SRC TR

- 10

- 129

HPV4 CHEMICAL DATA ADEQUA CY REVIEW

1,2

- Benzisothiazol

- 3(H)

- one,1,1

- dioxide, sodium salt , CAS No. 128

- 44

- 9

Prepared by:

Justine Von Runnen, Chris Janson

, Teresa Manyin and Joan D. Garey

Chemical, Biological, and Environmen tal Science 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

Elizabe th Margosches

, Alternate Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

July 23 , 2010HPV4 Chemical Data Adequacy Review

1,2

- Benzisothiazol

- 3(H)

- one,1,1

- dioxide, sodium salt, CAS No. 128

- 44

- 9

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 22202Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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1 A. PREPARATION OF THE DATA ADEQUACY ASSESSMENTS

A

- 1. Source material used for assessment

For this unsponsored chemi cal, 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

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

sciences with a concentration on biomedicine

T OXNET

- N ational L ibrary of M edicine data bases 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)

- Carcinogenicit y and mutagenicity test results for over 8000

**•** chemicals) ;

**•** Hazardous Substances Data Bank (HSDB)

- Comprehensive, peer

reviewed toxicology data for about 5000 chemicals ;

**•** Genetic Toxicology Data Bank (GENE

- TOX)

- Peer

- reviewed genetic toxicology test data for over 3000 chemicals.

TSCATS

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

Registry of toxic effects of chemical substances (RTECs)

Environmental Fate Database (EFDB)

ECOTOX

- EPA’s peer

- rev iewed ecotoxicology databaseDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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

Agency for Toxic Substances and Disease Registry (ATSDR) website

National Toxicology Program (NTP) website

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

European Chemical Substances Information System (ESIS)

- source of IUCLID Datasets

Any available IUCLID Datasets found through the ESIS website and robust summaries submitted to EPA by former spon sors on the assigned chemicals were retrieved. Original sources were obtained for any relevant toxicity data found for the assigned chemicals.

While IUCLID D ataset s sometimes contained studies of interest, certain study descriptions contained insuffici ent detail to assess adequacy. In these cases, the primary source of the study, if available and accessible, was obtain ed and review ed.

A

- 2. Evaluating data adequacy

Using standard OECD testing guidelines and professional judgment as guides, availab le 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 Klim isch 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 o f 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. NOTE: If no relevant studies were identified for the chemical , only the data availability/adequacy summary table was completed for the report

.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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3 B

- 1. TOXICITY TO AQUATIC VERTEBRATES

Summary of endpoint: In a dequate

Study 1: Acute toxicity study in fathead minnows

Title: Acute Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas)

Th e study is: Adequate Not Adequate

This study is considered inadequate due to missing important study details.

TEST SUBSTANCE

Identity: 1,2

- Benzisothiazol

- 3(H)

- one,1,1

- dioxide, sodium salt, CAS No. 128

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- 9

Remarks: None stated

METHOD

M ethod/guideline followed: None stated

Test type: Flow

- through

GLP compliant?: Not stated

Year study was performed: 1988

Test species: Fathead minnow ( Pimephales promelas )

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Not stated

Test method/conditions remarks: Not stated

RESULTS

Endpoint value(s): The 96

- hour LC 50 value for fathead minnows was 18

, 300 mg/L.

Results remarks: None statedDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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4 CONCLUSIONS

96h LC 50 = 18

, 300 mg/L

STUDY RELIABILITY

[ 3 ] Not reliable

.

REFERENCE

ECOTOX Reference No. 12859. Geiger, D.L., D.J. Call, and L.T. Brooke. Acute Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas) Volume IV. Source: Ctr.for Lake Superior Environ.Stud., Volume 4, Univ.of Wisconsin

- Superior, Superior, WI

. 355 p p

. Publication Year: 1988.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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5 B

- 2. ACUTE TOXICITY

Summary of endpoint: A dequate

Study 1: Acute oral and i.p. toxicity studies in rats and mice

Title: Toxicological Studies with Sodium Cyclamate and Saccharin

The study is: Adequate Not Adequate

This study was considered inadequate due to lack of important study details , n on

- standard method; limited number of test substance concentrations tested; and non

- standard s train used.

TEST SUBSTANCE

Identity: S odium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Acute

GLP compliant?: Not stated

Year study performed: 1968

Test species: Wistar rats, mongrel rats and mice

Number of anima ls/sex/dose: Not stated

Route of administration: Oral and intraperitoneal

Vehicle: Not stated

Doses/concentrations tested: Not stated

Statistical methods used: The LD 50 was calculated by the method of Mi ller & Tainter (1944).

Test method/conditions remarks: Single doses of sodium cyclamate, sodium saccharin or

sodium cyclamate/sodium saccharin (10:l) mixture were given to rats, mice and rabbits by intragastric intubation or by intravenous or intraperitoneal injection. The animals were observed

for a minimum of 5 days after dosing for mortality and signs of toxicity.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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6 RESULTS

Endpoint value(s):

Oral

LD 50 (mouse) = 17.5 g/kg

LD 50 (mon grel rats) = 17.0 g/kg

LD 50 (Wistar rats) = 14.2 g/kg

Intraperitoneal

LD 50 (mouse) = 6.3 g/kg

LD 50 (mon grel rats) = 7 .1 g/kg

Results remarks: The acute oral toxicity varied with the species and strain of animal

. In Wistar rats the cyclamate/saccharin combination was less toxic than either of the components, but this

was not the case with mice or mongrel rats. Dietary ad ministration of cyclamate or saccharin throughou t g estation did not appear to alter the intraperitoneal toxicity of any of the three test

materials to the offspring. Feeding of the cyclamate/saccharin combination did cause some lessening of susceptibility, although the differences were small and far less than one order of

magn itude.

CONCLUSIONS

The acute toxicity of the materials was shown to vary with the strain of animal and the fat

content of the diet.

STUDY RELIABILITY

[ 3 ] Not reliable.

REFERENCE

Taylor, JD et al., 1967. Toxicological Studies with Sodium Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp. 313

- 327.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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7 Study 2: Acute oral toxicity stud y in rats

Title: Acute toxicity tests on 113 environmental chemicals

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: S odium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Acute

GLP compliant?: Not stated

Year study performe d: 1989

Test species: Wistar rats; 5 weeks of age obtained from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan).

Number of animals/sex/dose: 10

Route of administration: Gavage

Vehicle: G um arabic

Doses/concentrations tested: Not stated ( at least 6 increasing dose levels)

Statistical methods used: The LD 50 was calculated using the Litchfield and Wilcoxon method.

Test method/conditions remarks: Clinical signs and symptoms were monitored during the experimental period. Animals were given re gular laboratory chow 6 h ou r s after a single

administration and the number of dead animals was counted until the 14 th day. Anatomical examination was performed immediately after death or at terminal sacrifice on day 14.

RESULTS

Endpoint value(s): LD 50 = 1280 and 1580 mg/kg for males and females, respectively.

Results remarks: There were no acute toxic symptoms or change in tissues observed.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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8 CONCLUSIONS

The acute oral LD 50 was 1280 mg/kg for male rats and 1580 mg/kg for female rats.

STUDY RELIABILIT Y

[ 2 ] Reliable with restrictions.

REFERENCE

Has egawa et al. 1989. Acute T oxicity Tests on 113 Environmental Chemicals. Sci Rep Res Inst

Tohoku Univ Ser C Med : 36 (1

- 4) : 10

- 16.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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9 B

- 3. REPEATED

- DOSE TOXICITY

Summary of endpoint: Inadequate

Study 1 : Rep eated

- dose toxicity stud y in rats

Title: Toxicological Studies with Sodium Cyclamate and Saccharin : 38

- day rat feeding study

The study is: Adequate Not Adequate

Non

- standard method; no LOAEL/NOAEL reported; and limit ed number of test substance

concentrations tested.

TEST SUBSTANCE

Identity: S odium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type : R epeated

- dose feeding study

GLP compliant?: Not stated

Year study performed: 1967

Test species: Rats

Number of animals/sex/dose: 14/sex

Route of administration: Diet

Vehicle: Not stated

Exposure duration: 2 generations

Doses/concentrations tested: 0.5% (~ 401 mg/kg/day)

Test method/conditions remarks: F our groups of 14 male and 14 female individually

- housed

rats, weighing 75

- 100 g, received diets containing no added sweetener or 0.5 % sodium saccharin, sodium cyclamate or sodium cyclamate/sodium saccharin (10:l) mixture. Food and

water were available ad lib itum for 38 days. Survival, behavior, general physical appearance and stool condition were noted daily. On day 39, the surviving animals were killed off and sectionsDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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10 of renal and hepatic tissues were fixed in formalin and stained with haematoxylin eos in for microscopic examination.

RESULTS

Endpoint value(s): Not stated

Results remarks: It is evident that compared with the controls, there was a marked reduction in growth rate and food intake in all three test groups. Efficiency of food utilization was comparable among the groups, as was survival. Despite the fact that some diarrhea was noted in all groups, there were no overt differences in behavior among the animals.

