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Diss Factsheets

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 31, 1998 - July 20, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
EC Number:
418-570-8
EC Name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
Cas Number:
25383-07-7
Molecular formula:
C11H20NO5P
IUPAC Name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
Details on test material:
- Name of test material (as cited in study report): Fosfomycin PEA salt
- Physical state: white crystalline powder
- Lot/batch No.: MH 188
- Expiration date of the lot/batch: March 2, 2001
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A.
- Age at study initiation: (F0) 9 wks
- Weight at study initiation: (F0) Females: 200-225 g
- Housing: Makrolon cages measuring (mm) 425x266x159h, type 3D.
- Diet (e.g. ad libitum): Ad libitum (GLP 4 RF 25)
- Water (e.g. ad libitum): Ad libitum (municipal water main, filtered)
- Acclimation period: about 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 15-20 per hour (filtered on HEPA 99.99%)
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 h light (7 a.m. - 7 p.m.), artificial lighting.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(deionized water)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Every day, appropriate amounts of test article were weighed in suitable containers, mixed with vehicle and solubilized by magnetic stirring to obtain solutions at the concentrations of 5, 10 and 20 mg/ml. All formulates were administered within 4 hours of the preparation.

VEHICLE
- Concentration in vehicle (test article): 0, 5 , 10, 20 mg/ml
Details on mating procedure:
- M/F ratio per cage: 1/2 in pre-mating and mating periods: 0/1 and 0/1+litter in pregnancy and lactation periods, respectively; 1/1 in growing period.
- Mating period: 16 hours at a time (7 evenings/week for a maximum of 21 evenings).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (analized every morning from each female).
- After successful mating each pregnant female was caged (how): As soon as copulation had been ascertained, the females were allocated to the respective group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration checks were performed by RBM twice during the study. Results were as expected.
Duration of treatment / exposure:
During 14 days before the start of the mating period and continued during pregnancy up until day 21 of lactation.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested (by gavage).
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0: 28 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were established on the basis of a 4-week oral toxicity study in rats in which the highest dose, 500 mg/kg/day, induced too severe alterations, mainly in the liver, for use in pregnancy. The middle dose, 150 mg/kg/day, induced mild toxicological effects; 200 mg/kg/day was therefore considered the appropriate highest dose for pregnant females.
- Rationale for animal assignment (if not random): During the acclimatization period the fitness of the group was assessed and the entire group was deemed fit. Before treatment was started, all animals were weight. Animals in the extremes of the weight distribution and animals showing signs of illness were excluded from the study. From the remaining animals, the required number of rats were allocated to the dosage groups by means of a computerized randomization program.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day throughout the week, weekends included
- Cage side observations checked: mortality and symptomatology (clinical signs and behaviour)

DETAILED CLINICAL OBSERVATIONS: Yes (clinical signs and behaviour)
- Time schedule: three times a day (in the early morning, in the early afternoon and in the late afternoon) throughout the week, weekends included

BODY WEIGHT: Yes
- Time schedule for examinations: on the day before the start of treatment (day 0) and weekly thereafter until the end of the mating period. During pregnancy they were weighed on days 0, 7, 14 and 20 and during lactation on days 0 (day of parturition), 7, 14 and 21.

FOOD CONSUMPTION:
- Food consumption for each animal was recorded once a week throughout the pre-mating period, on days 7, 14 and 20 of pregnancy and on days 7 and 14 of lactation.

OTHER:
VAGINAL BLEEDING.
Oestrous cyclicity (parental animals):
The estrous cycles were checked during the acclimatization period on all females received in order to select the females with normal cycles for the study. Observation continued daily on the selected females during the pre-mating and mating periods until day 0 of pregnancy.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: At birth and during the lactation period:
External abnormalities, number of live and stillbirths, mortality, sex determination, weight, appearence of fur, eruption of incisors, eye opening, pinna detachment, surface righting reflex, pinna refles, corneal reflex, pupullary reflex, traction reflex, grip reflex, chimney test.
On day 21 of lactation, F1 offspring were killed except 1M and 1F of each litter: left alive and weighed weekly, checked daily for symptomatology and mortality. They were observed for balano-preputial gland cleavage and for vaginal opening. At weeks 7-8 from birth: Inclined Plane and Water Y Maze behavioural test were performed.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals: The females were killed at the end of the lactation period (day 21 of lactation). Females without a sperm-positive vaginal smear and not found to be gravid were killed 25 days after the end of the mating period. The females with a sperm-positive vaginal smear, which did not give birth were killed at day 25 of the presumed pregnancy.

GROSS NECROPSY
- Gross pathology examination was performed on all females. Organs with gross alterations were fixed in 10% buffered neutral formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: ovaries, uteri (with oviductus, cervix and vagina)
The following organs were removed and fixed in 10% buffered neutral formalin: ovaries, uteri with oviductus, cervix, vagina and pituitary gland.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 21 days of age.

