and Carcinogenesis Studies of Hexachloroethane (C AS No. 67

- 72

- 1) in F344/N Rats (Gavage Studies). NIH Publication No. 89

- 2816

Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. Salmonella mutagenicity test results for 250 chemicals Environ. Mutagen. Vol. 5 (Suppl 1) (1983) 3

- 142Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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90 Study 3: Yeast gene mutation assay

Title: Tetrachloroethane, Pentachloroethane, and Hexachloroethane: Genetic and Biochemical

Studies

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , C AS No. 67

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Remarks:

METHOD

Method/guideline followed: Not stated

Test type: in vitro

GLP compliant?: Not stated

Year study was performed: Not stated

Test species: Saccharomyces cerevisiae

Metabolic activation: With and without

Statistica l methods used: Statistical parameters were determined on data separately obtained on groups of five animals for a single liver microsomal fraction; all the calculations were done using Wilcoxon’s rank method as reported by Box et al. and Student’s t test

.

Concentrations tested:

Test method/conditions remarks:

Yeast strain

- Saccharomyces cerevisiae strain D7, obtained from Prof. F.K. Zimmerman, was used to measure the frequency of mitotic gene conversion at the trp locus, mitotic recombination

betwe en the centromere and the ade2 locus, and point reverse mutation at the ilv locus. In this work, we considered the mitotic gene conversion and reverse point mutation, which are sufficient to give indications of genetic effects.

Media

– To 1 liter of deio nized water, we added 10 g of yeast extract (Difco), 20 g of Bacto Peptone (Difco), 200 g of glucose for the cultures in logarithmic growth phase, and 20 g for the cultures in stationary phase.

Suspension test procedure

– Samples of 5 x 10 8 cells, grown i n 20% glucose were harvested during the logarithmic growth phase when cells contained a high level of cyt. P450; they wereDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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91 incubated in 4 ml of phosphate buffer, pH7.4, in the presence of various concentrations of HCE

for 2h at 37 C. Cells were washed and plated on selective media to ascertain trp+ convertants and ilv+ revertants. The same experimental procedure was performed with stationary phase cells in the presence and absence of S9 fraction.

Animals and preparation of microsomal fraction

– Swiss alb ino mice (CD1 strain), aged 2 months, were maintained on a standard laboratory diet prior to use. The chemicals were given in a single i.p. injection in corn oil. Two doses, 70 and 35% of the LD50, were used in the experiments. Mice were sacrificed 24 h ours after the treatment by cervical dislocation after stunning. Livers were removed aseptically and homogenized, at 4 ml/g, in 0.1 M Na+/K+ phosphate buffer, pH 7.4, containing KCl 1.5% (w/v), and the S9 fractions were prepared according to

Paolini et al. The post

- mitochondrial supernatants were poured into clear centrifuge tubes and centrifuged at 105,000 g for 60 min at 4 C. The resultant microsomal pellet was resuspended in 0.01 M Na+/K+ phosphate buffer, pH 7.4, using a hand

- driven potter Elvejhem ho mogenizer, and the resuspended pellet was centrifuged at 105,000 g for 60 min at 4 C. The resultant washed microsomes were resuspended as before and put on ice for the daily experiments.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration: No ne specified

Results remarks: Hexachloroethane induced a significant increase (P ≤ 0.01) of gene conversion only in logarithmic growth phase cells. Reverse point mutation remained unaffected under the same experimental conditions. Higher concentrations than 15 mM could not be tested because of the low solubility of HCE.

CONCLUSIONS

Hexachloroethane induced a significant increase of genetic effects in cells from the logarithmic growth phase, which contain a high level of cyt. 450.

STUDY RELIABILITY

( 2 ) Reliable

- missing some stud y details.

REFERENCE

Bronzetti G, Morichetti E, Del Carratore R, Rosellini D, Paolini M, Cantelli

- Forti G, Grilli S, Vellosi R, Teratog Carcinog Mutagen. 1989;9(6):349

- 57

.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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92 Study 4: Replicative DNA synthesis assay ( in vivo)

Title: The in vivo

- in vitr o replicative DNA synthesis (RDS) test with hepatocytes prepared from male B6C3F1 mice as an early prediction assay for putative nongenotoxic (Ames

- negative) mouse hepatocarcinogens

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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Remarks: Considered a mouse hepatocarcinogen. Purchased from Wako Pure Chemical Industries (Osaka)

METHOD

Method/guideline followed: Not stated

Test type: in vi vo

- in vitro

GLP complia nt?: Not stated

Year study was performed: Not stated

Test species: Male B6C3F1 mice

Metabolic activation:

Statistical methods used: The statistical significance of differences in hepatocyte viability was

checked with Student’s t

- test or the Welch t est (α = 0.05).

Concentrations tested:

Test method/conditions remarks: Male B6C3F1 mice were selected for the study because this strain is most commonly used in carcinogenicity studies. The mice were obtained from Charles River Laboratories and hous ed 4 to 5 animals per group in polycarbonate cages with hardwood

- chip bedding in an air

- conditioned environment at 20 to 24 C, 40 to 70% humidity and with a 12 hour light/dark cycle. All animals were given standard rodent food, MF (Oriental Yeast, Tokyo) and tap water ad libitum and quarantined for at least 1 week prior to dosing. For assessment of spontaneous RDS incidences in hepatocytes from mice of different ages, 5, 6, 7, 8, 9 and 10 week

- old animals were used, and for performance of mouse RDS test c oncerning putative nongenotoxic mouse hepatocarcinogens and noncarcinogens, 8

- week

- old animals were employed.

Mice were administered test chemicals by single oral gavage. Simple acute toxicity testing wasDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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93 first conducted using 4 or 5 mice, and the MTD of each sample was set at about half the LD50, with a limit of 2000 mg/kg. Subsequently, 4 or 5 mice per group were exposed to MTD and 1⁄2 MTD dosages. After 24, 39 and 48h, hepatocytes were prepared at each set time.

Hepatocyte isoloation and measurement o f RDS induction

– The procedures were principally conducted according to our methods for rat

- hepatocyte preparation (Uno et al., 1992 a,b), essentially in line with those reported by other researches (Williams et al., 1977; Mirsalis et al., 1985; Doolittle et al., 1987a). After anesthetizing with 5% pento

- barbital sodium (Pitman

Moore, Mundelein, IL), the portal veins were cannulated with 25G needles and the livers perfused in situ with Hanks’ balanced salt solution without Ca 2+ and Mg 2+ , containing 0.5 mM EGTA (Sigma) and 10 mM Hepes (Dojindo Laboratories, Kumamoto), pH 7.2t o 7.3, at 37C and a flow rate of 6 ml/min for a total of 6 ml. Subsequently, 0.05% collagenase type IV solution (Wako) containing 10 mg/ml of BSA (Iwai Chemical, Tokyo), 50 μg/ml of t rypsin inhibitor (Sigma) 560 μg/ml of calcium chloride and 10 mM Hepes, pH 7.5, maintained at 37 C, was perfused for 6 min at 6 ml/min. Perfused livers were minced and filtered, and hepatocytes were suspended in Hanks’ balanced salt solution. Hepatocyte viability was determined using the conventional trypan blue exclusion test (ICN Biomedicals, Costa Mesa, CA). Aliquots of 5 x 10 4
CO2 in Williams’ medium E (ICN Biomedicals, Costa Mesa, CA) containing 370 kBq/ml of [methyl

3 H]thymidine (NEN Research Produ cts, Boston, MA). Lab

- Tek chamber slides were dipped in photographic NR

- M2 emulsion (Konica, Tokyo), exposed at 4 C for 7 to 10 days and stained with hematoxylin solution (Wako). RDS incidences were calculated as the percentage [methyl

3 H]thymidine

- incorporation cells relative to 2000 hepatocytes counted per animal. For binuclear mouse hepatocytes, Doolittle et al. (1987a, b) have recommended counting as single labeled hepatocytes. However, we here counted each labeled nucleus, since it was difficult to clearly distinguish binuclear hepatocytes in observing slide samples.

Judgement criteria

– When the RDS incidence value was 0.4% or more at 24, 39 or 48 h after exposure to a chemical, the chemical was judged as positive. An incidence of less than 0.4% was judged to be negative.