Gross and microscopic examination of representative survivors revealed a number of

in flammatory and hydropic lesions in the liver and kidney s of the rats of the three test groups. These may have been associated either with the physical effects of diarrhea and water loss or

with some endemic disease in these rats. Three rats in each of the four groups had died during the course of the study. Calculation of the amount of material consumed daily indicated that intake of either saccharin or cyclamate alone or of the combination was approximately 400 mg/kg/day.

CONCLUSIONS

Overall, i t is evide nt that compared with the controls, there was a marked reduction in growth

rate and food intake in all three test groups. Efficiency of food utilization was comparable among the groups, as was survival. Despite the fact that some diarrhea was noted in all groups, there

were no overt differences in behavior among the animals.

STUDY RELIABILITY

[3] Not reliable.

REFERENCE

Taylor, JD et al., 1967. Toxicological Studies with Sodium Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp. 313

- 327.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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11 Stud y 2: Repeated

- dose toxicity study in dogs

Title: Toxicological Studies with Sodium Cyclamate and Saccharin : 11

- month dog study

The study is: Adequate Not Adequate

Non

- standard method; no LOAEL/NOAEL reported; limited number of test substance concentrations tested; and non

- standard species was used.

TEST SUBSTANCE

Identity: S odium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type : O ral repeated

- dose

GLP c ompliant?: Not stated

Year study performed: 1968

Test species: Dogs

Number of animals/sex/dose: 4 groups

Route of administration: Gavage

Vehicle: Not stated

Exposure duration: 11 months

Doses/concentrations tested: 0.065 g/kg

Test method/condition s remarks: Groups of four dogs received weekly 6 daily doses of sodium saccharin (0.065 g /kg). A control group received the basic diet only. The materials were administered by intragastric intubation in equal volumes of water. The doses selected were equiv alent in terms of "sweetness”.

The dogs were observed daily for survival, general physical condition, behavior, food and water consumption and character and frequency of excreta. The animals were weighed weekly and laboratory determinations, including ren al and hepatic function tests, were made initially and after 1, 2, 4, 9 and 11 months of dosing, at which time the surviving dogs were killed off and

gross examinations were carried out.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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12 RESULTS

Endpoint value(s): Not stated

Results remarks: During the initial 6 months of the study, all dogs behaved normally. General physical condition, appetite and response to stimuli appeared to be n ormal throughout this period. During the second half

- year, however, one dog on the saccharin diet showed marked

anorexia with an ensuing general debilitation. This dog steadily deteriorated and died from what appeared to be general ina ni tion. Post

- mortem examination revealed no abnormal organ pathology.

A bout month 10 of the study, all dogs developed soft, poorly

- fo rm ed st ools without frank diarrhea. This condition did not appear to affect food or water consumption or general physical appearance but was evident throughout the last 2 months of the experiment.

B odyweight, hemoglobin, total leukocyte count, non

- protein nitr ogen and phenol

sulphonphthalein retention were within normal limits and total erythrocyte count, b ilirubin and urinary analyses were also unaffected. The gross and histological appearance of the viscera at

necr o psy were within nor mal limits.

CONCLUSIONS

None stated.

STUDY RELIABILITY

[3] Not reliable.

REFERENCE

Taylor, JD et al., 1967. Toxicological Studies with Sodium Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp. 313

- 327.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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13 B

- 4. GENE MUTATIO NS

Summary of endpoint: Adequate

Study 1: Dominant lethal assay in mice

Title: T he mutagenic ity of sodium saccharin (S

- Na)

. I. Dominant

- lethal test

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Remarks : Not stated

METHOD

Method/guideline followed: Not stated

Test type: Dominant

- lethal test ( in vivo )

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Mice

Metabolic activation: Not applicable

Concentrations tested: 2000 mg/ kg (i.p.), 5000 mg/kg (p.o.) (single dose); 5 x 400 mg/kg (i.p.), 5 x 1000 mg/kg (p.o.) (repeated dose)

Statistical methods used: Chi

- square test

Test method/conditions remarks: Male mice were treated with a single dose of 2000 mg/kg (i.p.) or 5000 mg/ kg (p. o.) of sodium saccharin ( S

- Na ) , 250 mg/kg (i.p.) of ethyl

methanesulfonate or 3.0 mg/kg (i.p.) of mitomycin C, or repeated doses of 5 x 400 mg/kg (i.p.)

or 5 x 1000 mg (p.o.) of S

- Na at 12

- h intervals. After treatment with S

- Na, EMS and MC, these

ma les were mated to 4 females per week for 6 consecutive weeks (single dose) or for 2 consecutive weeks (repeated dose). All females were autopsied on day 12 of pregnancy and scored for corpora lutea, implants, early and late dead embryos and live fetuses. Results obtained were tested using χ 2

- test and calculated dominant lethality.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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14 RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not applicable

Results remarks: In the S

- Na i.p. group, a significant decrease (P < 0.01) in the number of live fetuses per corpora lutea or implantations was observed at 1

- 2 weeks after treatment. Furthermore, in both groups of i.p. and p.o. repeated dose of S

- Na, implantations and live fetuses per corpora lutea decreased with statistical signif icance (P < 0.001), while frequencies of dominant lethality of EMS and MC agreed with previous reports.

CONCLUSIONS

The test substance was considered to be mutagenic.

STUDY RELIABILITY

[2] Reliable with restrictions

.

REFERENCE

Masubuchi, M. et al. T he mutagenicity of sodium saccharin (S

- Na). I. Dominant

- lethal test. Mutation Research, 54 (1978) 218

- 219.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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15 Study 2: Dominant lethal assay in mice

Title: Induction of Dominant Lethals in Mice by Sodium Saccharin

The study is: Adequ ate Not Adequate

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Remarks: Madhurin is the trade name of sodium saccharin manufactured by M/s Sarabhai

Chemicals Ltd.

METHOD

Method/guideline followed: Not stated

Test type: Dominant

- lethal test ( in vivo )

GLP compliant?: Not stated

Year study performed: Not stated

Test species: CBA strain m ice

Metabolic activation: Not applicable

Concentrations tested: 172 mg

Statistical methods used: Chi

- square test

Test method/ conditions remarks: Male mice of CBA strain, 10

- 12 weeks old, with average body weight of 25 g, were used for treatment and female mice of 101 strain of the same age group with body weight ranging from

20

- 25 g were used for mating. Twelve tablets of Madh urin weighing 172 mg were dissolved in

100 m L of distilled water (1

- 72%) and each male mouse was allowed to drink the water. All the males were tested for their litter size (before allowing them to drink the water containing sodium saccharin) which was fo und to be on an average six. The treatment was continued for 30 days. Twenty

- four hours after the treatment ceased, each treated male was mated with three normal females of 101 strain. The vaginal plugs were examined every morning and the females which had mated were replaced by fresh females. The matings were continued for a period of four weeks. The mated females were dissected on the fifteenth day of gestation and the number of live and dead embryos were recorded. Control observations were made on comparable females mated to males which were given distilled water without sodium saccharin.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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16 RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not applicable

Results remarks: The ratio of the number of dead embryos to that of the total implants is expressed as percentage of dominant lethals. The percentage of the dominant lethal was taken as the criterion in assessing the damage caused to the male germ line by sodium saccharin. The peak effect period seems to be the fourth week followed by the third and second weeks of mating. It is of interest to note that with each week, the percentage of intrauterine deaths increased with the maximum in the fourth week. Also, the dead embryos per female were highest in the fourth week followed by third and second weeks. The results obtained, when compared week

- wise with that of the control data were statistically significant. The results obtained by Majumdar and Sanjeeva Rao et al (1971) in Drosophila by feeding have clearly

shown tha t all the pre

- meiotic stages of germ plasm are highly susceptible to sodium saccharin. Even though the results of chronic exposure (30 days) are difficult to interpret, in terms of the susceptibility of the stages of spermatogenesis, a comparison of the data obtained in Drosophila and mice clearly indicated the mutagenic potential of sodium saccharin in the pre

- meiotic stages of germ plasm.

Sodium saccharin thus appears to be a potential mutagenic agent and the results caution a more

careful consideratio n of its use in medicine and food industry.

CONCLUSIONS

The test substance was considered to be mutagenic.

STUDY RELIABILITY

[1] Reliable.

REFERENCE

Rao, M.S. and Qureshi, A.B. Induction of Dominant Lethals in Mice by Sodium Saccharin.

