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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No increase in revertant colonies was obtained with Sucralose when tested using various strains of Salmonella and E. coili in the absence and presence of metabolic activation.  The test substance was determined to be non-mutagenic.

The positive results observed in a L5178Y TK mouse lymphoma mutagenesis assay at 10,000 ug/ml (with and without S9) and at 7500 ug/ml (with S9), are likely a consequence of test conditions that far exceed guideline concentrations, resulting in toxicity through biologically irrelevant concentrations. Tests conducted in accordance with modern test guidelines are usually limited to a maximum concentration of 2000 ug/ml, which would provide a negative result in the current study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The current study predates the adoption of the OECD testing guidelines.

GLP compliance:

not specified

Remarks:

Predates GLP

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (Sucralose)
- Source and lot/batch No.of test material: C/327/9470/02
- Supplied by Tate and Lyle


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 0-4 degrees C, protected from light

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Solutions of each test material were prepared in sterile distilled water.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

16, 80, 400, 2000, 10000 ug/plate

Vehicle / solvent:

sterile distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10^-6

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: two

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

Sucralose was tested for mutagenic activity to bacteria according to OECD guideline No. 471. No increase in revertant colonies was obtained with Sucralose when tested using various strains of Salmonella in the absence and presence of metabolic activation. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The current test predates the adoption of OECD test guidelines but follows the same scientific principles

Deviations:

yes

Remarks:

Only two strains of Salmonella were used.

GLP compliance:

not specified

Remarks:

Predates GLP

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch Number: P3/49-557

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli, other: E coli W3110 (pol A+) and p3478 (pol A-)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

0.5ug,1ug,10ug,100ug, 500ug,1000ug/plate.
The four lowest doses exhibited little or no toxicity, the higher doses exhibited some toxicity.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

N-dimethylnitrosamine

methylmethanesulfonate

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable):1*10^8 cells / plate

DURATION
Cell cultures in molten agar with appropriate concentrations of test substance were poured on minimal agar plates incubated for 48 hours at 37 degrees C and scored for the number of colonies growing per plate.

NUMBER OF REPLICATIONS: One

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Not specified
- Any supplementary information relevant to cytotoxicity: The four lowest doses showed very little or no toxicity. The other doses may have either exhibited complete or slight toxicity.

Key result

Species / strain:

E. coli, other: W3110 (pol+) W3478 (Pol-)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Report indicates very little toxicity or no toxicity was observed with the four lowest doses. The other doses may have either exhibited complete or slight toxicity.

Conclusions:

Sucralose was tested for mutagenic activity to bacteria according to OECD guideline No. 471. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Principles of the test are equivalent to TG490, however the study predates modern guidelines

Deviations:

yes

Remarks:

maximum dose levels and cytotoxicity quantification methods deviate significantly from recent guidelines,extremely high concenterations may influence results through non-compound related effects such as precipitation and osmolarity.

GLP compliance:

yes

Type of assay:

other: L5178Y TK mouse lymphoma mutagenesis assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C327/9470/02
- Purity 100% +/- 2


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 Degreec C with dessication, Protected from light


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in sterile deionised water

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 Liver microsomes

Test concentrations with justification for top dose:

Without S9 - 10,000ug/ml, 7500ug/ml, 5625ug/ml, 4219ul/ml, 3164ug/ml, 2373ug/ml, 1780ug/ml, 1335ug/ml.
With S9 - 10,000ug/ml, 7500ug/ml, 5625ug/ml, 4219ul/ml, 3164ug/ml, 2373ug/ml, 1780ug/ml, 1335ug/ml, 1001ug/ml, 751ug/ml.

Study Predates modern guidelines, the only concentrations applicable to current guidelines are 1780ug/ml, 1335ug/ml, 1001ug/ml, 751ug/ml.