RESULTS

Genotoxicity result: Positive for RDS induction

Cytotoxic concentration: None specified

Results remarks:

Time (h) / Treatment dose (mg/kg) / RDS incidence / cell viability

24 / 1,000 / 0.23 ± 0.29 / 52 ± 16.0

39 / 1,000 / 1.21 ± 0.46 / 56 ±10.7 48 / 1,000 / 0.15 ± 0.04 / 71 ± 2.3 24 / 2,000 / 0.24 ± 0.19 / 55 ± 10.0 39 / 2,000 / 0.35 ± 0.19 / 49 ± 8.8 48 / 2,000 / 0.19 ± 0.12 / 62 ± 7.2Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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94 CONCLUSIONS

Hexachloroethane tested positive for hepatocyte RDS

STUDY RELIABI LITY

(3) Not reliable

REFERENCE

Miyagawa, M. et al. The in vivo

- in vitro replicative DNA synthesis (RDS) test with hepatocytes prepared from male B6C3F1 mice as an early prediction assay for putative nongenotoxic (Ames

negative) mouse hepatocarcinogens . Mutation Research 343 (1995), 157

- 183.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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95 Study 5 : Bacterial and yeast gene mutation assay

Title: Mutagenicity Screening in Yeast

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, P lainfield, NJ, under the specifications of MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not specified

Test type: Ames assay

GLP compliant?: Not specified

Year study performed: Not state d

Test species: Saccharomyces cerevisiae strain D4 and Salmonella typhimurium TA98, TA100, and TA1535, TA1537, TA1538

Metabolic activation: With and without

Concentrations tested: 0.1 μg to 500 μg per plate

Statistical methods used: Not stated

Test me thod/conditions remarks: The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically

- induced physiological effect at the high

- dose level. The low dose was in all cases, below a concentration that demonstrated any toxic effect. DMSO was used to prepare 2.5 percent stock solutions of this compound. The dose range employed for the evaluation was from 0.1 μg to 500 μg per plate. Positive and solvent controls using both direc tly active positive chemicals and those that require metabolic activation were run with each assay. The results were presented as revertants per plate for each indicator strain employed in this assay.

RESULTSDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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96 Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not specified

Results remarks: The results of the tests conducted in the absence or the presence of the rat liver activation were all negative.

CONCLUSIONS

The test substance was n ot mutagenic

.

STUDY RELIABILITY

[2 ] Reliable with restrictions. The study appears generally acceptable, although it is missing information including guideli ne followed and GLP compliance.

REFERENCE

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of

hexachloroeth ane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187

- 199.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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97 Study 6 : DNA repair assay with primary hepatocytes

Title: DNA Repair Tests of 11 Chlorinated Hydrocarbon Analogs Final Report EPA Contracts

The study is: Adequate Not Adequate

This study is not adequate for the genetic toxicity endpoint due to lack of sufficient information

n ecessary to assess the toxicity.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Liquid

METHOD

Meth od/guideline followed: Not specified

Test type: DNA repair assay

GLP compliant?: Not specified

Year study performed: ~ 1983

Test species: Hepatocyte primary cultures derived from B6C3F1 mice and Osborne

- Mendel

Metabolic activation: Not specified

Conc entrations tested: 10 - 2 to 10 - 9 molar

Statistical methods used: Not specified

Test method/conditions remarks: A minimum of three mouse HPC/DNA repair assays

and three rat HPC/DNA repair assays were performed.

Carrier for test substance

- DMSO

HPC/DNA R epair Assay

– The HPC/DNA repair test will be performed according to the methods developed by Williams. Immediately after washing, the test chemical and 10 μCi/ml tritiated thymidine, 60

- 80 Ci/mM, are added to the culture in 4 ml of Leibovitz (L15) medium

. Five logarithmically decreasing concentrations are tested on triplicate coverslips. Additionally, appropriate positive and negative controls, a solvent control, and an untreated cell control are run in parallel with the test sets.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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98 The HPC will be incu bated at 37 C for a total of eighteen to twenty hours in presence of the test chemical in [ 3 H]

- Tdr

- L15. Each coverslip is removed from its well and rinsed in three successive 100 ml washes of PBS. Coverslips are then immersed with the cell surface up in 2 ml of 1% sodium citrate (Fisher) in a clean 6 well dish for 10 min to allow the nuclei to swell, which permits better quantification of nuclear grains. Finally, the cells are fixed in three 30 minute changes of ethanol:glacial acetic acid (3:1), air dri ed, and mounted cell surface up on glass slides with Permount (Fisher). Slides are placed in slide grip holders and dipped, in total darkness, into

NTB

- 2 emulsion (Eastman Kodak), prewarmed for 1 hr at 45 C. Slides are dried overnight by suspending the s lides, in their holders, from a rack in a light

- tight box. Slides are placed in cardboard slide boxes which are wrapped in foil and stored at 4 C. After 7 days, autoradiographs are developed in D19 (Eastman Kodak) for 4 minutes, placed in a stop batch of acidified tap water for 30 seconds, immersed in fixer (Eastman Kodak) for 10 min, and washed in running tap water for 5 min.

Slides are stained in Harris’ alum hematoxylin and counterstained with eosin. The cells are then dehydrated through 100% ethanol, air dried and coverslipped with Permount.

Results of the HPC/DNA repair test are quantified by determining the net increase in nuclear grains induced by a test chemical. An Artek Model 880 electronic counter with microscopic attachment is used for grain counting.

Only those cells which were viable at the time of fixation, indicated by swollen nuclei, and those evenly coated with emulsion are scored. Between 5 to 20 cells randomly selected from each quadrant of the coverslip are counter. The number of cells to be scored depends on the nuclear/cytoplasmic ratio obtained.

Counts are obtained for each nucleus; background is determined by counting 3 nuclear sized areas adjacent to the nucleus. Net nuclear grain counts are calculated by subtracting the hig hest cytoplasmic count from the nuclear count.

RESULTS

Genotoxicity result: Inconclusive

Cytotoxic concentration (if applicable): Not specified

Results remarks: The results from all these experiments cannot be interpreted due to the high background cou nts.

CONCLUSIONS

Findings were inconclusive d ue to the high background counts

.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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99 STUDY RELIABILITY

[4] Not assignable. The study lacked sufficient information to assess the toxicity.

REFERENCE

TSCATS submission (OTS0509403, Document ID 40+8324292, produced 09/02/83). Naylor

Dana Institute 1983. DNA Repair Tests of 11 Chlorinated Hydrocarbon Analogs Final Report

EPA Contracts with Cover letter 1983.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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100 B

- 7. CHROMOSOMAL ABERRATIONS STUDY

Summary of endpoint: Adequate

Study 1 : Cytogenetic assay with CHO cells

Title: Toxicology and Carcinogenesis Studies of Hexachloroethane (CAS No. 67

- 72

- 1) in F344/N Rats (Gavage Studies)

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , C AS No. 67

- 72

- 1

Remarks: molecular weight

– 236.7

METHOD

Method/guideline followed: Testing was performed as reported by Galloway et al. (1985, 1987)

Test type: Chinese hamster ovary cytogenetics assay

GLP compliant?: Not stated

Year study was perfo rmed: Not stated

Test species: Chinese hamster ovary (CHO) cells

Metabolic activation: With and without

Statistical methods used: Statistical analyses were conducted on both the slopes of the dose

response curves and the individual dose points. An S CE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chanc e occurrence at two dose points is less than 0.001. Chromosomal aberration data are presented as percentage of cells with aberrations. As with SCEs, both the

dose

- response curve and individual dose points were statistically analyzed. A statistically sig nificant (P<0.003) trend test or a significantly increased dose point (P<0.05) was sufficient to

indicate a chemical effect.

Concentrations tested: 10

– 1,000 μg/mL

Test method/conditions remarks:

Hexachloroethane was sent to the laboratories as coded aliquots from Radian CorporationDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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101 (Austin, TX). The study chemical was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatic exchanges (SCEs) and chromosomal aberrations both in the presence and absence of Aroclor 1254

- ind uced male Sprague Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine (BrdU)

- substituted DNA. Each test consisted of concurrent solvent and positive controls and of

at least three doses of the study chemical; the high dose was limited by toxicity or solubility but did not exceed 5 mg/ml.

In the SCE test without S9, CHO cells were incubated for 26 hours with the study chemical in McCoy’s 5A medium supplemented with 10% fetal bovine serum , L

- glutamine (2mM), and antibiotics. BrdU was added 2 hours after culture initiation. After 26 hours, the medium containing the study chemical was removed and replaced with fresh medium plus BrdU and colcemid, and incubation was continued for 2 more hou rs. Cells were then harvested by mitotic

shake

- off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the chemical, serum

- free medium, and S9 for 2 hours. The medium was then removed and replaced with m edium containing BrdU and no study chemical; incubation proceeded for an additional 26 hours, with colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9.

