SRC TR-10-128

HPV4 CHEMICAL DATA ADEQUACY REVIEW

Hexachloroethane, CAS No. 67-72-1

Prepared by:

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

Chemical, Biological and Environmental Center

SRC, Inc.

7502 Round Pond 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

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

July 23, 2010
HPV4 Chemical Data Adequacy Review

Hexachloroethane, CAS No. 67-72-1

July 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 22202
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 toxicity 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

Chromosomal 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 data:

PubMed - Bibliographic database that contains references to journal articles in the life 

sciences with a concentration on biomedicine

TOXNET -National Library of Medicine databases on toxicology, hazardous chemicals, 

environmental health, and toxic releases; 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 Substances Data Bank (HSDB) - Comprehensive, peerreviewed toxicology data for about 5000 chemicals;

• Genetic Toxicology Data Bank (GENE-TOX) - Peer-reviewed genetic 

toxicology test data for over 3000 chemicals.

TSCATS - Unpublished 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 database
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 INCHEM 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 obtained for any relevant toxicity data found for the assigned chemicals. 

While IUCLID Datasets sometimes contained studies of interest, certain study 

descriptions contained insufficient detail to assess adequacy. In these cases, the primary source 

of the study, if available and accessible, was obtained and reviewed.

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 into 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 summarizing 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 inadequate, a basis for that 

conclusion is supplied. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

3

B-1. TOXICITY TO FISH

Summary of endpoint: Adequate

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. 67-72-1

Remarks: None

METHOD

Method/guideline followed: None stated

GLP: Not stated

Year study performed: 1978

Test type: Acute Fish Toxicity 

Test species: Bluegill (Lepomis macrochirus)

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 = 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 Chemical Data Adequacy Review CAS No. 67-72-1

4

96-hour LC50 = 0.98 ppm (0.85 – 1.10)

NOEC < 0.22 ppm 

Results remarks: None stated

CONCLUSIONS 

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 Chemical Data Adequacy Review CAS No. 67-72-1

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 including information regarding 

study methodology.

TEST SUBSTANCE

Identity: Ethane, hexachloro-, CAS No. 67-72-1

Remarks: None

METHOD

Method/guideline followed: None stated

GLP: Not stated

Year study performed: 1978

Test type: Acute Fish Toxicity 

Test species: 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 stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

6

CONCLUSIONS 

96-hour LC50 = 2.50 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 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

Remarks: None

METHOD

Method/guideline 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 

Concentrations 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-volumes 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 CaCO3), and alkalinity (as CaCO3) were determined for control, an 

intermediate, and the 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 CaCO3 (45.-45.5), and 41.8 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

8

mg/L as CaCO3 (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).

Biological 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 rearing 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 counted 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 more 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. Environmental Protection 

Agency (1975). 

Chemical methods – Chemical analyses for hexachloroethane was performed with a HewlettPackard 5730A gas chromatograph equipped with a Model 3552A data system and a 63Ni 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 on 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 determine 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 SpearmanKarber 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 stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 Contamination and Toxicology 12: 661-666.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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 Toxicity 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 program 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 stock 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 each compound to establish a range of toxicity and a full scale static test was then run to 

determine the 96-hour LC50. The control fish were exposed to the same amount of methanol as 

in the highest toxicant concentration. Filtered plant air was passed through 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 Chemical Data Adequacy Review CAS No. 67-72-1

11

Samples were collected and verified by Gas Chromography 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 was 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 ppm 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 (LC50) within the 96-hour exposure period. Log probit paper was used 

to calculate percent mortality versus concentration. A standard LC50 was determined according 

to the test methods described by the Committee on Methods of Toxicity Tests with Aquatic 

Organisms (1975).

Reviewer’s note: 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 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

Remarks: 98% purity; Lot: ED080787

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

Concentrations 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 

measured 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 tested 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 and 96 hours, with alternating replicated analyzed at 24, 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 

Results 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 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 Laboratory: Center for Lake Superior Environmental 

Studies, University of Wisconsin-Superior. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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 Fattig 

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 verify 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. During each diluter cycle, flow-splitting chambers divided each of five exposure 

concentrations and a control into duplicate exposure tanks. Exposure tanks in were glass aquaria 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

15

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: 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 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 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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: 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 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 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. During each diluter cycle, flow-splitting chambers divided each of five exposure 

concentrations and a control into duplicate exposure tanks. Exposure tanks in were glass aquaria 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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: 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 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 Chemical Data Adequacy Review CAS No. 67-72-1

18

Study 8: 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: 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 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. During each diluter cycle, flow-splitting chambers divided each of five exposure 

concentrations and a control into duplicate exposure tanks. Exposure tanks in were glass aquaria 
Draft HPV4 Chemical 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.

