Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 22 Mar 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The test material is only administered from gestation day 6 – 15, whereas treatment from gestation day 5 to the day prior to scheduled death (usually gestation day 20) of animals is recommended in the appropriate OECD TG. Furthermore, no analytical purity of the test material is available.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The test material is only administered from gestation day 6 – 15, whereas treatment from gestation day 5 to the day prior to scheduled death (usually gestation day 20) of animals is recommended in the appropriate OECD TG. No analytical purity is given.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol
EC Number:
226-159-8
EC Name:
1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol
Cas Number:
5306-85-4
Molecular formula:
C8H14O4
IUPAC Name:
(3R,3aR,6S,6aR)-3,6-dimethoxy-hexahydrofuro[3,2-b]furan
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 8 – 10 weeks (females)
- Weight at study initiation: 201 – 225 g (females)
- Housing: The male rats were housed individually in grid-bottomed cages suspended over paper-lined trays, except when females were introduced for mating. During acclimatization and until pairing for mating, the females rats were housed in groups of five in stainless steel cages with grid-bottoms suspended over paper-lined bedding. After mating, the female rats were individually housed in solid-bottomed polypropylene cages with graded softwood sawdust provided as bedding.
- Diet (e.g. ad libitum): pelleted SQC Rat and Mouse No. 3 expanded diet during acclimatization and pregnancy and SQC Rat and Mouse No. 3 expanded and reground diet whilst paired for mating (Special Diets Services Limited, Essex, UK)
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 22
- Humidity (%): 46 – 62
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Feb 1993 To: 22 Mar 1993

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The test article was formulated daily for dosing as solutions in purified water. The high dose level concentration (30 mg/mL) was prepared by dissolving an accurately weighed quantity of test article in the appropriate volume of vehicle. The low and intermediate dose level concentrations were then prepared by dilution of the high dose level concentration.

VEHICLE
- Concentration in vehicle: 30, 100 and 300 mg/mL
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- 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:
Day 6 - 15 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
Until Day 20 of gestation / post mating
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical observations were recorded daily from Day 0 of pregnancy.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Days 0, 6 to 15 and on Day 20 of pregnancy.

FOOD CONSUMPTION: Yes
- Food consumption was measured over the following periods: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15 and 15 to 20 of pregnancy.

