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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.34 (One-Generation Reproduction Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): FeEDDHMANa (trade name: Boliken Fe)
- CAS nr: 84539-53-7
- EC nr: 283-041-9
- Chemical name: N,N'-Bis(2-hydroxy-4-methylphenyl)ethylendiamine diacetic acid, ferric-sodium complex.
- Physical state: Red brown powder
- Analytical purity: 100 % (UVCB)
- Lot/batch No.: 954243
- Storage condition of test material: At room temperature, dry in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: (P) Males: 4 weeks, Females 12 weeks
- Weight at study initiation (pre-mating period): (P) Males: 165-218 g, Females: 265-330 g
- Housing: individually, in polycarbonate cages containing purified sawdust bedding (Woody SPF, Type BK10/20 or during lactation Type 3-4 was supplied by B.M.I., Helmond, The Netherlands). During mating, 1 female was caged together with 1 male in suspended stainless steel cages with wire mesh floors.
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBA, Type R-03-18-K, Oud Turnhout, Belgium), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C
- Humidity: 55 %
- Air changes: 15 air changes/hour
- Photoperiod: 12 hours dark/12 hours light

IN-LIFE DATES: From: 13-Mar-1996 (males), 08-May-1996 (females) To: 09-Aug-1996 (males), 08-Aug-1996 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations of the test item in water (w/w) were prepared daily immediately prior to dosing. Formulations were stirred prior to and during dosing procedures.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 7 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in a cage with sawdust bedding material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations were collected during the beginning of treatment, at an intermediate time and during the end of the dosing period. Samples of the highest and lowest concentrations were analysed to check stability (first analysis only) and homogeneity. Accuracy of preparation was determined for all concentrations.
Duration of treatment / exposure:
Parental males were treated until days 122 to 138 of the treatment period.
Parental females were treated until days 48 to 65 post coitum.
Frequency of treatment:
1x/day
Details on study schedule:
- Age at mating of the mated animals in the study: 16 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 750 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
28 rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose level selection was based upon the information obtained from previous oral toxicity studies using rats, i.e. a 28-day oral toxicity study (LSR 87/AKB005/802) and a teratogenicity study (NOTOX 158759).
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: once daily for clinical signs, twice daily for incidences of mortality

BODY WEIGHT:
- Time schedule: weekly for males and females
Mated females were weighed on days 0, 7, 14 and 20 of pregnancy and days 1, 7, 14 and 21 of the lactation period.
Live pups of one litter were weighed individually according to sex on the morning after birth (day 1) and on days 4, 7, 14 and 21 of the lactation period.

FOOD CONSUMPTION
- Time schedule: weekly for males and females
During the mating period analysis of food consumption was suspended.
Food consumption of mated females was measured during days 0-7, 7-14 and 14-20 of pregnancy and weekly thereafter starting on day 1 of the lactation period.
Oestrous cyclicity (parental animals):
No data on estrous cyclicity are given in the report.
Sperm parameters (parental animals):
No data on sperm parameters are given in the report.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- Litters were adjusted to 4 pups/sex or as near as possible; excess pups were killed and discarded. Litters of less than 8 pups remained intact.

PARAMETERS EXAMINED
The following parameters were examined in F1 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:
For external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals
- Maternal animals: all surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
The following tissues were prepared and examined microscopically: cervix, coagulation gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes, uterus and vagina.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed when weaned on day 21 of the lactation period.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. All animals were sexed.
Statistics:
For assumed normally distributed variables, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data were assumed not to be normally distributed.
The exact Fisher-test was applied if the variables could be dichotomised without loss of information.
All tests were two-tailed.
Reproductive indices:
Fertility index, conception index, gestation index, sex ratio (F1)
Offspring viability indices:
Live birth index, viability index, weaning index and overall survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
not examined
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:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the 750 mg/kg bw/day group, 1/28 males and 4/28 females died during the study. At this dose level, hunched posture, piloerection and emaciated and/or pale appearance were noted. A few males of the 200 mg/kg bw/day group showed hunched posture.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain and food consumption were decreased in animals of both sexes at 750 mg/kg bw/day and in males at 200 mg/kg bw/day. This decrease was dose-related. During the lactation period, body weight gain of females at 750 mg/kg bw/day showed a marked increase. Body weight ratios were decreased during the first 5 weeks of treatment for males and during the first 3 weeks for females at 750 mg/kg bw/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the absence of concomitant histopathological findings, a slight decrease of the fertility and conception indices was noted at 750 mg/kg bw/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related macroscopic findings at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There was no histopathological evidence of toxicity or infertility.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity: based on clinical signs, incidences of mortality, and changes in body weight and food consumption at higher dose levels
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: reproductive toxicity, fertility: based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
An increased mortality rate during post natal days 0-4 was seen at 200 and 750 mg/kg bw/day. A slight increase was noted at 50 mg/kg bw/day as well. The majority of the post natal loss was attributable to a few litters of each group, including 1, 3 and 3 litters in the low, mid and high dose groups, respectively. For further details, please refer to the table in the section "Any other information on results incl. tables".

CLINICAL SIGNS (OFFSPRING)
There were no unexpected clinical signs seen among pups of any dose group.

BODY WEIGHT (OFFSPRING)
Reduced body weights were noted during days 4 to 21 of lactation in pups of the high dose group (750 mg/kg bw/day).

GROSS PATHOLOGY (OFFSPRING)
There were no test item related macroscopic findings.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

POSTNATAL LOSS AND VIABILITY

 

Endpoint

Dose Group [mg/kg bw/day]

 

0

50

200

750

 

Dams with total litter loss at FLC [n]

1

0

0

0

(Pups lost [n])

(13)

 

 

 

Dead pups at FLC [n]

19

3

5

3

(Litters [n])

(5)

(3)

(3)

(1)

Postnatal loss PND 0-4 [n]

2

21

39

39

(Litters [n])

(1)

(7)*

(7)

(6)

Viability index (%)

99.4

94.9

90.4

85.4

 

 

 

 

 

FLC: first litter check

*: including 1 female with total pup loss (15/15)

 

CHEMICAL ANALYSIS

For the nominal concentrations of 0, 10, 40 and 150 mg/g, the concentrations analysed were in agreement with the concentrations prepared in this study. The test item was found to be mixed homogeneously through the vehicle and to be stable for 4 hours at ambient temperature.

Applicant's summary and conclusion

Executive summary:

In a one-generation reproduction toxicity study (NOTOX B.V., 1997), FeNa-EDDHMA in distilled water was administered to 28 Wistar rats/sex/dose level by single oral gavage (5 mL/kg bw) at dose levels of 50, 200 or 750 mg/kg bw/day. A concurrent control group was treated with the vehicle only. Treatment commenced 10 weeks prior to mating for males and 2 weeks prior to mating for females and continued for both sexes until at least the end of the lactation period. Pregnant females were allowed to litter normally. On day 4 of lactation, each litter was adjusted to 4 males and 4 females or as near as possible. The surviving offspring was euthanised as soon as possible after weaning.

The primary effect of treatment with the test item on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in male and female animals at 750 mg/kg bw/day. These signs of test item-related toxicity were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxcity in parental animals was 50 mg/kg bw/day under the conditions of this study.

In the offspring, increased post natal loss and reduced viability were noted during PND 0 -4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. With special regard to the low incidence and unusual distribution pattern of findings noted at 50 mg/kg bw/day, the NOAEL for developmental toxicity was 50 mg/kg bw/day under the conditions of this study.

Based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day, the NOAEL for reproductive performance/fertility was 200 mg/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a one-generation reproduction toxicity study (OECD 415).