Death 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 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 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

Photoperiod: 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 Chemical Data Adequacy Review CAS No. 67-72-1

20

Study 9: Acute toxicity study in bluegill sunfish

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: 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 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 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 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 the 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 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 

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 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 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 Chemical Data Adequacy Review CAS No. 67-72-1

24

Study 11: 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 0.56g.

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 

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 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 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: Thurston et al., Water Res 19(9): 1145-55 (1985).
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 mykiss); 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 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 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 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 Chemical Data Adequacy Review CAS No. 67-72-1

27

RESULTS

Endpoint value(s): The 96-hour LC50 value for rainbow trout was 1.18 mg/L. 

Results remarks: No mortality was observed 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: Thurston et al., Water Res 19(9): 1145-55 (1985).
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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

Exposure 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 24 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 

Agency (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 chamber resulting in 

chamber loadings ranging from 1.3 to 4.1 g/L. Five exposure concentrations and a control were 

used.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

29

Statistical Analysis: The LC50 concentrations were calculated by using the Trimmed SpearmanKarber method for estimating median lethal concentrations.

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 0.84 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] Reliable 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. Carlson, 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 Chemical Data Adequacy Review CAS No. 67-72-1

30

B-2. TOXICITY TO AQUATIC INVERTEBRATES

Summary of endpoint: Adequate

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. 67-72-1

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 stated

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 monitoring: 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 maintained 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, and 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 with 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

31

Lake Superior water which was filtered (5 um) and aerated. All chemical stock solutions were 

prepared by 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 neoprene 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 daphnids 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

pH 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 with 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 Laboratory: Center for Lake Superior Environmental 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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: Richter, J.E., S.F. 

Peterson, and C.F. Kleiner. 1983. Source: Arch.Environ.Contam.Toxicol. 12(6):679-684 

(OECDG Data File).
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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 LaboratoryDuluth, 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 animals 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 gravid 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 that 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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

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: Thurston et al., Water Res 19(9): 1145-55 (1985).
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

Remarks: None

METHOD

Method/guideline 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 stated

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-hour LC50 = 0.99 ppm (0.74 – 1.28)
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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): Syracuse Research Corporation. 

1978. Results of Continuous Exposure of Fathead Minnow Embryo to 21 Priority Pollutants.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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, hexachloro-, CAS No. 67-72-1

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 Monitoring: Not stated

Statistical Methods: Not stated

Method remarks: None stated

RESULTS

Nominal concentrations: Not stated

Measured concentrations: Not stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

38

Element value: 

24-hour EC50 = 99.2 ppm

48-hour EC50 = 67.3 ppm

72-hour EC50 = 91.7 ppm

96-hour EC50 (chlorophyll a) = 87.0 ppm

96-hour EC50 (cell count) = 93.2 ppm

Remarks: None

CONCLUSIONS

96-hour EC50 (chlorophyll a) = 87.0 ppm

96-hour EC50 (cell count) = 93.2 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 Chemical Data Adequacy Review CAS No. 67-72-1

39

Study 2: Acute toxicity in the saltwater algae

Title: Algae – saltwater species (Skeletonema costatum) 

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. 67-72-1

Remarks:

METHOD

Method/guideline followed: Not stated

Test type: Acute

GLP: Not stated

Year: 1978

Species/strain/supplier: Saltwater algae (Skeletonema costatum)

Element Basis: Not stated

Exposure Period: 96 hours

Analytical Monitoring: Not stated

Statistical Methods: Not stated

Method remarks: None stated

RESULTS

Nominal concentrations: Not stated

Measured concentrations: Not stated

Element value: 

24-hour EC50 > 18 ppm

48-hour EC50 = 8.47 ppm (6.20 – 10.5)

72-hour EC50 = 8.34 ppm (5.47 – 14.3)
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

40

96-hour EC50 (chlorophyll a) = 8.57 ppm (5.48 – 15.9)

96-hour EC50 (cell count) = 7.75 ppm (3.85 – 17.1)

