Short Chain Fructo Oligosaccharides

Endpoint summary

Description of key information

The key studies (Manish et al 2018 and Clevenger et al 1988) and the supporting studies observed no oral toxicity for repeated exposure.

The NOAEL for ScFOS for these two key study were reported below:

NOAEL (90 days) ≥ 9000 mg/kgbw/day (highest dose tested) - rat  (Manish et al 2018)

NOAEL (2 years) ≥ 2170 mg/kg bw/day for males and 2664 mg/kg bw/day for females (highest dose tested) – rat (Clevenger et al 1988) 

 

In a subchronic study of Manish et al 2018, ScFOS was administered orally by gavage to Wistar rats for a period of 90 consecutive days at the level doses of 0, 2000, 5000, and 9000 mg/kg/day. The goal was to assess the systemic toxicity potential days and to determine the reversibility of effects following 28 days recovery period.  No treatment-related clinical signs or mortalities were observed. Similarly, no treatment-related toxicologically or biologically significant changes in body weight, feed consumption, ophthalmological findings, neurological effects, hematology, clinical chemistry, urinalysis, and gross pathological findings were noticed. However, statistically significant increase in weight of cecum (without correlative microscopic change) was noted at all the test item-treated groups in males and females and was considered to be a trophic effect and not a toxic effect in rats. In conclusion, ScFOS up to 9000 mg FOS/kg body weight/day is considered safe in Wistar rats without any adverse toxicological findings when administered for 90 consecutive days. The “No Observed Adverse Effect Level (NOAEL)” established was 9000 mg ScFOS/kg body weight/day.

Clevenger et al. (1988) performed a combined 104-week chronic toxicity and carcinogenicity study with 100 4-week-old male and female Fischer 344 rats using scFOS. The animals were acclimatized for 7 days, then randomized into 4 groups of individually caged rats that received scFOS in their diet at concentrations of 0, 8000, 20,000, and 50,000 ppm, equivalent to 0, 341, 854, and 2170 mg/kg bw/day, respectively, for male rats and 0, 419, 1045, and 2664 mg/kg bw/day, respectively, for female rats. In conclusion, under the test conditions, no dose-related effects on survival, growth, hematology, blood chemistry, organ weights, or nonneoplastic lesions were observed. The results of this study indicate that ScFOS does not produce chronic toxicity in rats. The NOAEL was 50,000 ppm, the highest concentration tested, equivalent to 2170 mg/kg bw/day for males and 2664 mg/kg bw/day for females.

In addition, two subchronic studies performed on rats(Takeda and Niizato 1982). The study was carried out on male Wistar rats for 6 weeks. Groups of 18 rats, were administered single daily doses of 1.5, 3, or 4.5 g/kg bw/day of ScFOS (test) or sucrose and glucose (controls). It was concluded that no treatment-related toxicity occurred in any of the scFOS-treated groups; the no observed adverse effect level (NOAEL) for orally administered scFOS in this study was the highest dose tested, 4500 mg/kg bw/day. Another study was carried out on male Wistar rats. The test substance was scFOS and the controls were sucrose, glucose, and sorbitol. The animals were given ad libitum access to feed in which 5% sucrose and 5% starch were replaced with 5 or 10% of the test substances. There was no evidence of greater toxicity of ScFOS than the sorbitol or sugars used as controls even at concentrations as high as 10% of the diet and the NOAEL in this study was the highest dietary concentration, 10%, equivalent to approximately 7500 mg/kg bw/day.

Finally, Tokunaga et al. (1986) studied the safety of repeated-dose intake of ScFOS. Groups of n = 6 male Wistar rats weighing 40-50 g were given ad libitum access to a control diet containing 67% cornstarch, or one of 3 diets in which cornstarch was replaced by 10% scFOS, 20% scFOS, or 20% glucomannan as a fiber reference group. The authors did not establish a NOAEL in this study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

FOS (FOSSENCE TM) used for the experiments was provided by Tata Chemicals Limited (Tamilnadu, India) and stored at ambient conditions (>15°C to <25°C).

