1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline

Version / remarks:

24 June 1993

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

EC Number:

259-952-2

EC Name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Cas Number:

56038-13-2

Molecular formula:

C12H19Cl3O8

IUPAC Name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Provided by sponsor, Batch B041997SCM, 99.2% purity
- Purity test date: 21 September 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated at 1-10 degrees C
- Stability under test conditions: stable in aqueous solution for at least one year when stored below 21 degrees C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No reactivity - vehicle was water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For each dose group, the test article was dissolved in a small quantity of the measured vehicle (water). Additional vehicle was added to make up the required concentration before stirring until homogeneous.


FORM AS APPLIED IN THE TEST (if different from that of starting material) : Dissolved in water.

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl.NZW/Kbl BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 4-5 months
- Weight at study initiation: at least 2.5kg
- Housing: individually in floor pens with soft wood chips
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22 degrees C
- Humidity (%): 40-80%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):10/14

IN-LIFE DATES: From: 22 November 1998 To: 23 December 1998

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For each dose group, the test article was dissolved in a small quantity of the measured vehicle. Additional vehicle was added to make up the required concentration before stirring until homogeneous. The solutions were stored refrigerated (1-10 degrees C) until dispensed to the animal house. Solutions were prepared weekly and dispensed as daily aliquots.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Purified Water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration of test article formulation tested before the start of treatment and for the last week of dosing. The concentrations of the analysed formulations ranged between 98 and 104% of nominal.

Details on mating procedure:

Mating occured at the suppliers facility 3 days prior to delivery of animals to Covance.

Purchased timed pregnant

Duration of treatment / exposure:

Day 7 to Day 19 of gestation (inclusive)

Frequency of treatment:

Daily

Duration of test:

12 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

175 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

350 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose level was selected on the basis of the results of a range-finding study of embryo-foaetal development in the rabbit and the limit dose recommendations of the ICH and OECD guidelines. The low and intermediate dose levels were comparable to the dosages evaluated in a previous rabbit embryo-foetal development study. Individual dose volumes were adjusted according to the latest recorded body weight.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily & one hour after dosing.
- Cage side observations: observable signs of ill health or toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 4, 7, 9,12,15,19, 24 and 29 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded daily from Day 4 to 29 of gestation and reported on the body weight intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 29
- Organs examined: Macroscopic examination

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

Fetal examinations:

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

Statistics:

Data were processed, where appropriate, to give litter mean values, group mean values and standard deviations.

Body weight, body weight gain, and food consumption were analysed using one-way analysis of variance (ANOVA). Pairwise comparisons with control were made using Dunnett's test. A regression test was performed to determine whether there was a linear relationship between increasing dose and response. Levene's test for equality of variances among the groups was also performed, and in all cases this showed no evidence of heterogeneity.

The numbers of corpea lutea, implantations, number of foetuses per female, percentage of male foetuses, litter weight, placental weight and foetal weight were analysed using non-parametric methods.

Gravid uterus weight was analysed using Analysis of Covariance and Dunnett's test using the corrected body weight on Day 29 as covariate.

The proportions of females having pre-and post-implantation loss, early and late intrauterine deaths and the number of litters having malformations and variations were analysed using the Cochran-Armitage test for dose-response and the Fisher exact test for pairwise comparisons. The tests were interpreted with one-sided risk for increased incidence with increasing dose.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Soft faeces were seen in 6 high dose animals, 2 intermediate dose, and one control animal, one high dose animal had coloured faeces. These findings were observed on single days during late gestation and were considered to be indicitive of treatment. At necropsy three high dose animals had a moderately distended caecum or colon - an occurance observed in other studies where high doses of a poorly absorbed substance are administered through gavage. One other animal had a mottled liver and a moderately distended gall bladder.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One low dose animal was found dead on day 13 of gestation, necropsy findings indicate that this was due to a dosing error.

One high dose animal was killed for humane reasons on day 10 of gestation due to an injury to the thoratic area of its back.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effects on bodyweight occured in the low or intermediate dose groups.

