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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxicity to reproduction of the test item on rats was determined in a GLP study similar to OECD guideline 416. No adverse effects on mating performance, fertility or other reproductive effects were observed in either generation at treatment levels up to 30,000 ppm in a two generation reproductive study in rats. The NOAEL for reproductive endpoints was determined to be 30,000 ppm.

Dietary administration of sucralose at concentrations of 3,000, 10,000, and 30,000 ppm to male and female rats was associated with reduced food intake and bodyweight gain and increased water consumption at the mid and high dose groups during the two-generation reproductive study. A number of intergroup differences in absolute and relative organ weight were observed in F0 and F1 adults, most significantly a dosage related weight increase of the full and empty Caecum. These intergroup differences in organ weight were considered unlikely to be of toxicological significance; therefore, the NOAEL for general toxicity was determined to be 10,000 ppm.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-1985

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 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Study predates OECD Guidelines - similar in general principle to OECD 416

Deviations:

not specified

Principles of method if other than guideline:

The high intensity sweetener, TGS (Sucralose), was administered continuously in the diet at concentrations of 3000, 10000 and 30000 ppm (equivalent to 0.3%, 1.0% and 3.0% w/w) to groups of 30 male and 30 female rats of the Charles River CD strain (Sprague Dawley) throughout two successive generations. A fourth group, serving as control, received basal diet without the test material.

F0 animals were treated for 10 weeks before pairing twice in succession. The first pairing produced the F1A litters which were discarded at weaning. After the second pairing, males and females from the F1B litters were selected to form the F1 generation and were treated for 10 weeks before being paired, twice in succession to produce F2A and F2B offspring. These offspring were discarded after weaning.

Parental F0 and F1 animals were subjected to a detailed necropsy procedure and selected tissues were weighed and retained in fixative.

Observations:
- General conditions and mortality
- Body weight
- Food consumption and food conversion efficiency
- Test compound intake
- Water intake
- Oestrous cycles
- Mating performance and fertility
- Gestation length and gestation index
- Litter size and viability
- Physical development
- Sex ratio
- Auditory and visual function in offspring

Terminal Observations:
Organ weights
Necropsy
Macropathology

GLP compliance:

yes

Limit test:

no

Justification for study design:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: Selected by the Sponsor
- Route of administration: Diet

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by Sponsor. Batches:167001 - I-700 (97.9% pure), 167002 JJI-900 (97.7% pure), 167002 I-700 (97.7% pure), 163003 I-601 (99.4% pure)
- Purity: see above
- Purity test date: 26/06/1985

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature until 31/5/84, refrigerated at 4 degrees C thereafter
- Stability under test conditions: Stable for the duration of treatment, analysed by Sponsor - satisfactory

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: solid test substance incorporated into diet at 5%; further diluted with diet to meet target concentrations formulated on a weekly basis. Analysis of formulated diets was undertaken to show that substance was present at concentrations within +/- 10% of the target levels at Week 1 (start of study) and Week 11 (pairing of F0-F1A). Formulated diet was shown to be stable for 15 days at 23 degrees C..

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Solid mixed in Diet.

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5-6 wks
- Weight at study initiation: (P) Males: 162-210g; Females: 120-165g
- Housing: Plastic cages (RC1, RM2 modified, RB3) North Kent Plastics Limited. Cage population varied at different study stages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 58-69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From : 23 March 1984 To: 3 May 1985

Route of administration:

oral: feed

Vehicle:

other: Diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):weekly
- Mixing appropriate amounts of test substance with Spratts laboratory diet No.2
- Storage temperature of food: Ambient

Details on mating procedure:

- M/F ratio per cage: one to one
- Length of cohabitation: up to 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 1 of pregnancy
- After 21 days of unsuccessful pairing, replacement of first male by another male with proven fertility was placed with the female for another period of up to 7 days.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged: Individually in RB3 cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Spot checks for achieved concentrations were performed for the diets prepared for all dosage groups at the start of the first generation and at the start of mating using HPLC methods.

Duration of treatment / exposure:

10 weeks (70 days) minimum

Frequency of treatment:

daily in food

Details on study schedule:

- F1 parental animals not mated until after a treatment duration of 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were weanlings.
- Age at mating of the mated animals in the study: P0 ~ 15/16 weeks; F1 ~10/11 weeks.

Dose / conc.:

3 000 ppm

Dose / conc.:

10 000 ppm

Dose / conc.:

30 000 ppm

No. of animals per sex per dose:

30 male, 30 female

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Determined by Sponsor

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly. Females - weekly until mating detected and on days 1, 7, 14, 21 and 25 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

The percentage of females showing regular or other types of oesterous cycles before paring was recorded using daily vaginal smears.

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- maximum of 8 pups/litter - 4 male, 4 female, as nearly as possible - up to a maximum of 30 of each sex. Excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1a/F1b / F2a/ F2b ] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, pina unfolding, hair growth, tooth eruption, eye opening.

GROSS EXAMINATION OF DEAD PUPS:
yes, complete gross necropsy

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals parental were sacrificed once B litters were weaned
- Maternal animals: All surviving animals were sacrificed after the B litters were weaned.