NOEC = 1.0 ppm

Remarks: None

CONCLUSIONS

96-hour EC50 (chlorophyll a) = 8.57 ppm 

96-hour EC50 (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 Chemical Data Adequacy Review CAS No. 67-72-1

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 

Identity: Hexachloroethane, CAS No. 67-72-1

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/Selenastrum 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 stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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] Not 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 Chemical Data Adequacy Review CAS No. 67-72-1

43

B-4. ACUTE TOXICITY

Summary 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: 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 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 oral toxicity

GLP: Not stated

Year: Not stated

Species/Strain: Sprague-Dawley rats

Sex: Male and female

No. of animals per sex per dose: Not stated

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

Route of administration: Gavage
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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/kg (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 (methylcellulose)

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 Chemical Data Adequacy Review CAS No. 67-72-1

45

Study 2: Acute oral 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 oral 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: 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 stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

46

Remarks: 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) : 187-199.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 the 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

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 toxicity

GLP: Not stated

Year: Not stated

Species/Strain: Sprague Dawley rat

Sex: Male 

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 of deaths at each dose level: Not stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 

hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187-199.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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), 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 oral toxicity

GLP: Not stated

Year: Not stated

Species/Strain: Hartley guinea pigs

Sex: Male 

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: 4970 mg/kg (4030-6150)

Number of deaths at each dose level: Not stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 

hexachloroethane in laboratory animals. Am. Ind. Hyg. Assoc. J. 40 (3) : 187-199.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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/kg
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 

rabbits.

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 Chemical Data Adequacy Review CAS No. 67-72-1

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 important study details.

TEST SUBSTANCE 

Identity: Hexachloroethane, CAS No. 67-72-1

Remarks: Purity: 99.8%

METHOD

Method/guideline followed: Not 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/conditions 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 tissues 

and organs were examined. 

RESULTS

Endpoint value(s): Not specified
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 organto-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 nominal 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 observation period as compared with controls. No gross 

lesions were observed although two of the four remaining exposed animals showed exposurerelated microscopic changes. The lesions in both animals were a subacute diffuse interstitial 

pneumonitis of minimal 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 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

Remarks: Colorless, crystalline material that 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 not specified

Number of animals/sex/dose: Groups of 5

Route of administration: 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 stomach tube, with 

suspensions of the chemical in aqueous methyl cellulose solution.

RESULTS

Endpoint value(s): LD50 > 3.16 g/kg

Results remarks: The rat oral LD50 > 3.16 g/kg indicative of a low order of acute toxicity by 

this route. Signs of intoxication were observed in animals receiving the highest dosage level, 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 acute toxicity by ingestion, as indicated by an oral LD50

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, Document 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 insufficient number of animals tested.

TEST SUBSTANCE 

Identity: Hexachloroethane, CAS No. 67-72-1

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-

hours. 

RESULTS

Endpoint value(s): LD50 > 3.16 g/kg

Results remarks: The LD50 value is considered to be a low degree of toxicity of skin absorption. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

58

The animals were normal in appearance, behavior, and weight gain during the 14-day posttreatment observation 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 at 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 submission (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 Chemical Data Adequacy Review CAS No. 67-72-1

59

Study 9: Acute inhalation toxicity study 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 important study details.

TEST SUBSTANCE 

Identity: Hexachloroethane, CAS No. 67-72-1

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 methods 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 absorption were 

chiefly those of central nervous system involvement, and the guinea pigs appeared more 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 after 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 submission (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 Chemical Data Adequacy Review CAS No. 67-72-1

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 it is not of the minimum 

(28-day) duration.

TEST SUBSTANCE

Identity: Ethane, hexachloro-, CAS No. 67-72-1

Remarks: molecular weight – 236.7. > 99% pure

METHOD

Method/guideline followed: 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 (females) 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/concentrations tested: 0, 187, 375, 750, 1500 or 3000 mg/kg

Exposure duration: 12 doses over 16 days

Statistical methods: 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 hexachloroethane 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 weights 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-related 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 corticomedullary 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 Hexachloroethane (CAS No. 67-72-1) in F344/N Rats (Gavage 

Studies). NIH Publication No. 89-2816 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro-, CAS No. 67-72-1

Remarks: molecular weight – 236.7. > 99% pure

METHOD

Method/guideline followed: Not stated

Test type: Thirteen-week study

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/concentrations 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 five 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 per 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 than 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 increased 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 dose, 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 dose 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 the 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 was 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 of 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 cells). 