In the chromosomal aberration test without S 9, cells were incubated in McCoy’s 5A medium with the study chemical for 8 hours; colcemid was added, and incubation was continued for 2 hours. The cells were then harvested by mitotic shake

- off, fixed, and stained with Giemsa. For

the chromosomal aberra tion test with S9, cells were treated with the study chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with colcemid present for the final 2 hours. Cells were harvested in the same

manner as for the treatment without S9.

For the SCE test, if significant chemical

- induced cell cycle delay was seen, incubation time was lengthened to ensure a sufficient number of scorable cells. The harvest time for the

chromosomal aberration test was based on the cell cycle information obtained in the SCE test; if cell cycle delay was anticipated, the incubation period was extended approximately 5 hours.

Cells were selected for scoring on the basis of good morphology and completeness of karyt otype (21 +/

- 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. For the SCE test, 50 second

- division metaphase cells were usually scored for frequency of SCEs per cell from each dose; 100 first

- divis ion metaphase cells were scored at

each dose for the chromosomal aberration test.

Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, a nd cells containing 10 or more aberrations).

RESULTS

Genotoxicity result: PositiveDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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102 Cytotoxic concentration: None specified

Results remarks: In cytogenetic tests with cultured Chinese hamster ovary (CHO) cells, no

induction of chromosomal aberrations wa s observed after treatment with up to 500 μg/mL hexachloroethane in the absence of S9 or 1000 μg/mL in the presence of Aroclor 1254

- induced male Sprague Dawley rat liver S9. In these tests, treatment with hexachloroethane caused a considerable delay in ce ll cycle time, indicating toxicity. Also, precipitation of the study

chemical occurred at several concentrations. Hexachloroethane did not induce sister chromatid exchanges (SCEs) in CHO cells in the absence of S9; with S9, doses of 330 μg/mL

hexachloroe thane and above resulted in a significant increase in SCEs as well as toxicity, as evidenced by cell cycle delay; precipitation of hexachloroethane occurred at all doses of 330 μg/mL and above.

CONCLUSIONS

Based on findings from this study, the test su bstance was not considered to be mutagenic.

STUDY RELIABILITY

(1) Valid without restrictions

REFERENCE

National Toxicology Program (1989). NTP Technical Report on the Toxicology and Carcinogenesis Studies of Hexachloroethane (CAS No. 67

- 72

- 1) in F344/ N Rats (Gavage Studies). NIH Publication No. 89

- 2816

Galloway, S., Armstrong, M., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A., Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., Margolin, B., Resnick, M., Anderson, B., and Zeiger, E. Chromosome aberration and sister chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. Molec. Mutagen. Vol. 10 (Suppl 10) (1987) 1

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- 8

. REPRODUCTIVE TOXICITY

Summary of endpoint: Adequate

Study 1: Reproductive/development al toxicity study in rats

Title: Teratology Study in Rats

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Test substance is a crystalline white solid wit h a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Plainfield, NJ, under the specifications of MIL

- H

- 235B, National Stock Numb er 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not specified

Test type: Teratology

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Sprague

- Dawley

Number of animals/sex/dose: 8 groups of 22 pr egnant females/dose (5 groups for inhalation; 3 groups for oral)

Route of administration: Vapor (Inhalation) or Gavage

Exposure duration: Day 6 through Day 16 of gestation

Doses/concentrations tested: oral doses of 50, 100, 500 mg/kg; exposed to vapors at 15, 48 or 260 ppm

Vehicle: Air/Corn oil

Statistical methods used: Student’s t

- testDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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104 Test method/conditions remarks: Teratology studies were carried out in pregnant Sprague

Dawley rats given hexachloroethane by inhalation or by gavage from day 6 throug h day 16 of gestation. The day of finding sperm in the vaginal smears of rats was considered Day 0 of gestation. Twenty

- two pregnant rats per dose were used and all adult animals were sacrificed on day 20 of gestation by intracardiac injection of sodium pentobarbital. The reproductive tracts were exposed by laparotomy and the number of corpora lutea, implantation sites, and resporption sites were recorded. The fetuses were removed, examined for viable conceptuses, gross abnormalities, sex, weight, and b ody length. All grossly abnormal fetuses as well as eight normal appearing fetuses from each dam were fixed in either Bouin’s fluid or 95% ethyl alcohol. These were further studied by freehand section for soft tissue abnormalities (2 male and 2 female) o r after Alizarin Red S staining for skeletal malformations (2 male and 2 female).

Five groups of 22 pregnant female rats were given daily oral dosages form day 6 through day 16 of gestation of hexachloroethane (50, 100, 500 mg/kg), corn oil (5 mL/kg) and aspirin (250 mg/kg). Doses were adjusted according to individual daily body weights. Animals were housed in groups of 2 in suspended wire cages, food and water was freely available except for a daily 6

- hour period when both were removed to emulate the gr oups receiving hexachloroethane by

inhalation.

Three groups of 22 pregnant female rats were exposed from day 6 through day 16 of gestation to the vapors of hexachloroethane at concentration of 15, 48 and 260 ppm. All animals were observed for signs of to xicity and were weighed daily.

RESULTS

Endpoint value(s): Not stated

Results remarks:

Hexachloroethane caused slight to moderate tremors during days 15 and 16 of gestation in pregnant rats receiving 500 mg/kg hexachloroethane orally. Tremors were a lso observed during days 12 through 16 of gestation in pregnant rats receiving hexachloroethane, 260 ppm, by

inhalation.

Body weight gain of pregnant rats was significantly lower than the controls starting at day 8 of gestation for those receiving 500 mg /kg hexachloroethane orally and 260 ppm by inhalation, and day 14 for those exposed to 48 ppm.

There were no significant skeletal or soft tissue anomalies in fetuses exposed in utero to hexachloroethane. Fetuses from aspirin treated dams showed lower bod y weights and such malformations as hydrocephalus, spina bifida, and cranioschesis.

Fetuses from females treated with either 500 mg/kg hexachloroethane orally or aspirin showed significantly lower gestation indices and number of alive fetuses per dam. Th ese groups also showed higher fetal resorption rates when compared with corn oil controls. Maternal and fetal parameters of all other groups of treated animals appeared to be within normal limits. TheseDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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105 studies showed that hexachloroethane at dosages tox ic to the dams did not produce a teratogenic effect. Dosages of hexachloroethane that were toxic to the dams resulted in a slight slowing of fetal development.

Teratology rats receiving oral (500 mg/kg) or respiratory exposure (48 and 260 ppm) to HCE had an increased incidence of mucopurulent nasal exudates when compared with controls. The inflammatory exudates within the lumen of the nasal turbinates was observed in 85% of the 48 ppm group and in 100% of the 260 ppm exposed animals. About 70 percent of the orally exposed rats had evidence of upper respiratory tract irritation with approximately 20 percent having subclinical pneumonitis. Controls showed approximately 10% with a similar upper respiratory irritation and with subclinical pneumonitis.

CONC LUSIONS

Hexachloroethane at dosages toxic to the dams did not produce a teratogenic effect. Dosages of hexachloroethane that were toxic to the dams resulted in a slight slowing of fetal development.

STUDY RELIABILITY

[2] Reliable with restrictions. Th e study appears generally acceptable, although it is missing information including guideline followed and GLP compliance.

REFERENCE

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am . Ind. Hyg. Assoc. J. 40 (3): 187

- 199.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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106 B

- 9 . DEVELOPMENTAL TOXICITY

Summary of endpoint: Adequate

Study 1: Reproductive/developmental toxicity study in rats

Title: Teratology Study in Rats

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

- 72

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Remarks: Test substance is a crystalline white solid with a molecular weight of 236.74, bp 186.8 C and density of 2.09. Obtained from Pine Bluff Aresenal, Arkansas, identified as technical grade and supplied by Hummel Chemical Company, Plainfield, NJ, under the specifications of MIL

- H

- 235B, National Stock Number 6810

- 00

- N00

- 001. Approximately 99.8% pure.

