6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide, potassium salt

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1973-Dec 01 through 1976-Dec-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Scientifically reliable study with sufficient information for evaluation and assessment considering the date of study and report. Compared to current guideline requirements fewer reproductive and developmental parameters were examined but F1b and F2b and F3a/b generations were included and examined including parameters of subacute toxicity and teratogenicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1976

Report date:

1976

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

3 generations instead of two, including teratogenicity parts, top dose exceeded current limit dose of 1000 mg/kg bw/day

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

Study performed prior to implementation of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-1,2,3-oxathiazine-4(3-H)-one-2,2-dioxide potassium salt

IUPAC Name:

6-methyl-1,2,3-oxathiazine-4(3-H)-one-2,2-dioxide potassium salt

Constituent 2

Reference substance name:

6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide, potassium salt

EC Number:

259-715-3

EC Name:

6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide, potassium salt

Cas Number:

55589-62-3

Molecular formula:

C4H5NO4S.K

IUPAC Name:

potassium 6-methyl-2,2,4-trioxo-3,4-dihydro-1,2λ⁶,3-oxathiazin-3-ide

Constituent 3

Reference substance name:

Acesulfame potassium

IUPAC Name:

Acesulfame potassium

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report):Hoe 0-95 K
- Substance type: sweetener
- Physical state: crytsalline powder
- Analytical purity: 99.8%
- Impurities (identity and concentrations): Flouride < 1 pm, calcium 10 - 20 ppm
- Lot/batch No.: 4 batches not further specified
- Stability under test conditions: stable

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Civo colony, Zeist, The Netherlands
- Age at study initiation: jung adult (P) x 12 wks; weanling (F1a/1b)x10 wks; weanlings (F2a/2b) x 10 wks, weanlings (F3a/3b) x 4 wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing: 5 rats/cage
- Use of restrainers for preventing ingestion: not applicable
- Diet: ad libitum (standard laboratory stock diet)
- Water: ad libitum (tap water)
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24-26° C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h /12 h

IN-LIFE DATES:
From: 1973-Oct-(day not stated)
To: not stated

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 - 3 weeks
- Mixing appropriate amounts with (Type of food): laboratory stock diet
- Storage temperature of food: ambient temperature

Details on mating procedure:

- M/F ratio per cage:1:1
- Length of cohabitation:3 weeks
- Proof of pregnancy: no data
- After successful mating each pregnant female was caged (how): individually until litters were weaned
- Any other deviations from standard protocol: see further details on study schedule

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

Through three succesful generations (F0, F1a/b, F2 a/b, F3a/b). See further details on study design

Frequency of treatment:

Daily

Details on study schedule:

See further details on study design

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.3%, 1.0%, 3.0% (0, 3000, 10000, 30000 ppm (corresponding to about 150, 500, 1500 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

F0: 20 males per group, 30 females per group
Further details see further details on study design

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
From a previous sub-chronic toxicity study with rats it appeared that the feeding of Hoe 0-95 K at the very high dietary level of 10% only caused slight growth depression, caecal enlargment and increased relative weights of the liver and kidneys, not accompanied by histo-logical changes
No adverse effects an foetuses were noticed when the compoundwas administered to pregnant rats at levels up to 3% in the diet.

- Other:
The study started with 80 male and120 female, newly weaned, albino rats from the CIVO-colony of Wistar origin (F0 -generation).
The animals were housed in suspended, screen-bottomed stainless steel cages in a well ventilated room at 24-26° C. Groups of five rats of the same sex were housed in each cage. Each of the diets was fed to20 males and 30 females. Diets and tap water were constantly available. Individual body weights were recorded at week 0, 2, 4, 6, 8, 10, 12 and20. At week 12 and 20 the rats were mated in groups of 5 males and 10females on the same diet, to produce two successive litters (Fla and Flb respectively). After a mating period of three weeks the females were caged individually until the litters had been weaned. Records were made of the number of pups in each litter and of the total weight of the litter at day 1, 10 and 20. Litters containing more than eight siblings were randomly culled to eight, in order to equalize the stress of lactation among the dams.
At weaning age the F1a-litters were used to select the rats for a chronic toxicity/carcinogenicity study with the same test compound. The remaining weanlings were discarded.
The reproduction study was continued with 10 male and 20 female parent rats (F0) and the remaining 10 males and 10 females of each group ware killed at week 21 to examine the weights and the histological appearance of the liver, kidneys and caeca.
At weaning age of the F1b-litters 10 males and 20 females of each diet group were selected from as many litters as possible and maintained on the diet their parents had received. The Flb-rats were used to produce the F2a- and F2b-litters, the F2b-rats for rearing the F3-,andF3b-litters. Observations on litters were the same as described for theF0 -generation. From the F2b- and F3a-litters, 15 females and 5 males of each dosage group were used for teratological studies
After weaning their second litters, the mothers were sacrificedand the uterus was treated with ammonium sulphide solution for stainingand counting implantation sites.