Marked body weight losses (ranging up to 710 grams) were observed in four of the high dose animals which aborted their litter and in one high dose animal that suffered total litter loss in utero. Two other high dose animals with a high incidence of interuterine deaths also had a significantly lower body weight. The mean body weight of the high dose animals with live foetuses on day 29 of gestation was slightly less than controls during the treatment period and this difference remained evident until termination.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Periods of low food intake (0-17 g/day) were observed in five high dose animals that lost litters and two animals with a high incidence of late interuterine deaths. In animals with live litters at day 29 of gestation, the mean food intale during the treatment period was slightly lower than that of the controls in all treatment groups, but there was no dose response relationship observed. The slightly lower intakes observed between days 19 and 24 may have been associated with the slight gastro-intestinal disturbance seen during this period.

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 specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three high dose animals were observed to have a moderately distended caecum or colon and further animals was seen to have a mottled liver and a moderately distended gall bladder.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group four animals aborted, no abortions were observed in the low and intermediate dosing groups.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Two high dose animals had a high incidence of late resorptions and one had total early resorptions. The mean post-implantation loss of the remaining high dose animals was lower than that of the controls. In the intermediate dose group post implantation loss was slightly higher than the controls but the difference was not statistically significant and the incidence was similar to controls from other recent studies. In the low dose group post-implantation loss was less than that of the controls.

The incidence of pre-implantation loss showed no dose related trends.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One animal from the high group suffered total early resorptions and two had a high incidence of late resorbtions.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

One animal from the high group suffered total early resorptions and two had a high incidence of late resorptions.

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

number of abortions

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other:

Description (incidence and severity):

Three high dose animals had a moderately distended caecum or colon.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No dose related trends and no significant differences from control animals, and the historical control range.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant difference in the distrubution of sex.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter weight reflected the lower mean number of foetuses per female with the difference from control being statistically significant in the low and high dose levels but were withint the control range for historical controls.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Externals and visceral malformations were observed in 7 controls, 3 low dose, 7 intermediate dose and 4 high dose foetuses.

One high dose foetus had marked lenticular lesions, as did one low dose and 4 control animals. Further eye malformations were observed in one low dose and two intermediate dose animals.

In view of the isolated nature of the malformations, their distribution within the groups and the presence of similar malformations in control foetuses, they were considered to be unrelated to treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations occured in 4 control, 6 low, 5 intermediate, and 2 high dose foetuses. High dose malformations were extra and branched ribs and fused thoracic vertebral centra. Similar effects were observed in three low dose and one intermediate and one control animal. There was no dose response relationship.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Externals and visceral malformations were observed in 7 controls, 3 low dose, 7 intermediate dose and 4 high dose foetuses. Three high dose foetuses, two intermediate dose and two control animals exhibited cardiovascular malformations. In view of the isolated nature of the malformations, their distribution within the groups and the presence of similar malformations in control foetuses, they were considered to be unrelated to treatment.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The numbers of external, visceral and skeletal variations showed no dose-related trends and the differences from the controls were not statistically significant.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: eye

skeletal: rib

skeletal: vertebra

visceral/soft tissue: cardiovascular

visceral/soft tissue: eye

Description (incidence and severity):

The numbers of external, visceral and skeletal variations showed no dose-related trends and the differences from the controls were not statistically significant.

