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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
publication
Title:
Unnamed
Year:
1986

Materials and methods

Principles of method if other than guideline:
Assessing the effect of EG on fertility and general reproductive performance in male and female rats.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult nulliparous Fischer 344 rats
- Housing: two per cage in stainless-steel wire cages; during mating, each male was housed with 2 females; after mating and during lactation, the females were housed individually in plastic showbox cages with hardwood chips for nesting.
- Diet: Purina Formulab, ad libitum
- Water: city water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Administration of EG to the F0 rats of both sexes started at approximately 7 weeks of age.
Fresh diet was prepared every 2 weeks with the percentage of test item (EG) adjusted, based on the group mean body weight and food consumption, so as to maintain a relatively constant dosage level. However, the concentration of EG in the diet was not changed during gestation or during the first week of lactation, but was reduced two- and three-fold during the second and third weeks of lactation, respectively, to adjust for increased food consumption by the dams. This change in concentration was based on earlier unpublished results from the laboratory. Increased food consumption during lactation has since been reported in another study performed at the laboratory.
Details on mating procedure:
At approximately 100 days of age, 10 males were added to 20 females in each dosage group. The F1 and F2 rats were treated as described for the F0 animals until approximately 100 days of age, at which time the animals were cohabited. Brother and sister matings were avoided for each generation.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 generations
Frequency of treatment:
daily
Details on study schedule:
The date of parturition and the number of live and dead newborn were recorded for each litter. The appearance and behavior of dams and pups were observed daily. Litter size was randomly reduced to 10, if necessary, on Day 4 postpartum. Offspring were weighed as litters at 4 and 14 days and individually at 21 days postpartum, the day they were weaned. F1 rats were randomly selected within each dosage group for the next mating. Each litter was represented except for those conceived very late in the mating period.
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
30
Control animals:
yes, concurrent no treatment
Details on study design:
Two untreated diet control groups, designated 0.0A and 0.0B, were included to estimate the variation between 2 groups treated alike.

Examinations

Parental animals: Observations and examinations:
Body weights and diet consumption were recorded weekly except during gestation and lactation.
Litter observations:
Offspring were weighed as litters at 4 and 14 days and individually at 21 days post partum, the day they were weaned. F1 rats were randomly selected within each dosage group for the next mating.
Postmortem examinations (parental animals):
Necropsies were performed on five males and five females randomly selected from each dosage level of the F2 parents and the F3 weanlings. Microscopic examinations were performed on sections of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries.
Statistics:
Continuous data such as body weights were compared by analysis of variance validated by Bartlett's test for homogeneity of variance. Duncan's multiple range test was used to identify individual mean differences when indicated by a significant F value. Where Bartlett's test indicated heterogeneous variances, t tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the method of Weil (Weil, 1970). Discontinuous data such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the X2 test and by Fisher's exact test. The following reproductive indices were calculated and evaluated statistically by the previously described non parametric methods: fertility index (male and female), days from first mating to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pups alive at birth), 0 to 4-day survival index, 4 to 14-day survival index, 4 to 21day survival index. The last four indices are summarized in the tables as means for ease of understanding and presentation, although the nonparametric statistical methods did not include a comparison of means.
Reproductive indices:
yes

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Throughout the study there was no effect of EG treatment on body weight gain or diet consumption, nor was there any mortality among parental rats.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no reproductive effects associated with EG treatment doses up to 1000 mg/kg bw/d via diet.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related effect was observed for any of the indices. Also, EG treatment did not affect neonatal body weight at days 4, 14, or 21 post partum.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related histopathologic findings in F2 parents or in F3 weanlings. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. Unilateral hydronephrosis occurred in another high-dose F2 male. In addition, mild focal tubular hyperplasia was observed in one high-dose male F3 pup but was also diagnosed in two control male pups.
Other effects:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: there were no treatment-related effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Reproductive Indices

     1.0 g/kg bw/d  0.2 g/kg bw/d  0.04 g/kg bw/d  0.0A  0.0B
 F0 -> F1  Fertility index (%)  100  90  100  90  90
   Male  95  90  90  75  90
   Female  100  100  100  100  100
 F1 -> F2  Fertility index (%)  100  100  90  90  90
   Male  85  95  85  90  85
   Female  100  100  100  100  94
 F2 -> F3  Fertility index (%)  100  90  100  80  80
   Male  90  75  85  80  70
   Female  100  100  100  100  100

Neonatal body weight at day 21

     1.0 g/kg bw/d  0.2 g/kg bw/d  0.04 g/kg bw/d  0.0A  0.0B
 F1 pups  males  30.6 +/- 4.5  30.9 +/- 4.9  30.7 +/- 6.4  30.6 +/- 3.6  27.9 +/- 4.3
   females  29.0 +/- 4.5  29.2 +/- 4.5  29.5 +/- 4.7  27.9 +/- 3.3  27.0 +/- 3.5
 F2 pups  males  32.8 +/- 3.5  30.9 +/- 5.8  29.3 +/- 4.7  30.0 +/- 4.0  28.8 +/- 4.3
   females  30.8 +/- 3.4  30.2 +/- 4.9  28.8 +/- 3.8  28.5 +/- 3.1  27.5 +/- 3.4
 F3 pups  males  30.2 +/- 4.0  30.9 +/- 4.0  30.9 +/- 4.0  32.0 +/- 3.9  30.2 +/- 4.6
   females  28.6 +/- 3.8  28.2 +/- 3.4  29.7 +/- 4.0  30.1 +/- 3.5  27.7 +/- 3.9

Applicant's summary and conclusion

Conclusions:
In conclusion, there were no reproductive effects associated with the inclusion of as much as 1000 mg/kg bw/d of test item in the diet.