WATER CONSUMPTION No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: For necropsy, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Organs or tissues showing severe macroscopic abnormalities were removed and fixed in buffered formal saline.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: fetal weights (live fetuses), fetal sexes (live fetuses)
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [one third per litter]
- Skeletal examinations: Yes: [two thirds per litter]
- Head examinations: Yes: [one third per litter]
Statistics:
Group means and standard deviations were calculated, were appropriate, and data was subjected to an analysis of variance of Kruskal-Wallis testing. Where significance was achieved using analysis of variance, each treated group was compared with the control group using Dunnett´s test. Where significance was achieved using Kruskal-Wallis test, each treated group was compared with the control group using Dunn´s multiple comparison test.
Indices:
Pre-implantation loss = (a – b) / a x 100 (a: number of corpora lutea; b: total number of implantation sites)
Post-implantation loss = (b – c) / b x 100 (b: total number of implantation sites; c: number of live fetuses)
Sex-ratio = (number of male fetuses / total number of fetuses x 100) / (number of female fetuses / total number of fetuses x 100)
Historical control data:
There is no information available for historical control data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only clinical changes observed (alopecia, tip of tail missing and a soft, round, raised area in the middle of the abdomen) were minor in nature and commonly observed for this strain of rat in these laboratories.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the female rats died or was killed prematurely during the course of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment at any dose level on maternal bodyweight. Mean bodyweights were similar in all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment at any dose level on maternal food consumption. The amount of food consumed was similar in all groups.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Moreover, there were no treatment-related macroscopic abnormalities observed at necropsy. One female of the intermediate dose group (100 mg/kg bw/day) showed abnormal colored edges of the placental membranes. The fetuses of this rat were of low bodyweight and most had abnormalities. As this was an isolated finding, it was considered incidental and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Of the 24 female rats in each group that were mated at the start of the study, there were only three animals found to be not pregnant at termination (two in the control group, one in the intermediate dose group).
Other effects:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea was similar in all groups and was within the background range.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No relevant treatment related effects were observed.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on fetal weights. Mean fetal weights (male, female and combined) of the treated groups were similar to those of the control group. The fetuses of 100 mg/kg bw/day dose group of one femal rat with abnormal colored edges of placental membranes were of low body weight and most had abnormalities. As this was an isolated finding, it was considered incidental and unrelated to treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex ratio of the fetuses was similar in all groups except in the intermediate dose group (100 mg/kg bw/day), where the ratio (45 : 55) was marginally outside of background range (46 : 54 to 53 : 47). However, since this was isolated to the intermediate dose group, it was not considered to be treatment-related.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group, two fetuses from separate litters were observed with microphthalmia . In the lowest dose group (30 mg/kg bw/day), none of the fetuses had major abnormalities. In the intermediate dose group (100 mg/kg bw/day), the incidence of major abnormalities observed was unusually high. However, only two litters contained affected fetuses and in one of these, five of seven fetuses were affected. The major abnormalities seen were short body, restricted movement of the hindlimbs (probably as a consequence of the short body) and protruding tongue. In the high-dose group (300 mg/kg bw/day), one fetus was observed with major external/visceral abnormalities. These were domed head, short body and restricted movement of the limbs (probably as a consequence of the short body). As there were no dose-related trends and since all the major abnormalities were of a type and incidence that can occur spontaneously in this strain of rat, none was considered to be related to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Major abnormalities
In the intermediate dose group (100 mg/kg bw/day), micrognathia, cleft palate, short mandible retarded ossification of the long bones and mis-shapen long bones in both fore- and hindlimbs were observed. In all other dose groups, no major skeletal abnormalities were observed. As there were no dose-related trends and since all the major abnormalities were of a type and incidence that can occur spontaneously in this strain of rat, none was considered to be related to treatment.
There was significant (p<0.05) intergroup variation as indicated by the Kruskal-Wallis test in the incidence of 7 lumbar vertebrae. However, since the incidence in the treated groups was lower than in the control group and since there was no statistically significant differences and since the incidences were all within the background range, this was considered not of toxicological relevance. There was also a significant (p<0.01) increase in the incidence of retarded ossification of the 3rd sternebrae in fetuses in the intermediate dose group in comparison with the control group. However, as this effect was not seen in the lower dose group or the higher dose group, this finding was considered fortuitous and untreated to treatment.

Minor abnormalities
There was a slight, but not statistically significant, increase in the incidence of fetuses in the intermediate dose group in comparison with the control group as far as the following minor abnormalities were concerned: retarded ossification of the temporal bones, two or less candal centra vertebrae, retarded and non-ossification of the second sternebrae, retarded ossification of the fourth sternebrae, retarded non-ossification of the pubic bones, retarded ossification of the ischia and non-ossification of the metatarsais. The incidences for all these minor abnormalities, except retarded ossification of the ischia, were slightly above background ranges, but were not statistically significantly different from controls. Furthermore, there were no dose-related trends, so these findings could not be definitely associated with treatment.