- CAS no. 308066-66-2
- Composition (on dry basis):
> FOS: 95.46%
a) 1-Kestose (GF2): 40.23%
b) Nystose (GF3):47.15%
c) 1F-Fructofuranosylnystose (GF4): 8.08%
> Other sugars 4.50%
a) Glycerol: 1.40%
b) Fructose: 0.36%
c) Arabitol: 0.36%
d) Glucose: 0.63%
e) Sucrose: 1.75%
- Physical appearance: Fine white powder
- Moisture content (as per CoA): 2.26%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Young adult healthy male and female Wistar rats (HsdHan TM) bred at Eurofins Advinus were used.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Wistar rats from Eurofins Advinus were examined for good health and the suitability for the study and acclimatized for 5 days before the start of the treatment. All animals were housed (two/cage) with a temperature of 20–24°C, relative humidity 65–67%, and 12 h light and 12 h dark cycle. All animals were fed ad libitum with a standard diet (Teklad Global 14% protein rodent maintenance pellet diet manufactured by Envigo Laboratories,Venray, The Netherlands) and filtered water.
Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object. At the commencement of the treatment, the weight variation of rats did not exceed +20% of the mean body weight in each sex and group.
These 7- to 9- week-old rats were randomly distributed to different groups by the body weight stratification method using Provantis™ software (Version 8.7.3, Instem LSS, Staffordshire ST15OSD, United Kingdom).

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Details on oral exposure:

The dose volume employed was 15 mL/kg body weight.
The dose volume was calculated for individual animals on the first day of the treatment period (day 1) and was adjusted according to the most recent body weights recorded during the treatment period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for FOSSENCE TM contents thrice during the treatment period, that is, day 1 and on months 2 and 3.
High Performance Liquid Chromatography (HPLC) method using refractive index detection was validated at Eurofins Advinus. Validated HPLC method was used for the stability evaluation and concentration verification of
prepared dose formulation samples.

Duration of treatment / exposure:

90 consecutive days for the tested groups (G1, G2, G3 and G4)
118 consecutive days for the recovery groups (G1R and G4R). The vehicle or test item solution was not administered to the recovery groups for 28 days following the 90-day dosing period.

Frequency of treatment:

Once daily

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

9 000 mg/kg bw/day (nominal)

Remarks:

G4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 (control)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R (Control recovery)

Dose / conc.:

9 000 mg/kg bw/day (nominal)

Remarks:

G4R (high dose recovery)

No. of animals per sex per dose:

10 rats per sex per dose for G1 (control), G2,G3 and G4
5 rats per sex per dose for recovery groups such as control recovery (G1R) and high dose recovery (G4R)

Control animals:

yes

yes, concurrent vehicle

Details on study design:

The Fossence TM was mixed in Milli-Q water and administered through oral gavage route at the dose levels of 2000, 5000, and 9000 mg/kg/day (n=10 rats/sex/group) for 90 consecutive days. In addition, two recovery groups such as control recovery and high dose recovery were included (n=5 rats/sex/group).
The vehicle or test item solution was not administered to the recovery groups for 28 days following the 90-day dosing period.

Observations and examinations performed and frequency:

All rats were observed once daily for changes in appearance, behavior, clinical/toxic signs, and neurological changes and twice daily for morbidity and mortality.

Detailed clinical examination was done prior to the test article administration on day 1 and at weekly intervals thereafter during the treatment period.

Individual body weight and feed consumption were recorded prior to test article administration on day 1 and at weekly intervals thereafter till the end of the experiment period. Fasting body weight was recorded prior to termination.

Ophthalmological examination and functional observation battery were carried out. Ophthalmological examination was performed with an ophthalmoscope (direct ophthalmoscope, New York, USA; WelchAllyn) prior to start of treatment, end of the treatment period, and at the end of the recovery period.

The neurobehavioral examinations such as home cage observations, handling observations, open-field observations, sensory observations, neuromuscular observations, and physiological observation (rectal temperature) were conducted during 13th week of the treatment period for all the rats in main toxicity groups and during last week of the recovery period for the rats in recovery groups.

