Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Reference 2

Reference Type:

publication

Title:

MULTIGENERATION REPRODUCTION STUDY OF ISOMALT IN RATS

Author:

Waalkens-Berendsen, D.H. et al.

Year:

1990

Bibliographic source:

Fd Chem. Toxic. 28(1):11-19

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats of both sexes were fed with diets containing 2.5, 5 or 10% test item or diets containing 10% maize starch (control group) throughout 3 successive generations. An additional group received diet containing 10% sucrose. For each generation, two litters were reared until they were at least 3 weeks old. From the second litter of the F3 generation, 10 animals were used for autospy and histopathology at the age of 4 weeks.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

EC Number:

908-700-6

Cas Number:

64519-82-0

Molecular formula:

6-O-alpha-D-Glucopyranosyl-D-sorbitol: C12H24O11 1-O-alpha-D-Glucopyranosyl-D-mannitol: C12H24O11

IUPAC Name:

Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Cpb:WU (Wistar Random)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: newly weaned rats, not further specified
- Housing: 5 animals of the same sex per cage in suspended screen-bottomed stainless steel cages (premating periods); one female/one male per cage during mating, singly or with litter during gestation and lactation
- Diet: Institute´s powdered cereal based open-formula diet supplemented with 2.5% casein, 0.1% D,L-methionine and 10% maize starch (control groups) addition of 10% maize starch allowed the incorporation of the test and control substance at·the expense of starch, ad libitum
- Water: ad libitum
- Acclimation period: 9 - 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): approx. 50
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: diets were freshly prepared once every week
- Mixing appropriate amounts with: stock diet; the materials were thoroughly mixed into the carrier by means of a mechanical blender (Lodige) at levels of 0 (control), 2.5, 5 or 10% test item or 10% sucrose.

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights, food consumption and nominal dietary test item concentration at the end of prepairing period. Test item intake was calculated separately for all three parental generations and the lowest test item intake for each dietary concentration was selected as the achieved dose. The dietary concentration of 2.5% resulted in test item intakes of at least 1140 and 1400 mg/kg bw/day in males and females, respectively. Dietary concentrations of 5% resulted in test item intakes of at least 2350 and 2900 mg/kg bw/day in males and females, respectively. The dietary concentration of 10% resulted in test item intakes of at least 4600 and 5900 mg/kg bw/day in males and females, respectively. A table detailing the calculation of test item intake is attached to this endpoint study record.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 3 weeks
- After 1 week, the first male was replaced by another male so that a mximum of three different males were available for each female.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneous distribution of test item and sucrose in the diets were determined by analysing five samples taken at five different locations in the food container from one batch of each of the diets after one day and after 14 days of storage at room temperature. From each freshly prepared batch a sample was taken and stored in a freezer at -20 °C. In samples of the batches produced on day 75, 182, 271, 361, 439, 475 and 550 the content of test item and sucrose was determined by High Performance Liquid Chromatography (HPLC).

The results of the determinations in the first batch of the diets made, show that the actual dietary levels of test item and sucrose were close to the intended levels, indicating proper preparation of the diets (Table 1). The analysis of five samples taken from each diet immediately after preparation showed a coefficient of variation which is generally less than 5%. This is indicative of satisfactory homogeneous distribution of the test and control item in the diets. The figures obtained after 14 days of storage at room temperature show that the test item and sucrose are stable under the conditions of storage.

Results of the determinations of test item and sucrose in the diets during the course of the experiment are presented in Table 2. The figures generally showed some deviation from the intended levels. This is attributed to the analytical procedure applied rather than to insufficient mixing or to adding an incorrect amount of test item or sucrose to the diets. In the diets without any sucrose added an average amount of about 1% sucrose was found. The origin of this amount of sucrose is not known.

Duration of treatment / exposure:

(P) Males and Females: 12 and 21 weeks before the 1st and 2nd mating, respectively and throughout the study
(F1/P1): Selected rats were kept on the control or test diet of their parents for 12 and 21 weeks before the 1st and 2nd mating, respectively, and throughout the study.
(F2/P2): Selected rats were kept on the control or test diet of their parents for 12 and 21 weeks before the 1st and 2nd mating, respectively, and throughout the study.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- P1 parental animals not mated until 12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were at weaning age.
- Age at mating of the mated animals in the study: first mating: 15 weeks, second mating: 24 weeks

- P2 parental animals not mated until 12 weeks after selected from the F2 litters.
- Selection of parents from F2 generation when pups were at weaning age
- Age at mating of the mated animals in the study: first mating: 15 weeks, second mating: 24 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.5 other: %

Remarks:

25 000 ppm (at least 1140 and 1400 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data at the end of the premating period)

Doses / concentrations 2

Dose / conc.:

5 other: %

Remarks:

50 000 ppm (at least 2350 and 2900 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data at the end of the premating period)

Doses / concentrations 3

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (at least 4600 and 5900 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data at the end of the premating period)

No. of animals per sex per dose:

P generation: 100 males and females (20 males and females were used to produce F1 litters)
P1 generation: 20 males and females
P2 generation: 20 males and females
F3: 10 males and females

Control animals:

yes

other:

Details on study design:

- Other:
Study design:
The P0 generation consisted of 100 males and females per group. After 12 weeks on the test and control diets, the first mating occurred. Pups were weaned at day 21 and 50 males and females were used for a 8 week feeding study. The remaining pups were discarded at 3 – 5 weeks of age. The mating procedure was repeated 9 weeks after the first mating by using the same combination of mating partners. From this F1/P1 generation, 20 males and females were used to produce the F2/P2 generation. For this, the selected rats were weaned at day 21 and kept on the diets of their parents for further 12 weeks before mating. The offspring of the first mating was discarded and 20 females and males of the second mating were used for the production of the F3 generation. As with the F1-generation, the selected F2/P2 generation rats were fed with the various diets for 12 weeks after weaning and subsequently mated. After 21 weeks of exposure, the second mating procedure started. Pups from the first F3 offsping were discarded 3 weeks after birth. Weanlings from the second mating procedure were used for gross and microscopic evaluation after receiving the diets for 4 weeks (10/sex).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during the premating and mating periods. No weighing was performed during gestation and lactation periods.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/week: Yes, during premating periods
- For each generation, food consumption was recorded weekly during the premating period of 12 weeks. Food intake was not measured during the mating and lactation periods.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 1 post partum: yes
- Maximum of 8 pups/litter.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, other: viability index at birth and day 4, litter size
F3: haemoglobin level

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed after weaning of their second litter.
- Maternal animals: All surviving animals were killed after weaning of their second litter.

GROSS NECROPSY
- Gross necropsy consisted of: not specified
- The uteri of females were stained with ammonium sulphide for implantation sites.

Postmortem examinations (offspring):

SACRIFICE
- The F1 and F2 offspring not selected as parental animals were sacrificed when they were 3 weeks old.
- The F3 offspring of the first mating was sacrificed at the age of 3 weeks whereas 10 weanlings of the second mating were sacrificed 4 weeks after weaning (males: 28 days after weaning, females: 29 days after weaning) for detailed gross and microscopic examinations.

GROSS NECROPSY
- The following organs were weighed in F3 offspring: adrenals, brain, caecum, heart, kidneys, liver, ovaries, spleen, testes, thymus and thyroid

HISTOPATHOLOGY / ORGAN WEIGTHS
Tissues of all the major systems from the F3 generation including kidneys, liver, lungs, heart, stomach, epididymides, prostate, urinary bladder, pituitary, thyroid were examined microscopically.

Statistics:

Mean values and standard errors were calculated from the examined parameters. Statistical analyses were performed using ANOVA and Dunnett´s test (changes in body weights, food intake, haemoglobin levels and relative organ weights), Student´s t test (mean litter size at birth and pup weight) or chi-square test (viability and lactation indices).

Reproductive indices:

The following parameter were determined in the P, P1 and P2 generation for the first and second mating:
1. fertility index (%): (no. of pregnant females / no. of mated females) x 100
2. gestation index (%): (no. of females with live fetuses / no. of pregnant females) x 100
3. lactation index: (no. of live pups at day 21 / no. of live pups at day 4)

Offspring viability indices:

viability index on day 1 (%): (no. of pups born alive / total no. of born pups) x 100
viability index on day 4 (%) : (no. of live pups at day 4 / total no. of live pups on day 1) x 100%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormalities of condition or behaviour were observed in any of the groups during the pre-mating and mating period.
Several pregnant P0-females had difficulties in delivering their first litter and to a lesser extent also in delivering their second litter. This abnormality was observed in all groups; a specific test item effect was not apparent. The abnormalties included uncapability to delivery a complete litter, leaving one or more unborn pups in utero. As a result of this abnormality 20 out of 100 females died or had to be killed during this period. There was no dose-response in the incidence of these mortalities. The highest number of females (n = 7) which died/had to be killed was noted in the sucrose group.
In an attempt to determine the cause of these abnormalities, it was noted that the clock for regulating the light/dark cycle of 12 hours was out of order. As a result, the rats had been exposed to light continuously for several days during pregnancy. Since no other explanation could be found the continuous lighting was probably responsible for the irregularities observed. This is supported by the fact that no abnormalities in delivering their litter were observed in pregnant females of the next generations when the clock had been repaired.

Two P0 males died or had to be killed in extremis; one at 2.5 and one at 10% test item, respectively. In the absence of a dose-response these isolated mortalities were considered non-treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

As a result of delivery problems 20 out of 100 females died or had to be killed during this period. There was no dose-response in the incidence of these mortalities. The highest number of females (n = 7) which died/had to be killed was noted in the sucrose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

females: no effects
males: minimally decreased body weight at 10% test item (day 172: 426.5 g vs. 437.4 g in the control)

Since the differences a) were only small; b) did not occur in both sexes simultaneously; c) were not consistent in various generations, it is questionable whether the test item really affected growth rate at the dose levels tested.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The fertility index of parental dams was low in all groups, especially after the first mating, probably as a result of the failure of the automatic light control. No dose-dependency was noted; the lowest fertility was noted in the sucrose group both after the first and second mating.

