Sodium [[α,α'-(ethylenediimino)bis[2-hydroxybenzene-1-acetato]](4-)]ferrate(1-)

Toxicity to reproduction

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996-03-13 to 1997-01-07

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:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: FeEDDHMANa
- Chemical name: N,N'-Bis(2-hydroxy-4-methylphenyl)ethylenediamine diacetic acid, ferric-sodium complex
- Physical state: red brown powder
- Analytical purity: 100% (UVCB); 65% based on Certificate of Analysis (File code: RCD-TNA-95010)
- Expiration date of the lot/batch: not indicated
- 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 or test system 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.

VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water
- Stability in vehicle: Stated as being stable for at least 96 hrs (other NOTOX project)
- Concentration in vehicle: not reported
- Amount of vehicle (if gavage): 5 mL/kg b.w./day. Actual dose volumes were calculated according to the latest body weight.

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.
The time of onset, degree and duration were noted. Clinical signs were recorded under NOTOX Project 173565, which has been generated for the purpose of reporting only. Animals showing pain, distress or discomfort, which was considered not transient in nature or which was likely to become more severe, were sacrificed for humane reasons. The time of death was recorded precisely as possible. Cage debris of pregnant females was examined to detect abortion or premature birth. Signs of difficult or prolonged parturition were recorded. In the clinical signs tables, findings for each animal are presented as highest weekly scores.

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.

OTHER:
The females were allowed to litter normally. The day that partirition began was designated day 0 of lactation. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pubs or apparently inadequate lactation or feeding, were recorded.

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:
duration of gestation, 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 (F0-males were killed between days 122 to 138 of the treatment period)
- Maternal animals: all surviving animals (F0-females were killed between days 48 to 65 post coitum)

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
- F1-offspring were killed on the day of culling the litters (day 4 of lactation) or when weaned on day 21 of lactation.
- 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.

Food consumption of males receiving 50 mg/kg/day and of females receiving 50 or 200 mg/kg/day remained in the same range as controls, before and after correction for the body weight.
Food consumption was decreased with statistical significance compared to controls, in males receiving 200 mg/kg/day from day 57 of treatment until termination and in males receiving 750 mg/kg/day from the beginning until the end of treatment. Following correction for the body weight, relative food consumption of males receiving 750 mg/kg/day showed a statistically significant decrease in comparison with control males between days 1 to 36 of treatment.
The food consumption of females treated at 750 mg/kg/day was noted as decreased during the premating period and until the end of the gestation period. During the period of lactation the food consumtion of females treated at 750 mg/kg/day was similar as control females. Relative food consumption was decreased during the premating period and during the first week of the gestation period only. In all cases the difference with control females was statistically significant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

A slight delay of the precoital time in the 750 and 200 mg/kg dose groups was observed.
For all males and females that were paired, mating could be confirmed, resulting in 100 percent mating for each dose group. The fertility and conception indices of the 200 and 50 mg/kg dose groups were comparable with the control group. In the 750 mg/kg dose group these indices were considered to be slightly low, although the difference with controls did not achieve a level of statistical significance.
The gestation index was 100% in all treatment groups and were not affected by treatment with FeEDDHMANa.
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)

1

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

2

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)
Adverse effects on the F1-offspring consisted of an increased mortality rate during postnatal days 0-4 seen across the treatment groups (please refer to the table 1 in the section "Any other information on results incl.tables". 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 2 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. Signs that were more frequently observed among the pups of all treatment groups during the first litter check (FLC), post partum phase and last litter check (LLC), consisted of hypothermia, no milk in the stomach and small appearance. These signs normally precede the death of non-viable pups and were considered not to represent a distinct toxic effect caused by the test substance.

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

Comparison of the relative number of live and dead pups during the various phases of lactation (i.e. first litter check, post partum days 0-4 and post partum days 5-21) revealed a statistically significant increase in post natal loss during days 0-4 in all treatment groups. The Viability Indices of these groups were correspondingly low. However, it was noted that the number of pups found dead at the first litter check (FLC) of control dams, were relatively high in comparison with the treatment groups. In accordance with these findings a relatively low Live Birth Index was noted in control litters. The following number of decedents among the F^-offspring was noted when calculating the total number of deaths during days 0-4 Post partum including the FLC:

Table 1: TOTAL DEAD PUPS DURING FLC & DAYS 0-4 P.P.

	GROUP 1 0 MG/KG	GROUP 2 50 MG/KG	GROUP 3 200 MG/KG	GROUP 4 750 MG/KG
Litters affected	5	8	9	6
Total dead pups	21	24	44	42
Mean	0.9	0.9	1.7	2.2
N (litters)	24	27	26	19

When comparing the total number of dead pups over the first 4 days of lactation, including the FLC, the mean number of dead pups per litter in the 50 mg/kg dose group equals the number in the control group. At the 200 and 750 mg/kg dose level, the mean number of deaths per litter revealed a dose-related increase when compared to the control group. A similar pattern was seen in the Overall Surviving Indices of the treatment and control groups. However, it must be considered that 2 females of the 750 mg/kg dose group died on the first day of lactation and that therefore 26 of 29 of their pups were killed in extremis.

After culling of all litters to 4 male and 4 female pups (if practically possible), the low mortality rate seen among pups of the treatment groups was comparable to that of the control group. This was also reflected by the comparable Weaning indices in control and treated groups.

Table 2: 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

Conclusions:

Based on the mortality observed in animals of the high dose group, and the reduced body weight gain and food consumption seen in animals of the mid- and high-dose groups, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established. Due to the increased post natal loss and reduced viability index in the treatment groups observed on post natal Days 0-4, the developmental NOAEL could, in fact, not be established. However, based on the results of the 28-day and 90-day study with FeEDDHMANa, it cannot be excluded that anaemia and/or impaired renal function may have been present that finally caused the observed litter losses. On the other hand, the finding in the low dose group consisted of only one female with total litter loss (15 pups) and 6 other females with 1 dead pup/litter which was within normal limits and might have occurred by chance. Therefore, a developmental NOAEL of 50 mg/kg might be considered; effects at higher levels were considered to be closely related to (subclinical) maternal toxicity.

Executive summary:

In an one-generation reproduction toxicity study (NOTOX B.V., 1997), the structural analogue FeEDDHMANa (read across) 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).