Clinical Chemistry: All rats were fasted overnight before blood collection on day 91 for main toxicity groups and on day 119 for recovery groups. The blood was collected for all groups by retro-orbital plexus puncture with the help of a fine capillary tube under isoflurane (Abbott Laboratories, Illinois, USA) anesthesia. The hematological, coagulation, and clinical chemistry parameters listed in Table 1 (see "any other information on material and methods incl.tables") were determined using the ADVIA 2120 hematology system (Bayer Health Care LLC, Tarrytown, New York, USA), Start-4 coagulation analyzer (Diagnostica stago, 92600 Asnieres, France), and Dimension RxL Max clinical Chemistry System (Dade Behring Inc., Newark, Delaware, USA), respectively.

Urine was collected on day 91 from the main toxicity groups and on day 119 from the recovery animals in urine collection tubes. Each rat was placed in a specially fabricated cage overnight (water allowed) and next morning the collected urine was analyzed. The urinalysis parameters listed in Table 1 (see "any other information on material and methods incl.tables") were analyzed.

Sacrifice and pathology:

At the end of the treatment (day 91) and at the end of the recovery period (day 119), all rats were fasted overnight (approximately 16 h). On the day of necropsy, all rats were weighed (fasting body weight), exsanguinated under isoflurane anesthesia, and subjected to detailed necropsy.
The gross pathological changes, if any, were recorded for each rat. Necropsy observation included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs, and tissues. The organs and tissues listed in Table 2 (see "any other information on material and methods incl.tables") from all rats were collected and fixed using 10% Neutral Buffered Formalin (monosodium phosphate dihydrate, disodium hydrogen phosphate, and formaldehyde, all from Rankem, Gurugram, Haryana, India).
The organs marked with X were weighed. The paired organs were weighed together and combined weight was presented. The organ weight ratios as percentage of body and brain weight were calculated based on the fasted body weight and brain weight.

Histopathological examination was carried out on all the preserved organs and tissues of vehicle control (G1) and 9000 mg FOS/kg/day (G4) group rats. In addition, all gross lesions from all the animals were examined microscopically.
There were no test item-related changes observed at 9000 mg/kg/day dose group and hence, histopathological evaluation was not carried out for lower dose (2000 mg FOS/kg/day and 5000 mg FOS/kg/day) groups and recovery groups. The tissues were processed for routine paraffin embedding and 4–5 mm sections were stained with Mayer’s hematoxylin and eosin stain.

Statistics:

The individual data were subjected to statistical analyses.
The analyzed data were expressed as mean +/- SD. All quantitative variables like body weight, feed consumption, laboratory investigation (hematology, coagulation, and clinical chemistry), and organ weight data were tested for normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test) within the group before performing one-way analysis of variance (ANOVA) modeling by treatment groups. When the data were found to be nonoptimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and control group was done using Dunnet’s test when the overall treatment ‘F’ test was found significant. All analyses and comparisons were evaluated at the 5% (p < 0.05) level. The neurological observations (neuromuscular observation/body temperature/ body weights) was calculated and analyzed by SYSTAT Statistical package Version 12.0 using one way ANOVA and t test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no relevant clinical signs noticed at any of the dose levels in this study.
in this study.Few clinical signs such as sparse hair loss/local alopecia in four rats (control recovery male—1, high dose male—1, and high dose recovery female—2) and lacrimation in one rat (high dose female) were observed during the experimental period. The clinical sign of sparse hair loss/ local alopecia was observed randomly in 4/100 rats and hence considered as incidental findings. The clinical sign of lacrimation (slight in nature) was also considered to be an incidental finding due to its isolated occurrence.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were unaffected at 2000 and 5000 mg/kg/day doses in males and at all the doses tested in females as compared to the control group. The statistically significant lower body weights were observed on day 90 (p < 0.05) at 9000 mg/kg/day when compared to the control group in males. The body weights were slightly lower (without statistical significance) at 9000 mg/kg/day in both main and recovery group males for the most part of the treatment period from week 7 till the end of the treatment period and considered partially reversible at the end of the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was unaffected at 2000 mg/kg/day (G2) in males and females as compared to the control group. The statistical significant changes (decrease) in feed consumption were observed at
the doses of 5000 and 9000 mg/kg/day (p < 0.05) in males and females during the treatment period and considered reversible during the recovery period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

