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Effects on fertility

Link to relevant study records
Reference
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
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
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)
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)
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
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.

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
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
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
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
Reproductive effects observed:
not specified

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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
One well performed and reported study; the NOAEL was 750 mg/kg bw for effects on fertility.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a one-generation reproduction toxicity study (Reijnders, 1997), EDDHMA-Fe was administered to 28 Wistar rats/sex/dose level by single oral gavage 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 after weaning.

The primary effect on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in both sexes at 750 mg/kg bw/day. These signs 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.

Examination of the reproduction performance of males and females revealed a slight decrease of the 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 EDDHMA-Fe to parental rats at dose levels up to 750 mglkg/day produced no histopathological evidence of toxicity to reproduction or infertility. As such the NOAEL for reproductive performance/fertility was established at 750 mg/kg bw/day.

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. Effects at higher levels were considered to be closely related to (subclinical) maternal toxicity.

Data Waiving for 2 -generation study:

The performance of a two-generation reproductive toxicity study is scientifically unjustified. Signs of reproduction and developmental toxicity were found in the one-generation study only at concentrations with parental toxicity. In the developmental toxicity study no adverse effects on development were observed at all up to an exposure level of 1000 mg/kg bw/day (see below). In three repeated dose toxicity studies with oral administration (two 28-day studies and one 90-day study), no adverse effects on the reproduction organs were seen (no organ weight changes and no adverse histopathological findings).

In summary, it is very unlikely that EDDHMA-Fe will cause adverse reproductive and/or developmental toxicity effects in concentrations without maternal systemic toxicity in a two-generation reproductive toxicity study and conducting such a study is thus scientifically not justified. The available one-generation study and developmental toxicity study are sufficient for hazard assessment and risk characterisation.


Short description of key information:
A one-generation reproduction toxicity study (Reijnders, 1997) was performed with the test item in rats. Examination of the reproduction performance of males and females revealed a slight decrease of the fertility and conception indices in the 750 mglkg dose group. See below.

Justification for selection of Effect on fertility via oral route:
Well performed and reported GLP study

Effects on developmental toxicity

Description of key information
In a key developmental oral toxicity study (Reijnders, 1996), the NOAEL for developmental effects was established at the high dose level of 1000 mg/kg bw/day based on the absence of embryo-/foetotoxic or teratogenic effects. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - July 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Data on batch no. given; no full info on the composition of the test compound. Relatively well conducted and reported study according to guideline/standard. There were limitations, however, in the duration fo the treatment period (in comparsion to current OECD guideline) and details were missing such as acceptance criteria for the analyses
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
dosing from day 6 up to and including day 16 rather than from day 6 to day prior to planned sacrifice
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: ca. 8 weeks (not indicated whether this was at delivery or at start)
- Weight at study initiation: 219-300 g
- Fasting period before study: no
- Housing: in groups during acclimitisation; 3 females to 1 male during mating; thereafter individually
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: at least 5 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22 (with fluctuations; no further info)
- Humidity (%): ca. 55% (with fluctuations; no further info)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 March (day 0 = day of mating) To: ca. 3 weeks later (day 21 = day of gestation)
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily immediately prior to dosing. Formulations were shaken vigorously and stirred prior to and during dosing procedures


VEHICLE: distilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the first week (9 April 1996) and the last week (18 April 1996) samples were analysed to check stability and homogeneity
(low and high conc) and accuracy (all levels). Samples were analysed using HPLC. No acceptance criteria were indicated.
Accuracy was within 90-110% except for one high conc sample (84%); homogeneity was within 90-110%; and stability
measurements did not differ by more than 10%.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: until mating had occurred (no further info), and until 96 mated females had been obtained (out of 120)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility (no info).
- Further matings after two unsuccessful attempts: no info
- Verification of same strain and source of both sexes: males from stock were used that had been succesfull in previous studies
(no further info)
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
From day 6 to day 16 (inclusive) of pregnancy
Frequency of treatment:
Daily gavage
Duration of test:
Animals were killed on day 21 of gestation
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 mg/kg bw day
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary study at same dose levels (reported separately)
- Rationale for animal assignment (if not random): at random (no further info)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (including cage debris to detect abortion or premature birth)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6-17, and 21of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: On days 0, 3, 6, 9, 13, 17 and 21 of gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: thoracic and abdominal examinations, including ovary and uterine horn

