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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 June 1993 - 1 July 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Objective of study:

absorption

other: Clearance

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to compare the absorption and overall systemic exposure (by AUC comparison) of rats to sucralose after administration of sucralose either in the diet or by oral gavage, at dose levels used in previous safety studies. The study design was such that sucralose was administered for a period of 28 days and the absorption characteristics following both forms of administration were measured at the beginning and end of the test period. A direct comparison of relative systemic exposures from this study will aid in the interpretation of the findings in previous studies and should give support to the no-effect level (NOEL) obtained in the original gavage study. In addition, the level of absorption of sucralose following gavage and dietary administration in the rat model over a 28-day period provides important toxicokinetic information applicable to the assessment of the overall safety of sucralose.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

259-952-2

EC Name:

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

Cas Number:

56038-13-2

Molecular formula:

C12H19Cl3O8

IUPAC Name:

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

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor provided test material, batch 92-SC-157
- Expiration date of the lot/batch: Not provided
- Purity: 99.6%
- Purity test date: 19/3/1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated at 4 Degrees C, protected from light.
- Stability under test conditions: Sample analysed by sponsor upon completeion of study, found to be satisfactory
- Solubility and stability of the test substance in the solvent/vehicle: Analytical results show that the test material was stable from time of preparation to completion of dosing in both the diet and gavage solution.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:

Diet: A mixture of 37.5% powdered test material and diet was added to further quantities of diet to provide the required concentrations. Batches of premix and each test diet were prepared weekly.

Gavage: Solutions of test material in distilled water were prepared weekly so that each animal recieved a dose volume of 10ml/kg bodyweight.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

CD-1

Details on species / strain selection:

Sprague-Dawley rats were selected for the study since it has been shown in previously conducted studies using this test substance that animals of this species have a reduced bodyweight gain in comparison to controls when fed high levels of sucralose in the diet, whereas no reduction was seen in the same species administered sucralose by gavage. Therefore, rats were administrated sucralose in the diet and by gavage to ascertain the exposure of the rat to this compound following these two forms of administration.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: Approximately 28 days
- Weight at study initiation: males:125-196g, females 101-146g
- Housing:Individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C +/- 2
- Humidity (%): 50% +/-10%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12 hour light, 12 hour dark

Administration / exposure

Route of administration:

other: Oral: Gavage & Oral: Diet

Vehicle:

water

Remarks:

For Oral: Gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of test material in distilled water were prepared weekly so that each animal recieved a dose volume of 10ml/kg bodyweight.

DIET PREPARATION
- Rate of preparation of diet (frequency): Batches of premix and each test diet concentration were prepared weekly.
- Mixing appropriate amounts with (Type of food): A mixture of 37.5% powdered test material and rodent diet (SDS modified maintenance diet) was added to further quantities of diet to provide the required concentrations.

Duration and frequency of treatment / exposure:

Sucralose was administered daily to the rats either by gavage or the treated diet for 28 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4 000 mg/kg bw/day

Remarks:

Gavage

Doses / concentrations 2

Dose / conc.:

1 333 mg/kg bw/day

Remarks:

Gavage

Doses / concentrations 3

Dose / conc.:

1 333 mg/kg bw/day (nominal)

Remarks:

Dose calculated from 1% in diet for a period of 28 days - based on findings of a 2 year carcinogenicity study.

Doses / concentrations 4

Dose / conc.:

4 000 mg/kg bw/day (nominal)

Remarks:

Dose calculated from 3% in diet for a period of 28 days - based on findings of a 2 year carcinogenicity study.

No. of animals per sex per dose / concentration:

A total of 24 male and 24 female animals was used for each of the two dosages in the gavage phase of the study.

A total of 18 male and 18 female animals were used for each of the two dose levels in the dietary administration phase of the study

Two groups of 6 male and 6 female rats each were used as controls . One group had access to control diet only and the other had access to control diet and was dosed by gavage once daily with dose vehicle.

