SRC TR-10-129

HPV4 CHEMICAL DATA ADEQUACY 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 Environmental 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

Elizabeth Margosches, Alternate Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

July 23, 2010
HPV4 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 22202
Draft HPV4 Chemical Data Adequacy Review CAS No. 128-44-9

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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
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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. NOTE: If no relevant studies were identified for the chemical, only the 

data availability/adequacy summary table was completed for the report.
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B-1. TOXICITY TO AQUATIC VERTEBRATES

Summary of endpoint: Inadequate

Study 1: Acute toxicity study in fathead minnows

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

The 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. 128-44-9

Remarks: None stated

METHOD

Method/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 LC50 value for fathead minnows was 18,300 mg/L. 

Results remarks: None stated
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CONCLUSIONS

96h LC50 = 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 pp. 

Publication Year: 1988.
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B-2. ACUTE TOXICITY

Summary of endpoint: Adequate 

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, non-standard 

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

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

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 animals/sex/dose: Not stated

Route of administration: Oral and intraperitoneal

Vehicle: Not stated

Doses/concentrations tested: Not stated

Statistical methods used: The LD50 was calculated by the method of Miller & 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. 
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RESULTS

Endpoint value(s): 

Oral

LD50 (mouse) = 17.5 g/kg

LD50 (mongrel rats) = 17.0 g/kg

LD50 (Wistar rats) = 14.2 g/kg

Intraperitoneal

LD50 (mouse) = 6.3 g/kg

LD50 (mongrel 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 administration of cyclamate or saccharin 

throughout gestation 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 

magnitude.

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.
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Study 2: Acute oral toxicity study in rats

Title: Acute toxicity tests on 113 environmental chemicals

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Acute

GLP compliant?: Not stated

Year study performed: 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: Gum arabic

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

Statistical methods used: The LD50 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 regular laboratory chow 6 hours after a single 

administration and the number of dead animals was counted until the 14th day. Anatomical 

examination was performed immediately after death or at terminal sacrifice on day 14. 

RESULTS

Endpoint value(s): LD50 = 1280 and 1580 mg/kg for males and females, respectively.

Results remarks: There were no acute toxic symptoms or change in tissues observed.
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CONCLUSIONS

The acute oral LD50 was 1280 mg/kg for male rats and 1580 mg/kg for female rats.

STUDY RELIABILITY

[2] Reliable with restrictions.

REFERENCE

Hasegawa et al. 1989. Acute Toxicity Tests on 113 Environmental Chemicals. Sci Rep Res Inst 

Tohoku Univ Ser C Med: 36 (1-4): 10-16.
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B-3. REPEATED-DOSE TOXICITY

Summary of endpoint: Inadequate

Study 1: Repeated-dose toxicity study 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 limited number of test substance 

concentrations tested.

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Repeated-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: Four 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 libitum for 38 days. Survival, behavior, general physical appearance and 

stool condition were noted daily. On day 39, the surviving animals were killed off and sections 
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of renal and hepatic tissues were fixed in formalin and stained with haematoxylin eosin 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

inflammatory and hydropic lesions in the liver and kidneys 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, 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.

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.
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Study 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: Sodium saccharin, CAS No. 128-44-9

Remarks: Not stated

METHOD

Method/guideline followed: Not stated

Test type: Oral repeated-dose

GLP compliant?: 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/conditions 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 

equivalent 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 renal 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.
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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 normal 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 inanition. Post-mortem examination revealed no abnormal organ 

pathology.

About month 10 of the study, all dogs developed soft, poorly-formed stools 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. 

Bodyweight, hemoglobin, total leukocyte count, non-protein nitrogen and phenolsulphonphthalein retention were within normal limits and total erythrocyte count, bilirubin and 

urinary analyses were also unaffected. The gross and histological appearance of the viscera at 

necropsy were within normal 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.
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B-4. GENE MUTATIONS

Summary of endpoint: Adequate 

Study 1: Dominant lethal assay in mice

Title: The mutagenicity of sodium saccharin (S-Na). I. Dominant-lethal test 

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

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 

males 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 χ2

-test and calculated dominant lethality. 
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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 significance (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. The mutagenicity of sodium saccharin (S-Na). I. Dominant-lethal test. 

Mutation Research, 54 (1978) 218-219.
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Study 2: Dominant lethal assay in mice

Title: Induction of Dominant Lethals in Mice by Sodium Saccharin

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

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 mice 

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 Madhurin weighing 172 mg were dissolved in 

100 mL 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 found 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.
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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 that 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 consideration 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 Journal of Medical Research, 60 (1972) 599-603.
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B5. CHROMOSOMAL ABERRATIONS

Summary of endpoint: Adequate 

Study 1: Bone marrow cytogenetic assay in mice

Title: The mutagenicity of sodium saccharin (S-Na). II. Cytogenetic studies

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

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/conditions remarks: After i.p. injection of sodium saccharin (S-Na; 4000 mg/kg), 

metaphase plates were analyzed at 6, 24 or 48 h. Dose-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) and 
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gaps (3.3%) were scored. In the case of repeated dosing, although aberrant cells increased 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 concentrations 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. 

