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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Multigeneration study (no guideline followed), rats:

NOAEL general toxicity = 100 000 ppm (equivalent to ca. 4600 and 5900 mg/kg bw/day in parental males and females, respectively)

NOAEL reproductive toxicity = 100 000 ppm (equivalent to ca. 4600 and 5900 mg/kg bw/day in parental males and females, respectively)

NOAEL Offspring = 100 000 ppm (equivalent to ca. 4600 and 5900 mg/kg bw/day in parental males and females, respectively)

Link to relevant study records

Reference

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

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

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

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

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)

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)

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).

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%

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: 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.

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

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

Critical effects observed:

no

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

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

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

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

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

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

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

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

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

 

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4 600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In accordance with Regulation (EC) No 1907/2006, Annex VII, Section 8.7.1 Column 1, no screening study for reproductive/developmental toxicity reproductive study (OECD 421 or 422) is required because pre-natal developmental toxicity studies (Annex IX, 8.7.2) and a multigeneration reproductive toxicity study (Annex IX, Section 8.7.3) are available for

Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol (CAS 64519-82-0).

A multigeneration study in rats was performed with dietary administration of Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol. No guideline was followed but the study was performed according to GLP (Sinkeldam, 1982). The test item was fed to rats of three successive generations at concentrations of 2.5, 5.0 and 10.0% in the diet. A group of rats fed a diet containing 10% sucrose served as an additional control group. Resulting test item intake was retrospectively calculated based on mean body weights and food consumption at the end of pre-pairing period. 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. 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 initial mating was of 100 rats of each sex in each group. For subsequent matings 20 rats of each sex from each group were used. For each generation two litters were reared until they were at least 3 weeks old. Clinical signs, body weights, food consumption and fertility indices were determined for parental animals of all generations. Litter size, general pup condition, litter weights, sex ratio at birth, growth rate and mortality of pups were also recorded. The second litter of third generation rats (F3b) was given detailed gross and microscopic examinations 4 weeks after weaning.

Feeding the test item to rats for three successive generations did not induce any adverse effects on fertility, reproductive performance or development compared with control animals fed diets containing maize starch and sucrose instead of the test item. For the F3 offspring, a marked treatment-related change was an increase in the relative weight of the caecum (filled and empty), in the group fed 10% test item. There was also an increase in the relative weight of the filled caecum in males of the 5% test item group. These findings were not accompanied by diarrhoea or histological changes in the caecum. In the absence of any adverse effects, the observed NOAEL for general and reproductive toxicity as well as for offspring development in this study was 4600 and 5900 mg/kg bw/day for males and females respectively.

 

In conclusion, the data on reproductive toxicity of Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol do not indicate adverse effects of the substance on male or female reproductive function, fertility or development of the offspring.

Effects on developmental toxicity

Description of key information

Developmental toxicity study (equivalent or similar to OECD 414), rats:

NOAEL maternal animals: 100 000 ppm, corresponding to ca. 7970 mg/kg bw/day (highest dose tested)

NOAEL fetuses: 100 000 ppm, corresponding to ca. 7970 mg/kg bw/day (highest dose tested)

NOAEL fetal abnormalities: 100 000 ppm, corresponding to ca. 7970 mg/kg bw/day (highest dose tested)

Developmental toxicity study (equivalent or similar to OECD 414), rabbits:

NOAEL maternal animals: 100 000 ppm, corresponding to ca. 4800 mg/kg bw/day (highest dose tested)

NOAEL fetuses: 100 000 ppm, corresponding to ca. 4800 mg/kg bw/day (highest dose tested)

NOAEL fetal abnormalities: 100 000 ppm, corresponding to ca. 4800 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2001

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

Cpb:WU (Wistar, random)

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
- Weight at study initiation: 153 to 204 g
- Fasting period before study: no
- Housing: in supsended steel cages fitted with wire-mesh floors (4 females/cage during acclimatization, individually during pregnancy)
- Diet: stock diet for rats, modified with casein, d1-methionine and 10% maize food starch. The modification allowed the incorporation of up to 10% test item at the expense of maize starch (see Table 1). Food was offered ad libitum, to the control and all test item groups. A restricted feeding control group received 80% of the amount consumed by the control group animals from day 1 onwards.
- Water: source not specified (ad libitum)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): ≥ 40
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Oct 1981 To: 25 Nov 1981

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): not specified
- Storage temperature of food: not specified

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights and food consumption at the end of the period of organogenesis. The calculation of test item intake is detailed in Table 3.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual levels of test item in the diet were analysed once during the study and were found to equal or close to the target concentrations (see Table 2).