Indian Jou rnal of Medical Research, 60 (1972) 599

- 603.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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17 B5. CHROMOSOMAL ABERRATIONS

Summary of endpoint: Adequate

Study 1 : Bone marrow cytogenetic assay in mice

Title: The mutageni city of sodium saccharin (S

- Na). II. Cytogenetic studies

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Bone marrow cytogenetic assay ( in vivo )

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Mice (males)

Metabolic activation: Not stated

Concentrations tested: 4000 mg/kg (i.p. ; single dose); 0

- 1600 mg/kg ( i.p.; repeated dose)

Statistical methods used: Not stated

Test method/condi tions remarks: After i.p. injection of sodium saccharin ( S

- Na ; 4000 mg/kg), metaphase plates were analyzed at 6, 24 or 48 h . D ose

- response relationship was studied after

the 5 repeated i.p. injections of various amounts of S

- Na (0

- 1600 mg/kg) at intervals of 12 h.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not stated

Results remarks: Frequencies of aberrant cells with breaks and/or gaps began to increase at 6 h, reached a peak at 24 h and decreased thereafter. At 24 h cells with breaks (3.6%, P < 0.01) andDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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18 gaps (3.3%) were scored . In the case of repeated dosing, although aberrant cells increase d as the

dose was raised (from 5 x 400 to 5 x 800 to 5 x 1600 mg/kg )

, a clear dose

- response relationship

was not obtained.

CONCLUSIONS

High concentration s of S

- Na induced chromosome aberrations in vivo

.

STUDY RELIABILITY

[2] Reliable with restrictions

.

REFERENCE

Masubuchi, M. et al. The mutagenicity of sodium saccharin (S

- Na). II. Cytogenetic studies.

Muta tion Res earch, 54 (1978) 219

.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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19 Study 2 : In vitro mammalian chromosomal aberration test

Title: The mutageni city of sodium saccharin (S

- Na). II. Cytogenetic studies

The study is: Adequate Not Adequate

TEST SUBSTANCE

Id entity: Sodium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: in vitro

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Chinese hamster cells

Metabolic activation: N ot stated

Concentrations tested: 10, 15, 20, 25, 30 mg/L

Statistical methods used: Not stated

Test method/conditions remarks: Chinese hamster cells were treated with 20 mg/mL of sodium saccharin ( S

- Na ) for 6, 24 or 48 h, and scored at metaphase. Dose

- response relationship

was studied after treatment with various concentrations of S

- Na for 24 h.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not stated

Results remarks: At 24 and 48 h, cells showed a significant incr ease (P < 0.001) in the rate of breaks and gaps (17.0

- 19.5% and 25.0

- 28.5%, respectively). Aberrant cells increased as the dose was raised to 10, 15, 20, 25 and 30 mg/mL

.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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20 CONCLUSIONS

High concentration s of S

- Na induced chromosome aberrations in vitro

.

STUDY RELIABILITY

[2] Reliable with restrictions

.

REFERENCE

Masubuchi, M. et al. The mutagenicity of sodium saccharin (S

- Na). II. Cytogenetic studies.

Muta tion Research, 54 (1978) 219

.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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21 Study 3 : In vitro mammalian chromosomal aberration test

Tit le: Mutagenicity of sodium saccharin to human cells in vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed : Not stated

Test type: in vitro

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Cultured human embryo cells

Metabolic activation: Not stated

Concentrations tested: 10

- 20 mg/mL

Statistical methods used: Not stated

Test me thod/conditions remarks: Cultured human embryo cells were exposed to the test

substance.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not stated

Results remarks: Sodium saccharin was mutagenic

.

CONCLUSIONS

High conce ntration s of S

- Na induced chromosome aberrations in vitro

.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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22 STUDY RELIABILITY

[2] Reliable with restrictions

.

REFERENCE

Ashby, J. Mutagenicity of sodium saccharin to human cells in vitro . Mutation Research, 222 (1989) 299

- 300.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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23 B6. GENETIC TOXICI TY

- OTHER

Study 1: Unscheduled DNA synthesis assay

Title: Presence of unscheduled DNA synthesis in cultured human cells after treatment with

sodium saccharin

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Unscheduled DNA synthesis ( in vitro )

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Cultured human cells

Metabolic activation: Not stated

Concentrations tested: Not stated

Statistical methods used: Not stated

Test method/conditions remarks: Sodium saccharin was tested for possible genetic activity in cultured human cells by measuring the induction of chromosome aberrations and unscheduled DNA synthesis. The fibroblast cul tures prepared from tissues of aborted fetuses were treated

with 0.1

- 10 mg/mL of sodium saccharin for 24 hours.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not stated

Results remarks: There was a considerable increa se in the rate of chromatid breaks and gaps, but no significant amount of chromosome aberrations was produced at the lower concentrations

of 0.1

– 1 mg/mL. On the other hand, sodium saccharin was effective at inducing unscheduled DNA synthesis in cultured human cells. At 24 h of incubation in ADM, cultured cells wereDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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24 exposed to sodium saccharin dis solved in ADM for 1.5h. The level of UDS was evaluated by counting the silver grains over slightly labeled nuclei in the non

- S cells. In control cultures, the mean number of silver grains was 1.3 per nucleus, while it was 1.7, 2.1 and 3.5 per nucleus at concentrations of 1, 5, and 10 mg/mL respectively. There was a marked dose

- response relation ship between the level of UDS and the conc e ntration of sodium sacch arin.

CONCLUSIONS

The test substance induced unscheduled DNA synthesis in this assay.

STUDY RELIABILITY

[2] Reliable with restrictions

.

REFERENCE

Ochi, H. and Tonomura, A. Presence of unscheduled DNA synthesis in cultured human cells

after treatment with sodium saccharin. Mutation Research 54 (1978), 208.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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25 B7. REPRODUCTIVE TOXICITY

Summary of endpoint: A dequate

Study 1: R eproductive toxicity study in rats

Title: Long

- Term Toxicity of Sodium Saccharin in the Rat

The study is: Ad equate Not Adequate

This study was considered inadequate due to the testing of only once test substance concentration (5% in diet), and a NOAEL/LOAEL was not determined.

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

- 44

- 9

Remarks: The two batches of sodium saccharin (Lot Nos. GS

- 1233 and S

- 1022) used were manufactured by the Maumee process and contained no detectable amount of o

- TS ( ortho

toluenesulfonamide; limit of detection = 0.05 ppm). The total amount of organic s oluble impurities of Lot No. GS

- 1233 was approximately 5 ppm. This lot of saccharin was fed for 25 weeks while the F0 generation was on test and during the first week that the F1 animals were on

test. Lot No.S

- 1022, which was used during the remainder of the study, contained a maximum of 13 ppm organic soluble impurities. Both batches contained 40

- 50 ppm of water soluble impurities.

METHOD

Method/guideline followed: Not stated

Test type: 2

- G eneration repeated

- dose toxicity

GLP compliant?: Not stat ed

Year study performed: 1979

Test species: Sprague

- Dawley rats; animals were randomly distributed between the various treatment groups and were 32 days of age when the study commenced.

Number of animals/sex/dose: 50

Route of administration: Diet

Ve hicle: C orn oil

Exposure duration: 2 generationsDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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26 Doses/concentrations tested: 5% in diet

Test method/conditions remarks: The F 0 or parent generation animals consisted of groups of 50 male and 50 female (except where noted) Charles River, Caesaean

- derive d, Sprague

- Dawley rats. The basal diet was Master Laboratory c ubes to which 4% corn oil (w/w) was added after the cubes were ground. Analysis of several batches of feed during the study identified background levels of heavy metals, an average of 20 ppb o f nitrosamines, trace amount of 10 halogenated hydrocarbons ( total content 12 ppb), but no detectable amounts of aflatoxin or

aftlatoxin

- like compounds.

The study consisted of a treatment group administered 5% sodium saccharin in the diet as well as

a control group.

After 90 days on test, the F0 animals were mated on a one

- to

- one basis within treatment groups for a period of 1 week. The liter size was reduced to eight pups (approximately four males and four females) at 4 days post partum. At 21 days of age, the pups were weaned, placed in stock banks and fed their respective parents’ diet. At 28 days of age, animals were randomly selected for either the F1 portion of the chronic feeding study or a subacute study. Thus, the pups were exposed to the t est chemicals in utero , during lactation via the dams’ milk, and for the remainder

of their lifetime via the diet.

All animals were examined twice daily for general health status and signs of toxicity. Individual body weight and feed consumption were m easured weekly, at which time a superficial clinical examination was performed on each animal. Urine samples were obtained from approximately 10 rats per group at 6

- month intervals from the control and treatment groups. Blood samples

were obtained from t he tail aorta of approximately 10 rats per group , under light anesthesia. The blood samples were obtained from the F 0 animals at 4.5 weeks (Day 0 on test), 21, 44.5, 57, 65, 69, 96 and 98 weeks of age; the F 1 animals were sampled at 4.5, 18.5, 32.5, and 4 5.5 weeks of age. Where possible, the same 10 rats were sampled for each observation. A complete hematological examination was performed in duplicate, which included: erythrocyte enumeration (RBC); hematocrit (HCT); hemoglobin determination (Hb); mean co rpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC); total leukocyte enumeration (WBC); differential leukocyte count including absolute values; and occasionally hematopoietic organs (bone marrow, lymph node and spleen imprints) were ex amined in cases where hematologic

involvement was observed in moribund animals.