METHOD

Method/guideline followed: Not specified

Test type: Teratology

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Sprague

- Dawley

Number of animals/sex/dose: 8 groups of 22 pregnant females/dose (5 groups for inhalation; 3 groups for oral)

Route of administration: Vapor (Inhalation) or Gavage

Exposure duration: Day 6 through Day 16 of gestation

Doses/concentrations tested: oral doses of 50, 100, 500 mg/kg; exposed to vapors at 15, 48 or 260 ppm

Vehicle: Air/Corn oil

Statistical methods used: Student’s t

- testDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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107 Test method/conditions remarks: Teratology studies were carried out in pregnant Sprague

Dawley rats given hexachloroethane by inhalation or by gavage from day 6 through day 16 of gestation. The day of finding sperm in the vaginal smears of rats was considered Day 0 of gestation. Twent y

- two pregnant rats per dose were used and all adult animals were sacrificed on day 20 of gestation by intracardiac injection of sodium pentobarbital. The reproductive tracts were exposed by laparotomy and the number of corpora lutea, implantation sites, and resporption sites were recorded. The fetuses were removed, examined for viable conceptuses, gross abnormalities, sex, weight, and body length. All grossly abnormal fetuses as well as eight normal appearing fetuses from each dam were fixed in either B ouin’s fluid or 95% ethyl alcohol. These were further studied by freehand section for soft tissue abnormalities (2 male and 2 female) or after Alizarin Red S staining for skeletal malformations (2 male and 2 female).

Five groups of 22 pregnant female rat s were given daily oral dosages form day 6 through day 16 of gestation of hexachloroethane (50, 100, 500 mg/kg), corn oil (5 mL/kg) and aspirin (250 mg/kg). Doses were adjusted according to individual daily body weights. Animals were housed in groups of 2 in suspended wire cages, food and water was freely available except for a daily 6

- hour period when both were removed to emulate the groups receiving hexachloroethane by

inhalation.

Three groups of 22 pregnant female rats were exposed from day 6 through day 16 of gestation to the vapors of hexachloroethane at concentration of 15, 48 and 260 ppm. All animals were observed for signs of toxicity and were weighed daily.

RESULTS

Endpoint value(s): Not stated

Results remarks:

Hexachloroethane caused sli ght to moderate tremors during days 15 and 16 of gestation in pregnant rats receiving 500 mg/kg hexachloroethane orally. Tremors were also observed during days 12 through 16 of gestation in pregnant rats receiving hexachloroethane, 260 ppm, by

inhalation.

Body weight gain of pregnant rats was significantly lower than the controls starting at day 8 of gestation for those receiving 500 mg/kg hexachloroethane orally and 260 ppm by inhalation, and day 14 for those exposed to 48 ppm.

There were no significan t skeletal or soft tissue anomalies in fetuses exposed in utero to hexachloroethane. Fetuses from aspirin treated dams showed lower body weights and such malformations as hydrocephalus, spina bifida, and cranioschesis.

Fetuses from females treated with e ither 500 mg/kg hexachloroethane orally or aspirin showed significantly lower gestation indices and number of alive fetuses per dam. These groups also showed higher fetal resorption rates when compared with corn oil controls. Maternal and fetal

parameter s of all other groups of treated animals appeared to be within normal limits. TheseDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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108 studies showed that hexachloroethane at dosages toxic to the dams did not produce a teratogenic effect. Dosages of hexachloroethane that were toxic to the dams resulted i n a slight slowing of fetal development.

Teratology rats receiving oral (500 mg/kg) or respiratory exposure (48 and 260 ppm) to HCE had an increased incidence of mucopurulent nasal exudates when compared with controls. The inflammatory exudates within th e lumen of the nasal turbinates was observed in 85% of the 48 ppm group and in 100% of the 260 ppm exposed animals. About 70 percent of the orally exposed rats had evidence of upper respiratory tract irritation with approximately 20 percent having subclin ical pneumonitis. Controls showed approximately 10% with a similar upper respiratory irritation and with subclinical pneumonitis.

CONCLUSIONS

Hexachloroethane at dosages toxic to the dams did not produce a teratogenic effect. Dosages of

hexachloroethan e that were toxic to the dams resulted in a slight slowing of fetal development.

STUDY RELIABILITY

[2] Reliable with restrictions. The study appears generally acceptable, although it is missing information including guideline followed and GLP complianc e.

REFERENCE

Weeks MH, Angerhofer RA, Bishop R, Thomasion J, Pope CR (1979) The toxicity of hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3): 187

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109 B

- 10

. CARCINOGENICITY

Study 1: Chronic carcinogenicity study in rats

Title: NTP Technical Report on the Toxicology and Carcinogenesis Studies of

Hexachlor o ethane in F344/N Rats

TEST SUBSTANCE

Identity: Ethane, hexachloro

- , CAS No. 67

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- 1

Remarks: molecular weight

– 236.7; > 99% pure

METHOD

Method/guideline followed: Not s tated

Test type: Two

- year study

GLP compliant?: Yes

Year study was performed: ~1984

Test species: Fischer 344 Rats . The male and female F344/N rats used in these studies were produced under strict barrier conditions at Charles River Breeding Laborat ories under a contract to the Carcinogenesis program. Breeding stock for the foundation at the National Institutes of Health Repository. The rats shipped for study were progeny of defined microflora

- associated parents that were transferred from isolators to barrier

- maintained rooms. Animals were shipped to the study laboratory at 5 weeks of age. Male rats were quarantined at the study laboratory for 15 days and females for 14 days. Thereafter, a complete necropsy was performed on five

animals of each s ex to assess their health status. The rodents were placed on study at 7 weeks of age. The health of the animals was monitored during the course of studies according to the protocols of the NTP Sentinel Animal Program.

Number of animals/sex/dose: groups of 50/sex

Route of administration: Gavage

Vehicle: corn oil

Doses/concentrations tested: 0, 10 or 20 (males); 0, 80 or 160 (females)

Exposure du ration : 5 days/week for 103 weeks

Statistical methods: Survival Analyses: The probability of survival was estimated by the product

- limit procedure ofDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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110 Kaplan and Meier (1958) and is presented in the form of graphs. Animals were censored from

the surviva l analyses at the time they were found to be dead from other than natural causes; animals dying from natural causes were not censored. Statistical analyses for a possible dose

related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) life table test for a dose

- related trend. When significant survival differences were detected, additional analyses using these procedures were carried out to determine the time point at which significant differences in th e surivival curves were first detected. All reported P values for the survival analysis are two

- sided.

Calculation of Incidence: The incidence of neoplastic or nonneoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators include only those animals for which the site was examined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors) prior to histologic sampling, or when lesions could have appeared at multiple sites (e.g. Lymphomas), the denominators consist of the number of animals on which a necropsy was performed.

Analysis of Tumor Incidence: The majority of tumors in this study were considered to be incidental to the cause of death or not rapidly lethal. Thus, the primary statistical method used was a logistic regression analysis, which assumed that the diagnosed tumors were discovered as

the result of death from an unrelated cause and thus did not affect the risk of death. In this approach, tumor prevalence was modeled as a logistic function of chemical exposure and time. Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated if it did not significantly enhance the fit of the model. The dosed and vehicle control groups were compared on the basis of the likelihood score test for the regression coefficient of dose. This method of adjusting for intercurrent mortality is the prevalence analysis of Dinse and Lagakos (1983) , further described and illustrated by Dinse and Haseman (1986). When tumors are incidental, this comparison of the time

- specific tumor prevalences also provides a compariso n of the time

- specific tumor incidences (McKnight and Crowley, 1984).

In addition to logistic regression, alternative methods of statistical analysis were used. These include the life table test (Cox, 1972; Tarone, 1975), appropriate for rapidly lethal t umors, and

the Fisher exact test and the Cochran

- Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overall proportion of tumor

- bearing animals.

Tests of significance include pairwise comparisons of each dosed group with vehi cle controls

and a test for an overall dose

- response trend. Continuity

- corrected tests were used in the analysis of tumor incidence, and reported P values are one

- sided. The procedures described above also were used to evaluated selected nonneoplastic le sions.

Historical Control Data: Although the concurrent control group is always the first and most appropriate control group used for evaluation, there are certain instances in which historical control data can be helpful in the overall assessment of tumo r incidence. Consequently, control tumor incidences from the NTP historical control data base (Haseman et al., 1984, 1985) are included for those tumors appearing to show compound

- related effects.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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111 Analysis of Continuous Variables: For the hematologic, s erum chemical and relative organ weight data, the nonparameteric multiple comparison procedures of Dunnet (1980) and Williams (1971, 1972) were used to assess the significance of pairwise differences between dosed and vehicle control groups. Jonckheere’s test (Jonckheere, 1954) was used to evaluate the significance of dose

- response trends.

Test method/conditions remarks:

Groups of 50 male rats were administered 0, 10 or 20 mg/kg hexahloroethane in corn oil via gavage, 5 days per week for 103 weeks. Gro ups of 50 female rats were administered 0, 80 or 160 mg/kg on the same schedule.

Animal maintenance: Animals were housed five per cage. Cages were rotated during the studies.

Feed and water were available ad libitum.