Control animals:

yes, concurrent vehicle

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels used in previous safety studies
- Rationale for animal assignment (if not random): Random

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled (delete / add / specify): blood, plasma,
- Time and frequency of sampling: Blood samples were taken on Day 1 from gavage dose groups and Day 7 from dietary administration groups and then again from the same animals on Day 28. Blood samples were taken on Day 7, and not Day 1, from rats administered dietary sucralose due to the poor palatability of diet containing sucralose. Previous sucralose dietary administration studies have shown that due to the poor palatability there is an initial severe aversion to the test diet on Day 1, with a resultant decrease in food consumption (LSR Report No 85/MSP040/334). Afterwards, a degree of acclimatisation takes place although not to control levels. Thus, the collection of blood samples on Day 1 for the dietary administration groups would have given unrepresentative low levels of sucralose in plasma.
- Other: On the designated study days, blood samples were taken over a 24-hour dosing interval with a maximum of 3 blood samples taken from each rat, in order to minimise the physiological disturbance to the animal by removal of blood. Blood samples were obtained from a tail vein of the rat. Blood was collected into heparinsed tubes and then centrifuged to provide plasma.

Statistics:

In order to obtain estimates of areas under the plasma concentration-time curves to 24 hours, the trapezoidal approach was used on the mean values at each time point. Values recorded as below the level of quanitification were input as 0.1 ug/ml (half-way between zero and the limit) except for time 0 which was taken as zero on Day 1. Variances were also obtained using standard statistical techniques allowing comparisons between the groups to be carried out.

The mean AUC24 and their variances were dose adjusted to 1 mg/kg. Chi-square tests for heterogeneity were carried out between the dose-adjusted estimates for the two increasing dose levels. Comparisons were also made between the sexes and between the days for each dose level separately. These comparisons were performed for the oral gavage and dietary data separately. In addition, comparisons were made between the two routes of administration on Day 28.

The plasma concentrations which gave the maximum mean concentration of sucralose were used to estimate Cmax. Analysis of variance was carried out on the dose adjusted values for each route separately with dose level, sex, day of sampling and their interactions as factors. The significance level of the dose level group effect for the dose adjusted variable can be considered as a test for proportionality. A further analysis was performed on Day 28 data to make a comparison between the two routes.

Results and discussion

Preliminary studies:

In all previous dietary administration studies with sucralose, rats hae shown a reduction in food intake and decrease in bodyweight gain associated with the poor palatability of sucralose. In contrast, when sucralose was administrated by oral gavage, thus avoiding diet palatability effects at a dose level of at least 2000 mg/kg/day for 13 weeks, there were not statistically significant decreases in food intake or bodweight gain.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Analysis of plasma revealed that both gavage and dietary-treated animals were exposed systemically to high levels of sucralose throughout the 28-day period.

Main ADME results 2

Type:

distribution

Results:

No systemic accumulation of sucralose was observed following high dose administration.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

See Tables 1 and 2.

The AUCs on Day 7 for animals administered sucralose in the diet at both the 1% and 3% levels were similar to the matched gavage doses of 1333 and 4000 mg/kg/day on Day 1. This is apparent since the actual intakes of the 1% and 3% groups were approximately equivalent to 1200 and 3600 mg/kg/day. A similar comparison of the normalised AUC values for the gavage and dietary fed animals on Day 28 also revealed similar exposures with no statistically significant differences. Based on these data, it is clear that systemic sucralose exposure of the rat is similar at comparable dosages following either gavage or dietary administration.

The relative AUC of sucralose at the higher dose levels was reduced in comparison to the lower doses. At the higher dose levels, the larger amount of poorly absorbed compound like sucralose in the gut would be expected to decrease the extent of intestional absorption and could also reduce the level of nutrient absorption.

For both dietary and gavage administration, the higher dose levels was associated with singificantly lower normalised AUCs, therefore, the evidence suggests that the AUC is not proportional to dose administered.