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation day 0 to 21

Frequency of treatment:

daily, 7 days/week

Duration of test:

throughout pregnancy (day 0 to 21)

Dose / conc.:

2.5 other: %

Remarks:

corresponding to 25 000 ppm (ca. 1970 mg/kg bw/day, calculated from mean bw and food consumption)

Dose / conc.:

5 other: %

Remarks:

corresponding to 50 000 ppm (ca. 4120 mg/kg bw/day, calculated from mean bw and food consumption)

Dose / conc.:

10 other: %

Remarks:

corresponding to 100 000 ppm (ca. 7970 mg/kg bw/day, calculated from mean bw and food consumption)

No. of animals per sex per dose:

22 - 23 females/dose

Control animals:

yes

yes, plain diet

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment: Mated fermales were assigned to each group in rotation. The sequence was continued from where it ended on the previous day until each group contained the desired number of mated females.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: general condition and behaviour were checked

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 21 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Ovaries and empty uteri as well as placentas of live fetuses were weighed

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Both soft tissue and skeletal examinations were carried out on the foetuses of the control and high dose group as well as of foetuses of the restricetd feeding group.

Statistics:

For the statistical analyses of differences in degree of ossification between the test and control groups the Student t-test was applied to transformed ossification values (DgOt) expressed in degrees and calculated by the formula:
DgOt = arcsine √DgO°

Statistical analysis of differences in body weight, food consumption, organ weights, litter data, foetus weights and lengths and placenta weights was carried out by applying analysis of co-variance, followed by Dunnett's multiple comparison tes, whereas skeletal and visceral anomalies were evaluated by the Chi-square test.

Indices:

Percentage pre-implantation loss (PRIL) was calculatde for each litter by the formula:
PRIL = number of corpora lutea - number of implantation sites / number of corpora lutea x 100%

Percentage post-implantation loss (POIL) was calculated for each litter by the formula:
POIL = number of implantation sites - number of live young / number of implantation sites x 100%

The degree of ossification of foetus skeletons (DgO) was calculated for each litter by the formula:
DgO = number of bones without ossification (or with incomplete ossification) / numbers of bones examined

Historical control data:

Historical control data on reproduction data and on fetal malformations and variations (external, skeletal and visceral) are provided in the report.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Some females in each of the groups showed focal baldness. No signs of ill-health nor any reaction to the treatment were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights and weight gain during pregnancy were similar for both the controls and all test item-fed females. The body weights of pregnant females which had access only to restricted amounts of food stayed significantly behind as compared with the controls. During the full gestation period these animals gained only 60% of the mean weight gain of control animals.

Corrected body weight gains were similar in all groups with diet offered ad libitum. In the restricted feeding group corrected body weight gain was only 16% of that of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food intake was similar in all test item groups and the controls.
Since, the animals of the restricted-feeding group were provided daily with only 80% of the food consumed by the controls during the preceeding day, these animals consumed practically all the food offered. However, since the amounts of food were calculated on the basis of body weights, the actual food intake per animal in this group appeared to be even less than 80% of the food consumed by controls.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no consistent or dose-related differences in food efficiency between the test item groups and the controls.
The grand means of the restricted-feeding group were lower than the control value.

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:

no effects observed

Description (incidence and severity):

Gravid and empty uterus weights were significantly decreased in the restricted feeding group only.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At autopsy a minor degree of haemorrhagic nasal discharge was noticed in one dam of the 5.0% test item and in two dams of the restricted-feeding group. One female of the 10.0% test item group showed diarrhoea and one control animal exhibited a periocular haemorrhage. Focal baldness was observed in several dams in all groups.
No gross maternal organ changes were seen in in dams of any of the groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of early and late resorptions were similar in the test item groups and the control. A slight increase of the number of resorptions and through that of post-implantation loss was observed in the restricted­ feeding group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The feeding of restricted amounts of stock diet to pregnant females resulted in decreased maternal weight gain and consequently, in lower uterus weights. The effects on foetuses were principally of an embryotoxic nature as expressed by increased numbers of resorptions, smaller foetuses, decreased foetus weights and retardation in bone development. It is therefore concluded that restriction of the diet of pregnant rats during gestation induces embryotoxic effects.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7 970 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Foetal weights as well as foetal rump lengths were significantly decreased in the restricted-feeding group only.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Spina bifida was observed in 3 foetuses of one litter in the high dose group. Further, one foetus of the high dose group and one foetus of the restricted-feeding group showed a ringtail. No other externally visible malformations were observed.