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 Chemical Data Adequacy Review CAS No. 67-72-1

65

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 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

66

Study 3: Repeated-dose 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 repeated-dose endpoint as it is not of a sufficient duration.

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

Test type: Repeated-dose oral

GLP compliant?: Not stated

Year study 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 serum: 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 recorded 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 weight 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 included 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 nephrocalcinosis 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 significantly 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 significant when compared to controls.

CONCLUSIONS

NOEL = 100 mg/kg/day

LOEL = 320 mg/kg/day

STUDY RELIABILITY 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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) : 187-199.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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 specified

Test type: Subchronic repeated-dose toxicity

GLP compliant?: Not stated

Year study performed: 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 administration: 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 studies were conducted using 2000-liter glass and stainless steel dynamic 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 weeks. 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 chemistries 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 subchronic 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 period 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 necropsied 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 transluscent 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 day 

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 dependent 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 (high, 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 hours 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 necropsy was performed on all animals that died 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 compliance 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 secured 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 

pressure (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 differential 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 effects 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 of the fourth and fifth week. Challenging these animals with 

an interadermal injection of 0.1 suspension of hexachloroethane in saline produced no 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

72

sensitization response. The liver-to-body weight ratio was significantly higher than control.

Body weight gain 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 found 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-, spleenand testes-to-body ratios in the male rat and the liver 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 exposure-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 guinea 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 on 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. 

Pulmonary function studies – The mean values in all groups of dogs were not different from pretest control values and were within reported normal limits.

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

Excess mucus 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 female 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 

subclinical 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 exposed at 48 and 15 ppm 

HCE. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 exudate 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. moderate 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 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 Chemical Data Adequacy Review CAS No. 67-72-1

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: Hexachloroethane

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, 1960). 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-
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

75

day feeding 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 chemistry 

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 phosphatase 

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, weighed 

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. Weights 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 to 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 were 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 control 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 laboratory, 

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 trend 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 significant 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 ingestion 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 male 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

76

rats receiving 500 mg/kg/day. These gross observations 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 color 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 

examination 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 incidence 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 frequent 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 Chemical Data Adequacy Review CAS No. 67-72-1

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: Hexachloroethane, CAS No. 67-72-1

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

Vehicle: 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 appearance 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 and 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

78

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

RESULTS

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

Results 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 food 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 the 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 less 

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 hypetocytes 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 Dow 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

79

Chemical Company 1984. Hexachloroethane: Results of a 16-week toxicity study in the diet of 

CDF Fischer 344 rats with cover letter. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro-, (HCE), CAS No. 67-72-1

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 grams); 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 statistically 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 adulterant 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 monitored 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, 

and 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 third 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 13th week of the study. Hematologic 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 were 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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, mammary 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, thyroid 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 

from 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 days 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 study. 

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%, 100% 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: 

Diet 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 calculated 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 pattern – 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 until the rats were 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 activity 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 food 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 increased 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 atrophy 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 that 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 blood also decreased in an apparent first-order manner with halflives 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 concentration 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 HCE/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 Chemical Data Adequacy Review CAS No. 67-72-1

84

STUDY RELIABILITY 

[1] Reliable without restrictions.

REFERENCE

Gorzinski SJ, Nolan RJ, McCollister SB, Kociba RJ, Mattsson JL., Drug Chem Toxicol. 

1985;8(3):155-69.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

Remarks: 

METHOD

Method/guideline followed: Not stated

Test type: in vitro Ames test

GLP compliant?: Not stated

Year study was performed: ~ 1983

Test species: Salmonella 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 indicated 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 suitable 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 upper 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 once 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 108 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 poured 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 controls, 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 procedure 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 maintained 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 incubated 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 histidine-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 activation
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

87

STUDY RELIABILITY

(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. 1984. Investigations of the Species Sensitivity and Mechanism of Carcinogenicity 

of Halogenated Hydrocarbons Final Report.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

88

Study 2: Bacteria reverse mutation assay 

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-, CAS No. 67-72-1

Remarks: molecular weight – 236.7. 

METHOD

Method/guideline followed: Test was performed 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

Metabolic 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 modifications 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 tester 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 supplemented 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 triplicate 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 determination 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 hamster liver S9. 