In the absence of a dose dependency and because the effect was not reproducible in the P1 and P2 matings, the low fertiliy indices were considered non-treatment-related.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general and reproductive toxicity

Effect level:

>= 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

general and reproductive toxicity

Effect level:

>= 5 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

P1 generation: one female of the 2.5% group died/was killed in extremis. No further mortalities occurred.
P2 generation: no mortalities occurred

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

P1 generation
females: transiently slightly reduced body weight at 5% and 10% test item, occasionally increased body weight at 5% test item
males: minimally decreased body weight at 10% test item, trend towards decreased body weight at 5%

P2 generation
females: no effects
males: occasionally increased body weight at 5% test item and 10% sucrose

Since the differences a) were only small; b) did not occur in both sexes simultaneously; c) were not always dose-related; d) were not consistent in various generations and e) were sometimes present already at the time of weaning (day 0), it is questionable whether the test item really affected growth rate at the dose levels tested.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

P1 and P2 generation: caecal enlargement was observed at 10% test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general and reproductive toxicity

Effect level:

>= 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

general and reproductive toxicity

Effect level:

>= 5 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

After the first- and, to a lesser extent - also after the second mating of the P0 parents, the survival of pups during lactation was lower - and hence pup mortality was higher - than in breedings of the next generations parent rats. Since the phenomenon occurred in all groups, the controls included, the relatively low pup survival is not related to the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Congenital defects:
For one F1 pup at 10% sucrose anophthalmia of one eye was noted.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 5 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F2 generation: there was a statistically significant reduction in the number of pups per litter at 5% test item, but only after the first mating. Since the effect did not occur in the high dose and was not found in other generations, the decrease in mean litter size is considered an incidental finding.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

F3 offspring: Haemoglobin levels, determined at the end of the four-week feeding period were similar is all groups.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a considerable and statistically significant increase in the relative weight of both the filled and empty caecum in the group fed 10% test item, both in males and females (non-adverse). Also at the 5 % feeding level caecum weight tended to be increased, but the differences with the controls reached the level of statistical significance only for the filled caecum weight of males.

Caecal enlargement is known to be induced by a variety of substances which are not fully digestible, such as dietary fiber, raw potato starch, modified starches, lactose, pectins, alginates, etc., it is considered to be an adaptive rather than a pathological change. In the present study, the enlargement is most likely due to the presence in the large bowel of non-digested test item, which was not digested in the small intestines and is fermented in the large intestines by the gut flora.

Male F3 pups of the sucrose group showed a statistically significant increase in relative kidney weight. This increase was not accompanied by histopathological changes and therefore considered non-adverse.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

F3 offspring: incidence and degree of the histopathological changes were similar in all groups

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Congenital defects:
F2 offspring: one pup of the sucrose group had a thick and paralysed hind leg
F3 offspring: one pup of the sucrose groups had a missing foot, which was most probably amputated shortly after birth; one pup at 5% test item with a hydrocephalus, one pup at 10% test item with anophthalmia of one eye

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

2

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 5 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

3

Key result

Dose descriptor:

NOAEL

Generation:

other: F3

Effect level:

>= 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

4

Key result

Dose descriptor:

NOAEL

Generation:

other: F3

Effect level:

>= 5 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Analytical results of test substance concentration in diet (first preparation)

Nominal concentration (%)	Actual concentration (%) Dietary admix prepared 06 Dec 1979
	mean ± SD (n = 5)	after storage (14 days)
0.0	not detectable (< 0.1%)	not detectable (< 0.1%)
2.5	2.38 ± 0.13	2.3
5.0	5.06 ± 0.09	5.02
10.0	10.40 ± 0.23	10.5

 

 

Table 2. Analytical results of test substance concentration in diet (subsequent preparations)

Sampling date	Nominal test item concentration (%)
	0	2.5	5.0	10.0
	Actual test item concentration (%)
19 Feb 1980	0	2.5	5.4	9.2
06 Jun 1980	0	3.0	5.7	10.2
03 Sep 1980	0	2.4	6.0	10.3
02 Dec 1980	0	1.9	4.6	8.6
19 Feb 1981	0	2.5	5.3	11.8
26 Mar 1981	0	1.8	4.0	8.4
09 Jun 1981	0	208	3.0	8.7
Mean	0	2.4	4.9	9.6

 

Applicant's summary and conclusion

Conclusions:

In the conducted study, exposure to up to 10% of the test item in diet of 3 successive generations did not induce adverse effects on fertility, reproductive performance or development compared with control animals fed with 10% maize starch. Therefore, the test item is considered not to induce toxic effects on reproduction.