RESULTS

Endpoint value(s): None stated

Results remarks: The general health of the animals throughout the study was good. The only

apparent treatment

- related effect was a mo ist, but well

- formed fecal pellet excreted by rats consuming the diet containing saccharin, a common observation when 5% sodium saccharin is

fed.

Rats from both generations fed diets containing 5 % sodium saccharin had decreased growthDraft HPV4 Chemical Data Adequacy Review CAS No. 128

- 44

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27 rates

. There wer e no treatment

- related effects upon reproduction, longevity, or hematological parameters. The animals were free of the bladder parasite Trichosomoides crassicauda . The incidence of bladder tumors in male rats fed the 5 % saccharin diet was significantly increased in both generations compared to their respective control groups. An evaluation of individual feed consumption data for animals fed saccharin

– containing diets did not reveal any statistical difference between the amount of feed consumed by animal s which had bladder tumors and

those that did not. The incidence of bladder tumors in the female rats fed 5% saccharin diet was not significantly different from that in control animals.

CONCLUSIONS

The median time

- to

- death (time until 50% of the anima ls have died) was 679 ± 22 and 731 ± 25 days for F0 males and females, respectively, and 684 and 727 days for F1 males and females, respectively.

STUDY RELIABILITY

[3] Not reliable.

REFERENCE

Arnold, DL et al., (1980). Long

- Term Toxicity of ortho

- Tolu enesulfonamide and Sodium Saccharin in the Rat. Toxicol. Appl. Pharmacol. 52:113

- 52.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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28 Study 2 : R eproductive toxicity study in rats

Title: Effects of in utero and postnatal sodium saccharin exposure on the nutritional status of the young rat. I. Effec ts at 30 days post

- birth

The study is: Adequate Not Adequate

This study is not adequate because only one dose

- level was tested ; NOAEL/LOAEL not provided

.

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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- 9

Remarks: 99.2% pure. Obtained from PMC Specialties Group, Inc. (Cincinnati, OH USA). Purity information was provided by the supplier and confirmed by NMR. The impurities in saccharin from this source have been characterized (Riggin and Kinzer, 1983 ) and none of the impurities was found to be present at a level exceeding 1 ppm of the saccharin.

METHOD

Method/guideline followed: Not stated

Test type: Reproductive/Developmental toxicity

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Sprague

- Dawley rats

Number of animals/sex/dose: Sufficient to attain 6 litters per dosing group

Route of administration: Diet

Vehicle: Not stated

Exposure duration: Exposure began 4

- 6 weeks prior to mating

Doses/concentrations tested: 7. 5% of diet

Test method/conditions remarks: Animals and husbandry

– 54 male and 90 female Sprague

- Dawley rats (Charles River Breeding Laboratories, Inc., Kingston, NY, USA) were purchased when they were 4 weeks old. Additional rats were purchased for hea lth screening, which showed no hematological, serological or histopathological abnormalities. Breeding animals were housed individually in polycarbonateDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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29 cages with corncob bedding. Litters were housed with their mothers until weaning. Pups were

weaned a t 21 days of age, and thereafter housed by litter and sex in suspended stainless

- steel cages. The animal room was maintained at a temperature of 71 +/

- 5 ° F and a humidity of 50 +/

- 20%, and was on a 12

- hour light/dark schedule.

Diets and drinking water

– Purina Rodent Chow No. 5002 (meal form, Ralston

- Purina Co., St. Louis, MO, USA) and distilled water were provided ad lib itum

. Sodium saccharin ( NaS ) was mi x ed in the diet at a level of 7.5% by weight. The dietary level of saccharin was analysed by

HPLC before each batch of test diet was fed. The average measured concentration of NaS in the

diet was 7.3%.

Experimental design

– The experimental conditions for this study were designed to simulate a

two

- generation bioassay previously conducted with NaS (S choeing et al., 1985). After a 2

- week adaptation period during which they were fed Purina Rodent Chow No. 5002 ad lib itum , 6

- wk

old male and female F 0 rats were randomly assigned by weight to an experimental group (7.5% NaS, group 1) or a control group (u ntreated diet, group 2) (Martin et al., 1984). Mating was

started after 4

- 6 w ee k s of test diet feeding. Males were generally used for mating only until they had successfully mated with two females. Our objective was to obtain six litters per group,

wher e each litter in group 1 was matched with a litter in group 2 having the same birth date. To that end, extra F 0 rats were mated. The six matched litters from each group were used for urine collection and analyses, serum lipid and vitamin measurements, bi ochemical evaluation of bladder epithelial cells and liver vitamin analyses. Litters not selected for these primary analyses were used for other analyses including hematology and serum clinical chemistries.

F 0 males and non

- mated females were killed, wit hout autopsies, at the end of the mating period or as soon as it was apparent that the females were not pregnant. When possible, litters were randomly culled to four males and four females at day 4 after birth. Pups were weaned at 21 days of age and fed the same diet as the parental rats. F 0 female rats were killed after their litters

had been weaned. F 1 rats were killed when 30 days old.

Urine and feces collection

– 24

- day

- old pups from matched litters were individually placed in metabolism cages with food and water provided ad lib. After a 72

- hour period of acclimatization to the cages, urine was collected over three consecutive 24

- hour intervals. Urine was collected in 2 mL toluene; glass wool was used above the collection tube to prevent contamina tion of the urine by diet or feces. Urine specimens were frozen at

- 20 ° C until analysis. Analyses included volume (by weight); osmalality (by freezing point depression), pH, sodium, potassium, carbon dioxide and chloride, urea, creatinine, calcium, magn esium, ammonium, phosphate and

saccharin. Feces were also collected and moisture content was determined from the difference in weight after drying in an oven at 52 ° C. Fecal saccharin concentration was also measured. At the end of the final collection p eriod, after spending a total of 6 days in the metabolism cages, the

rats were killed.

Observations and measurements

– The rats were observed twice daily for evidence of overt toxicity and/or morbidity and mortality. For F 0 rats, food and water consumpti on were measured

over 7

- day intervals. Body weight measurements and detailed physical examinations were performed on the same days as food and water consumption determinations. Food and waterDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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30 consumption measurements for F 0 rats began with a 3

- day interv al immediately after weaning (day 21), followed by daily measurements while the rats were in the metabolism cages. Pups in each litter were weighed as a group by sex immediately after birth and at 4, 7 and 14 days after

birth. The animals were then indiv idually weighed on days 21, 24, 27 and 30. The health status of the pups was also determined on these days.

Biochemical analyses

– Blood for hematology was collected from the abdominal aorta of a

number of non

- mated females during the mating period and f rom various dams of non

- matched litters at birth and on days 13 and 21 of lactation. Blood samples were collected for hematology from some culled pups at 4 days post

- birth. These females and pups were killed following blood

collection.

Milk was collecte d manually from several dams anesthetized with Nembutal on day 13 of lactation. The dam and its litter were killed after milk collection, and the milk was analyzed for saccharin. No other analyses were performed on these animals.

At 30 days of age, F 1 r ats were killed under penthrane anesthesia. The pH of freshly voided urine was determined immediately prior to anesthesia. Blood specimens were collected at the time of killing from the abdominal aorta of pups in the matched litters, for the measurement of serum levels of cholesterol, triglycerides

, high

- density lipoprotein

, vitamin C

, vitamin A and

vitamin E

, and folate. Blood from the pups in the extra litters was used for hematology and clinical chemistries, including blood urea nitrogen (BUN), creati nine, sodium, potassium, chloride, carbon dioxide, calcium and phosphorus, which were analyzed by the same methods as

those used for urine.

The urinary bladder was removed at the time of killing and inverted over a wood applicator stick, so that epithelia l cells could be scraped off using a No. 11 scalpel blade. The cells were homogenized in phosphate buffered saline, using a motor

- driven Teflon pestle, and analy z ed for DNA, cholesterol, phospholipid and protein. Livers were weighed, homogenized and anal y z ed for vitamin A, vitamin A palmitate, vitamin E and folate. Caeca were weighed with and without

contents.

Statistics

– Statistical analyses were performed using a generalized linear model. Duncan’s multiple range test was used for multiple comparison s of means. In addition to treatment

- related effects, the data were also analyzed for inter

- litter variability and sex

- related differences.

RESULTS

Endpoint value(s): None stated

Results remarks:

Results in F0 animals

– Body

- weight gain during the pre

- mating period was significantly depressed in both male and female rats treated with 7.5% NaS: after 4 w ee k s , body weights were 10% less than those of control rats. The mean body weight of NaS

- treated females remained significantly below that of control a nimals throughout gestation and the first 2 w ee k s of lactation. However, weight gain during pregnancy was equivalent for the two groups and NaS

-Draft HPV4 Chemical Data Adequacy Review CAS No. 128

- 44

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31 treated females actually gained more weight than controls during lactation, so that there was no

difference bet ween the groups on day 21 post

- birth. Basal diet consumption (g/rat/day) was significantly less in NaS

- fed animals, except during w ee k 2 and 3 of gestation and w ee k 1 post

birth when consumption was similar in the two groups. NaS

- treated rats consistentl y consumed more water (g/rat/day) than control rats. NaS intake was approximately 1. 0 g/rat/day in females and 2.0 g/rat/day in males prior to mating and 2.0 g/rat/day in pregnant females. The NaS

concentration in milk of NaS

- treated females was 2.9 mg/m L on day 13 of lactation. No NaS

was detected in the milk of control rats.