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1. Group mean body weight (kg)

Days of Gestation	Control	175 mg/kg/day	350 mg/kg/day	1000 mg/kg/day
4	3.58	3.54	3.53	3.50
7	3.64	3.57	3.58	3.55
8	3.67	3.57	3.60	3.54
9	3.68	3.57	3.58	3.54
12	3.66	3.54	3.54	3.55
15	3.62	3.49	3.54	3.53
19	3.63	3.57	3.59	3.51
24	3.74	3.74	3.76	3.58
29	3.91	3.85	3.86	3.71
% body weight change Days 4-29	9.2	8.8	9.3	6.0
% body weight change Days 7-19	-0.3	0	0.3	-1.1
% body weight change Days 19-29	7.7	7.8	7.5	5.7

 

Table 2. Group mean food intake

Days of Gestation	Control	175 mg/kg/day	350 mg/kg/day	1000 mg/kg/day
4-7	181	172	173	166
7-8	165	92	117	117
8-9	151	124	111	113
9-12	124	109	113	118
12-15	69	70	77	81
15-19	81	93	98	74
19-24	153	159	166	114
24-29	140	161	157	146
Mean intake (g/day) Days 4-29	129	130	133	117
Mean intake (g/day) Days 7-19	102	94	99	94
Mean Intake (g/day) Days 19-29	147	160	162	130

 

Table 3. Group mean caesarian data

Uterine/Implantation Data	Control	175 mg/kg/day	350 mg/kg/day	1000 mg/kg/day
Number of females with live fetuses at Day 29 gestation	22	20	21	15
Mean # of corpora lutea per female	13.3	12.0	12.8	11.7
Pre-implantation loss
Mean %	11.8	15.0	18.2	15.7
# dams affected	15	13	10	9
Early intrauterine deaths
Mean #	0.5	0.2	0.5	0.0
# dams affected	8	4	7	0
Late intrauterine deaths
Mean #	0.4	0.7	0.8	1.3
# dams affected	7	8	8	6
Dead fetuses
Mean #	0.1	0.0	0.0	0.1
# dams affected	2	0	0	1
Post-implantation loss
Mean #	8.1	7.5	11.2	12.3
# dams affected	12	10	13	6
Mean # of fetuses per female	10.7	9.5	9.3	8.6
Fetal Data-Females with Live Fetuses	Control	175 mg/kg/day	350 mg/kg/day	1000 mg/kg/day
# of male fetuses	117	94	107	67
# of female fetuses	119	96	89	62
% male fetuses	50.0	47.8	53.3	55.4
Mean litter weight (g)	394.7	360.0 *	345.6	308.8 **
Mean placental weight (g)	4.83	4.96	5.01	5.08
Mean fetal weight (g)	37.4	38.4	38.5	36.1
Mean fetal weight (g)-males	37.1	39.1	39.0	37.5
Mean fetal weight (g)-females	37.4	37.8	37.9	34.7

*p<0.05; **p<0.01

Table 4. Fetal Defect Data

Endpoint	Control	175 mg/kg/day	350 mg/kg/day	1000 mg/kg/day
# of fetuses examined	236	190	196	129
# of litters examined	22	20	21	15
External and Visceral Defects
# showing malformations	7	3	7	4
-Mean % of fetuses affected	2.8	1.7	3.7	3.0
-Number of litters affected	5	3	6	4
# showing variations	45	70	55	46
-Mean % of fetuses affected	17.3	37.5	30.9	36.6
-Number of litters affected	16	19	19	15
Skeletal Defects
# showing malformations	4	6	5	2
-Mean % of fetuses affected	2.2	3.4	2.8	1.5
-Number of litters affected	4	4	3	2
# showing variations	210	169	188	116
-Mean % of fetuses affected	89.3	89.1	96.8	90.8
-Number of litters affected	22	20	21	15
Total # of fetuses showing malformations	10	8	11	6
% of fetuses examined	4.2	4.2	5.6	4.7
# of litters affected	8	6	8	5

 

Applicant's summary and conclusion

Conclusions:

The developmental toxicity of the test item on rabbits was determined in a GLP study according to ICH harmonized tripartite guideline similar to OECD guidelines. Oral gavage treatment with sucralose at a limit dose of 1,000 mg/kg/day produced maternal toxicity, abortion, and increased numbers of interuterine deaths, but no teratogenicity. No maternal or embryo-foetal effects were seen at 350 or 175 mg/kg/day. Thus, this study indicates that sucralose is not teratogenic in rabbits.