Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Justification for study design:
Not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch no. not specified
Erythritol was obtained from Cerestar, Vilvoorde Belgium. The test material had a white, crystalline appearance and was reported to have a water content of <0.5% and a purity of 99.9% on a dry matter basis. HPLC analysis determined that the actual dietary concentrations varied between 95 and 110% of the nominal concentrations.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar-derived strain
Details on species / strain selection:
Cri:WI(WU)BR
Sex:
male/female
Details on test animals and environmental conditions:
Source : Charles River Wiga GmbH, Sulzfeld, Germany
On arrival, the rats were about 5 weeks old
Diets : provided as a meal mash in stainless steel cans. The food was covered by a perforated stainless steel plate to prevent spillage. Diet : CIVO cereal-based stock diet, food and water available ad libitum

Acclimatization :
Duration : 2 weeks
Rats housed in groups of 4
Male/female separated
Cages : Suspended stainless steel group cages with wire mesh floor and front under conventional conditions
Temperature : 19–24.7°C
Relative Humidity : 40 and 70%.
Photoperiod : 12h light/dark cycle
Diet : defatted soya bean meal (11.0%), fish meal (7.0%), meat and bone scraps (4.0%), whole ground wheat (36.0%) and maize (29.7%), brewer’s yeast (3.0%), alfalfa meal (3.0%), whey powder (2.0%), defatted bone meal (0.4%), salt and vitamin premixes (0.6%), and soya bean oil (3.0%). Analysis : contain crude protein (20.2%), crude fat (6.3%), crude fiber (3.3%), moisture (10.4%), calcium (0.87%), phosphorus (0.72%), magnesium (0.17%), as well as minerals, trace elements, and vitamins in adequate amounts.

Premating period and thereafter until termination :
Premating period : 10 weeks
Diet of the control group : 87.4% cereal-based stock diet, 10% wheat starch, 2.5% casein, and 0.1% d,l-methionine.
Diet of the treatment group : The three erythritol dose groups received the same diet, to which 2.5, 5, or 10% erythritol was added at the expense of wheat starch.
Initial body weight : 176.3 g for males (149.8 to 206.3 g) and 135.5 g for females (119.4 to 158.7 g)



Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: erythritol administered in diet
Details on exposure:
Erythritol mixed into the diet: 87.4% cereal-based stock diet, 10% wheat starch (to which 2.5, 5, or 10% erythritol was added at the expense of wheat starch), 2.5% casein and 0.1% d,l-methionine.

Animais received the test article for 10 weeks prior to mating, during mating, gestation, and lactation. During the premating period, food consumption measured weekly for each cages by weighing the feeders. Individual food consumption of all mated females was also recorded weekly during pregnancy and lactation.

Actual intakes, first and second generation :

first generation :
actual intakes lactation during premating : male : 1.4, 2.8, 5.95 g/kg body weight/day
actual intakes during premating : female: 1.69, 3.42, 6.68 g/kg body weight/day
actual intakes during gestation : female : 1.56, 3.27, 6.44 mg/kg body weight/day
actual intakes during lactation : female : 3.79, 7.43, 15.81 mg/kg body weight/day

second generation :
actual intakes during premating : male : 1.61, 3.33, 7.03 mg/kg body weight/day
actual intakes during premating : female : 2, 3.4, 8.46 mg/kg body weight/day
actual intakes during gestation : female : 1.54, 3.18, 6.64 mg/kg body weight/day
actual intakes during lactation : female : 3.38, 7.48, 16.45 mg/kg body weight/day

From Day 0 onward, the rats received the test diets with 0 (control), 2.5, 5, and 10% erythritol. There was no adaptation period with stepwise increasing levels.

Details on mating procedure:
Three days before the start of the study, all rats were weighed and allotted to four groups of 24 rats/sex/group by computer randomization on basis of mean body weight.
At the end of the 10-week premating period, the animals were paired, according to numerical sequence, by caging one female with one male of the same dose group (F0-generation). The next morning the females were checked for evidence of mating. Females not showing a vaginal plug or sperm cells were returned to the male. This procedure was continued for 3 weeks. The day of detection of a vaginal plug of sperm cells was considered to be Day 0 of gestation. Upon evidence of copulation, males were returned to their original group cage. Positive females were housed individually in nesting cages for the birth and rearing of their pups (F1-generation).