It appeared that after giving birth, the females in both groups were anemic, relative to the non

mated females, with decreases in red blood cell count (RBC), hemoglobin (Hgb), and hematocrit (HCT). There were no differences between the groups on the day of birth. The control females recovered by day 13 post

- birth, but the NaS

- fed females remained anemic until day 21.

Results in F 1 animals: litter size and survival and pup weight s

– Feeding 7.5% NaS did not adversely affect reproductive performance. Both the control and test groups had high success rates for matings and deliveries. There were no significant differences between groups in the number of male and female pups born pe r litter, few pups were stillborn in either group, and body weights of pups at birth were similar for the two groups. However, weight gain after birth was depressed in the NaS

- treated litters. NaS

- treated male and female pups weighed significantly less t han control pups at 4 and 21 days post

- birth. The study of survival was complicated by the fact that some litters were killed at birth or on day 13 post

- birth. The litters were culled to four males and four females on day 4 post

- birth as mentioned in the m ethods

section . Survival appeared to be slightly better in the control group than in the NaS group.

Results in matched litters

– body weights and food and water consumption

– Similar effects were generally observed in matched and non

- matched litters. After weaning, mean body weights of

NaS

- treated pups continued to be significantly less than those of control pups. When killed at 30 days post

- birth, male NaS treated rats weighed only 64% as much as control males and female

NaS treated rats weighed only 61% as much as control females. During the first 3 days after weaning, NaS

- treated animals ate less than one

- half as much diet as control animals and also consumed less water. Both groups progressively showed an increase in food consumption over

the fin al 6 days, but NaS

- treated animals consistently consumed significantly less than controls. However, when adjusted for body weight, the basal diet consumption of the NaS

- treated group on days 27 and 30 post

- birth was equivalent to or greater than that of t he control group (on day 27: 200 and 183 g/kg body weight/day for NaS

- treated males and females, respectively, versus 172 and 167 g/kg body weight/day for control males and females, respectively ; on day 30: 165 and 182 g/kg body weight/day for NaS

- treated males and females, respectively, versus 163 and 159 g/kg body weight/day for control males and females, respectively ) . Between days 25 and 30

post

- birth, water consumption was increased in NaS

- treated females on day 30 post

- birth (13.4 g/kg body weight/da y for males and 14.7 g/kg body weight/day for females).

Urine and feces analyses

– There were numerous differences between the groups. Urine analyses were performed separately on days 28, 29 and 30. However, for statistical analysis the

data from the 3 days were pooled, and the differences between groups were analysed by Duncan’s multiple range test. Compared with the controls, the urine of NaS

- treated rats showedDraft HPV4 Chemical Data Adequacy Review CAS No. 128

- 44

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32 increases in volume, sodium, magnesium, phosphate and ammonia. Urinary potassium, calcium

, creatinine, osmolality, urea, carbon dioxide, chloride and pH were decreased. The pH of freshly voided urine, obtained just prior to killing, was also decreased with NaS treatment (male NaS = 6.5; male control = 7.2; female NaS = 6.4; female control = 7 .4).

There was litt le difference in the weight of dry feces between the groups, but the feces of NaS

treated rats had a higher moisture content: approx. 50% for NaS

- treated rats and 30% for the control rats. The caecal content weight of NaS

- treated anima ls was 2

- 2.5 times that of control animals. The absolute weight of the empty caecum was also increased from 0.51 g in untreated males to 0.58 g in NaS

- treated males, although there was no difference with the females.

Bladder chemistry

– NaS treatment had no significant effect on the bladder content of cholesterol, phospholipid, DNA or protein. Also no differences were observed between the groups when the parameters were expressed in terms of amounts per μg DNA or per μg protein.

Liver chemistry

– Livers were collected from rats in the matched litters. Each liver was weighed, and then the entire liver was homogenized before aliquots were removed for the determination of vitamin A, vitamin A palimitate, vitamin E and folate. Possible variations

associate d with the distribution of the vitamins in the liver were thereby avoided. In animals of both sexes, there was a significant decrease in the hepatic folate concentration associated with NaS treatment. In addition, the concentration of vitamin E was signi ficantly increased in NaS

treated females, relative to controls, and the concentration of vitamin A palmitate was significantly increased in NaS

- treated males. Mean liver weights of NaS

- treated males and females were approximately half those of control an imals, and the liver weights in relation to body weights were also significantly reduced by NaS treatment. As a result, the total hepatic level of each of the vitamins was significantly lower in NaS

- treated animals than in controls.

Serum vitamins and li pids

– Compared with control levels, serum vitamin A and folate in the

NaS

- treated rats were significantly decreased and serum Vitamin E was increased. Vitamin C was significantly decreased only in NaS

- treated males. A 10

- fold increase in serum triglycer ides

was measured in NaS

- treated animals, and cholesterol was also significantly increased but to a much less extent. The concentration of high

- density lipoproteins was significantly lower in NaS

treated rats.

Hematology and serum chemistry in unmatche d litter s

– Because the volume of blood collected from animals in the matched litters was only enough for the analysis of serum vitamins and lipids, blood from rats in the other litters was used for hematology evaluation and additional

serum clinical chemi stries. Hematocrit evaluated in pups eliminated during the culling procedure on day 4 post

- birth, was significantly lower in the NaS group than in the control group. By day 30 post

- birth the difference between the groups was enhanced and significant

decr eases were also observed in RBC and Hgb levels of NaS

- treated rats.

There were significant differences between the groups for several of the serum chemistry analyses. A considerable increase in BUN was associated with NaS treatment, while creatinine

was decreased. Other changes were relatively minor. The serum NaS concentration was approximately 0.20 mg/ml in both male and female treated rats.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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33 Differences between male and female F 1 rats

– There was no sex difference in the number and

survival of newborn pups and in pup weights. Male and female rats did not differ in the amount

of food and water consumed. The NaS intake and excretion of male and females in the treatment groups were also similar.

Similar urinalyses values were obtained from males and fe males of each group. In controls, the urine of females had a significantly smaller volume and a higher pH compared with males. In the treated group, the urinary NaS concentration was significantly lower in females than in

males.

There were no sex

- associ ated differences in any of the bladder or liver chemistries. Female rats in the control group had significantly lower concentrations of serum vitamin C, vitamin A and folate than control males. They also had significantly higher levels of high

- density li poproteins

than NaS

- treated males. NaS

- treated females showed significantly higher serum concentrations of BUN, sodium and chloride than NaS

- treated males. Hematology values were similar for males and females of the same group. In general, relatively fe w sex

- related differences were observed in the treated and control groups.

Differences between F 1 litters

– For the various chemistry parameters evaluated, analyses were performed to determine whether significant differences existed between litters that r eceived the same treatment. The data for males and females were analy z ed separately. Inter

- litter

differences were numerous in both sex and in both NaS

- treated and control rats.

CONCLUSIONS

Overall, b ody weights were significantly depressed in NaS

- trea ted litters by 4 days after birth, and were 35% lower than controls by 30 days when the animals were killed

. NaS treatment of the offspring was associated with an increase in fecal moisture content and ca ec al content weight, changes in several urinary ana lytes, a 50% increase in serum cholesterol, a 10

- fold increase in serum triglycerides and decreases in serum and hepatic vitamins. In addition, NaS

treated dams and pups were anemic

. Relatively few differences between males and females were noted, but sig nificant inter

- litter differences existed. The num erous physiological changes

indi cate that 7.5% dietar y NaS exceeds the maximum tolerated dose for weanling rats.

STUDY RELIABILITY

[3] Not reliable.

REFERENCE

Garland, E.M. et al. Effects of in utero and postnatal sodium saccharin exposure of the nutritional status of the young rat. I. Effects at 30 days post

- birth. Food Chem Toxicology 29 (1991), 657

- 667.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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34 Study 3 : R eproductive toxicity study in rats

Title: Effects of in utero and postnatal sodiu m saccharin exposure on the nutritional status of the young rat. I. Effects at 30 days post

- birth

The study is: Adequate Not Adequate

This study is not adequate because reproductive measurements were not provided an d only one

dose

- level was tested.

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

- 44

- 9

Remarks: 99.2% pure. Obtained from PMC Specialties Group, Inc. (Cincinnati, OH USA). Purity information was provided by the supplier and confirmed by NM R. The impurities in saccharin from this source have been characterized (Riggin and Kinzer, 1983) and none of the impurities was found to be present at a level exceeding 1 ppm of the saccharin.