External variants were seen in several foetuses; subcutaneous haemorrhages or petechia occurred in one or a few foetuses in all groups, while one dismature foetus was observed in the high dose group. None of these findings were considered to be related to the treatment.

Although spina bifida occurred in 3 foetuses of the top-dose group only, the total number of foetuses with skeletal or visceral malformations in this group was similar to the number of affected control foetuses. In addition, the percentage of litters with one or more foetuses showing malformations was even higher in the control (4 out of 17) than in the high dose group (3 out of 19). Therefore, and since all 3 spina bifida foetuses originated from the same litter, no significance is attached to these findings.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were observed in 5 foetuses: in 2 foetuses from the control, in 2 from the high dose, and in 1 foetus from the restricted­ feeding group. Neither the type nor the incidence of the observed malformations indicated any treatment-relationship.

Minor skeletal anomalies and skeletal variants were observed in several foetuses in all groups or occasionally in one or a few foetuses.
The number of foetuses showing dislocation of one or several sternebrae was increased in both the high dose and the restricted-feeding group. Further, the incidences of supernumerary ribs and skull bones was increased in the restricted-feeding group only.

A slightly higher number of incompletely ossified cervical vertebral bodies in the high dose group was considered an incidental finding and reflects the variation in ossification normally found in skeletons of foetuses of the strain of rats used. For the remaining data on ossification degrees no differences were observed between the control and high dose group.
In the restricted-feeding group however decreased degrees of ossification were observed in several bones such as metacarpals, metatarsals and cervical thoracic vertebral bodies.

The somewhat higher number of dislocated sternebrae in the high dose group as compared with the control group, is not considered of significance since, firstly no other indications were noticed that might point to a skeletal effect of the test substance and secondly, the observed control values were rather low in comparison with those of historical control series.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in 7 foetuses: the control group revealed 2 foetuses with microphthalmia and 1 foetus with anophthalmia. They originated all three from different litters. In the high dose group spina bifida was observed in 2 foetuses from one litter. One of these foetuses also showed microdactyly of a hindlimb. Further, a double kidney was observed in one foetus from a different litter. The restricted-feeding group revealed only one foetus with visceral malformations consisting of right-sided testicular cryptorchism and left-sided testis atrophy.

The other observations listed,were either minor anomalies or visceral variants.

Neither meningial haemorrhages nor dilated brain ventricles were observed in the control group. However, since these anomalies are regularly being observed in our control series, these findings were considered to reflect the variation normally found, rather than to be related to the treatment. The other minor anomalies were about equally spread among the high dose, restricted-feeding and the control animals or occurred occasionally in one or a few foetuses.
No differences were noted in the nature of the observed visceral variants. Although the number of foetuses with unilaterally increased renal pelvic cavitation was slightly higher in the high dose group as compared with controls, the observed incidence is fully comparable with values observed in historical control series. Moreover, there were no other indications of a treatment-related soft tissue effect. Therefore, no significance was attached to the somewhat higher incidence of minor kidney variants.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7 970 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Key result

Developmental effects observed:

no

Table 2. Analytical results of test substance concentration in diet

Nominal concentration (%)	Actual concentration (%)
2.5	2.5
5.0	5.0
10.0	9.6

Table 3. Achieved test item intake

	Nominal concentration of test item in diet (%)
	0.0	2.5	5.0	10.0
Body weights (g, mean bw at end of organogenesis, day 16)	238.4	241.7	240.1	240.9
Food consumption (g/food/animal/day, mean food consumption at end of organogenesis, day 16)	18.5	19.0	19.8	19.2
Food consumption (g/food/kg bw/day, mean of means)	77.6	78.6	82.5	79.7
Test item intake (g/kg bw/day)	0.0	2.0	4.1	8.0
Test item intake (mg/kg bw/day)	0.0	1965.2	4123.3	7970.1

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Nov 1982 - 15 March 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

not details on diet preparation given

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2001

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ENKI-Konijnenfarm, Someren, The Netherlands
- Age at study initiation: approximately 8 months
- Weight at study initiation: 2920 to 4180 g
- Fasting period before study: no
- Housing: single housing in supsended steel cages fitted with wire-mesh floors and fronts
- Diet: modified standard laboratory rabbit diet (10% maize starch added), allowing incorporation of up to 10% test item at the expense of maize starch (see Table 1) (ad libitum)
- Water: unflouridated tap water (ad libitum)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 ± 3
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 Nov 1982 To: 15 Mar 1983

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): not specified
- Storage temperature of food: not specified
- Homogeneous distribution of the test substance in the diet was achieved by mixing the components in a Lödige mechanical blender.