Days 4 post-partum : litter adjusted to eight pups by eliminating extra pups by random selection to yields, as nearly as possible, four males and four females per litter.
Day 21 post-partum : pups weaned. After that, 24 male and 24 female pups of each dose group were selected at random and from as many litters as possible to rear the next generation. The remaining pups were discarded

Procedures followed to mate the F1-generation and to rear the F2-litters were the same as those described for the F0-generation.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The mean intake of erythritol was calculated per kilogram of body weight per day on the basis of the nominal dietary erythritol concentrations. Water consumption was not measured.
Duration of treatment / exposure:
10 weeks
Frequency of treatment:
At each meal, food and water available ad libitum
Details on study schedule:
Main steps of the study :
- Acclimatization : 2 weeks
- 3 days before the start of the study, all rats were weighed and allotted to four groups of 24 rats/sex/group kilo by computer randomization on basis of mean body weight.
- Premating : 10 weeks
- Mating after the premating. The next morning, if the females did not show a vaginal plug or sperm cells were returned to the male. Procedures continued for 3 weeks.
- Birth of the pups
- Days 4 post-partum : litter adjusted to eight pups by eliminating extra pups by random selection to yields, as nearly as possible, four males and four females per litter.
- Day 21 post-partum : pups weaned. After that, 24 male and 24 female pups of each dose group were selected at random and from as many litters as possible to rear the next generation. The remaining pups were discarded
- Sacrificed and gross examination of the F0 parent : at week 20
- The study was terminated at the end of weaning of the F2 pups.



Doses / concentrationsopen allclose all
Dose / conc.:
2.5 other: % at the expense of dietary starch
Dose / conc.:
5 other: % at the expense of dietary starch
Dose / conc.:
10 other: % at the expense of dietary starch
Dose / conc.:
0 other: % at the expense of dietary starch
No. of animals per sex per dose:
24 rats/sex/generation/dose group
Control animals:
yes, concurrent no treatment
yes, plain diet
Positive control:
Not specified

Examinations

Parental animals: Observations and examinations:
Body weights during premating period : all prospective parent males and females were recorded weekly
Body weights after mating period : males were weighed weekly until sacrifice / Mated females were weighed on Days 0, 7, 14, and 21 of gestation and during lactation on Days 1, 7, 14, and 21 postpartum. / Females which did not have a positive smear or vaginal plug and females which did not deliver a litter were not weighed after the end of the premating and gestation period, respectively.

Clinical signs and abnormal behavior : all animals were checked daily
Estrous cyclicity (parental animals):
Not specified
Sperm parameters (parental animals):
Not specified
Litter observations:
Clinical signs and abnormal behavior : all animals were checked daily
Litter size / number of male and female / number of pups with abnormalities : determined on days 1, 4 (before culling), 7, 14 and 21 postpartum
Body weights of entire litter : determined on days 1 4 (before culling), 7, 14 postpartum
Body weight of individual pups : on day 21, at weaning
Postmortem examinations (parental animals):
All parental animals of the F1-generation were sacrificed and necropsied
At termination, all surviving parental animals of the F0- and F1-generation were killed by decapitation under ether anesthesia and then examined for gross pathological changes.
Postmortem examinations (offspring):
Necropsy was performed on stillborn pups and pups dying during lactation.
Statistics:
clinicat findings were evaluated using Fisher's exact probability test; body weight and food consumption data were analysed using one way analysis of variance and Dunnett's multiple comparison tests; mating parameters were evaluated using Fisher's exact probability test; duration of gestation and litter size were evaluated using Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test; pup body weights were analysed by analysis of variance followed by Dunnett's multiple comparison test; implantations were analysed using Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test; pathologicai changes were analysed using Fisher's exact probability test. Ail tests were two-sided.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Transient diarrhea occurred in all F0-males and -females of the 10% erythritol group at the start of the study, but ceased in most animals with continued administration of erythritol.

Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gains were below those of controls in the F0-males and -females of the 10% erythritol group.
Males : the reduction of body weights was significant from Week 1 to 19
Female : this effect reached statistical significance in Week 4 only
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
10% erythritol : Food consumption of F0-males and -females was significantly below that of controls during the first week of treatment in the 10% erythritol group. Thereafter, it was significantly increased, in males through the end of the study in Week 19 and in females during Weeks 6–10 of the premating period and the first week of lactation
5% erythtritol : significant enhancing effect on food intake was seen only rarely (F0 females in Weeks 6, 9, and the first week of gestation)
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
tap water were provided ad libitum throughout the study but not measured
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Gross necropsy andmicroscopic examination of the parenteral reproductive organs also did not reveal treatment-related changes
Gross pathological findings:
no effects observed
Description (incidence and severity):
Minor abnormalities observed equally distributed among the different groups or occurred in one or a few rats only.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination did not reveal any histopathological changes that could be related to the treatment. The abnormalities observed were common findings in rats of this strain and age. Moreover, the lesions were about equally distributed among the test groups and controls, or they occurred only in one or a few animals.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
minor abnormalities were equally distributed among the different groups or occurred in one or a few rats only. Microscopic examination did not reveal any histopathological changes that could be related to the treatment. The abnormalities observed were common findings in rats of this strain and age. Moreover, the lesions were about equally distributed among the test groups and controls or they occurred only in one or a few animals.

Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
minor abnormalities were equally distributed among the different groups or occurred in one or a few rats only. Microscopic examination did not reveal any histopathological changes that could be related to the treatment. The abnormalities observed were common findings in rats of this strain and age. Moreover, the lesions were about equally distributed among the test groups and controls or they occurred only in one or a few animals.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating was successful in all pairs of each treatment group in each generation except in F0-rats of the 10% erythritol group in which 22 of 24 females were mated. Precoital time was slightly shorter in the F0- than in the F1-generation but it was similar among the different treatment groups.
There were no significant differences between groups with regard to the number of pregnant females (18–22 per group in the F0-generation).

The female fertility index varied from 75 to 83%
The fecundity index from 75 to 92%
Both indices were unaffected by the erythritol treatment and were within normal limits

The gestation time did not differ between the groups.
The gestation index varied between 91 and 100%.

Postimplantation loss was moderate and similar among the different treatment groups. A few stillborn pups were observed but the live birth index was close to 100% in all groups.

Effect levels (P0)

open allclose all
Key result
Effect level:
> 7 530 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Effect level:
> 7 670 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
Diarrhea was not observed in any rat of the F1-generation.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1-males and -females of the 10% erythritol group exhibited reduced body weights, the difference from controls being statistically significant for males during Weeks 0–8 and 17–18 and for females during Weeks 0–4. However, the rates of body weight gain of males and females of this dose group were not different from those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
10% erythtitol : Food consumption was consistently increased in F1-males and -females from the first measurement (about 2 weeks after weaning) to the end of the study (difference from controls statistically significant in all weeks except for males in Weeks 1 and 15)
5% erythtritol : significant enhancing effect on food intake was seen only rarely (F1-males in Weeks 8–10; and F1-females in Weeks 5–7 and 9, the last week of gestation, and the first week of lactation)

At the high-dose level, the erythritol intake amounts to about 6.5 g/kg body wt/day. However, in lactating females, it is more than twice as high.
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 specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Minor abnormalities observed equally distributed among the different groups or occurred in one or a few rats only.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination did not reveal any histopathological changes that could be related to the treatment. The abnormalities observed were common findings in rats of this strain and age. Moreover, the lesions were about equally distributed among the test groups and controls, or they occurred only in one or a few animals.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: estrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no significant differences between groups with regard to the number of pregnant females (17–21 per group in the F1-generation)

The female fertility index varied from 71 to 88%
The fecundity index from 71 to 88%
Both indices were unaffected by the erythritol treatment and were within normal limits

The gestation time did not differ between the groups
The gestation index varied between 91 and 100%.