METHOD

Method/guideline followed: Not stated

Test type: Re productive/Developmental toxicity

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Sprague

- Dawley rats

Number of animals/sex/dose: 22 females and 11 males per group in groups 1

- 4; and 11 females and six males per group in group s 5 and 6.

Route of administration: Diet

Vehicle: Not stated

Exposure duration: Exposure began 4

- 5 weeks prior to mating

Doses/concentrations tested: Group 1

– F0 = Control, F1 = Control from conception until day 90 of age. Group 2

– F0 = NA; F1 = 1% S odium Saccharin ( NaS ) (from conception until 90 days of age) Group 3

– F0 = NA; F1 = 3% NaS (from conception until 90 days of age) Group 4

– F0 = 7.5% NaS; F1 = 7.5% NaS (from conception until 90 days of age) Group 5

– F0 = 7.5% NaS; F1 = 7.5% NaS from con ception until day 30 of age, Control fromDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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35 day 30 to day 90 of age. Group 6

– F0 = Control, F1 = Control from conception until day 30 of age, 7.5% NaS from day 30 to day 90 of age.

Test method/conditions remarks:

Animals and husbandry

– 56 male and 110 f emale Sprague Dawley rats (Charles River Breeding Laboratories, Inc., Kingston, NY, USA) were purchased when they were 4 weeks old. Additional rats were purchased for health screening, which showed no hematological, serological or histopathological abnorm alities. Breeding animals were housed individually in polycarbonate cages with corncob bedding. Litters were housed with their mothers until weaning. Pups were weaned at 21 days of age, and thereafter housed by litter and sex in suspended stainless

- stee l cages. The animal room was maintained at a temperature of 71 +/

- 5 ° F and a humidity of 50 +/

- 20%, and was on a 12

- hour light/dark schedule.

Diets and drinking water

– Purina Rodent Chow No. 5002 (meal form, Ralston

- Purina Co., St. Louis, MO, USA) and distilled water were provided ad lib itum

. Sodium saccharin ( NaS ) was mi x ed in the diet at a level of 7.5% by weight. The dietary level of saccharin was analy z ed by HPLC before each batch of test diet was fed. The average measured concentration of NaS i n the

diet was within +/

- 6% of the nominal levels.

Experimental design

– Male and female rats were randomly assigned by weight to each experimental group: 22 females and 11 males per group in groups 1

- 4 and 11 females and 6 males per group in groups 5 an d 6. They were placed on the appropriate test diet at 6 w ee k s of age. Mating was started after 4

- 5 weeks of test diet feeding. Litters were randomly culled on day 4 after birth to four males and four females when possible. Pups were weaned at 21 days o f age and fed the same diet as the parental rats. Parental males were killed at the end of the mating period and female rats were killed after their litters had been weaned. Seven litters from each group 1

- 4 were killed when they were 30 days old to eval uate the dose

- response effects of NaS.

Also at 30 days, the diet for group 5 was changed from 7.5% NaS to control, and the diet for group 6 was changed from control to 7.5% NaS. Seven litters from each of groups 1, 4, 5 and 6 were then killed at 90 days of age to evaluate the reversibility of the effects of NaS. The decision to kill a litter at 30 or 90 days of age was based on its birth

- date. An attempt was made to kill litters from at least two groups, with no more than three litters per group, on a g iven day.

Observations and measurements

– The rats were observed twice daily for evidence of overt toxicity and/or morbidity and mortality. For F 0 rats, food and water consumption were measured for 1 week prior to mating and for 3 weeks during gestation. All food consumption data were corrected for the NaS content of the diet, and reflected the consumption of base diet only. Body weight measurements and detailed physical examinations were performed on the same days as food and water consumption determin ations and detailed physical examinations were performed before mating

. For rats of the second generation (F 1 ), food and water consumption were determined weekly after weaning except during week 4 post

- birth when some litters were killed

and others had th eir experimental diets changed. Pups in each litter were weighed as a group by sex immediately after birth and at 4, 7 and 14 days after birth and then individually at weekly intervals until the time of killing . The health status of the pups was also det ermined on theseDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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36 days.

Analyses

– F 1 rats were killed under penthrane anesthesia. Blood specimens were collected in hematology (hematocrit, hemoglobin, erythrocytes and leucocyte counts; mean corpuscular volume, hemoglobin, and hemoglobin concentration). The measurement of serum levels of cholesterol and triglycerides was carried out by the enzymatic method of Spayd et al

. using an Ektachem 700. Vitamins A and E were determined by a procedure similar to those of Driskell et al. (1982) and Catignani and Bieri (1983). A Quantaphase B

- 12/folate kit was used for folate

determination. Iron and iron

- binding capacity were measured according to Megraw et al. (1973) in pooled serum samples from litters of groups 1 and 3; males killed at 30 days post

- birth and

b oth males and females killed at 90 days post

- birth were evaluated. The adrenals, liver and full and emptied caecum were removed at the time of killing and weighed.

The urinary bladder was inflated in situ with 2% glutaraldehyde in 0.1 M

- cacodylate buffer (pH 7.4) and then removed and processed for light microscopy or for scanning electron microscopy and classified. SEM evaluations were performed on bladders from two males and two females per litter (from 14 males and 14 females per group) in groups 1 and 4 for both the 30

- and 90

- day sacrifice periods; from two males and two females per litter in groups 5 and 6 killed at 90 days; and from one male and one female per litter in groups 2 and 3 killed at 30 days. Bladders from

one male and one female in each litter were also examined by light microscopy.

Livers were examined for lipids both histologically and biochemically. For histological examination, a 3

- mm 3 piece of the left lateral lobe of the liver was put into cold picric acid

- p

formaldehyde fixative , and the rest of the liver was frozen in liquid nitrogen and then stored at

- 80 ° C. The fixed liver tissue was dehydrated and embedded in araldite 502 with DDSA and DMP resin in labeled gelatin capsules. 1

- μm section s w ere stained with toluidine blue and examined by light microscopy. I n addition, thin sections were stained with lead citrate and uranyl acetate and examined for lipid with a Philips 300 electron microscope. For biochemical determinations of cholesterol and triglyceride, the frozen live r was weighed and homogenized (1:20, w/v) in chloroform

- methanol (2:1, v/v) using a motor

- driven teflon pestle. Lipids were extracted by the method of Folch et al. (1957). Separate aliquots of the extract, each equivalent to 0.01 g liver tissue were anal ysed for triglycerides and cholesterol. Triglycerides were

determined in fresh extracts, while cholesterol was measured after storage of the extracts at

- 80 ° C for approximately 1 month.

Statist i c s

– Statistical analyses were peformed with a generalized linear model procedure. Duncan’s multiple range test was use for multiple comparison of means.

RESULTS

Endpoint value(s): None stated

Results remarks: NaS treatment was associated with lower body

- weight gains, increased water consumption and

decrease d (in females) or unchanged (in males) basal diet consumption. Significant differences were noted only between the control and the high

- dose groups, with the exception of aDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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37 significant increase in water consumption by the females treated with 1% NaS (51.6 g/rat/day v. 42.1 g/rat/day by control females). In all the groups, more than 80% of the females mated and at least 90% of mated females gave birth. Litter weights at birth were similar for all groups.

For male and female rats at w ee k 4 post

- birth, the values of body weight and food consumption (basal diet) decreased as the NaS concentration in the diet increased. R ats treated with 7.5% NaS weighed about 60% as much as control rats, and males fed 3% NaS weighed significantly less (approx. 10%) than con trols. Correspondingly, significant decreases in diet consumption were also observed at 3 and 7.5% dietary NaS. Water consumption in NaS

- treated groups did not differ significantly from that of control animals.

At week 12 post

- birth, body weights of 7.5 % NaS

- treated males and females remained significantly lower than controls. Subsequent to the change in diet at 30 days post

- birth, body weights of rats in group 5 (NaS  control) approached control values, and rats in group 6 (control  NaS) gained less weight than control rats. All male rats fed NaS including those switched from NaS to control diet consumed significantly less diet than controls during wk 6

- 12

post

- birth. Female rats in all NaS

- treated groups consumed similar amounts of diet as did

cont rols. Water consumption during w ee k s 6

- 12 post

- birth was significantly greater, compared with controls in groups 4 (7.5% NaS) and 6 (control  7.5% NaS).

In male and female rats killed at 30 days post

- birth, there was a decrease in both absolute and

rela tive liver weights related to the dose of NaS. At 90 days, males on the 7.5% NaS diet (groups 4 and 6) had lower absolute and relative liver weights than controls (group 1). Male rats switched from NaS to control diet (group 5) also had lower absolute li ver weights than control

rats. For female rats

, liver weights were lower than those of controls, on an absolute basis for those fed 7.5% NaS diet throughout the study, and on an absolute and relative basis for those

switched from the control to NaS diet. Data collected for the caeca and adrenals show that there were no differences at 30 days in the absolute weight of the empty caecum. At 90 days, all animals exposed to 7.5% NaS

, except females in group 5, had increased caecal weights compared with contro ls. On an absolute basis, adrenal weights from 7.5% NaS

– fed animals tended to be lower than those from control animals, but they were greater than those of control animals when expressed relative to body weight.

