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Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
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: 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.

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Critical effects observed:
not specified
Lowest effective dose / conc.:
30 000 ppm
System:
gastrointestinal tract
Organ:
other: Caecum

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
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

Results: F2 generation

General toxicity (F2)

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.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

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.