There were no abnormal ophthalmological findings noticed at any of the dose levels in this study.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test itemrelated biologically significant adverse effects observed in hematological and coagulation parameters of both the sexes across the groups. There were few statistically significant
differences in hematology parameters in FOS treated animals compared to controls included decreased hemoglobin at 5000 mg/kg/day in males; decreased mean corpuscular hemoglobin concentration at all FOS-treated groups in males and at 5000 and 9000 (main and recovery) mg/kg/day in females; increased mean platelet volume at 2000 and 5000 mg/kg/day in males and 9000 mg/kg/day recovery in males and females; decreased absolute eosinophils at 9000 mg/kg/day in males; and decreased reticulocytes (both absolute and %) at 9000 mg/kg/day recovery females.
In the coagulation parameters, decreased prothrombin time values at 9000 mg/kg/day recovery males were noted.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related biologically significant adverse effects observed in clinical chemistry parameters of both the sexes across the groups. There are occasional sporadic findings of statistically significant differences in the following parameters from FOS-treated rats compared to controls included decreased total cholesterol, total proteins, and globulin at 5000 and 9000 mg/kg/day in males and at 9000 mg/kg/day in females; alanine aminotransferase at 9000 mg/kg/day recovery in males; decreased potassium at 9000 mg/kg/day recovery in males; and increased alkaline phosphatase at 9000 mg/kg/day in females.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in the urinalysis parameters in treated rats compared to controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Increase in absolute and relative cecum weight (with and without content) was observed at 9000 mg/kg/day in both the sexes. However, this change was not associated with any microscopic changes and hence considered as test item-related non-adverse effect. The cecum weight change was completely reversed in the recovery males, whereas in females, it was partially recovered.
Similar increase in cecum weight was also present at 2000 and 5000 mg/kg/day in both the sexes and was attributed to test item administration.
All other statistically significant differences observed in organ weight and their ratios were considered incidental as the changes were minimal in magnitude and/or lacked the
microscopic correlation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross changes observed in male and female rats.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in neurological/functional examination carried out at the end of treatment period for the main toxicity treatment groups and at the end of recovery period for the toxicity recovery groups.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes observed in male and female rats at all the doses tested. All the microscopic findings observed in males and females at 9000 mg/kg/day dose were considered incidental/ spontaneous and not related to test item administration, as they were distributed randomly across the groups and/ or normally present in rats of this age. In addition, observed microscopic findings were comparable to vehicle control group.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

9 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: All evaluated parameters

Key result

Critical effects observed:

no

System:

other: All organs described in the table 2

Organ:

adrenal glands

aorta

bone

brain

colon

duodenum

gonad

heart

kidney

lacrimal gland

larynx

liver

lungs

mammary gland

mesenteric lymph node

oesophagus

ovary

oviduct

pancreas

pituitary gland

salivary glands

seminal vesicle

skin

spinal cord

spleen

sternum

stomach

testes

thymus

tongue

trachea

ureter

uterus

vagina

Stability and analysis of the test item in vehicle:

The stability of the test item in the vehicle at 65 and 650 mg/mL concentrations showed that the test item was found to be stable for 24 h at room temperature and 4 days at refrigerated temperature.

The dose formulations were analyzed in subchronic (90-day) study.

The results of dose formulation analysis indicated that the analyzed concentrations were found to be within +10% of the claimed concentration and the relative standard

deviation (%RSD) was less than 10% of the claimed concentration for FOS set (GF2, GF3, and GF4). For other sugars (Glycerol, Arabitol, Fructose, Glucose, and

sucrose), the analyzed concentrations were within +20% of the claimed concentration and the %RSD was less than 15% of the claimed concentration.

The control group did not show any peaks for test item contamination.

Conclusions:

The oral gavage administration of FOSSENCE TM up to 9000 mg FOS/kg body weight/day is considered safe in Wistar rats without any adverse toxicological findings when administered for 90 consecutive days. The “No Observed Adverse Effect Level (NOAEL)” established was 9000 mg FOS/kg body weight/day in this study under the test conditions employed.

Executive summary:

A subchronic study was conducted to assess the systemic toxicity potential of the FOSSENCE TM when administered orally by gavage to Wistar rats for a period of 90 consecutive days and to determine the reversibility of effects following 28 days recovery period. The doses employed in this study were 0, 2000, 5000, and 9000 mg/kg/day.