Vehicle / solvent:

sterile deionised water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension;
- Cell density at seeding (if applicable): 1x10^6 cells/ml

DURATION
- Preincubation period: 72hrs
- Exposure duration:4hrs
- Expression time (cells in growth medium):48hrs

SELECTION AGENT (mutation assays): selective media containing BUdR or Trifluorothymidine


DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Evaluation criteria:

Mutation frequency is determined by dividing the average number of colonies present in the restrictive media plates by the average numer of colonies x10^4 in the corresponding vehicle controls and multiplying the resulting quotient by a factor of two.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

10,000ug/ml without s9, 10,000ug/ml & 7500ug/ml with S9

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Limited biological relevance due to exessivley high test concentration.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Doses: Without S9 - 5,625ug/ml, 4219ul/ml, 3164ug/ml, 2373ug/ml, 1780ug/ml, 1335ug/ml. With S9 - 7500ug/ml ,5,625ug/ml, 4219ul/ml, 3164ug/ml, 2373ug/ml, 1780ug/ml, 1335ug/ml, 1001ug/ml, 751ug/ml.

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Lower, more biologically relevant concentrations show no evidence of mutagenicity.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Dose concentrations far exceed those reccomended in recent test guidelines and as such may result in observations of toxicology caused by secondary mechanisms with little biological relevance, such as changes on pH or osmolarity.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative suspension growth.

Remarks on result:

not determinable because of methodological limitations

Remarks:

Positive results observed at concentrations five times higher than current guidline maximums, the biological relevance of this result is in question.

Conclusions:

The genetic toxicity of the test item on mammalian cells was determined in a GLP study similar to the OECD guideline 490. In concentrations up to 2000 µg/ml no adverse effects were observed, which would provide a negative result in the current study.

Executive summary:

The positive results observed in a L5178Y TK mouse lymphoma mutagenesis assay at 10,000 ug/ml (with and without S9) and at 7500 ug/ml (with S9), are likely a consequence of test conditions that far exceed guideline concentrations, resulting in toxicity through biologically irrelevant concentrations. Tests conducted in accordance with modern test guidelines are usually limited to a maximum concentration of 2000 ug/ml, which would provide a negative result in the current study.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The genetic toxicity of the test item on mammalian cells was determined in a study according to the OECD guideline 474. It is concluded that, under the conditions of the test, there was no biologically significant evidence of treatment induced damage to chromosomes or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice after 24, 48, or 72 hours of exposure to sucralose.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1984 version of OECD 474

GLP compliance:

not specified

Remarks:

Predates GLP

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: From Sponsor / Batch Number 163003
- Purity: 99.4% (HPLC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 0-4 degrees C in the dark
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in distilled water up to maximum concentration of 330 mg/ml
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Dissolved in water

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Preparations of test compound were mixed with dosing vehicle (distilled water) to achieve appropriate concentrations

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 17-24g
- Housing: Polypropylene cages, single sex, groups of 5
- Diet: ad libitum
- Water:ad libitum
- Acclimation period: minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (+/-2)
- Humidity (%): 55% (+/-20%)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Distilled, sterile water.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Test compound dissolved in sterile distilled water on the day of use.

Duration of treatment / exposure:

Single dose, termination and sampling after 24, 48 and 72 hours.

Frequency of treatment:

single dose

Post exposure period:

24, 48 and 72 hours.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

5 animals per sex per dose per time point (24, 48 & 72 Hrs)

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Chlorambucil
- Route of administration: oral
- Doses / concentrations: 30 mg/kg in 10% ethanol

Tissues and cell types examined:

Both femurs of each mouse were dissected out following termination by carbon dioxide gassing and cervical dislocation.
Marrow cells were flushed into clean centrifuge tubes with 2.5 ml foetal calf serum, the suspension was centrifuged (1000 RPM, 5 minutes) and the bulk of supernatant removed.

Three smear slides were prepared from the cells of each animal and stained manually using the Schmid staining technique.

Slides were evalulated blind to eliminate bias, approximately, 2000 mature and polychromatic erythrocytes were scored for each animal, at least 1000 of each cell type will be scored for the presence of micronuclei.

Details of tissue and slide preparation:

Both femurs of each mouse were dissected out following termination by carbon dioxide gassing and cervical dislocation.
Marrow cells were flushed into clean centrifuge tubes with 2.5 ml foetal calf serum, the suspension was centrifuged (1000 RPM, 5 minutes) and the bulk of supernatant removed.

Three smear slides were prepared from the cells of each animal and stained using the Schmid staining technique.