Mutation Research, 54 (1978) 219.
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Study 2: In vitro mammalian chromosomal aberration test

Title: The mutagenicity of sodium saccharin (S-Na). II. Cytogenetic studies

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

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: Not 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 increase (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.
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CONCLUSIONS

High concentrations 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. 

Mutation Research, 54 (1978) 219.
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Study 3: In vitro mammalian chromosomal aberration test

Title: Mutagenicity of sodium saccharin to human cells in vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

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 method/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 concentrations of S-Na induced chromosome aberrations in vitro. 
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STUDY RELIABILITY

[2] Reliable with restrictions.

REFERENCE

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

(1989) 299-300.
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B6. GENETIC TOXICITY - 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. 128-44-9

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 cultures 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 increase 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 were 
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exposed to sodium saccharin dissolved 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 

relationship between the level of UDS and the concentration of sodium saccharin.

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.
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B7. REPRODUCTIVE TOXICITY

Summary of endpoint: Adequate

Study 1: Reproductive toxicity study in rats

Title: Long-Term Toxicity of Sodium Saccharin in the Rat

The study is: Adequate 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 (orthotoluenesulfonamide; limit of detection = 0.05 ppm). The total amount of organic soluble 

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-Generation repeated-dose toxicity

GLP compliant?: Not stated

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

Vehicle: Corn oil

Exposure duration: 2 generations
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Doses/concentrations tested: 5% in diet

Test method/conditions remarks: The F0 or parent generation animals consisted of groups of 

50 male and 50 female (except where noted) Charles River, Caesaean-derived, Sprague-Dawley 

rats. The basal diet was Master Laboratory cubes 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 of 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 test 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 measured 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 the tail aorta of approximately 10 rats per group, under light anesthesia. The 

blood samples were obtained from the F0 animals at 4.5 weeks (Day 0 on test), 21, 44.5, 57, 65, 

69, 96 and 98 weeks of age; the F1 animals were sampled at 4.5, 18.5, 32.5, and 45.5 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 corpuscular 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 examined 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 moist, 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 growth 
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rates. There were 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 animals 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 animals 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-Toluenesulfonamide and Sodium 

Saccharin in the Rat. Toxicol. Appl. Pharmacol. 52:113-52.
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Study 2: Reproductive toxicity study in rats

Title: Effects of in utero and postnatal sodium 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 only one dose-level was tested; NOAEL/LOAEL not 

provided.

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 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 health screening, which showed no hematological, serological 

or histopathological abnormalities. Breeding animals were housed individually in polycarbonate 
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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-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 libitum. Sodium saccharin (NaS) was 

mixed 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 (Schoeing et al., 1985). After a 2-week 

adaptation period during which they were fed Purina Rodent Chow No. 5002 ad libitum, 6-wkold male and female F0 rats were randomly assigned by weight to an experimental group (7.5% 

NaS, group 1) or a control group (untreated diet, group 2) (Martin et al., 1984). Mating was 

started after 4-6 weeks 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, 

where each litter in group 1 was matched with a litter in group 2 having the same birth date. To 

that end, extra F0 rats were mated. The six matched litters from each group were used for urine 

collection and analyses, serum lipid and vitamin measurements, biochemical 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.

F0 males and non-mated females were killed, without 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. F0 female rats were killed after their litters 

had been weaned. F1 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 contamination 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, magnesium, 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 period, 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 F0 rats, food and water consumption 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 water 
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consumption measurements for F0 rats began with a 3-day interval 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 individually 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 from 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 collected 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, F1 rats 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), creatinine, 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 epithelial 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 analyzed for 

DNA, cholesterol, phospholipid and protein. Livers were weighed, homogenized and analyzed 

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 comparisons 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 weeks, body weights were 

10% less than those of control rats. The mean body weight of NaS-treated females remained 

significantly below that of control animals throughout gestation and the first 2 weeks of 

lactation. However, weight gain during pregnancy was equivalent for the two groups and NaS-
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treated females actually gained more weight than controls during lactation, so that there was no 

difference between the groups on day 21 post-birth. Basal diet consumption (g/rat/day) was 

significantly less in NaS-fed animals, except during week 2 and 3 of gestation and week 1 postbirth when consumption was similar in the two groups. NaS-treated rats consistently 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/mL 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 nonmated 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 F1 animals: litter size and survival and pup weights – 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 per 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 than 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 methods

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 final 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 the 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/day 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 showed 
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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 little difference in the weight of dry feces between the groups, but the feces of NaStreated 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 animals 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 

associated 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 significantly increased in NaStreated 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 animals, 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 lipids – 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 triglycerides 

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 NaStreated rats. 

Hematology and serum chemistry in unmatched litters – 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 chemistries. 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 

decreases 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.
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Differences between male and female F1 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 females 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-associated 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 lipoproteins 

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 few sex-related differences were 

observed in the treated and control groups.