 

No treatment-related clinical signs or mortalities were observed. Similarly, no treatment-related toxicologically or biologically significant changes in body weight, feed consumption, ophthalmological findings, neurological effects, hematology, clinical chemistry, urinalysis, and gross pathological findings were noticed.

However, statistically significant increase in weight of cecum (without correlative microscopic change) was noted at all the test item-treated groups in males and females and was considered to be a trophic effect and not a toxic effect in rats.

 

In conclusion, the oral gavage administration of FOSSENCE TM up to 9000 mg FOS/kg body weight/day is considered safe in Wistar rats without any adverse toxicological findings when administered for 90 consecutive days. The “No Observed Adverse Effect Level (NOAEL)” established was 9000 mg FOS/kg body weight/day in this study under the test conditions employed.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

The chronic rat study was conducted by the Biosafety Research Center, Foods, Drugs and Pesticides (An-Pyo Center), Shizuoka-ken (437-12), Japan.

GLP compliance:

not specified

Specific details on test material used for the study:

Neosugar is a mixture of fructooligosaccharides such as 1-kestose (GF2), nystose (GF3) and 1F-1-fructofuranosyl nystose (GF4). The sugar composition of Neosugar is approximately 37% GF2, 21% GF3 and 12% GF4.
Neosugar provided by Meiji Seika Kaisha Ltd.

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 4 weeks of age
- Housing:
- Diet: ad libitum, modified NIH open formula rat and mouse diet (Oriental Yeast Co., Tokyo, Japan)
- Water: ad libitum
- Acclimation period: week of acclimation,


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24"C,
- Humidity (%): 50-60%.
- Photoperiod (hrs dark / hrs light): 12-hr light-dark cycle

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

104 weeks of treatment.

Frequency of treatment:

in diet

Dose / conc.:

8 000 ppm

Remarks:

in diet

Dose / conc.:

20 000 ppm

Remarks:

in diet

Dose / conc.:

50 000 ppm

Remarks:

in diet

No. of animals per sex per dose:

50 rats of each sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

After 1 week of acclimation, groups of 50 rats of each sex began receiving neosugar at concentrations of 0, 8000, 20,000, or 50,000 ppm in their diet. Because of the low toxicity of neosugar, the selection of the highest exposure concentration was based on the maximum volume of neosugar that could be incorporated as a food additive. The experiment was terminated after 104 weeks of treatment.

Observations and examinations performed and frequency:

Animals were observed at least twice daily. Body weights were determined weekly for the first 26 weeks and biweekly thereafter. Food consumption was determined weekly for all animals throughout the study. Food efficiency and neosugar intake were calculated from body weight and food consumption data.
At the termination of the study, surviving animals were anesthetized with ether, and blood samples were collected from the abdominal aorta. Hematology measurements were performed on all samples using a Coulter Counter Model SP (Coulter Electronics, USA). Blood smears fixed with methanol and stained with Wright-Giemsa were used for differential leukocyte counts and morphological evaluation. Blood chemistry measurements using serum were performed using a Centrifichem System 400 (Union Carbide Co., USA) except for Na, K, and Cl, which were measured with an automatic electrolysis analyzer (Toa Electronics, Tokyo, Japan). Brain, adrenals, heart, spleen, lungs, testes, liver, ovaries, and kidney weights were recorded for all animals surviving the length of the experiment. All animals, including those dying spontaneously or killed in a moribund condition, were necropsied. Tissues were fixed in 10% neutral buffer formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

At the conclusion of the study, feed conversion efficiency and scFOS intake were calculated and blood samples were taken. Hematology outcomes were hematocrit, hemoglobin, red-blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, total leukocyte count, and differential leukocyte counts. Clinical chemistry outcomes included sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, BUN, uric acid, creatinine, TC, albumin, total bilirubin, AST, ALT, ALP, and lactate hydrogenase. Brain, adrenal gland, heart, lung, spleen, liver, kidney, testis, and ovary weights were recorded and tissue samples were obtained for histopathological examination.

