Short Chain Fructo Oligosaccharides

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The potential toxic effects of the ScFOS was investigated in a GLP-compliant Reproduction/Developmental Toxicity Screening Test performed in accordance with OECD Guideline No. 421. The objectives of this study were to determine the potential toxic effects of Short Chain Fructo-oligosaccharides when given via diet for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were determined.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) for Short Chain Fructooligosaccharides were established: Parental, Reproduction and Developmental NOAEL: at least 1160 mg/kg bw/day ppm (corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively).

In addition, GRAS Notice for scFOS from the Office of Food Additive Safety (FDA) report from an unpublished study conducted by Henquin (1988) that dietary administration of scFOS at concentrations up to 20% [equivalent to approximately 10,000 mg/kg body weight/day (U.S. FDA, 1993)] did not result in developmental toxicity. GRAS Notice also reports from an unpublished study conducted by Sleet and Brightwell (1990) evaluating the maternal and developmental toxicity of ScFOS, that dietary concentrations up to 20% [equivalent to approximately 10,000 mg/kg body weight/day (U.S. FDA, 1993)] provided to rats during postcoitum days 0 to 15 did not result in treatment related adverse effects (e.g. diarrhea), or differences in pregnancy outcome or in utero development. 

These two studies are summarized in Carabin and Flamm, 1999

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Nov 2021 to 29 Jul 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 14 days
- Route of administration: The oral route of administration via dietary inclusion was selected because gavage corresponds to a bolus dose, which is not representative of the intended route of exposure. From a toxicokinetic standpoint, exposure via diet is more physiological.
- Basis for dose level selection: The dose levels were selected based on information described in literature on rat studies with Short chain Fructoologosaccharides (scFOS), via read-across, with toxicity studies with Fructooligosaccharides, and in an attempt to produce graded responses to the test item.
- Other considerations: The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Specific details on test material used for the study:

Identification: Actilight 950P Premium "Fructo-Oligosaccharides"
Substance: Short-chain fructo-oligosaccharides
EC No.: 908-300-1
Appearance: White powder
Purity: 95.4%

COMPOSITION:
scFOS is short chains of fructose molecules linked to a molecule of sucrose (glucose-fructose disaccharide). Thus, scFOS is a multiconstituent substance composed of three oligosaccharides: 1-kestose (GF2), nystose (GF3) and fructosyl-nystose (GF4).
With:
- TOTAL FOS: 95.4 expressed as dry matter
- Kestose (GF2): 39.2% FOS
- Nystose (GF3): 47.6% FOS
- 1F-Fructofuranosylnystose (GF4:) 13.6% FOS
- Glucose +Fructose + Saccharose : 4.6% expressed as dry matter

Species:

rat

Strain:

Wistar

Remarks:

Wistar-Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: males: 10-11 weeks old; females: 13-14 weeks old
- Weight at study initiation: 265 – 331 g males; 182 – 228 g females
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same administration group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam. Cages containing sterilized wooden fibers as bedding material were equipped with water bottles.
Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: standard powder rodent diet; ad libitum
- Water: tap water; ad libitum
- Acclimation period: 6 days

It is considered that there are no known contaminants in the water, food or housing materials that could interfere with the outcome of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 38 - 54
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 01 Dec 2021 To: 03 Feb 2022

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Diets were prepared freshly for use at room temperature for a maximum of 10 days. The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 10 days for supplementing food during the respective food consumption measurement interval.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear] referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing: females who have not shown evidence of mating were separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female was re-mated once with a male for a maximum of 7 days. A male of the same group having previously shown evidence of mating was used for re-mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet Preparation Sample Collection :
- Occasion: first week of treatment
- Concentration: All groups, 2 x approximately 5 g
- Homogeneity: Groups 2 and 4, 2 x approximately 5 g

Analytical method: LC-MS/MS

Duration of treatment / exposure:

Males: 29 days
Females: 42 – 64 days

Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.
Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day of scheduled necropsy.