Variants
In the intermediate dose group, there was a significant (p<0.05) increase in the incidence of retarded ossification of the interparietal and the occipital bones in comparison with the control group but all incidences were within background range. There was a slight increase in the incidence of retarded ossification of the parietals and the incidence was above background range but not statistically significant. However, there was no effect at the highest dose level and, therefore this was considered not to be treatment-related. At 30 mg/kg bw/day, there was a slight increase in the incidence of uni- or bilateral vestigial 14th rib in comparison with the control group and the incidence was above background range but not statistically significant. Again, this finding was considered not to be treatment-related. In the highest dose group, the incidence of ossification of the 5th sternebrae was marginally outside of background range (27%; background range: 11 – 26.9%). Because this increase was so small and not statistically significant, this observation was considered not to be treatment-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group, two fetuses from separate litters were observed with pre-ductal co-arctation. In the intermediate dose group (100 mg/kg bw/day), hypoplastic lung lobes were noted and a slight, but not statistically significant, increase in the incidence of fetuses with absent innominate arteria. In the high-dose group (300 mg/kg bw/day), one fetus was observed with umbilical hernia. As there were no dose-related trends and since all the abnormalities were of a type and incidence that can occur spontaneously in this strain of rat, none was considered to be related to treatment.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: No relevant treatment related effects were observed in the highest dose level.

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Skeletal examination of fetuses: Group mean data

Dosage mg/kg bw/day)

 

0

30

100

300

Key findings

Type of findings

No. of fetuses

Mean (%)

No. of fetuses

Mean (%)

No. of fetuses

Mean (%)

No. of fetuses

Mean (%)

 

Numbers of litters examined

 

22

 

24

 

23

 

24

 

Number of fetuses examined

 

226

 

228

 

221

 

241

 

 

Skull

 

Cleft palate

major

0

0.0

0

0.0

3

2.6

0

0.0

Occipital: not ossified

minor

1

0.4

0

0.0

0

0.0

0

0.0

Hyoid not ossified

variant

23

10.0

45

18.6

46

19.3

37

15.4

Occipital: retarded ossification

variant

10

4.3

12

4.8

26

11.5*

21

8.9

Interparietal: retarded ossification

variant

16

6.6

9

3.6

35

16.3*

27

11.2

Parietals: retarded ossification

variant

5

2.2

6

2.4

14

8.2

3

1.2

Frontals: retarded ossification

minor

1

0.4

0

0.0

4

3.0

0

0.0

Nasals: retarded ossification

minor

0

0.0

0

0.0

1

0.4

0

0.0

Temporal bones: retarded ossification

minor

3

1.3

0

0.0

13

5.7

5

2.0

Zygomatic arch: retardd ossification

minor

0

0.0

0

0.0

1

0.5

0

0.0

Mandible: retarded ossification

minor

0

0.0

0

0.0

1

0.4

0

0.0

Mandible: short

major

0

0.0

0

0.0

2

1.2

0

0.0

 

Sternebrae

 

1st: not ossified

minor

0

0.0

0

0.0

1

0.4

0

0.0

2nd: not ossified

minor

1

0.4

1

0.5

4

2.0

2

0.8

3rd: not ossified

minor

0

0.0

0

0.0

2

0.7

0

0.0

4th: not ossified

minor

0

0.0

0

0.0

1

0.4

0

0.0

5th: not ossified

variant

35

15.6

43

18.6

44

19.6

63

27.0

6th: not ossified

variant

18

7.8

7

3.4

17

9.2

8

3.2

1st: retarded ossification

minor

1

0.4

2

0.8

3

2.1

3

1.2

2nd: retarded ossification

minor

3

1.2

3

1.1

11

6.3

6

2.3

3rd: retarded ossification

minor

0

0.0

1

0.4

6

3.4*

1

0.4

4th: retarded ossification

minor

3

1.2

2

0.8

7

4.3

2

0.8

One or more: bilobed, bipartite or misaligned

minor

2

0.9

9

3.7

6

2.6

3

1.5

All normal

usual

179

79.3

178

78.8

162

74.0

172

70.5

*p<0.05

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in pregnant female Sprague-Dawley rats with the test material at dose levels of 30, 100, and 300 mg/kg bw/day administered from gestation days 6 to 15 no test item related toxicological findings in dams or fetuses were revealed. Under the conditions of the study, 300 mg/kg bw/day was considered as no observed effect level (NOEL) for both maternal and embryo-fetal toxicity of the test material.