Statistics:

A two-tailed Yates corrected Chi-square analysis was used to determine the significance of differences in survival and differences in the incidence of nonneoplastic lesions.
When pairwise comparisons of nonneoplastic lesions revealed a significant difference between treatment and control groups, logistic regression analysis was performed to test for dose-related trends. One-way analysis of variance (ANOVA) was used to assess the significance of differences in body weight, food consumption, food efficiency, organ weight, hematology, and blood chemistry. Where significant effects were found, the Student-Newman-Keuls (SNK) multiple range test for multiple comparisons among means was performed to determine where differences among the group means were located.
A one-tailed Yates corrected Chi-square test was used to determine the significance of differences in the: incidence of tumors. Logistic regression and Cochran-Armitage Chi-square tests for trend were used to determine significant dose-related trends. A significance level of P =< 0.05 was used in all analyses.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At the termination of the experiment, survival of male rats administered 0, 8000, 20,000 and 50,000 ppm neosugar was 88%, 72%, 68%, and 84%, respectively. The survival rates of the male 8000 and 50,000 ppm groups were comparable to the control group, but mortality in the 20,000 ppm group was significantly
higher than in controls. The survival of female rats administered 0, 8000, 20,000, and 50,000 ppm neosugar was 82%, 74%, 74%, and 88%, respectively. All female neosugar-treated groups had survival rates comparable to controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights of all male and female neosugar treatment groups were comparable to their respective controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Overall food consumption by neosugar-treated male rats was comparable to the control group. When overall food consumption data for females were analyzed by ANOVA, significant variation among groups was found; however, subsequent pariwise group comparisons by the SNK test procedure showed no significant intergroup differences involving the control group.
Neosugar intake by the 8000, 20,000, and 50,000 ppm groups was calculated to be approximately 341, 854, and 2170 mg/kg/day, respectively, for male rats and 419, 1045, and 2664 mg/kg/day, respectively, for female rats.

Food efficiency:

no effects observed

Description (incidence and severity):

Overall food efficiencies by neosugar-treated male and female groups were comparable to their control groups.

Description (incidence and severity):

Neosugar exposure had no significant effect on any of these parameters. In addition, neosugar treatment had no effect on differential leukocyte counts

Description (incidence and severity):

Male rats fed neosugar showed a slight but significant elevation of Na and Cl. Male rats fed 20,000 ppm neosugar had slightly elevated levels of blood glucose and creatinine. The male 50,000 ppm group had slightly decreased creatinine levels. All other parameters for male neosugar-treated rats were similar to control values. In females, all blood chemistry parameters were similar to controls except for a slight elevation of uric acid in the 8000 and 20,000 ppm groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Neosugar treatment had no effect on organ weights. Organ/body weight ratios also were similar among groups except for adrenal weight ratios of females. However, pairwise comparisons showed no significant differences involving the control group.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

As might be expected in aging rats, many types of nonneoplastic lesions were observed in all groups, including controls. Numerous lesions were present in liver, kidneys, adrenials, and lymph nodes. Other age-related lesions that occurred in the majority of animals were hyaline bodies in brain tissue, pigment deposits in the spleen, and atrophy of the thymus.
Stomach fibrosis, seminal vesicle atrophy, and decreased spermatogenesis were common in males, and pituitary and adrenal angiectasis were extremely common in females.
Nonneoplastic lesions were classified as slight, moderate, or marked. The majority of lesions were slight. Exceptions were chronic nephropathy in males, where 20-40% of these lesions were moderate in severity. In females, renal calcium deposits and protein casts and pigment deposits in the spleen were of moderate or marked severity in 15-40% of animals with these lesions.
Neosugar treatment did not affect the severity of these lesions except for renal protein casts in male rats. Renal casts of moderate severity were found in 0, 4, 7, and 6 rats in the male control, 8000, 20,000, and 50,000 ppm groups, respectively. One male in the 20,000 ppm group had renal casts of marked severity.
The incidence of the majority of nonneoplastic lesions was similar in all groups whether fed control diet or neosugar. However, pairwise comparisons revealed that some lesions occurred in greater or lesser incidence in neosugar groups than in controls.. It is apparent from the incidence data that simple pairwise comparisons alone were inadequate for determining a cause-effect relationship between neosugar treatment and the occurrence of a particular lesion. Therefore, additional intergroup comparisons were made, which included a quantitative dose-response trend analysis, comparison of the severity of lesions, and consideration of the background incidence of these lesions in historical controls.

