Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
March-August 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant 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
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.34 (One-Generation Reproduction Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts
EC Number:
283-041-9
EC Name:
Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts
Cas Number:
84539-53-7
Molecular formula:
non specified (UVCB substance)
IUPAC Name:
non specified (UVCB substance)
Constituent 2
Reference substance name:
EDDHMA-Fe
IUPAC Name:
EDDHMA-Fe
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)
- Batch No.: 954243 d.d. 19-10-1995
- 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: ca. 4 weeks, Females: 12 weeks
- Weight at study initiation: Mean group weight Males: 188-195 g, Mean group weights Females: 294-295 g
- Housing: from arrival untial allocation 5/group, 1:1 during mating, individually during lactation , 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: ca 21°C with occasional fluctuations (no further info)
- Humidity: 55% with occasional fluctuations (no further info)
- Air changes (per hr): 15
- Photoperiod: 12 hours dark/12 hours light

IN-LIFE DATES: From: 25-Mar-1996 (males) To: 09-Aug-1996 (necropsy of last F0 males)

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: most animals had mated within 2 days. First period lasted 8 days. One female of the mid and one female of the high dose group mated on days 5/6 of the 2nd mating period only
- Proof of pregnancy: vaginal plug and 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 by HPLC to check stability (first analysis only) and homogeneity. Accuracy of preparation was determined for all concentrations. Although no acceptance criteria had been indicated in the report, all levels measured were within 90-110% of the target/nominal concentration.
Duration of treatment / exposure:
Parental males were treated from 10 weeks before mating until days 122 to 138 of the treatment period.
Parental females were treated from 2 weeks before mating 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
- F1 animals not mated (one generation study); the F1 offspring was killed on the day of culling (day 4 of lactation) or when weaned(on day 21 of lactation)
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
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). Dose levels were 0, 50, 200 and 1000 mg/kg bw.
- Rationale for animal assignment: at random based on BW
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.

WATER CONSUMPTION: via subjective appraisal only
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
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. Offspring found dead or killed before day 14 of lactation were sexed and externally examined if practically possible. The stomach was examined for the presence of milk. Offspring found dead or killed on or after day 14 of lactation was subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs. All animals were sexed and descriptions of all macroscopic
abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals (on days 122 to 138)
- Maternal animals: all surviving animals (on 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
- 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
Offspring was subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs. All animals were sexed and descriptions of all macroscopic abnormalities were recorded. From the few lesions detected in this study (kidney dilatation in 7 pups and petechiae in the thymus of 1 pup) no sample was taken for possible histopathological examination, as this was considered not to yield additional information.
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
Offspring viability indices:
sex ratio, 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:
no effects observed

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)

Spontaneous deaths occurred among animals of the 750 mg/kg dose group only. One male was found dead on day 73 of treatment, 1 female on day 6 of gestation, 1 female on day 20 of gestation and 2 females died on the first day of lactation. Macroscopic findings a t necropsy did not indicate a possible cause of death.
Signs and symptoms that were considered to be an adverse effect of the test substance were hunched posture, piloerection, emaciation, and pale appearance. These findings were noted in males and females receiving 750 mg/kg/day. Hunched posture was also noted in a few males receiving 200 mg/kg/day. Other findings noted in control animals or treated animals, were considered not toxicologically relevant.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Compared to controls, statistically significant low body weight and body weight gain was seen in males receiving 200 mglkglday and in males and females receiving 750 mg/kg. In males of the 200 mg/kg dose group the decrease in body weight and body weight gain was seen from day 78 and 85 of treatment onwards, respectively, and lasted until termination. Males receiving 750 mg/kg showed decreased body weight and body weight gain from the first until the last week of treatment. Although the difference in body weight of females receiving 750 mglkglday and control females was not statistically significant on days 1 and 8 of the premating treatment period, body weights were considered as decreased during the entire period of treatment, i . e . from week 1 of the premating period until termination of treatment at the end of the lactation period. The decrease in body weight gain of females receiving 750 mg/kg/day was statistically significant in comparison with controls from the first week of treatment until day 14 of gestation. On day 20 of gestation the body weight gain of these females was equal to control females; a marked and statistically significant increase was seen during the lactation period. Food consumption was statistically significantly decreased compared to controls, in males receiving 200 mg/kg 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 body weight, relative food consumption of males receiving 750 mglkglday showed a statistically significant decrease in comparison to control males between days 1 to 36 of treatment. The food consumption of females treated at 750 mglkg was considered to be decreased during the premating period until the end of the gestation period. During the period of lactation the food consumtion of the females treated at 750 mglkg was similar to 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.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): via gavage