CONCLUSIONS

The test substance was not 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 

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) 3-142
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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-, CAS No. 67-72-1

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

Statistical 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 

between 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 deionized 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 108 cells, grown in 20% glucose were harvested 

during the logarithmic growth phase when cells contained a high level of cyt. P450; they were 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 albino 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 hours 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 homogenizer, 

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: None 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 study 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 Chemical Data Adequacy Review CAS No. 67-72-1

92

Study 4: Replicative DNA synthesis assay (in vivo)

Title: 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

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethane, hexachloro-, CAS No. 67-72-1

Remarks: Considered a mouse hepatocarcinogen. Purchased from Wako Pure Chemical 

Industries (Osaka)

METHOD

Method/guideline followed: Not stated

Test type: in vivo-in vitro

GLP compliant?: 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 test (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 housed 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 concerning 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 was 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 of 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 (PitmanMoore, Mundelein, IL), the portal veins were cannulated with 25G needles and the livers 

perfused in situ with Hanks’ balanced salt solution without Ca2+ and Mg2+, 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 trypsin 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 104

CO2 in Williams’ medium E (ICN Biomedicals, Costa Mesa, CA) containing 370 kBq/ml of 

[methyl3H]thymidine (NEN Research Products, 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 

[methyl3H]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 ± 7.2
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

94

CONCLUSIONS

Hexachloroethane tested positive for hepatocyte RDS

STUDY RELIABILITY

(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 (Amesnegative) mouse hepatocarcinogens. Mutation Research 343 (1995), 157-183.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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 specified

Test type: Ames assay

GLP compliant?: Not specified

Year study performed: Not stated

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 method/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 directly 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. 

RESULTS
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 not mutagenic.

STUDY RELIABILITY

[2 ] Reliable with restrictions. The 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 Chemical Data Adequacy Review CAS No. 67-72-1

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

necessary to assess the toxicity.

TEST SUBSTANCE

Identity: Hexachloroethane, CAS No. 67-72-1

Remarks: Liquid

METHOD

Method/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

Concentrations 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 Repair 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 Chemical Data Adequacy Review CAS No. 67-72-1

98

The HPC will be incubated at 37 C for a total of eighteen to twenty hours in presence of the test 

chemical in [3H]-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 dried, 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 slides, 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 highest 

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 counts.

CONCLUSIONS

Findings were inconclusive due to the high background counts. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 Chemical Data Adequacy Review CAS No. 67-72-1

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-, CAS 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 performed: 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 doseresponse curves and the individual dose points. An SCE 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 chance 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 

significant (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 Corporation 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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-induced 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 hours. 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 medium 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 S9, 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 aberration 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 karytotype 

(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-division 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, and cells containing 

10 or more aberrations).

RESULTS

Genotoxicity result: Positive
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

102

Cytotoxic concentration: None specified

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

induction of chromosomal aberrations was 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 cell 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 

hexachloroethane 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 substance 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) 1-176
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

103

B-8. REPRODUCTIVE 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. 67-72-1

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 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-test
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

104

Test method/conditions remarks: Teratology studies were carried out in pregnant SpragueDawley 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. 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 body 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) or 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 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 slight 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 significant 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 either 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 

parameters of all other groups of treated animals appeared to be within normal limits. These 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

105

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 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.

CONCLUSIONS

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. The 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 Chemical Data Adequacy Review CAS No. 67-72-1

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. 67-72-1

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 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-test
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

107

Test method/conditions remarks: Teratology studies were carried out in pregnant SpragueDawley 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. 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 body 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) or 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 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 slight 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 significant 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 either 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 

parameters of all other groups of treated animals appeared to be within normal limits. These 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 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.

CONCLUSIONS

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. The 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 Chemical Data Adequacy Review CAS No. 67-72-1

109

B-10. CARCINOGENICITY

Study 1: Chronic carcinogenicity study in rats

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

Hexachloroethane in F344/N Rats

TEST SUBSTANCE

Identity: Ethane, hexachloro-, CAS No. 67-72-1

Remarks: molecular weight – 236.7; > 99% pure

METHOD

Method/guideline followed: Not stated

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 Laboratories 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 sex 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 duration: 5 days/week for 103 weeks

Statistical methods: 

Survival Analyses: The probability of survival was estimated by the product-limit procedure of 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

110

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

the survival 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 doserelated 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 the 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 

comparison 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 tumors, 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 vehicle 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 lesions.

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 tumor 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 Chemical Data Adequacy Review CAS No. 67-72-1

111

Analysis of Continuous Variables: For the hematologic, serum 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. Groups 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 number 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 lesions. 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 Management System, the slides, paraffin blocks, and 

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

tissue audit. The slides, individual animal data records, and pathology tables were sent to an 

independent quality assessment laboratory. The individual animal records and tables were 

compared for accuracy, slides and tissue counts were verified, and histotechnique was evaluated. 

