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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Lack of information/clarity on dosing – which seems to have stopped at mating, rather than continuing to weaning · No information on stability and homogeneity of diet mixes, or whether any analyses of concentrations were conducted. No calculations of actual doses given. No other internal inconsistencies within the report were noted, although there is very little data present.

Data source

Reference
Reference Type:
review article or handbook
Title:
Unnamed
Year:
1971

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
no
Remarks:
Study was reported before any GLPs extant.

Test material

Constituent 1
Reference substance name:
Aspartame
EC Number:
245-261-3
EC Name:
Aspartame
Cas Number:
22839-47-0
IUPAC Name:
methyl L-alpha-aspartylphenylalaninate
Test material form:
solid: crystalline
Details on test material:
purity: 99.5%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch number of test material: various
- Purity, including information on contaminants, isomers, etc.: 100 %

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 90-100 days old at mating
- Housing: wire mesh cages
- Diet ad libitum: Purina Laboratory Chow
- Water ad libitum

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Aspartame incorporated in diet by thorough mixing in a twin-shell, Patterson-Kelley blender to provide the appropriate dietary level for each group. Fresh batches of diet prepared weekly.
Dosages adjusted on the basis of group mean body weight and food consumption determined for each sex at intervals during the pre-breeding phase of treatment.
Actual consumption determined in weeks 4 and 9 of pre-mating phase.
Details on mating procedure:
- 1M/2F per group used for first round of breeding (P1 generation).
· Males were rotated among females in each group at weekly intervals
· Males returned to individual cages
· Females placed in individual nesting boxes to produce F1A litters
· F1A litters reduced to 10 per litter 24 hr post partum
- Spare F1A offspring from this study were used for lifetime toxicity study
· 60M/60F controls
· 40M/40F from each dose group
- 10M/20F per treatment group used for second round of breeding (P2 generation)
· Except control females (n=19)
· Fed the appropriate diet for 9 wk (to 90-100 days of age)
· Bred as for P1 generation
· F2A litters reduced to 8 per litter.
Duration of treatment / exposure:
Start of dosing: Nine weeks before start of mating. Duration: Two parental generations, Two one-litter filial
generations
Frequency of treatment:
daily, 7 days each week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
4 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 Males 24 Females
Control animals:
yes, concurrent no treatment
Details on study design:
Food and water were available ad libitum throughout the study.
Prior to initiation of breeding, parental animals maintained in individual wire mesh cages and fed the appropriate diet for 9 wk (to 90-100 days of age).
- 1M/2F per group used for first round of breeding (P1 generation).
· Males were rotated among females in each group at weekly intervals
· Breeding period lasted 3 wk
· Males returned to individual cages
· Females placed in individual nesting boxes to produce F1A litters
· F1A litters reduced to 10 per litter 24 hr post partum
- Spare F1A offspring from this study were used for lifetime toxicity study E70
· 60M/60F controls
· 40M/40F from each dose group
- 10M/20F per treatment group used for second round of breeding (P2 generation)
· Except control females (n=19)
· Fed the appropriate diet for 9 wk (to 90-100 days of age)
· Bred as for P1 generation
· F2A litters reduced to 8 per litter.
· Five F2A litters were used for neonatal toxicity study E11
· Remaining litters used for evaluation of F2A weaning data
· All excess weanling and all P2 generation males and females sacrificed and
discarded.

Examinations

Parental animals: Observations and examinations:
- General observations: Before mating (weeks 1, 4 and 9). At reduction of F1A litters to 10 pups per litter.
- Food consumption: Weeks 1, 4 and 9 of premating phase
- Body weight: Pups at 24 hr and at weaning
Number of conceptions
Live births
Stillbirths
Size of natural and nursing litters
Deaths during lactation period
Gross signs of abnormality
Postmortem examinations (offspring):
F2A Pups (evenly divided as to sex if possible) were selected for
· Clinical laboratory studies
· Tissue preservation
· Histopathological evaluation
Ten pups per group (2 per litter) sacrificed 24 hr post partum
· Gross necropsy performed
· Tissues preserved for histopathology
Ten pups per group (2 per litter) sacrificed on pnds 5, 15 and 21
· Blood taken by arterial puncture of abdominal aorta for clinical laboratory
studies
· Gross necropsy performed
· Tissues preserved for histopathology
Corroborative studies undertaken using kidneys from concurrent F2A litters
Reproductive indices:
Reproduction indices calculated as:
· Fertility index = number of pregnancies/number of females
· Gestation index = number of full term litters born/number of pregnancies
· Live birth index = number of pups alive at 24 hr/number of pups at birth
· Lactation index = number of pups weaned/number of pups nursed

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Mortality:
no mortality observed
Description (incidence):
All P males and females survived their respective phases of study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights during pre-mating periods were generally comparable between groups.
Parental terminal body weights were generally comparable between groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Generally comparable between control and test groups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental observations among a few control and test animals during one or both generations:
· Bloody crust on eyes
· Red eyelids
· Lacrimation
· Wheezing
· Hunched appearance
· Rough fur
· Alopecia
· Soft faeces