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): not measured

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): not measured

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The majority of pairs mated during the first 2 days of the mating period. All pairs of the control group had mated within the first 4 days. The numbers may indicate a slight delay of the precoital time in the 750 and 200 mg/kg dose groups . For all males and females that were paired, mating could be confirmed, resulting in 100 percent mating for each dose group. In the 750 mg/kg dose group the fertility and conception indices were considered to be slightly low, although the difference with controls did not achieve the level of statistical significance. The gestation index was 100% in all treatment groups and was not affected bytreatment with FeEDDHMANa. The average number of implantation sites in pregnant females receiving 750, 200 or 50 mg/kg/day revealed slight variations. However, none of these variations were considered to exceed normal biological limits expected in control rats of the same age and strain.

ORGAN WEIGHTS (PARENTAL ANIMALS): not measured

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal lesions that were considered to be treatment-related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no microscopically observed lesions that were considered to be treatemnet-related.

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 mortality (750 mg/kg), and reduced body weight gain and food consumption (200 and 750 mg/kg bw)
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive toxicity, fertility: a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day. However, the poor physical condition of animals of this group might have been responsible for these effects

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 examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

GESTATION PERIOD
The duration of gestation of treated females was comparable to control females and ranged between 21.9 to 22.1 days.

VIABILITY (OFFSPRING)
Comparison of the relative number of live and dead pups during the various phases of lactation (i.e. first litter check (LFLC), post partum days 0-4 and 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 F1-offspring was noted when calculating the total number of deaths during days 0-4 Post partum including the FLC:
Total number of litters: 24, 27, 26, 19, for control, low, mid and high dose, respectively
Total number of litters affected: 5, 8, 9, 6, respectively
Total number of dead pups: 21, 24, 44, 42, respectively
Mean number: 0.9, 0.9, 1.7, 2.2, respectively
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 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 mgfkg dose group died on the first day of lactation and that therefore 26 of 29 of their pups were killed in extremis. After culling, the mortality rate was similar in all groups, controls included.

CLINICAL SIGNS (OFFSPRING)
There were no unexpected clinical signs seen among pups of any dose group and the incidences remained within biological normal limits. 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)
During the lactation period, pup body weight gain (in both males and females) was statistically significantly reduced from day 4 post partum until day 21 post partum in litters of the 750 rngjkg dose group when comparing with the control litters.

SEXUAL MATURATION (OFFSPRING): not examined.

ORGAN WEIGHTS (OFFSPRING): not examined.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic lesions, noted in pups at necropsy, included dilated renal pelvis, petechiae in the thymus and a missing tail tip. None of these findings was treatment-related. The observation "no milk i n the stomach" was related to premature death of the pups, i .e. still birth or shortly after birth, or inadequate feeding by the mother. The incidence was high in the 750 mg/kg dose group. This can be associated with the death of parental females numbers 205 and 220, which died immediately following partus. Cannibalism occurred in control and treated groups to the same extent.

HISTOPATHOLOGY (OFFSPRING): not examined

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: increased mortality (at 200 and 750 mg/kg bw) and reduced body weights during lactation (750 mg/kg bw); see also at remarks

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

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 mglkg was established. The NOAEL for fertility and reproduction effects was at least 750 mg/kg bw, the highest dose tested.
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 studies 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:

Three groups, comprising 28 male and 28 female Wistar rats, received FeEDDHMANa daily at a dose level of 50,200 or 750 mglkg/day by oral gavage. A similar group was treated with the distilled water as a vehicle control. 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. During the mating period (2 weeks), males and females were caged together on a one-to-one basis until mating was confIrmed. After mating both sexes were separated until termination.

Clinical signs were recorded daily during the treatruent period and until termination. Body weights and food consumption of males and females were determined weekly, commencing at the start of treatment, and for mated females on days 0, 7,14 and 20 of pregnancy and days 0, 7, 14 and days 0, 7, 14 and 21of lactation. During mating, analysis of food consumption was suspended. 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. Each litter was examined to determine the following

parameters: duration ofgestation; the number of live pups at the fIrst litter check (FLC) and daily thereafter during the lactation period; the number of still births and the litter size at the FLC; the sex of all pups; individual pups weights on days 1,4,7,14 and 21 oflactation; for each pup the external,

physical or behavioral abnormalities were recorded daily.