Details on distribution in tissues:

Not determined

Details on excretion:

Not determined

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

Cmax: 30.3 ug/ml (males); 32.2 ug/ml (females)

Remarks:

Gavage 4000 mg/kg dose group-Day 1

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

Cmax: 38.7 ug/ml (males); 35.4 ug/ml (females)

Remarks:

Gavage 4000 mg/kg dose group-Day 28

Toxicokinetic parameters 3

Key result

Toxicokinetic parameters:

Cmax: 15.4 ug/ml (males); 12.4 ug/ml (females)

Remarks:

Dietary 3% dose group-Day 7

Toxicokinetic parameters 4

Key result

Toxicokinetic parameters:

Cmax: 10.5 ug/ml (males); 9.7 ug/ml (females)

Remarks:

Dietary 3% dose group-Day 28

Toxicokinetic parameters 5

Key result

Toxicokinetic parameters:

Cmax: 18.2 ug/ml (males); 14.6 ug/ml (females)

Remarks:

Gavage 1333 mg/kg dose group-Day 1

Toxicokinetic parameters 6

Key result

Toxicokinetic parameters:

Cmax: 18.0 ug/ml (males); 12.8 ug/ml (females)

Remarks:

Gavage 1333 mg/kg dose group-Day 28

Toxicokinetic parameters 7

Key result

Toxicokinetic parameters:

Cmax: 6.2 ug/ml (males); 4.8 ug/ml (females)

Remarks:

Dietary 1% dose group-Day 7

Toxicokinetic parameters 8

Key result

Toxicokinetic parameters:

Cmax: 4.1 ug/ml (males); 4.2 ug/ml (females)

Remarks:

Dietary 1% dose group-Day 28

Toxicokinetic parameters 9

Key result

Toxicokinetic parameters:

Tmax: 0.5 hours (males and females)

Remarks:

Gavage 4000 mg/kg dose group-Day 1

Toxicokinetic parameters 10

Key result

Toxicokinetic parameters:

Tmax: 1 hour (males); 3 hours (females)

Remarks:

Gavage 1333 mg/kg dose group-Day 1

Toxicokinetic parameters 11

Key result

Toxicokinetic parameters:

Tmax: 1 hour (male); 0.5 hour (female)

Remarks:

Gavage 4000 mg/kg dose group-Day 28

Toxicokinetic parameters 12

Key result

Toxicokinetic parameters:

Tmax: 1 hour (male and female)

Remarks:

Gavage 1333 mg/kg dose group-Day 28

Toxicokinetic parameters 13

Key result

Toxicokinetic parameters:

AUC: 248.2 ug h/ml (males); 254.4 (ug h/ml (females)

Remarks:

Gavage 4000 mg/kg dose group-Day 1

Toxicokinetic parameters 14

Key result

Toxicokinetic parameters:

AUC: 106.6 ug h/ml (males); 97.7 ug h/ml (females)

Remarks:

Gavage 1333 mg/kg dose group-Day 1

Toxicokinetic parameters 15

Key result

Toxicokinetic parameters:

AUC: 266.4 ug h/ml (males); 241.7 ug h/ml (females)

Remarks:

Gavage 4000 mg/kg dose group-Day 28

Toxicokinetic parameters 16

Key result

Toxicokinetic parameters:

AUC: 126.7 ug h/ml (males); 95.2 ug h/ml (females)

Remarks:

Gavage 1333 mg/kg dose group-Day 28

Toxicokinetic parameters 17

Key result

Toxicokinetic parameters:

AUC: 266.6 ug h/ml (males); 208.2 ug h/ml (females)

Remarks:

Dietary 3% dose group-Day 7

Toxicokinetic parameters 18

Key result

Toxicokinetic parameters:

AUC: 187.8 ug h/ml (males); 177.8 ug h/ml (females)

Remarks:

Dietary 3% dose group-Day 28

Toxicokinetic parameters 19

Key result

Toxicokinetic parameters:

AUC: 112.5 ug h/ml (males); 82.1 ug h/ml (females)

Remarks:

Dietary 1% dose group-Day 7

Toxicokinetic parameters 20

Key result

Toxicokinetic parameters:

AUC: 80.4 ug h/ml (males); 73.5 ug h/ml (female)

Remarks:

Dietary 1% dose group-Day 28

Metabolite characterisation studies

Metabolites identified:

not measured

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Similarity in peak plasma concentrations on Days 1 and 28 for the gavage administration dose group is an indication that no systemic accumulation of sucralose takes place following repeated single oral doses up to 4000 mg/kg/day. This finding is reinforced by the AUC data (actual and normalised) which show no notable change betwee dose 1 and dose 28 at steady state and similarly between dietary treated animals AUC data on Day 7 and Day 28. These results indicate that the AUC characteristics of sucralose do not change appreciably following chronic high dose level or administration by either gavage or dietary administration, further showing that there is essentially no change in bioavailability or clearance.