Postimplantation loss was moderate and similar among the different treatment groups. A few stillborn pups were observed but the live birth index was close to 100% in all groups

Effect levels (P1)

open allclose all
Key result
Effect level:
> 7 530 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Effect level:
> 7 670 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Effect level:
> 3 765 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Effect level:
> 3 835 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
pup mortality in the 10% erythritol group seemed to differ from that of the control group, in the F0-generation because it was higher than the control group. But the results were no significant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The group mean body weight of the F1-pups (derived from F0-parents) was significantly decreased in the 10% erythritol group on Day 21 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
10% erythritol group : Food consumption was consistently increased in F1-males and -females of the 10% erythritol group from the first measurement (about 2 weeks after weaning) to the end of the study (difference from controls statistically significant in all weeks except for males in Weeks 1 and 15)
5% erythritol group : a significant enhancing effect on food intake was seen only rarely (F1-males in Weeks 8–10; and F1-females in Weeks 5–7 and 9, the last week of gestation, and the first week of lactation)
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
The sex ratio on Day 1 and Day 21 varied between 48 and 57% in the F0-generation (F1 pups)
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Visual examination of the pups revealed a few abnormalities such as small size (i.e., body weight of less than 75% of controls), subcutaneous hemorrhages at different sites, alopecia, and missing or dead tail tips. These findings were randomly distributed among the various groups or they occurred in one or a few pups only with the exception of smaller F1-pups in the 10% erythritol group which is in line with the lower pup body weights of this group
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The mean number of live pups per litter was slightly higher in the 10% erythritol group of both generations.

Effect levels (F1)

open allclose all
Key result
Generation:
F1
Effect level:
>= 7 530 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
viability
mortality
gross pathology
other: sex-ratio
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Generation:
F1
Effect level:
> 7 670 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
viability
mortality
gross pathology
other: sex-ratio
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Generation:
F1
Effect level:
> 3 765 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Generation:
F1
Effect level:
> 3 835 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
pup mortality in the 10% erythritol group seemed to differ from that of the control group, in the F1-generation (pup F2) because it was lower than the control group. But the results were no significant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In the F2 pups no difference was observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
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:
no effects observed
Description (incidence and severity):
The sex ratio on Day 1 and Day 21 varied between 50 and 57% in the F1-generation (F2 pups)
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Generation:
F2
Effect level:
> 7 530 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
value from test report (7.6 mg/kg bw/d in the publication)
Sex:
male
Basis for effect level:
viability
mortality
food consumption and compound intake
gross pathology
other: sex-ratio
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Generation:
F2
Effect level:
>= 7 670 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
value from test report (7.6 mg/kg bw/d in the publication)
Sex:
female
Basis for effect level:
viability
mortality
food consumption and compound intake
gross pathology
other: sex-ratio
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The effects seen in parental animais consisted of body weight changes which were due to the reduced calorie intake due to erythritol added to the diet at high levels. This was concluded based on the lower body weights despite higher intake of food in the erythritol groups. In addition, no toxicological effects were apparent in the treated animais.
Despite the reduced body weights in high-dose animais, no effects on fetal development or pup body weights were observed, nor were any reproductive parameters affected. Some statistical differences were noted in pup mortality rates; however, the differences were contradictory, being increased in one generation but decreased in the next, or appeared only at lower dose.
As a result, the authors concluded that erythritol did not affect reproductive performance or fetal development in rats when administered in the diet at levels of up to 10% for 2 consecutive generations. Mean intakes of erythritol at the highest dose levels were reported to be 7.53 and 7.67 g/kg body weight/day for males and females, respectively.
Executive summary:

In an assay similar to OECD 416 (two generation reproductive toxicity study), the authors concluded that erythritol did not affect reproductive performance or fetal development in rats when administered in the diet at levels of up to 10% for 2 consecutive generations. Mean intakes of erythritol at the highest dose levels were reported to be 7.53 and 7.67 g/kg body weight/day for males and females, respectively.