At termination of the study, parental males and females were euthanized and macroscopic examinations recorded. Samples ofthe cervix, coagulation gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes, uterus vagina and all gross lesions were taken from all animals. Tissues from animals ofthe control and high dose groups and of animals found dead were further processed and subjected to histopathological examination. The surviving offspring was euthanized as soon as possible after weaning. Each pup was subjected to an external and internal macroscopic examination.

In the 750 mglkg dose group, 1 out of 28 males and 4 out of 28 females died during the study. Clinical signs related to ill health were noted in males and females of the 750 mglkg dose group. These signs consisted of hunched posture, piloerection and emaciated and/or pale appearance. A few males of the 200 mglkg dose group showed hunched posture. Body weight gain and food consumption were noted as decreased in animals receiving 750 (both sexes) or 200 mglkg bw/day (males only). This decrease was dose-related. During the lactation period body weight gain of high dose females showed a marked increase. Food consumption: body weight ratios were decreased during the fIrst 5 weeks oftreatruent for males

and during the fIrst 3 weeks for females ofthe high dose group. After this period food consumption body weight ratios attained the same levels as controls.

Examination of the reproduction performance of males and females treated with FeEDDHMANa revealed a slight decrease ofthe fertility and conception indices in the 750 mglkg dose group. However, the fertility and conception indices in the control group were also low. Oral administration of FeEDDHMANa to parental rats at dose levels up to 750 mglkg/day produced no histopathological evidence of toxicity to reproduction or infertility.

The number ofdead pups at fIrst litter check was much higher for controls (19 pups) than for any treatment group (3, 5, and 3 dead pups for Groups 2, 3, and 4, respectively). This extremely high number was due, almost exclusively, to female 131 whose entire litter of 13 pups was found dead at the first litter check. The pup mortality in this control female may have been related to histopathological changes in the ovaries and uterus as observed in this animal. Higher mortality rates were seen among pups of dams of the 750, 200, and 50 mglkg dose groups during the first 4 days oflactation, and corresponding reductions in fetal viability were also noted. The post natal loss from Days 0 -4 of lactation included: 2 pups from 1 litter for Group 1, 21 pups over 7 litters for Group 2, 39 pups from 7 litters for Group 3, and 39 pups over 6 litters from Group 4. The majority of the post natal loss was attributable to a few litters of each group who had total losses or that had to be killed in extremis due to the death of the dam. In Group 2 (50 mglkg), 15 of the 21 dead pups came from a single litter (Dam 142). In Groups 3 (200 mglkg) and 4 (750 mglkg), 35 of the 39 dead

pups from each treatment group were each from 3 litters. Remarkably, one dam from each treatment group with total litter losses lost their pups over a few days and not all at once.

However, irrespective of the animals with total litter losses, an increase in the number of litters affected was observed on post natal Days 0 -4 when compared to controls, and as such, a treatment-related effect on post natal loss and viability cannot be excluded. There were no treatment related effects on the duration of gestation, live pups at fIrst litter check, breeding loss from Days 5-21, living pups on Day 21, or on the weaning index.

Fetal body weights were unaffected following treatment up to 200 mglkg. Pups from dams receiving 750 mglkg showed decreased body weights from day 4 until day 21 post partum. Hypothermia and no milk in the stomach were more commonly noted in all treatment groups than in the control group; this was almost exclusively seen in the litters that had increased pup mortality. Though these signs were isolated to a few litters from each treatment group, a treatment-related effect cannot be excluded.

CONCLUSION

The primary effect of FeEDDHMANa on parental animals was a poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption. This was seen at 750 mg/kg for both sexes, and with reduced severity at 200 mg/kg for males only.

Following treatment with FeEDDHMANa, parental rats ofthe 750 mglkg group showed a slight decrease in the fertility and conception indices. The poor physical condition of animals in the 750 mg/kg dose group might have been responsible for this effect. Oral administration ofFeEDDHMANa

to parental rats at dose levels up to 750 mg/ kg/day produced no histopathological evidence of toxicity to reproduction or infertility.

Adverse effects on the FI-offspring consisted of an increased mortality rate during post natal days 0-4 seen across the treatment groups, and reduced body weights during days 4 to 21 of lactation for pups of the high dose group only. 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.

Based on the mortality observed in animals of the high dose group, and the reduced body weight gain and food consumption seen in animals ofthe mid- and high-dose groups, a parental No Observed Adverse Effect Level (NOAEL) of 50 mglkg 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 studies with FeEDDHMANa, it cannot be excluded that anaemia and/or impaired renal function may have been present that fmally caused the observed litter losses. On the other hand, tbe 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.