Frequency of treatment:

Continuous - feeding study

Details on study schedule:

F1 animals were not mated nor treated directly with the test substance.

Dose / conc.:

100 mg/kg bw/day

Remarks:

Target dose level equivalent to 1000 ppm; actual test material intake: 104 and 123 mg/kg bw/day for males and females respectively; Group 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Target dose level equivalent to 5000 ppm; actual test material intake: 341 and 406 mg/kg bw/day for males and females respectively; Group 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Target dose level equivalent to 15000 ppm; actual test material intake: 1087 and 1233 mg/kg bw/day for males and females respectively; Group 4

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to first administration and at least once daily from start of administration onwards, up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 1 of treatment (prior to administration) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, quantitatively measured per cage.
- Time schedule: Weekly, except for males and females which were housed together for mating
and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 postcoitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Regular basis throughout the study

OTHER: On the day of scheduled autopsy, blood was drawn from the retroorbital sinus under isofluorane anesthesia; 150 μL serum was used for measurement of total T4, and the remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).

Oestrous cyclicity (parental animals):

- Frequency: Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage was continued for those females with no evidence of
copulation until termination of the mating period. End of Treatment - on the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
- Procedure: Estrous cycles was evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Sperm parameters (parental animals):

For all surviving males, the following assessments was performed:
Sperm samples was taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility was assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa per animal was recorded. Evaluation was performed for all samples.
One epididymis (right) was removed, weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 100 pups/litter (50/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia; T4 and/or TSH serum levels

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals: Following completion of the mating period (a minimum
of 28 days of administration).
- Maternal animals: Females which deliver: PND 14-16; Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27; Without evidence of mating: Approximately 24-26 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were collected: cervix, epididymis, mammary gland, parathyroid gland, pituitary gland, prostate gland, seminal vesicle gland including coagulation gland and fluid, thyroid gland, liver, ovaries, testes, uterus, vagina
The following tissues were prepared for microscopic examination: cervix, epididymis, mammary gland, prostate gland, seminal vesicle gland including coagulation gland and fluid, thyroid gland, ovaries, testes, uterus, vagina
The following organs were weighted: epididymis, parathyroid gland, prostate gland, seminal vesicle gland including coagulation gland and fluid, thyroid gland, liver, ovaries, testes

Postmortem examinations (offspring):

SACRIFICE AND GROSS NECROPSY:
- The surplus F1 offspring were sacrificed at PND 4 by decapitation. Sex was determined both
externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected. All remaining pups were euthanized on PND 14-16.
- The animals sacrificed 14-16 PND were subjected to postmortem examinations as follows: Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. The thyroid was collected from two pups per litter (from one male and one female pup)

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions was analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test were used to compare all groups. The above pairwise
comparisons were conducted using Fisher’s exact test whenever the overall test was significant.
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not
significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Reproductive indices:

Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Live birth index (%) = (Number of live offspring on Day 1 after littering/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%) = (Number of live female pups/Number of live pups) x 100
Percentage live males at First Litter Check (%) = (Number of live male pups/Number of live pups) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number of live offspring on Day 1 after littering) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 after culling) x 100
Post-implantation survival index (%) = (Total number of offspring born/Total number of implantation sites) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs noted in females occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and/or due to the transient nature, these were considered.
to be not test material-related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly higher body weight gain was observed in females at 1000 mg/kg bw/day compared to control between Days 1-3 of premating and between Day 9 of premating and the start of mating (Day 15 of dosing). As this did not result in any significant effects on body weights and as the effect was transient in nature, this was considered unrelated to treatment with the test material.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In females at 100 mg/kg bw/day, a reduced absolute food consumption was noted between Days 17-20 of post-coitum, and an increased relative food consumption was observed between Days 4-13 of lactation. These observations were considered unrelated to treatment with the test material as this occurred in absence of a dose-related response. The high absolute and relative food consumption of Male Cage No. 7 (1000 mg/kg bw/day) during Day 1-3 of premating was potentially the result of a weighing error as this high level of food consumption only occurred in this one cage during this single period. This incidental increase was considered not test material-related.