At 30 days, rats exposed to NaS showed a dose response with respect to hematology. Animals exposed to 7.5% NaS (group 4) were anemic at 30 days post

- birth, as indicated by the decrease in RBC, Hgb and HCT. Mean values for mean corpuscular volume, hemoglobin and hemoglobin concentration were con sistent with a diagnosis of iron deficiency. Rats of both sexes fed 1% NaS did not show any signs of anemia, and those fed 3% NaS had significantly decreased Hgb and HCT. At 90 days post

- birth, the values of these two parameters in rats exposed to 7.5% N aS throughout the study were still significantly below control values; however, the effect was less than that at 30 days post

- birth. In addition, no difference was observed in hematological parameters between the group switched from the 7.5% NaS to contro l diet at 30 days post

- birth and control group. Beginning NaS treatment at 30 days post

- birth did not produce anemia. The effects in the female rats were similar to those in the males.

Compared with the control group, serum cholesterol, triglycerides an d vitamin E were increasedDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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38 and serum vitamin A and folate were decreased in both males and females. Serum cholesterol, triglycerides and vitamin E in the 1 and 3% groups were slightly, and in some cases significantly,

lower than in controls. The decrease s in serum vitamin A and folate were dose

- related: a significant decrease was observed in males and females given NaS doses equal to or greater than 3 and 1%, respectively. At 90 days post

- birth, with continuous NaS treatment (group 4), the changes in ser um lipid concentrations were reversed. Serum triglyceride levels in males and females and serum cholesterol levels in males were lower than in controls. The lipid values for groups 5 and 6 were also significantly lower than or equivalent to those of the control group. Serum folate in group 4 was similar to that in control animals; elevated and reduced values were observed in groups 5 and 6, respectively. Serum vitamin A remained significantly depressed in all male groups treated with 7.5% NaS. However, there were no differences between the mean serum vitamin A values for the female groups. For both males and females in all groups treated with NaS, serum vitamin E was significantly below the control value.

Iron and iron

- binding capacity were measured in pooled serum samples from litters in groups 1 (control) and 4 (7.5% NaS). For male rats in group 4 killed at 30 days of age, the serum iron level was significantly reduced to a level that was approximately 20% of the control value and

iron

- binding cap acity was increased by 50% over the control value. Female rats could not be evaluated at 30 days of age because insufficient serum sample volume. At 90 days of age

, the serum iron level of control males was approximately half of that at 30 days, and NaS treatment was associated with only a 30% decrease, relative to the day

- 90 control value. Although iron

binding capacity was greater at 90 days for male rats fed 7.5% NaS, the difference was not statistically significant (p > 0.05). For female rats killed at 90 days, there was no significant difference between groups 1 and 3 in mean serum iron concentrations, but iron

- binding capacity was significantly lower in group 4 than in group 1.

A significant increase was observed in the hepatic concentration of tr iglycerides in males and females and cholesterol in females treated with 7.5% NaS. Examination of the livers by light microscopy showed an increase in the number and size of microvesicular vacuoles in hepatocytes from group 4 in comparison with group 1 at 30 days post

- birth. Although not quantitative, these findings are indicative of lipid accumulation. Examination by electron microscopy confirmed the increased cytoplasmic lipid in hepatocytes and also indicated the presence of decreased glycogen in rats fed 7.5% NaS.

The microscopic effects of NaS treatment on the bladder were negligible. Simple hyperplasia

was noted in the bladders of some 7.5% NaS

- treated rats at 90 days. Morphological changes in the urothelium were also observed by SEM in these gro ups, but the severity and incidence were

low.

CONCLUSIONS

Overall, m ost physiological consequences of NaS treatment in the weanling rat, including

anemia and reductions in serum folate and vitamin A concentrations were dose

- dependent. Serum vitamin E, c holesterol and triglyceride concentrations were decreased at the two lower

doses of NaS but were significantly increased with 7.5% NaS. The no

- effect level (NOEL) was similar for physiological effects and for bladder tumor production in two

- generation stu dies (1%Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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39 NaS in the diet). The reversibility of the effects of 7.5% NaS was examined in 90

- day

- old rats. The increases in lipids and vitamin E were reversible. Although values for hematological parameters and serum vitamin A remained significantly reduce d at 90 days, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% dietary NaS on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly r elated to the production of bladder tumors.

STUDY RELIABILITY

[ 3 ] Not r eliable

.

REFERENCE

Garland, E.M. et al. Effects of in utero and postnatal sodium saccharin exposure of the nutritional status of the young rat. I I

. Dose response and reversibilit y . Foo d Chem Toxicology 29 (1991), 669

- 679

.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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40 Study 4 : R eproductive toxicity study in rats

Title: Combined Chronic Feeding and Three

- Generation Reproduction Study of Sodium

Saccharin in the Rat

The study is: Adequate Not Adequate

This study is not adequate because many details are missing, including a description of all reproductive parameters measured, and NOAEL/LOAEL values are not identified.

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Rem arks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Combined chronic and 3

- generation reproduction study

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Charles River CD Rat

Number of animals/sex/dose: 48 offspring/sex/group

Route of administration: Diet

Vehicle: Not stated

Exposure duration: Not stated

Doses/concentrations tested: The dietary levels were 0, 0.01, 0.1, 1.0, 5.0 and 7.5% sodium

saccharin

Test method/conditions remarks: The carcinog enic and reproductive effects of sodium saccharin were studied in the Charles River

CD rat in a combined chronic and 3

- generation reproduction study. For the chronic study, 48 male and 48 female F1 weanling offspring per test group were selected and conti nued on the same test regiment as their parents. The dietary levels were 0, 0.01, 0.1, 1.0, 5.0 and 7.5% sodium saccharin. The rats were mated to produce 2 litters. Serial sacrifices were performed at 14 and 18 months. The study was continued until the number of survivors in a group fell to 20%Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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41 of the starting number. The last rats were killed approximately 28 months after the first weanlings were selected for the chronic study.

RESULTS

Endpoint value(s): None stated

Results remarks: There were no t significant differences between the test and control groups in hematological values, organ weights or survival. Average weaning weights were decreased in litters from parents receiving 5 and 7.5% sodium saccharin; other reproductive indices showed

scatt ered variations but these were not consistent for all generations. During the chronic study some of the initial weaning weight depression was overcome, but rats on 5 and 7.5% sodium saccharin had lower average body weights throughout the study. Histopath ological examination of the urinary bladders from rats on the chronic study longer than 18 months revealed an increased incidence of transitional cell carcinomas in the 7.5% sodium saccharin group.

CONCLUSIONS

None stated

STUDY RELIABILITY

[ 4 ] Not assi gnable

– results reported only as abstract

REFERENCE

Taylor, J.M. et al. Combined chronic feeding and three

- generation reproduction study o f sodium saccharin in the rat. Toxicol. Appl. Pharmacol. 29 (1974), 154.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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42 B

- 8. DEVELOPMENTAL TOXICITY

Summar y of endpoint: Inadequate

Study 1 : Developmental toxicity study in rats

Title: Histological examination of perinatal eye development in the rat after ingestion of sodium cyclamate and sodium saccharin during pregnancy

The study is: Ad equate Not Adequate

This study is not adequate for the developmental toxicity endpoint because only one dose level

of the test substance was administered.

TEST SUBSTANCE

Identity: Sodium saccharin, CAS No. 128

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Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: Developmental toxicity

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Wistar rats

Number of animals/sex/dose: 26

- 28 females/group

Route of administration: Die t

Exposure duration: From mating until day 20 of pregnancy

Doses/concentrations tested: 0

- 4%

Vehicle: Not stated

Statistical methods used: None statedDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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43 Test method/conditions remarks:

Groups of 26

- 28 female Wistar rats (obtained from Mus Rattus Cor p., Brunnthal, Munich and weighting 182

- 229 g) were mated with 3

- to 5

- month

- old males. The females were assumed to be pregnant when spermatozoa were found in a vagina smear. The pregnant rats were kept at an ambient temperature of 20

- 23 ° C in an atmosph ere of 60% mean relative humidity, with a 12

- hour electric light/dark cycle. They were given Altromin

- R powdered feed (supplied by Altromin Ltd., Lage/Lippe) and tap

- water ad lib . From mating until day 20 of pregnancy, they

were treated with feed contain ing 0

- 4% sodium saccharin, complying in each case with the standards of purity established in Germany for the marketed product. The average amount of test substance ingested daily was 98

. 8 mg sodium saccharin per rat. The mean daily intake of a control g roup of similar size was 24

. 4 g.

One half of the dams in each of the three groups were delivered by caesarian section on day 20 of pregnancy. The other animals were left to deliver their young spontaneously and rear them

for 3 weeks.