GROSS NECROPSY
- Gross necropsy consisted of a complete gross necropsy including adrenals, caecum (full and empty), epididymes, kidney, liver, lungs, ovaries, pituitary, prostate, seminal vesicles, spleen, testies, thymus, uterus.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated above were prepared weighed and retained in buffered saline solution.

Postmortem examinations (offspring):

SACRIFICE
- The F1A offspring and the F1B offspring not selected as parental animals and all F2 offspring were sacrificed after weaning.

GROSS NECROPSY
Gross necropsy consisted of a complete gross necropsy including adrenals, caecum, epididymes, kidney, liver, lungs, ovaries, pituitary, prostate, seminal vesicles, spleen, testies, thymus, uterus.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.

Statistics:

The significance of suggestive inter-group differences was tested using appropriate statistical tests. The following tests were used:
-Student's t-test: Bodyweights, Bodyweight change, Litter size
-Dunnetts' t-test: Absolute and relative organ weights
-Student's t-test after co-variant analysis against terminal bodyweight: Relative organ weights
-Mann-Whitney U-test: Food intake, water intake

Reproductive indices:

Litter size, Sex ratio, Oesterous cycles, pre-coital interval, mating performance and fertility, Oesterous cycles, pre-coital interval. Gestation length and index

Offspring viability indices:

Physical development: Pinna unfolding, Hair growth, Tooth eruption, Eye opening, live birth index, lactation index, group mean offspring weight,
Auditory and visual function.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related effects on clinical conditions were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

two P0 animals - one male (3000 ppm group) and one female (10,000 ppm group) were killed in extremis, this was not attributed to treatment related effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

All treated F0 males showed a significant reduction in bodyweight gain (p<0.001) from Week 10 onward. The 3000 and 10000 ppm group animals achieved approximately 90% of the control weight gain whereas in the 30000 ppm dose group this was reduced to 80%.

Bodyweight gain of females showed a significant dosage-related reduction in all treated groups during the maturation period before the first pairing. Mean weight gain was 88, 83 and 75% of the control value for the 3000, 10000 and 30000 ppm dose groups, respectively.

At pairing the group mean bodyweights of the treated females were 95, 93 and 87% of the control bodyweight for groups receiving 3000, 10000 and 30000 ppm, respectively. Despite some variation in weight gain between groups during the gestation and lactation phases, the overall patterns of bodyweight gain remained similar to that established for each group during maturation and at necropsy, treated females weighed 92, 93 and 85% of the controls for animals receiving 3000, 10000 and 30000 ppm, respectively.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was reduced for all treatment groups during the maturation period.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency for F0 males during the maturation phase was similar in the treated and control groups, in females this was slightly reduced in all treated groups during the maturation period.

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

no effects observed

Description (incidence and severity):

Mean water intake in treated males was similar to controls, F0 females in the treatment group showed a slight dose related increase in water intake.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Unaffected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Unaffected by treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

The majority of matings occured during the first oesterous cycle after pairing. Mating, conception and fertility indices were unaffected by treatment at both the F0-F1A and F0-F1B pairings. Gestation length was similar in all groups and for all pairings. All pregnant females with the exception of a 3000 ppm female with uterine implantation scars gave birth to live offspring and there was no evidence of dystocia at parturition.

Note that the increased ovarian weight seen in females at the high dose group was not associated with any significant trend of macroscopic changes and no impact on reproductive performance with no consistent dose response relationship.

Key result

Dose descriptor:

LOAEL

Effect level:

>= 30 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Remarks on result:

other:

Remarks:

increased Caecal weight

Key result

Dose descriptor:

LOAEL

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Lowest effective dose / conc.:

30 000 ppm

System:

gastrointestinal tract

Organ:

other: Caecum

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related effects on clinical conditions were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One F1 female from the 10000 ppm group was killed in extremis, this was not considered to be treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At selection of the F1 males, group mean bodyweights in the treated groups were reduced compared with that of controls (3000 ppm-95% of control; 10000 ppm-91% of control, p<0.01; 30000 ppm-89% of control, p<0.001). Bodyweight gain to pairing after 10 weeks of treatment and to termination after 26 weeks of treatment was significantly reduced in the 3000 and 30000 ppm groups but was only marginally affected in the 10000 ppm group.

At selection of the F1 females, the mean bodyweights were 95%, 94% and 91% of the control weights for animals receiving 3000, 10000, and 30000 ppm, respectively. At pairing, the group mean bodyweights of the treated females were 92%, 91% and 89% of the control bodyweight for groups receiving 3000, 10000 and 30000 ppm, respectively. Despite some variation in weight gain between groups during gestation and lactation phases, the overall patterns of bodyweight gain remained similar to that established for each group during maturation and at necropsy, treated females weighed 93, 96 and 91% of the controls for animals receiving 3000, 10000, and 30000 ppm, respectively.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Overall food intake of treated F1 males and females during the 10-week maturation period were approximately 2-5% lower in the treated groups than in the control group but inter-group differences were rarely significant and there was no clear association with dosage.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency of F1 animals was similar to the control group during the maturation period.