All tumor diagnoses, all target tissues, and all tissues from a randomly selected 10% of 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 between the laboratory and quality 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 diagnosis 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 diagnoses 

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).

Nonneoplastic 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 Clinical 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 rats 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 morphologic 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 larger 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 nucleolus 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 may 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

113

be derived either 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 was 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 increased 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 controls 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 renal 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: Pheochromocytomas 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 complex 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 malignant 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 studies, 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 

mineralization 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 without restrictions
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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. 89-2816 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

115

Study 2: Chronic carcinogenicity study in mice

Title: Carcinogenicity Toxicity study of Hexachloroethane

TEST SUBSTANCE 

Identity: Hexachloroethane, CAS No. 67-72-1

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 specified

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 followed 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 methods 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 vehicle 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, spleen, 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 control 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 hunched 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

week 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 

incidence 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 controls 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 observations 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 the 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

117

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

vehicle controls. For female 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 22/49 (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 

tubular 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 depression 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 with 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 bioassay 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. For 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 test, 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 Chemical Data Adequacy Review CAS No. 67-72-1

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 

appearance 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. 

Microscopically, 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 regenerative 

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 the 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 missing 

information including guideline followed and GLP compliance.

REFERENCE

Bioassay of Hexachloroethane for Possible Carcinogenicity, CAS No. 67-72-1. National Cancer 

Institute Carcinogenesis Technical Report Series No. 68 (1978).
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

119

B-11. SKIN IRRITATION

Study 1: Dermal irritation study in rabbits

Title: Skin Irritation Toxicity in Rabbits

TEST SUBSTANCE 

Identity: Hexachloroethane, CAS No. 67-72-1

Remarks: None stated

METHOD

Method/guideline followed: Not specified

Test type: Dermal

GLP compliant?: 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 propylene 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 Chemical Data Adequacy Review CAS No. 67-72-1

120

10% in Diamond paraffin oil (rabbit 3-180): The solution 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 times in a period of twentysix 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, somewhat 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 remarks: 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 #3-63
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 seven 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 possible 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 treatment 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, increasing 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 redness 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 prominence 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 hyperplasia 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 irritation, 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. It is possible a very faint follicular reaction had also 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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.

REFERENCE

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 Chemical Data Adequacy Review CAS No. 67-72-1

123

B-12. PHARMACOKINETICS AND METABOLISM 

Study 1: Pharmacokinetic study in rats

Title: Hexachloroethane: Tissue Clearance and Distribution in Fischer 344 Rats

TEST SUBSTANCE

Identity: Ethane, hexachloro- (HCE), CAS No. 67-72-1

Remarks:

METHOD

Method/guideline followed: No data

Test type: Pharmacokinetic

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 duration: 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 100 

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.

RESULTS
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 (half 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 was 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 clearance 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 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 

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 disproportionately 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 of HCE in male and female rat kidney would be useful

in assessing hazard.

STUDY RELIABILITY 

2 – Reliable with restrictions
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

125

REFERENCE

TSCATS submission (OTS0206444, Doc ID 878213746; received 06/29/83): Dow Chemical 

Company. 1978. Hexachloroethane: Tissue Clearance and Distribution in Fischer 344 Rats.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

126

Study 2: Pharmacokinetic study in rats

Title: Hexachloroethane: Tissue Clearance and Distribution in Fischer 344 Rats

TEST SUBSTANCE 

Identity: Hexachloroethane, CAS No. 67-72-1

Remarks: Not specified

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 males/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 study 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 toxicity 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 following 

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 toxicity. 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 HCE in animals exists and when possible it is ideal to study the fate 

and toxicity of a chemical under identical conditions.
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

127

Samples of blood, liver, kidney and fat were collected at necropsy, weighed and stored frozen. 

The samples were homogenized in 5 mL 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 chromatograph (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. Because 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 control 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 kidney 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 firstorder manner (t1/2 = 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: 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 this 

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 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. 
Draft HPV4 Chemical Data Adequacy Review CAS No. 67-72-1

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 increase 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 indicates 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 disappear 

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 (OTS0206444, 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 Chemical Data Adequacy Review CAS No. 67-72-1

129

CAS No. 67-72-1: 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