Differences between F1 litters – For the various chemistry parameters evaluated, analyses were 

performed to determine whether significant differences existed between litters that received the 

same treatment. The data for males and females were analyzed separately. Inter-litter 

differences were numerous in both sex and in both NaS-treated and control rats.

CONCLUSIONS

Overall, body weights were significantly depressed in NaS-treated 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 caecal content 

weight, changes in several urinary analytes, a 50% increase in serum cholesterol, a 10-fold 

increase in serum triglycerides and decreases in serum and hepatic vitamins. In addition, NaStreated dams and pups were anemic. Relatively few differences between males and females were

noted, but significant inter-litter differences existed. The numerous physiological changes 

indicate that 7.5% dietary 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.
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Study 3: Reproductive toxicity study in rats

Title: Effects of in utero and postnatal sodium 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 and 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 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: 22 females and 11 males per group in groups 1-4; and 11 females 

and six males per group in groups 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% Sodium 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 conception until day 30 of age, Control from 
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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 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 health screening, which showed no hematological, serological 

or histopathological abnormalities. 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-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 libitum. Sodium saccharin (NaS) was 

mixed in the diet at a level of 7.5% by weight. The dietary level of saccharin was analyzed by 

HPLC before each batch of test diet was fed. The average measured concentration of NaS in 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 and 6. They were placed on the appropriate test diet at 6 weeks 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 of 

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 evaluate 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 given day.

Observations and measurements – The rats were observed twice daily for evidence of overt 

toxicity and/or morbidity and mortality. For F0 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 determinations and detailed physical examinations were performed 

before mating. For rats of the second generation (F1), food and water consumption were 

determined weekly after weaning except during week 4 post-birth when some litters were killed 

and others had their 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 determined on these 
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days.

Analyses – F1 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 

both 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-mm3 piece of the left lateral lobe of the liver was put into cold picric acid-pformaldehyde 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 sections were stained with toluidine blue and 

examined by light microscopy. In 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 liver 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 analysed 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.

Statistics – 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 

decreased (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 a 
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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 week 4 post-birth, the values of body weight and food consumption 

(basal diet) decreased as the NaS concentration in the diet increased. Rats treated with 7.5% NaS 

weighed about 60% as much as control rats, and males fed 3% NaS weighed significantly less 

(approx. 10%) than controls. 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 

controls. Water consumption during weeks 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 

relative 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 liver 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 controls. 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 consistent 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% NaS 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 control 

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 and vitamin E were increased 
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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 decreases 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 serum 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 capacity 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 ironbinding 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 triglycerides 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 groups, but the severity and incidence were 

low.

CONCLUSIONS

Overall, most 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, cholesterol 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 studies (1% 
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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 reduced 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 related to the production of bladder tumors.

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. II. Dose response and reversibility. Food Chem Toxicology 

29 (1991), 669-679.
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Study 4: Reproductive 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. 128-44-9

Remarks: 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 carcinogenic 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 continued 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% 
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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 not 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 

scattered 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. Histopathological 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 assignable – results reported only as abstract

REFERENCE

Taylor, J.M. et al. Combined chronic feeding and three-generation reproduction study of sodium 

saccharin in the rat. Toxicol. Appl. Pharmacol. 29 (1974), 154.
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B-8. DEVELOPMENTAL TOXICITY

Summary 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: Adequate 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. 128-44-9

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: Diet

Exposure duration: From mating until day 20 of pregnancy

Doses/concentrations tested: 0-4% 

Vehicle: Not stated

Statistical methods used: None stated
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Test method/conditions remarks: 

Groups of 26-28 female Wistar rats (obtained from Mus Rattus Corp., 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 atmosphere 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 containing 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 group 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, 

mean 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 implantation sites 

demonstrated 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 behavior, 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 were killed with 

CO2, 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-week-old animals 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 fetuses 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

Endpoint value(s): Developmental NOAEL = 98.8 mg/day

Results remarks: The general study provided no indication of any primary embyrotoxic effect 

of the treatment and the histological examination showed that treatment with the sweetening 
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agents caused no ocular 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 particular, 

no lytic, necrotizing or vacuolating process of the ocular lenses were present. The fibers of the 

lenses showed no increase in circumference or disorientation. The epithelial cells of the lenses 

had reached their regular position and showed normal 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

Luckhaus, 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.
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B-9. CARCINOGENICITY STUDY

Study 1: Carcinogenicity study in rats

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

in Utero-Exposed Rat

TEST SUBSTANCE 

Identity: Sodium saccharin, CAS No. 128-44-9

Remarks: Not stated

METHOD

Method/guideline followed: 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 offspring 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 rats 
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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 smear.

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 ether, 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, pituitary 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 skin. 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 severely 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 

periods, 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, treatment 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 the 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 probably 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 doseor time-related trend.

The most significant pathologic finding in the study was the presence of 11 neoplasms of the 
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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 and 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 

transitional 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 was 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 on 

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 test 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 Pharmacology 54 (1980), 57-75.
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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