Logistic regression is a parametric statistical test that analyzes for dose-related trends. Positive and negative trends are denoted by the sign of the dose coefficient and Student’s t value. Of the 17 lesions with significant findings by pairwise comparisons, only 3 were common to both males and females, and for 2 of the 3 (lymph node granulation and stomach fibrosis), opposite trends were observed. However, as indicated by the P values, no significant positive or negative dose-related trend:, were detected by logistic regression for any of the lesions in either sex.
A borderline positive trend (P = 0.07) was noted for lymph node granulation and prostatic atrophy in males. In females, only two lesions, adrenal angiectasis and adrenal hyperplasia, showed a posiitive (although nonsignificant) dose-related trend. The severity of these particular lesions in males and females was not affected by neosugar treatment. Furthermore, their incidence in neosugar groups was within the historical control range. With regard to lymph node granulation, opposite dose-related trends were noted in males and females. Although most of th nonneoplastic lesions in historical control F-344 rats from this laboratory were quite common, there were also wide variations in occurrence in historical controls. Despite the wide historical range, the incidence of some lesions in the neosugar study controls was outside the historical control range. The incidence of prostatic atrophy in concurrent controls was lo%, whereas the historical mean was S8%, and the range was 15-100%. Similarly, the incidence of dilated gastric glands and lymph node granulation in concurrent male controls were at the lower extreme of the historical ranges. Consequently, lesions that were significantly increased in neosugar groups as compared by pairwise comparison to concurrent control groups and that had borderline dose-response trends (lymph node granulation and prostatic atrophy in males) were within the historical control range.

If neosugar treatment were responsible for the occurrence of a particular lesion, the severity of the lesion would be expected to increase with increasing dose. Most of the lesions observec were slight in severity in control as well as neosugar groups. Neosugar treatment had no effect on severity of any of the lesions where significant differences in incidence were observed by pairwise comparissons, which further supports the conclusion that the observed lesions were not related to neosugar treatment.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Pituitary adenomas, pheochromocytomas, thyroid C-cell adenomas, Langerhans' islet adenomas, and leukemias occurred commonly in control groups of both sexes. Interstitial cell tumors of the testis were extremely common in control male rats, with an incidence greater than 80%. In the female control group, mammary gland fibroadenomas and endometrial stromal polyps were common. All of these tumors are considered to be spontaneous in F-344 rats.

Neosugar treatment did not increase the incidence of rare tumors in male or female rats. The incidence of spontaneous tumors in neosugar-treated animals was comparable to their incidence in concurrent controls, with the exception of pituitary adenomas. In male rats, the incidence of pituitary adenomas for the 0, 8000, 20,000, and 50,000 ppm dose groups was 20'70, 26%, 38%, and 440/0, respectively. The average background rate of pituitary adenomas in control male F-344 rats in this laboratory is 31%, with a range of 17-49%. Thus, the incidence of this tumor in male rats used in this experiment was within the historical range. However, pairwise comparisons between neosugar treatment groups and the concurrent control group showed the 20,000 and 50,000 ppm dose groups to have a significantly higher incidence of pituitary adenomas. To test for dose-related trends, two widely accepted trend tests were performed on these data. The Cochran-Armitage Chisquare
test indicated a dose-response trend (P = 0.007), whereas logistic regression analysis showed no such trend (P = 0.51). In female rats, the incidence of pituitary adenomas for the 0, 8000, 20,000, and 50,000 ppm dose groups was 48%, 39%, 38'40, and 28%, respectively. The incidence of pituitary adenomas in females showed an apparent negative trend (opposite to that seen in males), but the difference between the high-dose group and control was not significant using a two-tailed Chi-square test (P = 0.06). The background rate in control female F-344 rats ranges from 24% to 49%, with a mean incidence of 40%.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: All parameters

Key result

Critical effects observed:

no

Conclusions:

Under the test conditions, no dose-related effects on survival, growth, hematology, blood chemistry, organ weights, or nonneoplastic lesions were observed.
The NOAEL was 50,000 ppm, the highest concentration tested, equivalent to 2170 mg/kg bw/day for males and 2664 mg/kg bw/day for females.