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
No indication of a compound-related effect on indices of:
· Fertility
· Gestation
· Live births
· Lactation

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 4 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No indication of a compound-related effect on:
· Mean litter size
· Mean pup weights at birth (24 hr)
At weaning, significant body weight suppression was observed among pups at 4,000 mg/kg/day but not at 2,000 mg/kg/day.
Description (incidence and severity):
Necropsy findings were similar between control and test litters.
No gross abnormalities observed among F1A pups examined:
· Three controls: Small number examined reflects survival during lactation and group size requirement for subsequent lifetime toxicity study and P2 generation.
· 34 at 2,000 mg/kg/day
· 28 at 4,000 mg/kg/day
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings among pups from a few control and test litters:
· Weak/thin appearance
· Hunching
· Wheezing
· Laboured respiration
· Squinting

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
>= 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No stillbirths among the five litters per group selected for this study.
· Litters had 5-7 male and 5 female pups per litter
· Mean body weights at 24 hr were 6-7 g
External appearance 24 post partum was normal
Observations during lactation period:
· Controls
o Two male and two female pups lost from one litter (presumably cannibalised); one female pup was hunched and thin, had bloody crust on nose
o Pups from one litter were unusually small at weaning
o One male pup from another litter found dead on pnd 4 (see intercurrent deaths, above)
· At 2,000 mg/kg/day: Pups from one litter were unusually small during lactation period
· At 4,000 mg/kg/day: One female pup lost (presumably cannibalised)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No indication of a compound-related effect on:
· Mean litter size
· Mean pup weights at birth (24 hr)
At weaning, significant body weight suppression was observed among pups at 4,000 mg/kg/day but not at 2,000 mg/kg/day.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology values:
· Haematocrit, haemoglobin and erythrocyte count were similar between groups.
o Slightly lower than in older animals; thought to be reflective of the age of the animals
· There was a general reduction in total leukocyte counts among treated groups at pnd 15 and 21
o Significant in males on pnd 15 and females on pnd 21
Blood chemistry values comparable between control and test groups.
· ALP values differed between 15 and 21 days postpartum
o This is attributed to normal variation for animals of this age.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathology observed in control or test pups killed by design 24 hr post partum or on pnd 5, 15 or 21.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No pathological alterations were detected in liver, heart or stomach.
· Small focal accumulations of polymorphonuclear cells were observed in the mucosa and submucosa of one pup at 2,000 mg/kg/day.
“Subtle histological alterations” were observed in kidney sections on pnd 15 and 21:
· Minimal to mild hypertrophy and vesiculation of nuclei in cells of tubules of the inner cortex, visible under low magnification as a basophilic band.
· Observed in all pups examined on pnd 15 and in 6/9 on pnd 21
· Less severe on pnd 21 or in pups from 2,000 mg/kg/day group.
· These changes were not considered to be compound-related.
· Epithelial lines cystic spaces, presumably representing dilated tubules, were also present in occasional control and test animals.
The authors state that: · “Assessment of the significance of these changes was rendered somewhat difficult owing to the normally changing morphology during this period of maturation”.
Corroborative analysis undertaken in other offspring at pnd 28-30.
· No significant differences in kidney morphology were noted between control and test groups.
· The nuclear hypertrophy and vesiculation observed in pups at pnd 15 and 21 were not detected in corroborative analysis.
· Interstitial nephritis and regenerative tubular epithelium were present in all groups, being at least as common in control as test pups.
· These changes were typically minimal to mild in degree.
· Spontaneous mineral deposits, tubular and pelvic dilatation were observed in small numbers of both control and treated pups.
· Minimal accumulation of inflammatory cells was detected in one control female and one male at 4,000 mg/kg/day.
o Considered to be an incidental finding.
Incidence of renal hypertrophy and/or vesiculation or renal tubular epithelial cells. The histological changes seen in the kidney appear to be consistent with normal histological processes during postnatal development. There is evidence of an effect of treatment at pnd 15-21, but this is transient and appears to have resolved by pnd 28-30. This result was discussed with the review team pathologist, who agreed that it was likely to be part of normal renal development and was unlikely to hold any toxicological significance.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Findings among 10M/10F pups per group:
· Lungs with dark red, abscessed or consolidated areas in:
o 11 control pups
o 11 pups at 2,000 mg/kg/day
o 13 pups at 4,000 mg/kg/day
· Dilatation of renal pelvis in two control pups.
· Greenish tinge to kidneys in:
o 2 control pups
o 5 pups at 2,000 mg/kg/day
o 2 pups at 4,000 mg/kg/day
· Dark red adrenals in
o 4 pups at 2,000 mg/kg/day
o 1 pup at 4,000 mg/kg/day
· Clear jelly-like substance covering salivary or submaxillary glands in
o 1 control pup
o 1 pup at 2,000 mg/kg/day
· Watery ovarian cysts in one female at 2,000 mg/kg/day

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2a
Effect level:
>= 2 000 mg/kg bw/day (nominal)
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

Applicant's summary and conclusion

Conclusions:
The NOAEL from these studies was determined to be 2000 mg/kg bw/day, based on the lower pup weights at weaning in both generations.