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

Endpoint summary

Description of key information

The repeated dose toxicity of the test item via oral application on rats was determined in a GLP study according to FDA guidelines.

It is concluded that the oral administration of sucralose to CD rats at dosages of up to 3000 mg/kg/day, for 26 weeks resulted in only a few minor treatment-related changes. These included a dosage-related enlargement of the caecum and a small increase in bodyweight-relative kidney weight at 3000 mg/kg/day. No histopathological changes were noted in either organ and the kidney weight changes did not correspond to any haematological or clinical chemistry changes. The dose related increase in water intake, slight decrease in adjusted bodyweight at 3000 mg/kg/day and caecal enlargement are not uncommon findings when high doses of poorly absorbed materials are administered to rats. Therefore, in the absence of any histopathological or other findings of an adverse nature, none of the changes observed are considered to be toxicologically significant.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Similar to OECD Guideline for Repeated Dose Toxicity Studies

Qualifier:

according to guideline

Guideline:

other: FDA Guidelines for the Safety Assessment of Direct food additives and colour additives used in food

Version / remarks:

1984

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 92-SC-157
- Expiration date of the lot/batch: not stated
- Purity test date: 16/03/1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated at 4 degrees C, protected from light.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stable/Soluble


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Daily preparation of a series of dilutions in osmotically purified water.

Species:

rat

Strain:

CD-1

Details on species / strain selection:

CD-1 rat selected due to regulatory acceptance, available historical control data, and it's use in previous gavage and dietary studies with sucralose.

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: 30-33 days
- Weight at study initiation: 102-145g (males); 96-121g (females)
- Fasting period before study: None
- Housing: Individual
- Diet: Ad libitum apart from over night before blood sampling.
- Water: Ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: Powdered rodent diet, LAD 2 - Special diet services. Water from public water supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55%
- Air changes (per hr): ~15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as this is the route of human exposure. Dosages were equally divided into two daily administrations. The dose was divided and administered during the dark cycle to ensure that sucralose was dosed at the time of maximal eating activity. Gavage administration was also chosen to avoid the palatability problems inherent with the dietary administration of sucralose.

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The sucralose was prepared for administration as a series of graded concentrations in purified water (obtained by reverse osmosis). Formulations for each group were prepared daily and divided into two equal aliquots, one for the second aministration that day, and one for the first administration the following day. Each aliquot was held in a refrigerator until required for administration. All formulations were prepared daily to provide the required daily dosages at a constant volume dosage, 10ml/kg bodyweight at each of the two daily administrations (i.e., 20ml/kg bodyweight/day).
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: Formulated to provide required daily dose at a constant volume dosage at each of the two daily administrations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined by a trial preparation made up using the same methods employed throughout the study. The stability of the test material in the vehicle was assessed for the lowest and highest concentration, after 24 and 48 hours storage at ambient temperature. In addition, samples of each formulation prepared for administration on Day 1 and on one occasion in each of Weeks 16 and 26 of treatment were retained and analysed for the test material. The method of analysis was a modification of that supplied by the Sponsor.

Analyses confirmed that the intended concentrations at each dose level were provided during the study.

Duration of treatment / exposure:

at least 26 weeks

Frequency of treatment:

Daily (7 days/week) approximately two hours after lights were switched off and approximately two hours before the lights switched on each day.

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Dose / conc.:

3 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 male, 20 Female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Sponsor selected dose levels.
- Rationale for animal assignment (if not random): Random

- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weeks 1, 4, 8, 12, 16, 20 and 24

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment, Weeks 16 and 24
- Dose groups that were examined: Groups 1 (Control) & 4 (High)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 25 weeks of treatment
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes
- How many animals: 10 male, 10 female
- Parameters examined: Packed cell volume, Haemoglobin concentration, erythrocyte count, total and differential Leucocyte count, platelet count. The following characteristics were calculated: mean cell haemoglobin, mean cell volume and mean cell haemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 25 weeks treatment
- Animals fasted: Yes
- How many animals: 10 male,10 female
- Parameters examined.: Alkaline phosphatase activity, ALT, AST, GGT, OCT, Urea nitrogen, creatine concentration, glucose concentration, total bilirubin concentration, total protein concentration, electrophoretic protein fractions, sodium, potassium, and calcium concentrations, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. Organs were weighed. External and cut surfaces of the organs and tissues were examined as appropriate. Abnormalities and interactions were noted and the required tissue samples preserved in the fixative.

HISTOPATHOLOGY: Yes
Adrenals; aorta (thoracic); brain; caecum; colon; duodenum; epididymides; eye and optic nerve, left; heart; ileum; ovaries; pancreas; pituitary; prostate; rectum; salivary gland-submandibular, left; sciatic nerve, left; skeletal muscle (thigh); spinal cord; spleen; jejunum; kidneys; liver; lungs with mainstem bronchi; lymph nodes; mammary gland-caudal; oesophagus; sternum; stomach; testes; thymus; thyroid with parathyroids; trachea; urinary bladder; uterus with cervix.

Tissues were examined for all animals of Control (Group 1) and High (Group 4) dose groups sacrified on completion of the scheduled treatment period and for all animals killed or dying during the study. Tissues reported at macroscopic examination as being abnormal were examined for all animals.

Other examinations:

Organ weights: Adrenals; brain; caecum (with contents); caecum (without contents); heart; kidneys; liver; lungs with mainstem bronchi; ovaries; pituitary; prostate; spleen; testes; thymus; thyroid with parathyroids; uterus with cervix

Statistics:

Standard deviations were calculated, as considered appropriate, using the sample statistic.

For food consumption, bodyweights (and any data derived from these), water consumption and organ weights, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.