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

no effects observed

Endocrine findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Epididymal sperm count was reduced, and the number of sperm cells with a normal morphology were slightly lower at 100 and 300 mg/kg bw/day compared to control. In absence of a dose-related response and/or as the difference was only minor, these findings were considered not test material-related. The apparent lower number of detached sperm heads in all groups administered with the test material compared to control (only statistically significant at 100 mg/kg bw/day) could be attributed to one control animal with a very high count of spermatids with detached heads. After excluding this male, the mean number of detached heads in the control group was 2. This was in the same range as all treated groups.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was one control couple and one couple treated at 300 mg/kg bw/day with no offspring, and one female treated at 100 mg/kg bw/day with one implantation site only. No abnormalities were seen in the reproductive organs of these males or females that could explain the lack of offspring. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material. Stage dependent qualitative evaluation of spermatogenesis in
the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
- Mating Index: Mating index was unaffected by treatment with the test material. All females showed evidence of mating resulting in mating indices of 100% for all groups.
- Precoital Time: Precoital time was considered not to be affected by treatment with the test material. All females showed evidence of mating within 4 days, except for one control female that
mated after 14 days.
- Number of Implantation Sites: Number of implantation sites was considered not to be affected by treatment with the test material. The mean number of implantation sites was 13.4, 11.8, 13.9 and 13.3 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The slightly reduced number of implantation sites at 100 mg/kg bw/day could be attributed to one female which had one implantation site only.
- Fertility Index: Fertility index was considered not to be affected by treatment with the test material. The fertility indices were 90, 100, 90 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One control female and one female at 300 mg/kg bw/day were not
pregnant. The non-pregnancy of the latter female was considered not test material-related due
to the incidental occurrence and in absence of a dose-related trend.
- Gestation Index and Duration: Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test material. Except for one female at 100 mg/kg bw/day, all pregnant females had live offspring. The failed pregnancy, which had only one implantation site, was considered unrelated to treatment with the test material due to the incidental occurrence and lack of a dose-related trend.
- Parturition/Maternal Care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The nature and incidence of observed clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be test material-related.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test material.

- Post-Implantation Survival Index: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by the test material. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 95, 93, 94 and 89% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The post-implantation survival index was slightly reduced at 1000 mg/kg bw/day compared to control. As the concurrent control group had a relatively high post-implantation survival index compared to the historical control data range, as the number of implantations and the live litter size at 1000 mg/kg bw/day were within normal limits and as the post-implantation survival index at 1000 mg/kg bw/day was within the historical control range this slight reduction was considered not related to treatment with the test material. For one female (100 mg/kg bw/day), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 14-16 days of) lactation.
- Litter Size: Litter size was considered not affected by treatment with the test material. Live litter sizes were 12.8, 12.2, 13.1, and 11.8 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
- Live Birth Index: Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test material. The live birth index was 100% for all groups.
- Viability Index: Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test
material. Viability indices were 100, 100, 98 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The slightly lower viability indices in the 300 and 1000 mg/kg bw/day groups could be attributed to two pups at 300 mg/kg bw/day that were found missing on PND 2 or 4 and to one missing pup at 1000 mg/kg bw/day on PND 2. A pale appearance was noted at first litter check for the pup at 1000 mg/kg bw/day. No clinical signs were observed for the two pups at 300 mg/kg bw/day before these went missing. Pups missing were most likely cannibalized. These missing pups were considered to be unrelated to treatment with the test material since the mortality incidence remained within the range considered normal for pups of this age.
- Lactation Index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test material. No
pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index
of 100% for all groups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Results of diet analysis

'The concentrations analyzed in the diet samples of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e., mean accuracies between 80% and 120%). The diet samples of Groups 2 and 4 were prepared homogeneous (i.e., coefficient of variation ≤ 10%).''