Executive summary:

In this study, the chronic toxicity of neosugar were examined in Fischer 344 rats.

In this aim, 50 rats (males and females) were fed diets containing 0, 8000, 20000, or 50000 ppm neosugar equivalent to 0, 341, 854, and 2170 mg/kg bw/day, respectively, for male rats and 0, 419, 1045, and 2664 mg/kg bw/day, respectively, for female rats for 104 weeks.

Rats were observed twice daily and feed consumption was measured weekly. Body weights were taken weekly for the first 26 weeks and every other week for the remainder of the study. At the conclusion of the study, feed conversion efficiency and scFOS intake were calculated and blood samples were taken. Hematology outcomes were hematocrit, hemoglobin, red-blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, total leukocyte count, and differential leukocyte counts. Clinical chemistry outcomes included sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, BUN, uric acid, creatinine, TC, albumin, total bilirubin, AST, ALT, ALP, and lactate hydrogenase. Brain, adrenal gland, heart, lung, spleen, liver, kidney, testis, and ovary weights were recorded and tissue samples were obtained for histopathological examination.

As a result, in this chronic rat study, survival of both sexes was unaffected by neosugar treatment. The only significant finding was a decreased rate of survival in the male 20,000 ppm dose group. However, this is not believed to be treatment related, since it was an isolated occurrence and no dose-response relationship was evident. Body weight gain, food intake, hematology, and organ weights of both sexes were likewise unaffected by neosugar treatment.

 

In the chronic rat study, survival of both sexes was unaffected by neosugar treatment. The only significant finding was a decreased rate of survival in the male 20,000 ppm dose group. This is not believed to be treatment related, since it was an isolated occurrence and no dose-response relationship was evident. Body weight gain, food intake, hematology, and organ weights of both sexes were likewise unaffected by neosugar treatment.

Sporadic increases and decreases in the incidence of some lesions were found by pairwise comparisons between control and neosugar treatment groups. However, if neosugar treatment were responsible for these significant findings, a dose-response relationship with regard to incidence and severity would be expected. But, logistic regression analysis of the incidence data showed no significant positive or negative dose-related trends. In addition, the severity of the lesions with significant findings was not increased by neosugar treatment. Any decrease in severity would be undetectable, since almost all lesions were very mild. It should be noted that of the 17 lesions with significant findings, only 3 were common to both males and females, and for 2 of the 3 (lymph node granulation and stomach fibrosis), opposite trends were observed. No biological basis for a sex difference was apparent. A survey of the historical control incidence of these lesions showed that these lesions are common and highly variable in aging F-344 rats. Consideration of all of these factors leads to the conclusion that neosugar treatment did not affect the incidence of nonneoplastic lesions.

A slight but significant increase in blood Na and Cl was observed in male neosugar-treated rats, which is suggestive of mild renal impairment. However, other indicators of renal function, including K, Ca, phosphorus, BUN, uric acid, creatinine, and albumin, were not affected by neosugar treatment.

 

In conclusion, under the test conditions, no dose-related effects on survival, growth, hematology, blood chemistry, organ weights, or nonneoplastic lesions were observed. The NOAEL was 50000 ppm, the highest concentration tested, equivalent to 2170 mg/kg bw/day for males and 2664 mg/kg bw/day for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 170 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- A 90-day repeated dose toxicity study performed on rat via oral route, under GLP compliance and in accordance with OECD TG 408, was flagged as a key study and assigned a Klimisch score of 1.

- A 2 years repeated dose toxicity study performed on rat via oral route, under GLP compliance and in accordance with a method similar to  OECD TG 453, was flagged as a key study and assigned a Klimisch score of 2.

Justification for classification or non-classification

The NOAEL for scFOS via the oral route were NOAEL ≥ 9000 mg/kgbw/day (highest dose tested) for the 90 days repeated toxicity study and NOAEL ≥ 2170 mg/kg bw/day for males and 2664 mg/kg bw/day for females (highest dose tested) for the 2 years repeated toxicity study.

The available studies do not point out any organ specifically targeted by ScFOS and according to the results of the several studies available there is no need to classify the scFOS according the CLP regulation.