Conduct of the 4-week feeding study with the F3b-litters
At weaning age of the F3b-litters, 10 males and 10 females of each diet group were selected from as many litters as possible, and continued on the same diet for a 4-week feeding study. Individual body weights were recorded weekly. The food consumption of each group was measured continuously.
In week 4 all surviving rats were killed by decapitation and examined grossly for pathological changes. The following organs were weighed: heart, kidneys, liver, spleen, brain, gonads, thymus, thyroid, adrenal and caecum (filled and empty). Samples of these organs and of a wide rang of other organs were fixed in a 4% neutralized formaldehyde solution.
Detailed microscopic examination was performed on all male and female rats of the highest dose group and of the control group. Haematoxylin and eosin stained paraffin sections of the organs weighed and also of the following organs were examined: lung, trachea, salivary glands, urinary bladder, skeletal muscle, thoracic aorta, oesophagus, gastro-intestinal tract (6 levels), pancreas and axillary and mesenteric lymph nodes.

Conduct of the teratology study
At weaning age of the F2b and F3alitters, 15 females and 5 males of each dosage group were selected and maintained on the various diets until used for teratology studies. When 12 weeks old, these selected animals were mated (1 male was caged with 3 females). Vaginal smears were taken daily to determine whether mating had occurred. The day of detection of spermatozoa in the vaginal smear was considered as day 0 of the gestation period. Then the pregnant females were individually caged.
On day 21 of pregnancy the dams were killed by decapitation. Both ovaries and uterus were removed. The number of corpora lutea were counted and the ovaries were weighed. The foetuses were removed from the uterus, dried of amniotic fluid, weighed, examined for macroscopic abnormalities, sexed and fixed in 70% ethanol for 4 days or in Bouin's fluid for at least one week. Intra-uterine dead embryos and foetuses and the number and position of the implantation sites in both uterine horns were recorded. The empty uterus was weighed.
Every third foetus, taken from the uterus was fixed in 90% ethanol after being eviscerated, skinned and stripped of most subcutaneous fat. Thereafter these foetuses were cleared and stained with Alizarine Red S according to the method of Dawson (1926) for examination of the skeleton.
The remaining foetuses, fixed in Bouin's fluid, were transferred to 70% ethanol before being cut into a number of slices according to the technique of Wilson (1965) for examination of the soft tissues.
All examinations for foetal abnormalities were carried out under a dissecting microscope. Soft tissue and skeletal examinations were restricted to the foetuses of the highest and lowest dose groups and the control group.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: F0: week 0, 2, 4, 6, 8, 10, 12 and 20

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

Oestrous cyclicity (parental animals):

No measured

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, testis and epididymis histopathlogy

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced (details see further details on study design)
- Maternal animals: All surviving animals (details se further details on study design)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. (details se further details on study design)

HISTOPATHOLOGY:
F3b litter: lung, trachea, salivary glands, urinary bladder, skeletal muscle, thoracic aorta, oesophagus, gastro-intestinal tract (6 levels), pancreas and axillary and mesenteric lymph nodes.

ORGAN WEIGHTS:
F0: liver, kidney, caecum (filled and empty)
F3b litter: heart, kidneys, liver, spleen, brain, gonads, thymus, thyroid, adrenal and caecum (filled and empty).

Postmortem examinations (offspring):

See further design of study design.

Statistics:

Student's t -test: all data except mortality, skeletal and visceral anormalities
Chi-square test: mortality, skeletal and visceral anormalities
Wilcoxon test: body weight F3b generation

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No abnormalities of condition or behaviour were observed in any ofthe diet groups, and in any of the successive generations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The body weights of males were decreased in the 3% group in the F0- and F1b-generation. This effect occurred also at the 1% level, but only in males of the F0-generation. The growth figures in females were generally comparable in all groups, but in the F1-generation slightly decreased body weights were observed in the highest dose group. There were no significantly decreased body weights in the highest dose groupof the F2-generation parent rats.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The percentage of females which casted a litter was very high in all groups throughout the study. The mean number of young per litter did not show any significant differences amongst the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 rats: A slight increase in the weight of the filled caecum was observedin males of the highest dose group. The relative weights of the liver and kidneys were comparable in the various groups

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross autopsy findings of the organs weighed did not reveal any abnormalities, attributable to the feeding of the test substance

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopical examination of the organsweighed did not reveal any abnormalities, attributable to the feeding ofthe test substance

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

2

Dose descriptor:

NOAEL

Effect level:

30 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

Remarks on result:

other: Generation: F3a/b (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
No effetc

CLINICAL SIGNS (OFFSPRING)
No efefct

BODY WEIGHT (OFFSPRING)
F3b: The body weight was slightly decreeased in males but not in females at the high dose.

ORGAN WEIGHTS (OFFSPRING)
F3b: no effect

GROSS PATHOLOGY (OFFSPRING)
No substance-related effect.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

2

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

2

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Results of the 4 -week study with F3b rats:

No deaths or abnormalities of condition or behaviour were observedduring the 4 -wek experiment in any of the groups.