Historical control data for Wistar Han rats (period 2018-2022):
Post-implantation survival index (%): mean = 91; P5 – P95 = 83-97 (n=111).

 

Table 1. Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.

Group No.	Dose level ppm	In-Life Reason	Histopathology
1	0	Not pregnant	No histopathological correlate
2	1500	One implantation site only	No histopathological correlate
3	5000	Not pregnant	No histopathological correlate
4	15000	n.a.	n.a.

n.a. = not applicable

 

Table 2. Developmental data summary

	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	1500 PPM	5000 PPM	15000 PPM
Total number of offspring born	115	110	118	118
Total number of uterine implantation sites	121	118	125	133
Number of live offspring on Day 1 after littering	115	110	118	118
Number of live offspring on Day 4 (before culling)	115	110	116	117
Number of live offspring on Day 4 (after culling)	72	72	72	80
Number of live offspring on Day 13 after littering	72	72	72	80
Post-implantation survival index (%)	95	93	94	89
(Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%)	100	100	100	100
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%)	100	100	98	99
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%)	100	100	100	100
(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100

 

Table 3. Reproductive performance summary

	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	1500 PPM	5000 PPM	15000 PPM
Females paired	10	10	10	10
Females mated	10	10	10	10
Pregnant females	9	10	9	10
Females with implantations only	0	1	0	0
Females with living pups on Day 1	9	9	9	10
Mating index (%)	100	100	100	100
(Females mated / Females paired) * 100
Fertility index (%)	90	100	90	100
(Pregnant females / Females mated) * 100
Gestation index (%)	100	90	100	100
(Females with living pups on Day 1 / Pregnant females) * 100

 

 

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Short Chain Fructooligosaccharides were established: at least 15000 ppm (corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively).

Executive summary:

The objectives of this study were to determine the potential toxic effects of Short Chain Fructo-oligosaccharides when given via diet for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 1500, 5000 and 15000 ppm, based on information described in literature on rat studies with Short chain Fructo-oligosaccharides (scFOS) and, via read-across, on toxicity studies with Fructo-oligosaccharides. The study design was as follows: 

Experimental design

Group No.	Target Dose (mg/kg bw/day)	Diet Concentration (ppm)	Number of animals
			Males	Females
1	0	0	10	10
2	100	1500	10	10
3	300	5000	10	10
4	1000	15000	10	10

 

During the lactation period, the following dietary concentrations were used since food intake would be considerably higher in lactating females (based on historical control data for relative food consumption the dietary concentrations were reduced by 1.5-times): 

Dietary concentrations during lactation

Lactation	Group 1	Group 2	Group 3	Group 4
Day 1 upto and including day of necropsy	0 ppm	1000 ppm	3333 ppm	10000 ppm

 

Chemical analyses of dietary preparations were conducted once during the study and confirmed that the diets containing test material were prepared accurately and homogenously. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, measurement of thyroid hormones (thyroxine (T4) in F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Parental results No parental toxicity was observed up to the highest target dose level tested. No test material-related changes were noted in any of the parental parameters investigated in this study (i.e., mortality, clinical appearance, body weight, food consumption, T4 thyroid hormone levels (in F0-males), macroscopic examination, organ weights, and microscopic examination).

Reproduction results No reproduction toxicity was observed up to the highest target dose level tested. No test material-related changes were noted in any of the reproductive parameters investigated in this study (i.e., mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results No developmental toxicity was observed up to the highest target dose level tested. No test material-related changes were noted in any of the developmental parameters investigated in this study (i.e., gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels (in PND14-16 pups) and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Short Chain Fructooligosaccharides were established: Parental, Reproduction and Developmental NOAEL: at least 15000 ppm (corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively).

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague Dawley rats (strain Crl CD (SD) BR).