Clinical Examinations and Pathology : All animals were observed two times per day, and clinical signs were recorded at least one time per month. Body weights were recorded one time per week for the first 14 weeks of the studies and one time every 4 weeks thereafter. Mean body weights

were calculated for each group. Animals found moribund and those surviving to the end of the studies were humanely killed. A necropsy was performed on all animals including those found dead. Some tissues were excessively autolyzed or missing, and thus, the n umber of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on the study.

During necropsy, all organs and tissues were examined for grossly visible le sions. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histopathologic examination of tissues was performed according to an inverse pyramid design (McConnel, 1983a,b). That is, complete histopathologic examinations were performed on all high dose and vehicle control animals and on low dose animals dying through month 21 of the study. In addition, histopathologic examinations were performed on all grossly visible lesions in all dose groups. Potential target organs for chemically related neooplastic and nonneoplastic effects were identified as adrenal glands (male), kidney, pituitary gland, and thyroid gland from the short

- term studies or the literature and were determined by examination of the pathology data; these target organs/tissues in the lower dose group were examined histopathologically.

When the pathology evaluation was completed by the laboratory pathologist and the pathology data entered into the Toxicology Data Ma nagement System, the slides, paraffin blocks, and

re sidual wet tissues were sent to the NTP Archives for inventory, side /block match, and wet

tissue aud it. The slides, individual animal data records, and pathology tables were sent to an independent quality assessment labor a tory. The individual animal records and tables were compared for accuracy, slides and tissue counts were verified, and histot echnique was evaluated.

All tu mo r diagnoses, all target tissues, and all tissues from a randomly selected 10% o f the animals were evaluated by a quality assessment pathologist. The quality assessment report and slides were submitted to the Pathology Working Group (PWG) Chairperson, who reviewed all target tissues and those about which there was a disagreement betw een the laboratory and qualityDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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112 assessment pathologists.

Representative slides selected by the Chairperson were reviewed by the PWG, which included the laboratory pathologist, without knowledge of previously rendered diagnoses. When the consensus diagnosi s of the PWG differed from that of the laboratory pathologist, t he laboratory pathologist was asked to reconsider the original diagnosis. This procedure has been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). The final dia gnoses represent a consensus of contractor pathologists and the NTP Pathology Working Group. For subsequent analysis of pathology data, the diagnosed lesions for each tissue type are combined according to the guidelines of McConnell et al. (1986).

Nonneo plastic lesions in the adrenal glands and kidneys were examined by the quality assessment pathologist and PWG because they were considered part of the toxic effect of the

chemical.

RESULTS

Endpoint value(s): No data

Results remarks: Body Weights and C linical Signs: Mean body weights of high dose male rats

were 5%

- 6% lower than those of vehicle controls after week 81; mean body weights of high dose

female rats were 5%

- 9% lower than those of vehicle controls between week 41 and week 101.

Dosed female ra ts were hyperexcitable.

Survival: No significant differences in survival were observed between any groups of either sex.

Pathology and Statistical Analyses of Results: This section describes the significant or noteworthy changes in the incidences of rats with neoplastic or nonneoplastic lesions of the kidney and adrenal gland.

Kidney

– Renal tubule hyperplasia and neoplasia were observed at greater incidences in dosed male rats. The combined incidence of adenomas or carcinomas in the high dose males was significantly greater than that in the vehicle controls. All the carcinomas and one of the adenomas, also in a high dose male, were seen grossly; one of the carcinomas metastasized. Microscopically, tubular hyperplasia and neoplasia represented a morpho logic continuum. Hyperplasia consisted of tubules that were slightly enlarged (up to approximately twice the normal diameter) and contained increased numbers of tubular epithelial cells that were stratified

and filled the tubular lumens. Adenomas were la rger than the hyperplasia and ranged in size from less than 1 mm up to approximately 4 mm. Adenomas were discrete masses consisting of variably sized, solid nests of cells and occasional tubular structures separated by a sparse

fibrovascular stroma. The adenomas were composed of moderately large, somewhat pleomorphic epithelial cells with round

- to

- ovoid nuclei with a prominent nucleolus and eosinophilic granular to clear vacuolated cytoplasm. One adenoma consisted of enlarged cells with prominent nucleol us and eosinophilic granular to clear vacuolated cytoplasm. One adenoma consisted of enlarged cells with prominent eosinophilic cytoplasm granules characteristic of renal “oncocytoma.” The histogenesis of the oncocytoma is uncertain but mayDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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113 be derived ei ther from the proximal tubule or collecting ducts of the kidney. Carcinomas were larger masses, up to 1 cm or more in diameter, and consisted of sheets of pleomorphic epithelial cells that, in some tumors, surrounded a central cystic area. Nephropathy wa s observed in nearly all males, but the severity was slightly increased in dosed males relative to vehicle controls; the overall average severity was mild in vehicle controls and mild to moderate in dosed males. The incidences of nephropathy were also inc reased in dosed females (vehicle control, 22/50; low dose, 42/50; high dose, 45/50), and severity of nephropathy was significantly (P < 0.01) increased in dosed females relative to vehicle controls; the overall average severity was minimal

in vehicle contr ols and minimal to mild in dosed females. Nephropathy in each sex consisted of tubular cell degeneration and regeneration, tubular dilation and atrophy, glomerulosclerosis,

interstitial fibrosis and chronic inflammation. Linear mineralization of the rena l papillae (2/50; 15/50; 32/50) and hyperplasia of the pelvic transitional epithelium (0/50; 7/50; 7/50) were increased in dosed males. The incidences of these lesions were not increased in females by

chemical administration.

Adrenal Gland: Pheochromocyt omas occurred at an increased incidence in low dose male rats and, to a lesser extent, in high

- dose male rats, relative to vehicle controls. Malignant pheochromocytomas occurred in one vehicle control, two low dose, and one high dose male rats,

and two co mplex pheochromocytomas occurred in high dose male rats. Pheochromocytomas were circumscribed masses of altered adrenal medullary cells that lacked the normal orderly arrangement in small packets separated by a sparse fibrovasular stroma. The cells were arranged in large solid clusters or irregular branching cords. The cells had small hyperchromatic nuclei or slightly enlarged vesicular nuclei with prominent nucleoli. The cytoplasm of the cells was often more basophilic than that of normal cells. The m alignant pheochromocytomas consisted of anaplastic pleomorphic cells that obliterated the adrenal gland. Since complex pheochromocytomas are simply variants of the usual pheochromocytomas which contain nervous tissue in addition to adrenal medullary cells , these neoplasms were combined with the others for purposes of analysis. The incidence of pheochromocytomas in low dose male rats was significantly greater than that in the vehicle control group.

CONCLUSIONS

Under the conditions of the 2

- year gavage st udies, there was clear evidence of carcinogenic activity of hexachloroethane for male F344/N rats, based on the increased incidences of renal neoplasms. The marginally increased incidences of pheochromocytomas of the adrenal gland may have been related to hexachloroethane administration to male rats. There was no evidence of carcinogenic activity of hexachloroethane for female F344/N rats administered 80 or 160 mg/kg by gavage for 103 weeks. The severity of nephropathy and incidences of linear

mineraliza tion of the renal papillae and hyperplasia of the transitional epithelium of the renal pelvis were increased in dosed male rats. The incidences of severity of nephropathy were

increased in dosed female rats.

STUDY RELIABILITY

1

– Reliable with out restr ictionsDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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114 REFERENCE

National Toxicology Program (1989). NTP Technical Report on the Toxicology and Carcinogenesis Studies of Hexachloroethane (CAS No. 67

- 72

- 1) in F344/N Rats (Gavage Studies). NIH Publication No. 89

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115 Study 2: Chronic carcinogenici ty study in mice

Title: Carcinogenicity Toxicity study of Hexachloroethane

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Purity: 98%

METHOD

Method/guideline followed: Not specified

Test type: Carcinogenicity

GLP compliant?: Not specified

Year study performed: ~ 1978

Test species: Osborne

- Mendel rats and B6C3F1 mice, Battelle Memorial Institute, Columbus, Ohio (rats) and Charles River Breeding Laboratories, Inc., Wilmington, MA (mice), 6 weeks old (rats), body weight not spe cified

Number of animals/sex/dose: 50/sex/dose level/species

Route of administration: Oral gavage

Vehicle: Pure corn oil (20 animals/sex)

Exposure duration: 5 days/week cyclically for 44 of 78 weeks in rats and continuously for 78

weeks in mice followe d by an observation period of 33 or 34 weeks (rats) and 12 or 13 weeks (mice)

Doses/concentrations tested: 250, 500 mg/kg/day (rats); 500 or 1000 mg/kg/day (mice). In week 9, the mice dose levels were increased to 600 and 1200 mg/kg/day.