GROSS NECROPSY
- Gross necropsy

HISTOPATHOLOGY / ORGAN WEIGTHS
Grossly abnormal tissues were fixed in a suitable fixative for histopathological examination, if necessary.
Statistics:
To compare control group with the treated groups:
to compare frequencies: Chi square, Fisher's exact test and Trend test.
to compare the mating distribution during the time the F0 generation was allowed for mating: Log-Rank test
to compare all the other data: Barlett's homogeneity of variance:
if homogeneous, ANOVA. If significant (p <0.05): Dunnett's multiple comp.
if not homogeneous, TRANSFORMATIONS (inverse, logarithm decimal, square and square root) were made:
if transformation was successfully: ANOVA and Dunnett's test.
if not homogeneous: Kruskal-Wallis non parametric ANOVA: if significant (p<0.05), Mann Whitney's "U" test.
Reproductive indices:
Pre-coital interval.
Mating performance and fertility: mating index, fertility index, pregnancy index and pregnancy period.
Individual and group values.
Offspring viability indices:
Mean survival time, birth index, viability index on day 4, weaning index, post-implantation losses, mean time (actuarial means) of appearance of the morphological and physical development data.
Individual and group values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All the animals belonging to the 100 and 200 mg/kg/day groups showed slight hypersalivation 10 min after treatment for about 1 h; this clinical sign started after about 3 weeks of dosing and continued up to the end of treatment (scheduled killing). No clinical signs were observed at 50 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of the 50 mg/kg/day group died on day 2 of the lactation period owing to wrong gavage. No other deaths were observed during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No biologically important effects were observed on the mean body weight gain of treated females in comparison with the control females during the pre-mating, pregnancy and lactation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly lower mean daily food consumption was found in the 200 mg/kg/day treated females in comparison with the control group during the pre-mating period and until day 7 of pregnancy. No effects were observed at 50 and 100 mg/kg/day.
Food efficiency:
not specified
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):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No changes were noted in the estrous cycles.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Only female parents were examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
No differences were found in mating and fertility indices or in the mean pre-coital time between the control and treated groups. The length of pregnancy was similar in all the experimental groups and no effects on parturition were observed at any dosage. No important differences were noted in the mean value per litter of implantations and of live born among the various experimental groups. The sex ratio was unaffected at all dosages.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: clinical signs; food consumption
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: clinical signs; food consumption

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A slightly lower birth index, viability index on day 4 and weaning index were observed at 200 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During lactation the body weight of pups of the treated groups, mostly at 100 and 200 mg/kg/day, was slightly lower than that of the control group and this continued up to day 56 of the post-weaning period in the case of young male and female rats belonging to the 200 mg/kg/day group.
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:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day dose
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No differences were noted in the % of success at the Chimney test and in the Inclined Plane test among the various experimental groups.
No important differences were noted in the Water Y Maze test among the various experimental groups in the 1st trials, while a longer mean time was observed in the 2nd retest of the 200 mg/kg/day group both for male and female rats.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

PRE-and POST-WEANING PHYSICAL DEVELOPMENT (OFFSPRING)
No important differences were noted in the developmental parameters among the various experimental groups, except for a delay in the mean time of vaginal opening of the 200 mg/kg/day group in comparison with the control group.
No differences were noted in the % of success at the Chimney test and in the Inclined Plane test among the various experimental groups.
No important differences were noted in the Water Y Maze test among the various experimental groups in the 1st trials, while a longer mean time was observed in the 2nd retest of the 200 mg/kg/day group both for male and female rats.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: postnatal survival; pup weight; vaginal opening; Water Y Maze test
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: postnatal survival; pup weight; vaginal opening; Water Y Maze test

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEL in one-generation reproduction toxicity study in female rats by oral route was 50 mg/kg/day both for mother and offspring (F0 and F1).
The NOAEL in one-generation reproduction toxicity study in female rats by oral route was 100 mg/kg/day both for mother and offspring (F0 and F1).
Executive summary:

In this study the effects of Fosfomycin PEA salt were assessed on the reproductive performance of female Sprague-Dawley Crl:CD (SD) BR rats when treated in the F0 generation and on the subsequent development of the F1 generation. The study followed the OECD Guideline 415. Twenty eight female rats/group were treated orally prior to mating, during mating and pregnancy and up to day 21 of lactation with the doses 0 (control), 50, 100 and 200 mg/kg/day. All females were allowed to give birth and rear their young (F1) up to day 21 of lactation, then were killed with their pups (except one male and one female pup per litter, which were subjected to some behavioural tests during the growing period).

The test article induced mild clinical signs during the treatment period (hypersalivation after dosing) in the 100 and 200 mg/kg/day groups and a lower mean daily food consumption at 200 mg/kg/day. A slightly lower mean body weight of pups with a slightly lower postnatal survival was found in the pups belonging to the 200 mg/kg/day group. A longer mean time in the 2nd retest of the Water Y Maze test and a delay in the mean time of vaginal opening possibly related to retarded development of these pups were also observed. At 100 mg/kg/day only slight lower pup body weight was observed. No interferences were noted in the reproductive performance at any dosage.

The NOEL (no-effect level) was considered to be 50 mg/kg/day both for the mothers and the offspring. The NOAEL (no-adverse effect level) could be considered 100 mg/kg/day for dams since at this dosage only hypersalivation was present. The same dose level (100 mg/kg/day) could be fixed as NOAEL for the offspring since the lowering body weight of these pups was no longer seen after weaning.