Sex:

female

Route of administration:

oral: feed

Vehicle:

other: in diet

Details on exposure:

Four groups of 24 to 27 pregnant females were administered scFOS from day 0 to 6 post coitum at 4.75% dietary concentration. On post-coital day 6, the dietary concentration of scFOS in the 4 groups was changed to 0, 5%, 10%, or 20%. On day 15, all of the animals were placed on an scFOS-free diet. A fifth group received an scFOS-free diet throughout the entire study.

Dose / conc.:

4.75 other: %

Remarks:

4.75% dietary concentration

No. of animals per sex per dose:

Four groups of 24 to 27 pregnant females

No treatment-related effects were observed during days 0 to 6 or during days 6 to 15. There was no mortality and no diarrhea was observed in any of the test animals. scFOS administered at 4.75% dietary concentration during days 0 to 6 did not affect body weight and body weight change in any of the groups, but, beginning on day 8, body weight was reduced dose-dependently in all 3 scFOS groups relative to controls. The 5% group exhibited a lower weight gain than the controls while the 10 and 20% groups actually lost weight. From day 11 to the end of the study, the body weight and body weight change were similar in all groups except the 20% scFOS group, which remained below controls. At

At necropsy, the findings in dams were unremarkable. The number of pups per litter, the sex ratio, and viability of both the embryo and the fetus were not affected by the dietary administration of scFOS. Litter and fetal weights were not significantly different except that the fetal weight of the 20% group was significantly greater than that of the controls. Structural development of the fetuses was unremarkable. The only treatment-related effect was the slightly lowered body weights of the dams, seen only in the 20% scFOS group. It was concluded that scFOS at dietary concentrations up to 20% does not cause adverse effects or adversely affect the pregnancy outcome or in utero development of the rat.

Conclusions:

It was concluded that scFOS at dietary concentrations up to 20% (equivalent to approximately 10,000 mg/kg body weight/day (U.S. FDA, 1993)) does not cause adverse effects or adversely affect the pregnancy outcome or in utero development of the rat.

Executive summary:

Sleet and Brightwell (1990; presented in Carabin and Flamm 1999) also evaluated possible maternal and developmental toxicity of scFOS using Sprague Dawley rats (strain Crl CD (SD) BR). Four groups of 24 to 27 pregnant females were administered scFOS from day 0 to 6 post coitum at 4.75% dietary concentration. On post-coital day 6, the dietary concentration of scFOS in the 4 groups was changed to 0, 5%, 10%, or 20%. On day 15, all of the animals were placed on an scFOS-free diet. A fifth group received an scFOS-free diet throughout the entire study. On day 20, the rats were sacrificed and their litters examined. About half of the fetuses were dissected and examined fresh prior to skeletal staining. No treatment-related effects were observed during days 0 to 6 or during days 6 to 15. There was no mortality and no diarrhea was observed in any of the test animals. scFOS administered at 4.75% dietary concentration during days 0 to 6 did not affect body weight and body weight change in any of the groups, but, beginning on day 8, body weight was reduced dose-dependently in all 3 scFOS groups relative to controls. The 5% group exhibited a lower weight gain than the controls while the 10 and 20% groups actually lost weight. From day 11 to the end of the study, the body weight and body weight change were similar in all groups except the 20% scFOS group, which remained below controls.

At necropsy, the findings in dams were unremarkable. The number of pups per litter, the sex ratio, and viability of both the embryo and the fetus were not affected by the dietary administration of scFOS. Litter and fetal weights were not significantly different except that the fetal weight of the 20% group was significantly greater than that of the controls. Structural development of the fetuses was unremarkable. The only treatment-related effect was the slightly lowered body weights of the dams, seen only in the 20% scFOS group. It was concluded that scFOS at dietary concentrations up to 20% does not cause adverse effects or adversely affect the pregnancy outcome or in utero development of the rat.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

From "Determination of the GRAS Status of the Addition of Short-Chain Fructooligosaccharides to Infant Formula as a Nutritional Supplement", FDA, july 2014

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Species:

rat

Strain:

Wistar

Sex:

female

Route of administration:

oral: feed

Vehicle:

other: in diet

Dose / conc.:

20 other: %

Remarks:

20% in diet

No. of animals per sex per dose:

Twelve female Wistar rats

The scFOS had no effect on the number of pregnancies but produced a reduction in body weight gain of the pregnant rats. This may be due to a lower caloric value for scFOS, decreased intake of food for this group, or diarrhea observed in the first week and softer stools in the second and third weeks.

the fetus and newborn weights were not affected. However, during the nursing period, a growth delay was observed for the male pups in the test group. This may be indicative of the restricted nutritional status of the lactating mothers. The study concluded that a diet containing 20% scFOS has no significant effects on the course of pregnancy in rats and on the development of their fetuses and newborns.

Conclusions:

The study concluded that a diet containing 20% scFOS ((equivalent to approximately 10,000 mg/kg body weight/day (U.S. FDA, 1993)) has no significant effects on the course of pregnancy in rats and on the development of their fetuses and newborns.

Executive summary:

Henquin (1988; presented in Carabin and Flamm 1999) demonstrated the lack of developmental toxicity of scFOS fed to rats at a 20% dietary concentration. Twelve female Wistar rats with a copulation plug were fed a diet containing 20% scFOS from day 1 to day 21 of gestation while 17 female Wistar rats with a copulation plug were fed a control diet. In the first 6 hours after birth, the litters were numbered, sexed, and weighed. Thirty-six hours after delivery, the newborns were equally distributed (9 neonates/mother) among the lactating mothers, which continued on their gestational diets.

The scFOS had no effect on the number of pregnancies but produced a reduction in body weight gain of the pregnant rats. This may be due to a lower caloric value for scFOS, decreased intake of food for this group, or diarrhea observed in the first week and softer stools in the second and third weeks. Despite the reduction in body weight gain of the pregnant rats, the fetus and newborn weights were not affected. However, during the nursing period, a growth delay was observed for the male pups in the test group. This may be indicative of the restricted nutritional status of the lactating mothers. The study concluded that a diet containing 20% scFOS has no significant effects on the course of pregnancy in rats and on the development of their fetuses and newborns.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 160 mg/kg bw/day

Species:

rat

Effects on developmental toxicity

Description of key information

The potential toxic effects of ScFOS were investigated in a GLP-compliant Reproduction/Developmental Toxicity Screening Test performed in accordance with OECD Guideline No. 421. The objectives of this study were to determine the potential toxic effects of Short Chain Fructo-oligosaccharides when given via diet for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were determined.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Short Chain Fructooligosaccharides were established: Parental, Reproduction and Developmental NOAEL: at least 1160 mg/kg bw/day pm (corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively).

In addition, GRAS Notice for scFOS from the Office of Food Additive Safety (FDA) report from an unpublished study conducted by Henquin (1988) that dietary administration of scFOS at concentrations up to 20% [equivalent to approximately 10,000 mg/kg body weight/day (U.S. FDA, 1993)] did not result in developmental toxicity. GRAS Notice also report from an unpublished study conducted by Sleet and Brightwell (1990) evaluating the maternal and developmental toxicity of ScFOS, that dietary concentrations up to 20% [equivalent to approximately 10,000 mg/kg body weight/day (U.S. FDA, 1993)] provided to rats during postcoitum days 0 to 15 did not result in treatment related adverse effects (e.g. diarrhea), or differences in pregnancy outcome or in utero development. 

These two studies are summarized in Carabin and Flamm, 1999.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 160 mg/kg bw/day

Species:

rat

Justification for classification or non-classification

Based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Short Chain Fructooligosaccharides were established: Parental, Reproduction and Developmental NOAEL: at least 1160 mg/kg bw/day (corresponding to an average test material intake of 1087 and 1233 mg/kg bw/day for males and females, respectively). 

Therefore, the ScFOS does not meet the CLP criteria for classification.