For all the dams, appearance, behavior, deaths, body weight and food consumption were recorded. Following surgical delivery, the numbers of implantations, fetuses and resorbed embryos were calculated, fetuses were inspected for external malformations, and litter weights,

m ean fetal weights litter, numbers of runts (fetuses < 3 g) and mean placental weights were recorded. Data recorded for the rest of the dams in each group included the duration of pregnancy, course of parturition, behavior during lactation, numbers of impl antation sites

demonstrate d at the end of the experiment by the staining of Salewski (1964), and pathological changes. For the offspring of these dams, records were kept of weights and sex ratios at birth and after 1, 2 and 3 wk, and of deaths and behavio r, time of eye

- opening, formation of fur, ability to run normally, macroscopic changes in the eye and direct papillary reflexes in response

to a flash

- light in a darkened room.

For the histological examination of the eyes, the fetuses and young animals we re killed with

CO 2 , and the heads were fixed in Bouin’s fluid. For better penetration of the fixation fluid into the area to be examined, the nose, lower jaw and back of the head were removed, the decalcification of bone necessary in the 3

- w ee k

- old animal s was achieved using sodium ehtylenediaminetetraacetate at 38 ° C. The subjects for examination were embedded, via methylbenzoate, in Paraplast, so that the microtomy yielded fronto

- parallel sections in the

cranio

- caudal series. The ocular region, from fe tuses and young from each dam, was cut in series or states, regularly including both lenses and the insertion of the optic nerves. The

sections were about 3

- 5 μm thick and were alternately stained with haemalum

- eosin and

Masson’s trichrome.

RESULTS

Endp oint value(s): Developmental NOAEL = 98

. 8 mg/day

Results remarks: The general stud y provided no indication of any primary embyrotoxic effect of the treatment and the histological examination showed that treatment with the sweeteningDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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44 agents caused no ocula r damage. The development of the eyes had followed a normal course in all the fetuses and young animals studied. No case of anophthalmia or microphthalmia occurred. The retina showed no unusual folding, the ocular lenses were of normal structure. In pa rticular, no lytic, necrotizing or vacuolating process of the ocular lenses were present. The fib er s of the lenses showed no increase in circumference or disorientation. The epithelial cells of the lenses had reached their regular position and showed nor mal mitotic activity. The vitreous body and the implantation of the optic nerves were normal.

CONCLUSIONS

Administration of sodium saccharin to pregnant rats is tolerated without embryotoxic effect.

STUDY RELIABILITY

[3 ] Not reliable.

REFERENCE

Lu ckhaus, G. & Machemer, L. Histological examination of perinatal eye development in the rat after ingestion of sodium cyclamate and sodium saccharin during pregnancy. Food Cosmet Toxicol. 16 (1978), 7

- 11.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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45 B

- 9. CARCINOGENICITY STUDY

Study 1: Carcinoge nicity study in rats

Title: Chronic Toxicity and Carcinogenicity to the Urinary Bladder of Sodium Saccharin in the

in Utero

- Exposed Rat

TEST SUBSTANCE

Identity: S odium saccharin, CAS No. 128

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Remarks: Not stated

METHOD

Method/guideline follo wed: Not stated

Test type : Chronic toxicity and carcinogenicity

GLP compliant?: Not stated

Year study performed: Not stated

Test species: Sprague

- Dawley rats

Number of animals/sex/dose: 48/sex/group

Route of administration: Diet

Vehicle: Not stated

Exposure duration: Varied

Doses/concentrations tested: 0.01, 0.1, 1.0, 5.0 and 7.5% of the diet

Test method/conditions remarks: Parental animals were weanling rats of the Charles River CD (Sprague

- Dawley) strain and were randomly distributed by weight to the test groups. The chronic test rats were the offspring of these treated parents. The parents were fed the diets continuously from weaning through mating at approximately 10 weeks of age, and through gestation to the weaning of their litters. The o ffspring were selected randomly from within a litter; the number selected from a litter from the chronic study was designed to give as equal representation of all available litters as possible and give a total of 48 males and 48 females per

test group.

Rats were observed daily for changes in behavior. Body weight and food consumption were measured weekly. The rats were mated twice. Standard hematological determinations (hemoglobin, hematocrit, red and white cell count and differential count) were made on 10 ratsDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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46 per sex per group after 14, 18, and 23 months. Hemoglobin was determined on a Fisher hemophotometer, hematocrit by the microhematocrit method, red and white cell counts on a Coulter Counter, and the differential count on a Wright’s stained sme ar.

Interim sacrifices of 4/sex/group were made at 14 months and 5/sex/group at 18 months. The rats for interim sacrifice were part of the original group. Treatment was continued until the number of survivors in a test group fell to 20% of the starting number. For this purpose, males and females were considered separately. When survival in the control group fell to 20% of the controls and all the remaining test rats were killed. Rats scheduled for sacrifice, or found moribund, were anesthetized with e ther, exsanguinated and subjected to thorough gross examination. The urinary bladder was ligated at the neck and inflated with a small volume of buffered formalin before excision. Other tissues taken for histopathologic study included eyes, brain, pituit ary gland, salivary glands, thyroid, adrenals, trachea, lungs, heart, liver, pancreas, esophagus, stomach, small and large intestines, kidneys, testes, prostate, seminal vesicles (or ovaries and uterus) spleen, bone marrow (sterna), skeletal muscles, and s kin. The liver, heart, spleen kidneys, and testes were weighed before fixation. The stomach and intestines were

washed out before fixation. All tumor masses were excised and fixed. Rats found dead were also necropsied and tissue preserved unless severe ly autolyzed. The fixed tissues were embedded in paraplast, sectioned at 6

- 7 um, and stained routinely with hematoxylin and eosin. All the tissues collected were examined microscopically. Statistical comparisons between groups were madding using Student ’s t

- test or chi square.

RESULTS

Endpoint value(s): Evidence of carcinogenicity

Results remarks: General condition of rats was good throughout the study.

There were a few significant differences in the mean hematological values at each of the time

pe riods, but no dose

- or time

- related trend; the differences were not considered to be biologically significant.

Deaths in the 0.01 and 0.1% sodium saccharin male groups occurred earlier and these groups

were terminated after 109

- 113 weeks. Otherwise, trea tment did not appear to affect survival. The remaining males were killed after 114

- 121 weeks when survival in the control group fell to 20% of the starting number. The number of control females killed terminally exceeded 20% of the starting number, but t he poor condition of several rats in this group necessitated sacrifice of all surviving females after 117

- 121 weeks on experiment.

Females of the 7.5% sodium saccharin group had a number of significantly decreased organ weights but these changes were pr obably related to the decreased body weight rather than a specific effect of the test substance. A few scattered significant differences in absolute or relative organ weights in the animals sacrificed serially at 15 and 18 months did not show a dose

or t ime

- related trend.

The most significant pathologic finding in the study was the presence of 11 neoplasms of theDraft HPV4 Chemical Data Adequacy Review CAS No. 128

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47 urinary bladder. Nine occurred in the 7.5% sodium saccharin group, one in the 5.0% sodium saccharin group, and one in the control group. They occurred primarily in males. The increased incidence of tumors in the 7.5% sodium saccharin group in comparison with control was significant. The earliest neoplasm was observed in a 7.5% male found dead at 103 weeks.

The two neoplasms in the control an d 5% groups were benign and classified as transitional cell polyps. Four of nine neoplasms at 7.5% were also benign, two as transitional cell polyps and two as transitional cell papillomas. There were five malignant tumors in the 7.5% group, all

transiti onal cell carcinomas. One showed focal squamous metaplasia; one extensive squamous metaplasia.

Incidences of hyperplasia were usually focal rather than diffuse and occurred most commonly in the dome and midportion of the urinary bladder. The lesion wa s composed of well differentiated epithelial cells. There was a significant increase in the incidence in the females of the 7.5% sodium saccharin group in comparison with the control females. The incidence in males of the 7.5% sodium saccharin group was increased, but not significantly, in comparison to the control

males.

The most frequently occurring non

- neoplastic lesions were chromic pulmonary disease with or without associated broncheictasis or lung abscess which occurred in a majority of animals o n study longer than 18 months; chronic nephritis which occurred in 53

- 82% of animals in the various groups and calyceal polyposis, which occurred in 38

- 71% of the animals in various groups. None of these were attributed to treatment.

CONCLUSIONS

The tes t substance has the potential to be carcinogenic.

STUDY RELIABILITY

[1] Reliable.

REFERENCE

Taylor, J.M. et al. Chronic toxicity and carcinogenicity to the urinary bladder of sodium

saccharin in the in utero

- exposed rat. Toxicology and Applied Pharma cology 54 (1980), 57

- 75.Draft HPV4 Chemical Data Adequacy Review CAS No. 128

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48 Summary of Endpoints

Endpoint Study Information

Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish Yes No Yes Acute Toxicity to Daphnia No No Yes Toxicity to Aquatic Plants No No Yes

Health Effects

Acute Toxicity Yes Yes No Repeated

- Dose Toxicity Yes No Yes

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity Yes No Yes Developmental Toxicity Yes No Yes