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights on gestation day 19 (end of organogenesis) and food consumption from gestation days 6 - 19 (period of organogenesis). The calculation of test item intake is detailed in Table 3.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual levels of test item in the diet were analysed twice during the study. In the 2.5% group, the actual levels of the test item in the diets were close to the intended level, but in the 5.0 and 10.0% group, the levels determined were slightly lower than the intended levels. This deviation is attributed to the analytical procedure applied, rather than to insufficient mixing or adding an incorrect amount of Palatinit to the diet (see Table 2).

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Ovulation was induced by intravenous injection of luteinizing hormone (Pregnyl, Organon, Oss, The Netherlands, 1 mL of a solution in saline containing 50 IU/mL). Subsequently, immediately after injection of Pregnyl, the females were artificially inseminated with about 40 million spermatozoa, freshly obtained from dummy copulations of male New Zealand White rabbits of proven fertility. The day of insemination was considered as day 0 of pregnancy.

- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: none, insemination referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation day 0 to 28

Frequency of treatment:

daily, 7 days/week

Duration of test:

throughout pregnancy (day 0 to 29)

Dose / conc.:

2.5 other: %

Remarks:

corresponding to 25 000 ppm (ca. 1100 mg/kg bw/day, calculated from mean body weights and food consumption on day 19)

Dose / conc.:

5 other: %

Remarks:

corresponding to 50 000 ppm (ca. 2200 mg/kg bw/day, calculated from mean body weights and food consumption on day 19)

Dose / conc.:

10 other: %

Remarks:

corresponding to 100 000 ppm (ca. 4800 mg/kg bw/day, calculated from mean body weights and food consumption on day 19)

No. of animals per sex per dose:

36 - 37 females/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: doses were based on on the results of an earlier, pilot oral embryotoxicity/ teratogenicity study in New Zealand White rabbits
- Rationale for animal assignment: inseminated fermales were assigned to each group in rotation. The sequence was continued from where it ended on the previous day until each group contained the desired number (22/group) of mated females. When, at autopsy, it turned out that the number of pregnancies was below the expected number in each of the groups, the insemination procedure was resumed for another 14- 15 animals/group in study Part II. Again the inseminated females were assigned to each group in rotation.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: general condition and behaviour were checked

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 19 and 29 of pregnancy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Ovaries and empty uteri as well as placentas of live fetuses were weighed

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter with intact skeleton, half per litter after decapitation
- Head examinations: Yes: half per litter

Both soft tissue and skeletal examinations were carried out on all foetuses of the control and high dose group.

Statistics:

For the statistical analyses of differences in degree of ossification between the test and control groups the Student t-test was applied to transformed ossification values (DgOt) expressed in degrees and calculated by the formula:
DgOt = arcsine √DgO°

Statistical analysis of differences in body weight, food consumption, organ weights, litter data, foetus weights and placenta weights was carried out by applying analysis of co-variance, followed by Dunnett's multiple comparison tes, whereas skeletal and visceral anomalies were evaluated by the Chi-square test.

Indices:

Percentage pre-implantation loss (PRIL) was calculatde for each litter by the formula:
PRIL = number of corpora lutea - number of implantation sites / number of corpora lutea x 100 %

Percentage post-implantation loss (POIL) was calculated for each litter by the formula:
POIL = number of implantation sites - number of live young / number of implantation sites x 100 %

The degree of ossification of foetus skeletons (DgO) was calculated for each litter by the formula:
DgO = number of bones without ossification (or with incomplete ossification) / numbers of bones examined

Historical control data:

Historical control data on reproduction data and on fetal malformations and variations (external, skeletal and visceral) are provided in the report.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One control animal suffered from rhinitis as was indicated by mucopurulent nasal discharge.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The control animal suffering from rhinitis died on day 17 of gestation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Some variation in body weights and weight gain was observed during the different periods, but there were no consistent or significant group differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake and food efficiency figures showed some variation, but no significant group differences were noted.