Any other information on results incl. tables

Clinical Signs: Two animals assigned to gavage phase were found dead but it was concluded that they died due to inadvertent administration of the test solution into the lung. No clinical signs were observed in any dose group via gavage or dietary administration other than salivation in all animals of the 4000 mg/kg/day gavage dose group lasting only about 30 minutes after dosing.

Bodyweight: After 28 days of treatment, mean group bodyweight gain for gavage administred animals were similar to those of the controls. A decrease in overall bodyweight gain of those administered sucralose in the diet was achieved after 28 days. In males, there was a 15% decrease in mean bodyweight gain in the 3% dietary group and a 9% decrease in the 1% dietary group in comparison to control. For females, there was an 11% decrease in bodyweight gain in the 3% dietary group and a 16% decrease in the 1% dietary group.

Food Consumption: The overall food consumption for animals dosed by gavage were similar to the controls over the 28 -day period. Those in the dietary group showed both a weekly and cumulative decrease in food intake in both sexes of the dose groups relative to controls.

Table 1 - Mean pharmacokinetic parameters following gavage and dietary administration of sucralose at two dosage levels to male rats

Parameter	Gavage				Diet
	1333 mg/kg		4000 mg/kg		1% (1175 mg/kg)	1% (801 mg/kg/day)	3% (3562 mg/kg/day)	3% (2583 mg/kg/day)
	Day 1	Day 28	Day 1	Day 28	Day 7	Day 28	Day 7	Day 28
Cmax (ug/ml)	18.2	18.0	30.3	38.7	6.2	4.1	15.4	10.5
Tmax (hours)	1.0	1.0	0.5	1.0	9.0	12.0	12.0	12.0
Cmin (ug/ml)	0.3	1.6	0.9	3.1	2.2	2.0	5.4	4.1
AUC (ug h/ml)	106.6	126.7	248.2	266.4	112.5	80.4	266.6	187.8

 

Table 2 - Mean pharmacokinetic parameters following gavage and dietary administration of sucralose at two dosage levels to male rats

Parameter	Gavage				Diet
	1333 mg/kg		4000 mg/kg		1% (1206 mg/kg)	1% (914 mg/kg/day)	3% (3580 mg/kg/day)	3% (2753 mg/kg/day)
	Day 1	Day 28	Day 1	Day 28	Day 7	Day 28	Day 7	Day 28
Cmax (ug/ml)	14.6	12.8	32.2	35.4	4.8	4.2	12.4	9.7
Tmax (hours)	3.0	1.0	0.5	0.5	12.0	12.0	12.0	12.0
Cmin (ug/ml)	0.3	2.1	1.0	3.3	1.6	1.9	4.8	4.0
AUC (ug h/ml)	97.7	95.2	254.4	241.7	82.1	73.5	208.2	177.8

Applicant's summary and conclusion

Conclusions:

To compare the absorption and overall systemic exposure of rats to sucralose, a GLP, well-documented, scientifically valid study was designed such that the test substance was administered to rats by either gavage or the diet for a period of 28 days and the absorption characteristics were measured at the beginning and end of the test period. Under the conditions of this study, it was concluded that overall systemic exposure to sucralose is similar following administration either as a bolus gavage dose or via the diet as demonstrated by the AUC data. The clearance characteristics of sucralose in the rat showed that even at relatively high concentrations, systemic accumulation does not occur. For the gavage treated animals, sucralose was esesentially totally cleared within the 24-hour dosing period. Additionally, it is clear from this study that the decrease in mean bodyweight gain seen with dietary administration is not produced by gavage doses despite having a similar systemic exposure. Thus, direct comparisons can be appropriately made between gavage and dietary administration studies with sucralose.