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

effects observed, treatment-related

Description (incidence and severity):

Mean water intake of the F1 animals was increased at the 10000 and 30000 ppm male treatmet groups and across all female treatment groups.

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:

effects observed, treatment-related

Description (incidence and severity):

After covariate analysis of organ weights and bodyweight, statistically significant effects recorded in both sexes were limited to increased weight of the caecum with its contents at all treatment levels and of the empty caecum and the kidneys at 30000 ppm. Empty caecal weight was also increased for males at 10000 ppm and kidney weight was increased in females receiving 10000 ppm. Ovarian weight was increased at the low dose and intermediate dose levels but not at 30000 ppm. Thymus weight was reduced in high dose males (30000 ppm) and in all treated groups of females but the reduction in weight did not appear to be dosage-related in females. Relative kidney weight however was increased in 30000 ppm males and females and in 10000 ppm females.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle was unaffected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Unaffected by treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive performance and fertility indexes were unaffected by treatment.

Key result

Dose descriptor:

LOAEL

Effect level:

>= 10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

organ weights and organ / body weight ratios

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight of F1A and F1B offspring at Day 1 post partum showed a slight, dosage-related reduction, but statistical significance was only achieved by 30000 ppm F1B offspring (p<0.01). A statistically significant, dosage-related reduction in bodyweight gain (day 1-25 post-partum) was recorded in all treated groups, for both the F1A and F1B litters.

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 examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Examination of offspring that died before weaning revealed absence of food in the stomach as the major finding.

Offspring killed after weaning presented a number of minor anomalies which have been previously recorded in historical control animals. No evidence of any adverse response related to treatment was observed.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Responses to auditory and visual stimuli were essentially similar in all groups.

Mean onset and completion times for pinna unfolding, hair growth, tooth eruption and eye-opening were essentially similar in all groups of animals.

The ratio of male to female offspring was unaffected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

LOAEL

Generation:

F1 (cohort 1A)

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Generation:

F1 (cohort 1B)

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition and apperance of offspring were unnaffected by treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F2 offspring viability was not affected by treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of F2 off spring at day 1 were slighty reduced but only significantly so in the 30,000ppm treatment groups.

The rate of body weight gain was slightly reduced for all treatment groups when compared to controls.

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 examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Examination of offspring that died before weaning revealed absence of food in the stomach as the major finding.

Offspring killed after weaning presented a number of minor anomalies which have been previously recorded in historical control animals. No evidence of any adverse response related to treatment was observed.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Responses to auditory and visual stimuli were essentially similar in all groups.

Mean onset and completion times for pinna unfolding, hair growth, tooth eruption and eye-opening were essentially similar in all groups of animals.

The ratio of male to female offspring was unaffected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

no

Treatment related:

no

Conclusions:

The toxicity to reproduction of the test item on rats was determined in a GLP study similar to OECD guideline 416. No adverse effects on mating performance, fertility or other reproductive effects were observed in either generation at treatment levels up to 30,000 ppm in a two generation reproductive study in rats. The NOAEL for reproductive endpoints was determined to be 30,000 ppm.

Dietary administration of sucralose at concentrations of 3,000, 10,000, and 30,000 ppm to male and female rats was associated with reduced food intake and bodyweight gain and increased water consumption at the mid and high dose groups during the two-generation reproductive study. A number of intergroup differences in absolute and relative organ weight were observed in F0 and F1 adults, most significantly a dosage related weight increase of the full and empty Caecum. These intergroup differences in organ weight were considered unlikely to be of toxicological significance; therefore, the NOAEL for general toxicity was determined to be 10,000 ppm.

Executive summary:

The toxicity to reproduction of the test item on rats was determined in a GLP study similar to OECD guideline 416. No adverse effects on mating performance, fertility or other reproductive effects were observed in either generation at treatment levels up to 30,000 ppm in a two generation reproductive study in rats. The NOAEL for reproductive endpoints was determined to be 30,000 ppm.

Dietary administration of sucralose at concentrations of 3,000, 10,000, and 30,000 ppm to male and female rats was associated with reduced food intake and bodyweight gain and increased water consumption at the mid and high dose groups during the two-generation reproductive study. A number of intergroup differences in absolute and relative organ weight were observed in F0 and F1 adults, most significantly a dosage related weight increase of the full and empty Caecum. These intergroup differences in organ weight were considered unlikely to be of toxicological significance; therefore, the NOAEL for general toxicity was determined to be 10,000 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The developmental toxicity of the test item on rabbits was determined in a GLP study according to ICH harmonized tripartite guideline similar to OECD guideline 416. 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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline

Version / remarks:

24 June 1993

Deviations:

no

GLP compliance:

yes

Limit test:

no

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.

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

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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

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.

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.

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

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

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

Abnormalities:

effects observed, treatment-related

Localisation:

other:

Description (incidence and severity):

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

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.

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

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.

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

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

 

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

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

Published literature indicates that the abortions and embryo-foetal loss observed should be considered secondary to reduced maternal food intake and weight loss.

Justification for classification or non-classification

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), the test substance should not be classified for reproductive toxicity since there were no adverse effects on sexual function, fertility or on development of the offspring.