Food efficiency:

no effects observed

Description (incidence and severity):

Apart from a somewhat lower food efficiency in all test item groups during the period of histogenesis (day 19-29) no substantial differences between groups were observed. This is considered a coincidental finding since no consistent or significant group differences were observed either in body weight gain or in food intake figures throughout the study

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 specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At autopsy, some minor gross changes of the genital tract such as ovary cysts, focal oedema or small focal haemorrhages were observed in one or a few animals in all groups. Minor symptoms of scabies were found in one rabbit of the mid and in one of the high dose group, while focal baldness was observed in another two animals of the same groups. Rhinitis, as was indicated by mucopurulent nasal discharge was found in one control and one low dose animal.
No gross changes were observed that could be attributed to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Abortion occurred in three animals: one control animal on day 24, one low dose animal on day 20, and one high dose animal aborted on day 16 of pregnancy. All three animals were killed after abortion but no relevant findings were observed at autopsy.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Post-implantation loss, comprising of both early and late resorptions plus foetal deaths, was calculated for each litter as a percentage of the total number of implants, the litters revealing only resorptions (two low dose litters) or dead foetuses (one high dose litter) were counted as 100%. Through that, mean post­implantation loss values were higher in both the low and high dose groups, where total litter losses were observed in two, respectively one litter.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled kill, two animals of the low dose group revealed only embryonic resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of early and late resorptions were similar in all groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead foetuses was slightly higher in the high dose group. This somewhat higher number was mainly caused by the total litter loss (4 dead foetuses) of a single animal.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 800 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Foetal weights as well as foetal rump lengths were simimlar in all groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One severely malformed foetus revealing an umbilical hernia and bilateral exophthalmia was observed in the low dose group. Acauda occurred in one foetus of the high dose group. No other externally visible malformations were observed.

Externally visible variants were seen in several foetuses and consisted mostly of a dysmature appearance. Further, one low dose foetus showed a small focal hypertrophy of the mandible. Although no macroscopically visible alterations were observed in foetuses of the control group, none of the observed findings were considered to be related to treatment, since there was no dose-response relationship. Moreover, the observed alterations are usually seen in the same frequency in other control series of the strain of rabbits used in this experiment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were limited to three foetuses of two different litters of the control group. The observed malformations consisted of misshapen thoracic vertebrae accompanied by three malformed ribs in one foetus, and a bifurcated rib in another foetus of the same litter. Further, a misshapen sternum was observed in one foetus of another litter.

The other observations were minor skeletal anomalies or skeletal variants and were observed either in several foetuses of both the control and the high dose group or occasionally in a single foetus.

No skeletal malformations, anomalies or variants were observed that could be related to the treatment

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations, including the above-mentioned acauda, were observed in 6 foetuses in total. Unilateral agenesis of the kidney and ureter occurred in one foetus of the high dose group, while another foetus of the same litter revealed partly fused liver lobes. Further, atrophy of one ovary was observed in one control and two high dose foetuses.

Minor visceral anomalies and visceral variants were observed in a number of foetuses of both the control and high dose group. Although some minor differences occurred in the incidences of liver anomalies and circulatory system variants, both the nature and the total incidences of the observed minor anomalies and variants were similar in both groups.

No foetal soft tissue anomalies or variants were observed that could be related to the treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Key result

Developmental effects observed:

no

Table 2. Analytical results of test substance concentration in diet

Nominal concentration (%)	Actual concentration (%) Diet prepared 04 Nov 1982	Actual concentration (%) Diet prepared 01 Dec 1982
0.0	0.0	0.0
2.5	2.4	2.4
5.0	4.6	4.4
10.0	8.8	9.3

Table 3. Test item intake

	Nominal concentration of test item in diet (%)
	0.0	2.5	5.0	10.0
Body weights (g, mean bw on end of organogenesis, day 19)	3980.9	3904.6	3917.5	3916.3
Food consumption (g food/animal/day, mean value during organogenesis (days 6-19)	188.2	176.5	169.6	186.4
Food consumption (g food/kg bw/day, mean of means)	47.3	45.2	43.3	47.6
Test item intake (g/kg bw/day)	0.0	1.1	2.2	4.8
Test item intake (mg/kg bw/day)	0.0	1130.1	2164.6	4759.6

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7 970 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies (Klimisch score 2), and are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity / teratogenicity