The significance of inter-group differences in haematology (excluding the incidence of morphological abnormalities evident in the blood smears) and blood chemistry was assessed by Student's 't'-test using a pooled error variance.

Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.

Unless stated, group mean values or incidences for the treated groups were not significantly different from the controls (p>0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No signs or changes in behavior indicative of a treatment related effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 7 deaths in treatment groups during the study, none were considered to be related to treatment with sucralose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statittically significant changes in the overall weight gain of sucralose treated animals when compared to controls.

Analysis of covariance conducted by Sponsor allowing for food consumption and initial bodyweight, showed a small (up to 4.9%) but statistically significant decrease relative to controls in adjusted body weight of males receiving the high dose only during weeks 23-26.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences in cumulative group mean food consumption when compared with controls although the mean total food intake per animal of the high dosage males was slightly higher (4.9%).

Food efficiency:

no effects observed

Description (incidence and severity):

Male and female rats receiving the high dose demonstrated a slight, not statistically significant reduction in group mean food conversion efficiency (Male 5.7% reduction, female 6.8% reduction)

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was higher (11-34%) in both sexes in a dose related manner amongst all treatment groups apart from the male low dose group; these differences were statistically significant in the medium and high dose groups.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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):

Treated animals had statistically significantly (p<0.01) higher absolute and bodyweight-relative full caecal weights, and significantly increased empty caecal weights with the exception of females in the low dose group; a clear dosage-relationship was apparent for the increased full and empty caecal weights, both on an absolute and bodyweight-relative basis for both sexes. The effect was larger for the full weights than for the empty weights, indicating a more marked effect on caecal contents than on caecal tissue.

The body weight-relative kidney weights of animals were slightly higher (p<0.05) than those of control animals in the 3000mg/kg/day dose group (highest dose group).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination revealed distended or elongated caecum among several animals of each sex in the 3000 and 1500mg/kg groups and males in the 750mg/kg group. These findings are consistent with the increased weights recorded for this organ among treated animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no changes which could be associated with the caecal effects noted at macroscopic examination or the slightly high bodyweight-relative kidney weights.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

LOEL

Effect level:

ca. 750 - <= 3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

organ weights and organ / body weight ratios

water consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

> 3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

organ weights and organ / body weight ratios

water consumption and compound intake

Remarks on result:

other:

Remarks:

In absence of any histopathological or other findings of an adverse nature, none of the changes observed are considered to be toxicologically significant.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

other: caecum

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Selected Organ Weights Relative to Bodyweights-Group Mean Values (%) for Animals Killed After 26 Weeks of Treatment

Organ	Males				Females
	Control	750 mg/kg/day	1500 mg/kg/day	3000 mg/kg/day	Control	750 mg/kg/day	1500 mg/kg/day	3000 mg/kg/day
Caecum-Full	0.874	1.427 a	2.006 a	2.327 a	1.394	1.671 a	2.031 a	2.600 a
Caecum-Empty	0.247	0.337 a	0.367 a	0.454 a	0.379	0.388	0.459 b	0.486 a
Kidney	0.748	0.741	0.790	0.791 b	0.794	0.777	0.801	0.849 b

Table 2: Water Consumption-Group Mean Values (ml/animal/day)

Week Number	Males				Females
	Control	750 mg/kg/day	1500 mg/kg/day	3000 mg/kg/day	Control	750 mg/kg/day	1500 mg/kg/day	3000 mg/kg/day
1	24	23	24	28 c	20	23 x	25 y	26 z
4	35	35	38	42 c	28	32	32	35 b
36	36	36	45 x	47 z	33	36	36	39
12	37	36	42	49 c	34	34	36	41
16	40	42	49 a	53 c	32	38	39 b	43 c
20	39	39	49 a	55 c	33	37	42 b	42 b
24	34	36	44 a	54 c	36	40	43	46 b

a=p<0.05; b=p<0.01; c=p< 0.001 (Dunnett’s Test)

x=p<0.05; y=p<0.01; z=p<0.001 (Behren’s-Fisher Test)

Conclusions:

The repeated dose toxicity of the test item via oral application on rats was determined in a GLP study according to FDA guidelines. None of the changes observed are considered to be toxicologically significant. It is concluded that repeated oral administration of Sucralose does not cause adverse effects in rats.

Executive summary:

It is concluded that the oral administration of sucralose to CD rats at dosages of up to 3000 mg/kg/day, for 26 weeks resulted in only a few minor treatment-related changes. These included a dosage-related enlargement of the caecum and a small increase in bodyweight-relative kidney weight at 3000 mg/kg/day. No histopathological changes were noted in either organ and the kidney weight changes did not correspond to any haematological or clinical chemistry changes. The dose related increase in water intake, slight decrease in adjusted bodyweight at 3000 mg/kg/day and caecal enlargement are not uncommon findings when high doses of poorly absorbed materials are administered to rats. Therefore, in the absence of any histopathological or other findings of an adverse nature, none of the changes observed are considered to be toxicologically significant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

gastrointestinal tract

Organ:

other: caecum

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Dosage related caecal enlargement, evident in this study as increased full and empty absolute and bodyweight-relative caecal weights and as macroscopically observed elongation or distension, is a consistent finding in rat studies involving oral administration of sucralose. Similar findings have been noted in rats administered large amounts of a broad range of carbohydrates, all of which are poorly absorbed from the gastrointentinal tract. Caecal enlargement is considered to be a physiological response to the increased osmolarity of the caecal contents and not to be of toxicological significance. This is supported by the lack of histopathological changes in this study.

Additional information

Oral route of exposure is most significant route to humans and most relevant repeated dose toxicity endpoint.

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 specific target organ toxicity following repeated exposures since no significant toxicity or histopathological effects were noted following repeated oral exposure in rats.