Statistical met hods used: Probabilities of survival were estimated by the product

- limit procedure of Kaplan and Meier (1958). Statistical analyses for a possible dose

- related effect on survival used the method of Cox (1972) for testing two groups for equality and used Tarone's (1975) extensions of Cox's methods for testing a dose

- related trend. One

- tailed P

- values have been reported for all tests except the departure from linearity test, which is only reported when

its two

- tailed P

- value is less than 0.05. The Cochran

- Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971, pp. 362

- 365), was also used.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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116 Test method/conditions remarks: Rats and mice were exposed for 44 of 78 weeks. Twenty animals of each sex were placed on test as the v ehicle controls, and twenty animals of each sex were placed on test as untreated controls from each species. From the first day, all animals were inspected daily for mortality. Body weights, food consumption, and data concerning appearance, behavior, signs of toxic effects, and incidence, size, and location of tissue masses were recorded at weekly intervals for the first 10 weeks and at monthly intervals thereafter. The presence of tissue masses was determined by observation and palpation of each animal. A necropsy was performed on each animal regardless of whether it died, was killed when moribund, or was sacrificed at the end of the bioassay.

Slides were prepared from the following tissues: subcutaneous tissue, lungs and bronchi, trachea, bone marrow, s pleen, lymph nodes, thymus, heart, salivary gland, liver, gallbladder and bile duct (mice), pancrease, esophagus, stomach, small intestine, large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, brain, muscle, tunica vaginalis, uterus, mammary gland and ovary.

RESULTS

Endpoint value(s): None stated

Results remarks:

In Osborne

- Mendel Rats:

Distinct dose

- related depression in mean body weight was evident in male rat groups. For female rats, low dose and contr ol groups exhibited similar growth patterns, but mean body weight of high dose rats was slightly depressed relative to the other female rat groups. Fluctuations in the growth curve may be due to mortality; as the size of the group diminishes, the mean body weight may be subject to wide variations. During the first year of the study the incidence of observed clinical signs was slightly increased in the treated rats when compared to the untreated controls. The signs observed with the most frequency were hunch ed appearance; reddened, squinted or lacrimating eyes; and abdominal urine stains. The incidences of these signs were comparable between low and high dose animals; however, the high dose females showed a higher frequency of abdominal urine staining from

we ek 4 until termination of the bioassay. During the second year of the study, behavior and appearance were comparable between treated and control animals. Respiratory abnormalities were observed in all groups during the latter part of the first year. The

in cidence of respiratory symptoms increased at gradual and comparable rates for all groups during the last six months of the study. Clinical observations associated with aging in laboratory rats were noted in comparable numbers of treated and untreated contr ols during the second year. These signs included: sores on the tail or other parts of the body, alopecia, discolored or rough fur, eye discharge or red crust around the eyes and palpable nodules and/or tissue masses. Isolated, apparently spontaneous obser vations noted in several treated rats included transient tremors, vaginal discharge and ataxia.

For male rats, the Tarone test indicated a significant (p < 0.001) association between increased dosage and accelerated mortality. Thirty

- eight percent (19/50 ) of the high dose and 48 percent (24/50) of the low dose males survived at least 90 weeks, compared to 70 percent (14/20) of theDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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117 untreated controls, and, despite the sacrifice of seven rats in week 60, 55 percent (11/20) of the

vehicle controls. For fema le rats mortality was significantly increased in the dosed groups compared to the untreated groups. The actual survival, however, was adequate for statistical analysis of late

- developing tumors as 48 percent (24/50) of the high dose, 54 percent (27/50) of the low dose, 70 percent (14/20) of the vehicle control, and 70 percent (14/20) of the untreated

control rats survived until the end of the test.

Pathology

– Each tumor type detected has been encountered previously as a spontaneous lesion

in Osborn

- Mendel rats and no appreciable difference in frequency was noted between the control

and treated rats.

With the exception of certain renal lesions, inflammatory, degenerative, and proliferative lesions

seen in control and treated rats were similar in number and kind to those naturally occurring lesions found in aged rats. In addition to the chronic inflammatory lesions of the kidney seen in control and treated animals, toxic tubular nephropathy was associated with compound exposure.

The lesion occurred in 22/4 9 (45%) low dose males, 33/50 (66%) high dose males, 9/50 (18%) low dose females and 29/49 (59%) high dose females and was characterized by degeneration, necrosis and the presence of large hyperchromatic regenerative epithelial cells. Overlying the

tubula r lesions were chronic interstitial nephritis and fibrosis, focal pyonephritis, tubular ectasia, cast formation, and focal glomerulosclerosis. Renal tubular

- cell adenomas were found in four

low dose male rats.

In B6C3F1 Mice

No distinct dose

- related depr ession in mean body weight was evident in males or females. Fluctuations in the growth curve may be due to mortality; as the size of the group diminishes, the mean body weight may be subject to wide variations.

There was no evidence of compound effect wi th regard to physical appearance or behavior of the treated mice during the first 34 weeks of the study. Signs often observed in group

- housed laboratory mice, particularly males, were observed at a comparable rate in all groups. These signs included sores on the body and/or extremities, a hunched appearance, localized alopecia, external genital irritation, and rough or stained fur.

A hunched or thin appearance was observed with greater frequency in the treated groups from

week 38 until termination of the b ioassay in week 91. The incidence of palpable nodules, tissue masses, or swollen areas was slightly greater in the treated mice than in the controls.

For both male and female mice there was no significant positive association between dose and mortality. F or males the survival was unexpectedly low in the control groups and the low dose group as only 25 percent (5/20) of the vehicle control, 5 percent (1/20) of the untreated control, and 14 percent (7/50) of the low dose mice survived until the end of the te st, compared to 58 percent (29/50) of the high dose mice. There were adequate numbers of females at risk from

late

- developing tumors as 68 percent (34/50) of the high dose, 80 percent (40/50) of the low dose, 80 percent (16/20) of the vehicle control, and 85 percent (17/20) of the untreated control

mice survived until the end of the test.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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118 Pathology

- Hepatocellular carcinomas occurred in 1/18 (6 percent) male untreated controls, 3/20 (15 percent) male vehicle controls, 15/50 (30 percent) low dose males, 31/49 (63 percent) high dose males, 0/18 untreated control females, 2/20 (10 percent) vehicle control females, 20/50 (40 percent) low dose females, and 15/49 (31 percent) high dose females. Microscopically, the hepatocellular carcinomas varied greatly in appearance. Some contained well

- differentiated hepatic cells that had a relatively uniform arrangement of the cords and others had very anaplastic liver cells with large hyperchromatic nuclei, often with inclusion bodies and with vacuolated pale cytoplasm . Arrangement of the neoplastic liver cells varied from short, stubby cords to nests of hepatic cells and occasionally pseudo

- acinar formation. Mitotic figures were often present. The hepatic neoplasms occurring in the control mice were not different in

ap pearance from those seen in the hexachloroethane

- dosed animals.

Toxic nephropathy occurred in 49/50 (98 percent) low dose males, 47/49 (96 percent) high dose males, 50/50 (100 percent) low dose females, and 45/49 (92 percent) high dose females. Microscopi cally, the nephropathy was characterized by degeneration of convoluted tubule epithelium at the junction of the cortex and medulla. Some affected tubules contained hyalin casts. Occasionally the damaged cells were replaced by enlarged dark staining regener ative tubular epithelium. At this stage, the kidney often showed infiltration of inflammatory cells, fibrosis, and calcium deposition.

CONCLUSIONS

There is no histopathologic evidence that hexachloroethane is carcinogenic in Osborne

- Mendel

rats under t he conditions of this experiment. Toxic tubular nephropathy was present in rats of both sexes at both dose levels, but not in the control rats.

Results of the histopathologic examination in mice indicate that hexachloroethane was carcinogenic, causing an increased incidence of hepatocellular carcinomas in male and female mice. This chemical also caused toxic nephropathy in mice of both sexes.

STUDY RELIABILITY

[2] Reliable with restrictions. The study appears generally acceptable, although it is missi ng information including guideline followed and GLP compliance.