Slides were evalulated blind to eliminate bias, approximately, 2000 mature and polychromatic erythrocytes were scored for each animal, at least 1000 of each cell type will be scored for the presence of micronuclei.

Evaluation criteria:

A total of ~2000 mature and polychromatic erythrocytes per animal were scored for the presence or abcence of micronuclei, the study aimed to score 1000 each of polychromatic and mature erythrocytes where possible, only deviating where there was a significant shift in the ratio of polychromatic to mature cells.

The counts for mature erythrocytes provide a check on the results of the younger cells, the ratio of mature cells to polychromatic cells was calculated for each animal as an indicator of gross toxicity.

Statistics:

Comparisons of the number of micronuclei scored in polychromatic cells between the control amd treatment groups were made using the Mann-Whitney U test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No Treated animals showed any adverse reactions to treatment, all animals survived to termination.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Frequencies of micronucleated polychromatic erythrocytes in treated animals at 24,48 and 72 hours post exposure were similar to those in concurrent controls, there were no significant differences observed between sexes in any group.

Statistical analysis indicated that there was a significant increase in the frequency of micronucleated polychromatic erythrocytes at a dosage of 1000mg/kg and 72hrs post exposure. This was caused by an unusually low occurance of micronuclei in the concurrent control animals (9/10 mice showed no micronuclei), no sucralose treated animals gave values in excess of 3.2 micronuclei per 1000 cells scored, therefore this apparent increase is not considered to to be of biological significance.

Statistically and biologically significant increases in the number of micronucleated polychromatic erythrocytes over control values were seen in positive control group animals given chlorambucil at 30mg/kg, demonstrating the sensitivity of the system.

Exposure period	24 hrs				48 hrs			72 hrs
Group	1	2	3	4	1	2	3	1	2	3
Mean Micronucleated cells / 1000 polychromatic cells (S.D)	0.7	0.7	0.4	23.7	0.7	1	0.5	0.1	1.2	0.6
Range	0.0-2.0	0.0-2.8	0.0-1.7	10.3-32.0	0.0-2.0	0.0-2.0	0.0-1.8	0.0-0.9	0.0-3.3	0.0-2.7
Ratio Polychromatic cells:mature cells	1	1	1	1	1	0.9	1	1	0.9	1

Group 1	distilled water
Group 2	1000mg/kg Sucralose
Group 3	5000mg/kg Sucralose
Group 4	Chlorambucil

Conclusions:

The genetic toxicity of the test item on mammalian cells was determined in a study according to the OECD guideline 474. It is concluded that, under the conditions of the test, there was no biologically significant evidence of treatment induced damage to chromosomes or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice after 24, 48, or 72 hours of exposure to sucralose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on a review paper by Brusick, D. et al. (2010), sucralose is not bio-reactive, is not metabolized to reactive intermediates and does not bioaccumulate in experimental animals or humans. These properties support the absence of adverse effects seen in the in vitro and in vivo genotoxicity studies, as well as the negative results in long term carcinogenicity studies conducted in rats.

Sucralose is currently approved for use in over 80 countries as a food ingredient. Comprehensive safety tests show no significant acute, subchronic or chronic toxicity at levels above expected human intakes. No adverse reactions were observed with sucralose intakes up to 16,000 mg/kg/day in mice or 10,000 mg/kg/day in rats-a dosage equivalent in sweetness to more than 1000 pounds of sucrose administered in a single day to a 165 pound adult (Brusick et al, 2010).

Additional information

Brusick et al (2010) found in a review of mugenicity studies with sucralose that all mutagenicity studies reported negative results except for the mouse lymphome assay which identified the results as equivocal. The 10 mg/ml is an extremely high concentration that may give rise to alterations producted through treatment conditions unrelated to direct DNA reactivity. In fact, under the current evaluation criteria that no longer relies on using fold increase rules but instead applies mutation frequency. When this new criterion was applied to the mouse lympohma study, Brusick (2010) found that the results would not be identified as positive with or without metabolic activation.

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 genotoxicity since both in vitro and in vivo mutagenicity assays using sucralose were considered to be negative under current criteria. In addition, a negative carcinogenicity study in rats further supports the non-genotoxic potential of the sucralose compound.