A pre-natal developmental toxicity study in rats was performed with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol (CAS 64519-82-0) via dietary admix equivalent or similar to OECD 414 and in compliance with GLP (Koeter, 1982). Groups of 22 and 23 female Wistar rats received the test substance daily from day 0 to 21 of gestation via the diet at concentrations of 2.5, 5 and 10% (corresponding to test item intakes of 1970, 4120 and 7970 mg/kg bw/day, calculated from mean bw and food consumption). Two groups served as controls: One control group received plain diet supplemented with 10% wheat starch ad libitum. A second control group received plain diet supplemented with 10% wheat starch at restricted levels (80% of the amounts consumed by the ad libitum-fed control group). On day 21 of gestation the animals were killed by decapitation and examined for maternal and foetal parameters. Neither treatment-related effects on reproduction parameters such as number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss, embryonic deaths, embryonic resorptions, fetal resorptions, live fetuses, dead fetuses nor systemic toxicity were observed in parental females and fetuses up to and including the highest dose concentration of 10%. No mortality occurred and necropsy and macroscopic examination did not show any treatment-related effects on parental females. The external/skeletal/visceral examination of fetuses did not reveal any treatment-related effects.

The feeding of restricted amounts of stock diet to pregnant females resulted in decreased maternal weight gain and consequently, in lower uterus weights. The effects on foetuses were principally of an embryotoxic nature as expressed by increased numbers of resorptions, smaller foetuses, decreased foetus weights and retardation in bone development. It is therefore concluded that restriction of the diet of pregnant rats during gestation induces embryotoxic effects.

The NOAEL for maternal/embryo-/fetotoxicity/teratogenicity in rats for Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol was found to be 10% (7970 mg/kg bw/day), the highest dose tested.

 

A pre-natal developmental toxicity study in rabbits was performed with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol (CAS 64519-82-0) via dietary admix equivalent or similar to OECD 414 and in compliance with GLP (Koeter, 1983). Groups of 36 and 37 female artificially inseminated New Zealand White rabbits received the test item daily from day 0 to 28 of gestation via the diet at concentrations of 2.5, 5 and 10% (corresponding to test item intakes of 1100, 2200 and 4800 mg/kg bw/day, calculated from mean bw and food consumption on day 19). One control group received plain diet supplemented with 10% wheat starch ad libitum. On day 29 of gestation the animals were euthanisied and examined for maternal and fetal parameters. Neither treatment-related effects on reproduction parameters such as number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss, embryonic deaths, embryonic resorptions, fetal resorptions, live fetuses, dead fetuses nor systemic toxicity were observed in parental females and fetuses up to and including the highest dose concentration of 10%. No mortality occurred and necropsy and macroscopic examination did not show any treatment-related effects on parental females. The external/skeletal/visceral examination of fetuses did not reveal any treatment-related effects. The NOAEL for maternal/embryo-/fetotoxicity/teratogenicity in rabbits forReaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitolwas found to be 10% (4800 mg/kg bw/day), the highest dose tested.

In a published supporting study in rats (Waakens-Berendsen, 1989), which followed no guideline, Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol was administered via dietary admixture to rats. Female Bay FB:30 rats were adapted to the test item by feeding them a diet containing a gradually increasing amount of test item for several days, prior to mating. Subsequently, they were mated. After mating the test item was fed continuously at concentrations of 2.5, 5 and 10% up to day 20 of pregnancy. In addition, one group of female Wistar rats was mated and fed 10% test item incorporated in the diet from day 0 up to day 20 of pregnancy, without previous adaptation. Finally, one group of untreated female Wistar rats served as controls. Half of the females in each group was selected for Caesarian section on day 20 of pregnancy. The other half was allowed to litter and rear their pups for 2 weeks (Wistar rats) or 3 weeks (Bay FB:30 rats). In Bay FB:30 females decreased body weight gain and food consumption were observed in the 5 and 10% group. Minor retardation in fetal development was observed in the high dose group only with the Bay FB:30 rats and was therefore considered to be related to maternal toxicity. In addition, a dose-related increase in the incidence of wavy ribs occurred in fetuses of the Bay FB: 30 rats. However, none of the observed effects were persistent in neonates. The test item appeared to have slight toxic effects in the dams of the Bay FB:30 strain but no toxicity in the offspring. In Wistar rats no toxicity and no effects on maternal performance or on embryonic, fetal or neonatal development were seen. It therefore seems that the Bay FB:30 strain is particular susceptible to the test item, while the standard Wistar rat strain, as in all previous studies, showed no adverse response to the test item even at the very high doses administered in this study. The test item, when fed at dietary levels up to 10%, did not induce structural or functional teratogenic effects in rats of either the Wistar or the Bay FB:30 strain.

In conclusion, the data on developmental toxicity of Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol do not indicate adverse effects on embryo-foetal development.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.