REFERENCE

Bioassay of Hexachloroethane for Possible Carcinogenicity, CAS No. 67

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- 1. National Cancer Institute Carcinogenesis Technical Report Series No. 68 (1978).Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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119 B

- 11

. SKIN IRRITATIO N

Study 1: Dermal irritation study in rabbits

Title: Skin Irritation Toxicity in Rabbits

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: None stated

METHOD

Method/guideline followed: Not specified

Test type: Dermal

GLP com pliant?: Not specified

Year study performed: ~ 1937

Test species: Rabbit (strain not specified)

Number of animals/sex/dose: 3/dose (sex not specified)

Route of administration: Dermal

Vehicle: Diamond paraffin oil, Deo base oil, 95% ethanol, and propyle ne glycol

Doses/concentrations tested: 1.5%, 5%, less than 10% (in 95% ethanol), 10%

Statistical methods used: Not specified

Test method/conditions remarks: Hexachloroethane was tested on rabbits for its action on the skin, using Diamond paraffin oil, Deo base oil, 95% ethanol and propylene glycol as media.

Exposure conditions: 1.5% in Diamond paraffin oil (rabbit 3

- 63): a. The oil solution was applied to the abdomen for periods of 1, 2, and 4 hours, after which it was thoroughly wiped off with cotton moistened with CCl4. b. The oil solution was also bandaged to the abdomen for 4 days.

1.5% in Diamond paraffin oil (rabbit 3

- 49): The solution was applied to one ear seven times in a period of 8 days.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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120 10% in Diamond paraffin oil (rabbit 3

- 180): The s olution was held in contact with the abdominal skin for 3 days, using cotton pads and bandages.

Diamond Paraffin Oil (rabbit 2

- 124): The oil was applied to the inside of one ear and to the abdomen seven times in a period of fifteen days. The abdomen was kept covered with a cloth bandage

10% in Deo base oil (rabbit 3

- 63): The solution was bandaged on the abdomen on two successive days.

5% in Deo base oil (rabbit 3

- 58): The oil solution was applied to one ear 17 times in a period of 22 days.

Deo base oil (rabbit 2

- 150): Thirteen times in a period of eighteen days the base oil was rubbed

on the abdomen for one minute.

Deo base oil (rabbit 2

- 137): The base oil was held in contact with the abdominal skin for a period of ten days by means of cotton pads held in place with cloth bandages. The applications were

renewed four times.

Deo base oil (rabbit 2

- 15): The Deo base oil was applied to the ear thirteen times in a period of 18 days.

Deo base oil (rabbit 2

- 137): The oil was applied to the ear fourteen time s in a period of twenty

six days.

Hexachloroethane

– saturated solution in 95% ethanol, somewhat less than 10%

- (rabbit 3

- 58): The solution was applied to the abdomen for a period of four days.

Hexachloroethane

– saturated solution in 95% ethanol, somew hat less than 10%

- (rabbit 3

- 63): The solution was applied to one ear twenty times in a period of thirty

- one days.

Hexachloroethane

– slightly less than 1.5% in propylene glycol

– (rabbit 2

- 283): The solution was held in contact with the abdomen by means of cotton pads for a period of 34 days.

Hexachloroethane

– slightly less than 1.5% in propylene glycol

– (rabbit 2

- 283): The solution was applied to the one ear 21 times in a period of 34 days.

RESULTS

Endpoint value(s): Values not specified

Results r emarks: The results for each media were as follows:

1.5% Hexachloroethane in Diamond paraffin oil applied to the abdomen for four hours There was no apparent reaction from Rabbit #3

- 63Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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121 1.5% Hexachloroethane in Diamond paraffin oil applied to the abdomen for four days There developed no apparent reaction.

10% Hexachloroethane in Diamond paraffin oil for 3 days A severe redness and some edema resulted. This cleared up in about 5

- 6 days.

1.5% Hexachloroethane in Diamond paraffin oil applied to the ear sev en times over a period of eight days A very severe reaction developed, hyperemia, marked follicular enlargement, and raised follicles end loss of hair. This reaction was apparently identical with that of the paraffin oil and completely masked any possibl e action of hexachloroethane.

Diamond paraffin oil applied to the inside of one ear and to the abdomen seven times over a period of fifteen days The ear became reddened after the third application, then a third layer of epidermis peeled off.

Further tre atment caused the ear to become thickened, heavy and stiff. The hair follicles became enlarged and protruded, making the ear quite rough. A heavy, dry scale covered the whole inside

surface of the ear. The skin of the abdomen quickly developed redness, i ncreasing intensity, and followed by a peeling of the epidermis. Toward the last of the treatment the skin became thickened in addition

to redness and scaliness.

10% Hexachloroethane in Deo base oil Contact for 2 days resulted in edema and severe redne ss followed by excoriation. A severe reaction was produced.

5 % Hexachloroethane in Deo base oil applied to one ear Under the treatment the ear developed hyperemia followed by thin exfoliation. After 15 days (12 applications) a slight follicular promine nce was apparent. The reaction progressed slowly; at 28 days, when killed, the ear showed a marked reaction, moderate hyperemia, moderate follicular enlargement with hair loss and exfoliation. Microscopic examinations showed evidence of an epithelial hyp erplasia of the ear.

Deo base oil An unspecified amount of the Deo base oil applied to the abdomen showed some redness and a fine scaliness toward the end of treatment over a period of eighteen days. When exposed for a period of ten days, marked irritati on, excoriation, and exudation followed by crustation had developed after three days of treatment. A marked hyperemia developed at the last. The whole cleared up within about a week. When applied to the ear, a faint exfoliation after nine applications ( over a twenty

- six day period) occurred.

Saturated solution of hexachloroethane in 95% ethanol (somewhat less than 10% ) There were no apparent results when applied to the abdomen. When applied to the ear, a slight hyperemia and a thin scale developed. I t is possible a very faint follicular reaction had alsoDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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122 appeared. Microscopic examination showed evidence of a very slight degree of epithelial hyperplasia.

Slightly less than 1.5% hexachloroethane in propylene glycol After three days of exposure to the abdomen of the rabbit, the abdomen was faintly reddened followed by a slight scaliness formation. This appearance persisted unchanged. No apparent reaction developed on the ear, and microscopic evaluations showed questionable indications of an epithelial hyperplasia.

CONCLUSIONS

The test substance had a very slight irritant action on the skin of a rabbit. This action was not strong enough to indicate a skin hazard for man.

STUDY RELIABILITY

[3] Not reliable. The study did not follow an OECD guideline , analytical monitoring was not conducted, an insufficient number of animals, as well as the species and the sex of the animals were not stated, no acclimatization period was noted, no LD 50 values were stated, and no

statistical methods were stated.

REFE RENCE

TSCATS submission (OTS0206444, Document ID 878213744, received 06/29/83). The Dow Chemical Company 1983. The Irritant Action of Hexachloroethane of Rabbits with Cover Letter

Dated 1937.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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123 B

- 12

. PHARMACOKINETICS AND METABOLISM

Study 1: Pharmac okinetic study in rats

Title: Hexachloroethane : Tissue Clearance and Distribution in Fischer 344 Rats

TEST SUBSTANCE

Identity: Ethane, hexachloro

- (HCE), CAS No. 67

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Remarks:

METHOD

Method/guideline followed: No data

Test type: Pharmacokine tic

GLP compliant?: Not stated

Year study was performed: 1978

Test species: Fischer 344 Rats

Number of animals/sex/dose: 20 males

Route of administration: Diet

Vehicle: Not stated

Doses/concentrations tested: 3, 30, 100 mg/kg/day

Exposure durat ion: 57 days

Statistical methods: No data

Test method/conditions remarks: Twenty male rats were administered the test substance in the diet at 100 mg/kg/day for 57 days. These animals were then placed on a control diet and sacrificed in groups of 3

- 4 rats each 0, 3, 6, 13, 22 and 33 days after being placed on the control diet and samples of fat, liver, kidney and whole blood were taken from HCE analysis.

Male and female rats (4/sex/dose) were administered the test substance in the diet at 3, 30 or 10 0 mg/kg/day for 110

- 111 days. Samples of fat, kidney, liver, and whole blood were taken from 4

rats/sex/dose level at the terminal sacrifice.

RESULTSDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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124 57 day test: The highest concentration of HCE was consistently found in the fat. The

concentration of HCE in the fat was approximately three times the concentration found in the kidney but over 100 times the concentration found in the liver and blood. The concentration of

HCE in the fat decreased from 303 +/

- 50 μg/g in an apparent first

- order manner (hal f life = 2.7 days). The concentration of HCE in the liver, kidney and blood also decreased in an apparent

first

- order manner with half

- lives very similar to the fat. The calculated half

- lives for the clearance of HCE from the kidney, liver and blood were 2.6, 2.3 and 2.5 days, respectively.