Body weights were slightly decreased in males of the highest dose group. In females body weights of all groups were comparable. Food consumption (during the first two weeks) and food efficiency were lowest in the top-dose group.

At 0.1% statistically significant differences with the controls occurred in the relative weights of the kidneys, testis and thymus. However, as these changes were not observedat the two higher feeding levels, they were considered incidental findings.

Filled caecum weights were slightly increased in males at 1.0% and 3.0% and in females at 3.0% Empty caecum weights tended to increase in these groups.

At autopsy, no pathological changes attributable to the ingestion of the test substance were observed, either in males or in females.

Microscopic examination did not reveal treatment-related changes.

Results of the teratology study with F2b and F3a rats:

There was no treatment-related effect on body weight/body weight gain and food intake.

There was no treatment-related or biologically relevant effect on necropsy, oragn weights or litter data.

In both generations, no significant differences in anomalies between test and control groups were observed. The anomalies noted were regularly observed in the strain of rats used.

Type and incidence of skeletal anomalies revealed no substance-relationship.

Applicant's summary and conclusion

Conclusions:

The multigeneration study with Acesulfame potassium in rats was carried in male and female Wistar rats at dietary levels of 0, 0.3, 1.0 and 3.0% for three successive generations, each comprising two consecutive litters. A teratogenicity study was conducted with 15 females per group of the F2b and F3a generations. Rats from the F3b generation were submitted to clinical and pathological examination. Pups from the F1a litters were used for a chronic toxicity/carcinogenicity study at the same dietary levels as the parents. Fertility, number of young per litter, birth weight, growth rate and mortality during the lactation period were not adversely affected and there were no indications of increased mortality in utero. Growth rate was slightly decreased in the top dose group of the F0 and F1 generations. In the teratogenicity studies, no adverse effects were seen in appearance, food consumption, autopsy of the dams, organ weights, or litter data; no visceral or skeletal abnormalities attributable to the treatment were observed.

The NOAEL for fertility and reproductive performance was 3.0% (30000 ppm corresponding to about 1500 mg/kg bw/day) a dose level exceeded the current limit dose of 1000 mg/kg bw/day.
The NOAEL for systemic parental toxicity was 1.0% (10000 ppm corresponding to about 500 mg/kg bw/day) due to slight reductions in body weight/body weight gain.
The NOAEL for pre-, post-natal developmental toxicity including teratogenicity was 3.0% (30000 ppm corresponding to about 1500 mg/kg bw/day) a dose level exceeded the current limit dose of 1000 mg/kg bw/day.

Executive summary:

Acesulfame potassium was investigated for its effect on fertility, reproductive performance, pre- and postnatal development in male and female Wistar rats at dietary levels of 0, 0.3, 1.0 and 3.0% in three successive generations, each comprising two consecutive litters. A teratogenicity study was conducted with 15 females per group of the F2b and F3a generations. Rats from the F3b generation were submitted to clinical and pathological examination. Pups from the F1a litters were used for a chronic toxicity/carcinogenicity study at the same dietary levels as the parents.

 

This study is assessed as appropriate and valid since it was performed comparable to an internationally accepted testing guideline with deviations that can be considered as minor. Reporting, assessment and data presentation in the study report was considered as appropriate considering the date of study and report.

Fertility, number of young per litter, birth weight, growth rate and mortality during the lactation period were not adversely affected and there were no indications of increased mortality in utero.

 

Growth rate was slightly decreased in the top dose group of the F0 and F1 generations. In the teratogenicity studies, no adverse effects were seen in appearance, food consumption, autopsy of the dams, organ weights, or litter data; no visceral or skeletal abnormalities attributable to the treatment were observed.

In the four-week feeding study with the F3b-generation rats body weights and food efficiency figures were slightly decreased in males at the highest dose level. The relative weights of the caecum were slightly increased in both sexes of the high-dose group and in males of the mid-dose group. These findings were considered to represent an adaptive condition. Gross and microscopic examination did not reveal any treatment-related pathological changes.

 

In the teratogenicity studies general appearance, food consumption and autopsy findings, organ weights 'and litter data were not adversely affected at any dose level. Visceral and skeletal examination of fetuses did not reveal any teratogenic effects attributable to the feeding of the test substance.

 

The only effects of the test substance which were considered treatment-related consisted of inconsistent, slight decreases in body weights in the top-dose group, and a slight increase in the weight of the caecum.

 

Conclusion:

The NOAEL for fertility and reproductive performance was 3.0% (30000 ppm corresponding to about 1500 mg/kg bw/day) a dose level exceeded the current limit dose of 1000 mg/kg bw/day.

 

The NOAEL for systemic parental toxicity was 1.0% (10000 ppm corresponding to about 500 mg/kg bw/day) due to slight reductions in body weight/body weight gain.

 

The NOAEL for pre-, post-natal developmental toxicity including teratogenicity was 3.0% (30000 ppm corresponding to about 1500 mg/kg bw/day) a dose level exceeded the current limit dose of 1000 mg/kg bw/day.