Short Chain Fructo Oligosaccharides

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

This 14-day dose range finding study was performed to select the dose levels for 90-day repeat dose study in rats (OECD 408)

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:

Wistar rats (HsdHan TM)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult healthy male and female Wistar rats (HsdHanTM) bred at Eurofins Advinus.

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for FOSSENCE TM contents from day 1 formulation samples.

Duration of treatment / exposure:

14 consecutive days

Frequency of treatment:

Once daily

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Dose / conc.:

9 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 rats per sex per dose

Control animals:

yes

yes, concurrent vehicle

Details on study design:

The doses used in this 14-days range finding study were selected based on the results of single dose toxicity study.

Observations and examinations performed and frequency:

The rats were observed for clinical signs or mortality, and body weights and feed consumption were measured.

Sacrifice and pathology:

All the rats were euthanized under isoflurane anesthesia and blood samples were collected by retro-orbital puncture on day 15 for clinical pathology (hematology, coagulation, and clinical chemistry), and organ weights.
Gross pathological examination were done on the same day (day 15).
Furthermore, based on the increased cecum weight observed in both sexes at all the doses tested, microscopic examination was performed on cecum, colon, duodenum, jejunum, and ileum from all dose group animals.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The slight decrease in feed consumption observed during treatment days 4–8 and 8–11 at 9000 mg/kg/day, however food consumption during days 11–14 was comparable to the control group. Hence slight decrease in food consumption that was observed during initial days of the treatment was considered as transient non-adverse finding.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item related changes observed in hematology and coagulation parameters

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item related changes observed in clinical chemistry parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item related changes observed in organs weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related changes observed in gross pathology.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes observed in cecum, colon, duodenum, jejunum, and ileum of both the sexes.

Details on results:

No clinical signs or mortality were observed at the tested dose levels of 2000, 5000, and 9000 mg/kg/day. The body weights were unaffected at all the doses tested. The slight decrease in feed consumption observed during treatment days 4–8 and 8–11 at 9000 mg/kg/day, however food consumption during days 11–14 was comparable to the control group. Hence slight decrease in food consumption that was observed during initial days of the treatment was considered as transient non-adverse finding. There were no test item related changes observed in hematology, coagulation, and clinical chemistry parameters as well as in organs weight and gross pathology. There were no test item-related microscopic changes observed in cecum, colon, duodenum, jejunum, and ileum of both the sexes. Fourteen-day repeat dose oral gavage administration of FOSSENCETM to Wistar rats did not cause any adverse toxicological changes on the evaluated parameters at 2000, 5000, and 9000 mg/kg/day doses.

Dose descriptor:

NOAEL

Effect level:

9 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: All evaluated parameters

Conclusions:

Fourteen-day repeat dose oral gavage administration of FOSSENCE TM to Wistar rats did not cause any adverse toxicological changes on the evaluated parameters at 2000, 5000, and 9000 mg/kg/day doses.

Executive summary:

A preliminary range finding study was conducted in ordertoselect the dose levels for 90-day repeat dose study in rats. In this 14-days toxicity test, the test item FOSSENCE TM was administered orally by gavage to Wistar rats for a period of 14 consecutive days. The doses employed in this study were 0, 2000, 5000, and 9000 mg/kg/day.

In 14-day studies, administration of the FOSSENCETM to Wistar rats did not cause any mortality or clinical signs and changes in body weights, feed consumption, and gross pathology at the doses of 2000, 5000, and 9000 mg/kg body weight.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

short chain Fructo-Oligosaccharides

EC Number:

908-300-1

Molecular formula:

C6H11O5(C6H10O5)nOH

IUPAC Name:

short chain Fructo-Oligosaccharides

Test material form:

solid: particulate/powder

Details on test material:

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%
- Heavy metals (Pb, As, Hg, and Cd) Below detection limits (Pb— 0.03 ppm, As—0.05 ppm, Hg— 0.01 ppm, and Cd—0.01 ppm)
- Microbial analysis Standard plate count: <10 cfu/g
Yeast count: <10 cfu/g
Mould count: <10 cfu/g
Enterobacteriaceae: 10 MPN/g
E.coli: Absent/10 g
Salmonella sp: Absent/100 g
Shigella sp: Absent/10g
- pH 5.48

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%

Test animals

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).

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

G2

Doses / concentrations 2

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

G3

Doses / concentrations 3

Dose / conc.:

9 000 mg/kg bw/day (nominal)

Remarks:

G4

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 (control)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R (Control recovery)

Doses / concentrations 6

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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.