Concentration of HCE in tissues after 110

- 111 days on test diet: The results from the 100 mg/kg/day dose level are consistent with the results from the clearance study. Although the

concentration of HCE in the fat w as lower than on day zero of the clearance study, these animals were fasted overnight prior to sacrifice and normal clearance of HCE may account for this difference. The concentration of HCE in the liver and kidney were higher than on day zero of

the clea rance study. However the concentration of HCE in the liver agreed with the expected concentration based on the later sacrifice intervals. This suggests the values for liver HCE on day zero were aberrantly low. The increased concentration in kidneys may be due to the increased mobilization of HCE from the fat as a result of the overnight fast. The concentration of HCE in male kidneys was higher than in female kidneys at all dose levels. As the dose level increased, there was a disproportionately large in crease in the concentration of HCE in male kidneys but a disproportionately small increase in the concentration of HCE in female kidneys. Thus the difference in concentration of HCE in the kidney of male and female rats was most striking at the 100 mg/kg/ day dose rate or when the data is expressed as a ratio of tissue/dietary concentration.

In both sexes there was a disproportionately large increase in the concentration of HCE in the fat with increasing dosage rates. In contrast, there was a disproportio nately small increase in the concentration of HCE in the liver and blood as the dosage rates increased.

Endpoint value(s): No data

CONCLUSIONS

HCE was rapidly cleared from the tissues of male rats with a half

- life of between 2 and 3 days.

Differences in the concentration of HCE in the kidneys of rats suggest that male rats should be

10 to 30 times more sensitive than females to the toxic effects of HCE. Tissue concentrations of HCE were not proportional to dose. Although this appears to be of limited toxicological significance, it does suggest that blood HCE analysis will be of limited value in assessing human exposure. Additional rat pharmacokinetic data is not needed at this time but data on the nature or

reason for the difference in accumulation o f HCE in male and female rat kidney would be useful in assessing hazard.

STUDY RELIABILITY

2

– Reliable with restrictionsDraft HPV4 Ch emical Data Adequacy Review CAS No. 67

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125 REFERENCE

TSCATS submission (OTS0206444, Doc ID 878213746; received 06/29/83): Dow Chemical Company. 1978. Hexachloroethane : Ti ssue Clearance and Distribution in Fischer 344 Rats.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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126 Study 2: Pharmacokinetic study in rats

Title: Hexachloroethane: Tissue Clearance and Distribution in Fischer 344 Rats

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67

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Remarks: Not spec ified

METHOD

Method/guideline followed: Not specified

Test type: Tissue Toxicity Study

GLP compliant?: Not specified

Year study performed: 1978

Test species: Fischer 344 Rats

Number of animals/sex/dose: 4/sex/dose (concentration in tissues); 3

- 4 male s/group (tissue clearance)

Concentrations tested: 3, 30, 100 mg/kg

Exposure duration: 110

- 111 days (concentration in tissues); 57 days (tissue clearance)

Statistical methods used: Not specified

Test method/conditions remarks: The objective of the stud y was to provide information on the fate of hexachloroethane (HCE) that would aid in the interpretation of existing toxicity data and in assessing hazard. Samples of fat, liver, kidney, and blood were obtained from Fischer rats

included in a subchronic to xicity study and analyzed for HCE. The results of these analyses

were used to calculate the rate at which HCE is cleared from the tissues and to determine the

effects of dose and sex on the distribution of HCE. These tissues were selected for the followi ng reasons. The greatest accumulation of most lipophilic chemicals occurs in fatty tissue, thus fat should provide the best estimate of maximum accumulation. Preliminary toxicity data indicated the liver and kidney were the primary target organs for toxi city. Blood was selected because it is the tissues best suited for repeated or human sampling and thus may be needed for subsequent

inter

- study comparisons. The study was included as part of a subchronic study because limited

information on the fate of H CE in animals exists and when possible it is ideal to study the fate and toxicity of a chemical under identical conditions.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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127 Samples of blood, liver, kidney and fat were collected at necropsy, weighed and stored frozen. The samples were homogenized in 5 m L of glass

- distilled hexane and centrifuged. The supernatant hexane was removed, diluted as required with additional hexane and analyzed. An automated sampler was used to inject the samples and standards in hexane directly into a Varian 3700 gas chromato graph (GC) equipped with electron capture detector. Area responses for the GC peaks were obtained using a Varian CDS 111 integrator. The extraction efficiency for HCE from blood, liver, kidney and fat into hexane were determined to be 0.986, 0.829, 1.000 and 0.711, respectively. Under the above analytical conditions, the retention time for HCE was 2.55 min and the limit of detection was 0.001 ug/g. An interference peak with the same retention time

as HCE was observed in some of the tissue blanks. Becau se of the sporadic nature of this peak, the data was not corrected for this interference. However, this interference could result in the concentration of HCE in some samples being over estimated by 0.015 μg/g and would be significant only when the samples contained very low concentrations of HCE.

RESULTS

Genotoxicity result: Not specified

Cytotoxic concentration (if applicable): Not specified

Results remarks:

Clearance of HCE from the Tissues: Twenty male rats were used to determine the rate at which HCE is cleared from the tissues. After 57 days on a diet providing 100 mg HCE/kg/day, the rats were placed on a control diet containing no HCE. These animals were sacrificed in groups of 3

- 4 rats each 0,3,6,13,22 and 33 days after being placed on the co ntrol diet and samples of fat, liver, kidney and whole blood taken for HCE analysis. The highest concentration of HCE was consistently found in the fat. The concentration of HCE in the fat was approximately three times

the concentration found in the kidn ey but over 100 times the concentration found in the liver and

blood. The concentration of HCE in the fat decreased from 303 +/

- μg/g in an apparent first

order manner (t1/2 = 2.7 days). The concentration of HCE in the liver, kidney and blood also

decre ased in an apparent first

- order manner with half

- lives very similar to the fat. The calculated

half

- lives for the clearance of HCE from the kidney, liver and blood were 2.6, 2.3, and 2.5 days respectively.

Concentration of HCE in Tissues after 110

- 111 Da ys on Test Diet: Samples of fat, kidney, liver

and whole blood were taken from 4 rats/sex/dose level at the terminal sacrifice. The concentration of HCE found in these tissues is presented in Tables 2 and 3. The results from the 100 mg/kg/day dose level are consistent with the results from the clearance study. Although the concentration of HCE in the fat was lower than on day zero of the clearance study, these animals were fasted overnight prior to sacrifice and normal clearance of HCE many account for t his difference. The concentration of HCE in the liver and kidney were higher than on day zero of the clearance study. However the concentration of HCE in the liver agreed with the expected concentration based on the alter sacrifice intervals. This sugge sts the values for liver HCE on day zero were aberrantly low. The increased concentration in kidneys may be due to the increased mobilization of HCE from the fat as a result of the overnight fast.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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128 The concentration of HCE in male kidneys was higher than in female kidneys at all dose levels. As the dose level increased, there was a disproportionately large increase in the concentration of HCE in male kidneys but a disproportionately small increase in the concentration of HCE in female kidneys. Thus the difference in concentration of HCE in the kidney of male and female rats was most striking at the 100 mg/kg/day dose rate or when the data is expressed as a ratio of the tissue/dietary concentration.

In both sexes there was a disproportionately large incr ease in the concentration of HCE in the fat with increasing dosage rates. In contrast, there was a disproportionately small increase in the concentration of HCE in the liver and blood as the dosage rate increased.

CONCLUSIONS

The tissue clearance data i ndicates that HCE is eliminated by the rat with a half

- life of between 2 and 3 days. Thus, upon repeated exposure, the concentration of HCE in the rat will plateau with 17 days. Because HCE is rapidly eliminated, reversible symptoms of toxicity should di sappear soon after the exposure is terminated.

STUDY RELIABILITY

[2] Reliable with restrictions. The study appears generally acceptable, although it is missing information including guideline followed and GLP compliance.

REFERENCE

TSCATS submission (O TS0206444, Document ID 878213746, received 06/29/83). Dow Chemical Company 1983. Hexachloroethane: Tissue Clearance and Distribution in Fischer 344

Rats 1978. Authors: Nolan and Karbowski. December 12, 1978. Performing Laboratory: Toxicology Research Laboratory Health and Environmental Research.Draft HPV4 Ch emical Data Adequacy Review CAS No. 67

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129 CAS No. 67

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- 1: Summary of Endpoints

Endpoint Study Information

Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish Yes Yes No Acute Toxicity to Daphnia Yes Yes No Toxicity to Aquatic Plants Yes No Yes

Health Effects Acute Toxicity Yes Yes No Repeated

- Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity Yes Yes No Developmental Toxicity Yes Yes No