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter, including sex
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (visceral); half per litter (skeletal)
Statistics:
If the variables can be assumed to follow a normal distribution, the Dunnett test (many-to-one t - t e s t ) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups. The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data can not be assumed to follow a normal distribution. The exact Fisher-test for 2x2
tables was applied if the variables could be dichotomized without loss of information. All tests were two-sided and in all cases
p<0.05 was accepted as the lowest level of significance. Macroscopic, skeletal and visceral findings were not subjected to
statistical analysis as this was considered not to provide any additional information.
Indices:
Pregnancy rate: (number of pregnant females/number of mated females) x 100
Pre-implantation loss: (number of corpora lutea - number of implantations) / (number of corpora lutea) X 100
Post-implantation loss: (number of implantations - number of live implants) / (number of implantations) X 100
Historical control data:
No info
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Number of mated females: 24 24 24 24 for the control, low, mid and high concentration group, respectively
Number of pregnant females: 21 24 23 22, respectively
Pregnancy rate (%): 88 100 96 92, respectively
Number of females with spontaneous deliveries: 1 1 0 1, respectively

MORTALITY: no

CLINICAL SIGNS: only 1 pregnant female receiving 1000 mg/kg/day showed a poor condition during the last week of pregnancy. There were no other clinical signs of toxicity or behavioural changes noted in this study.

BODY WEIGHT: following exclusion of non-pregnant females and females with spontaneous deliveries, body weight gain was reduced in the 1000 mglkg dose group from day 13 of pregnancy onwards.

FOOD CONSUMPTION: food consumption (excluding the non-pregnant females and females with spontaneous deliveries) in the 1000 mglkg group was reduced to a statistically significant level during days 9 to 13 and 13 to 17 of pregnancy. Food consumption was back to normal from days 17 to 21 of pregnancy, when treatment had ceased.

REPRODUCTION DATA: treatment of pregnant females with FeEDDHMANa did not reveal any adverse effect on the reproduction parameters recorded in this study. The number of corpora lutea, implantations and pre-and post implantation losses recorded in treated females were in the same range as control females. The statistically significant decrease in post-implantation loss recorded in the 1000 rng/kg group when compared to the control group, was considered not to be of toxicological relevance.

MACROSCOPIC EXAMINATION: Macroscopic observations at necropsy did not reveal test substance-related abnormalities.
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FOETAL MORTALITY: two foetuses of the 200 mg/kg/day dose group were found dead at necropsy. No test substance related mortality occurred. The incidence of embryonic and foetal deaths was comparable between the dose groups.

EXTERNAL EXAMINATIONS: macroscopic examination revealed no treatment-related abnormalities.

SEX RATIOS: sex ratios were comparable in treated and control groups.

FOETAL BODY WEIGHT: Statistically significant reductions in weights of live foetuses were seen on an individual basis in animals of the high concentration group. All foetuses of female 96 (1000 mglkg dose group) were smaller compared to the foetuses of other females in that group and in the control group, and was related to the poor physical condition and growth reduction noted in this female. This was considered to have influenced the slightly lower mean foetal weight in this dose group.

VISCERAL EXAMINATIONS: a number of anomalies was recorded in all groups, the types and group distribution of which did not suggest any association with treatment.

SKELETAL EXAMINATIONS: there was some intergroup variation in the incidences of the ossification parameters, but there were no consistent intergroup differences that were considered to represent toxicologically relevant adverse responses to treatment. Small numbers of foetuses with minor morphological changes were recorded in all groups, but their types and incidences did not suggest any dose-response relationship to treatment. The proportion of foetuses with supernumary (14th) rib(s) at the thoracolumbar border was unaffected by treatment.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The NOEL for maternal toxicity was 200 mg/kg bw based on reductions in BW gain and food intake; the NOAEL for developmental toxicity was 1000 mg/kg bw. Although a reduction in mean foetal BW was noted in animals of the high dose group, this reduction was very slight and considered to be mainly (but not exclusively) due to the poor condition of one female animal in this group.
Executive summary:

120 virgin females were mated with 40 males in order to obtain 4 groups of 24 pregnant females for the main study. From day 6 to day 16 of gestation inclusive, females of the treatment groups received daily oral administration of FeEDDHMANa at dose levels of 50, 200 or 1000 mg/kg body weight per day and females of the control group received daily oral administration of distilled water. Body weights and food consumption of females were determined at periodic intervals during the pregnancy period. On day 21 of gestation, all females were subjected to post-mortem examination and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horn were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live foetuses or still births , the weight and

sex of each live foetus and externally visible foetal macroscopic abnormalities. Alternate live foetuses of each litter were preserved in industrial methylated spirit or Bouin's fluid for examination of skeletal and visceral anomalies, respectively.

MATERNAL DATA: only 1 pregnant female of the 1000 mg/kg dose group showed signs of ill health during the last week of pregnancy. There were no other clinical signs of toxicity or behavioural changes noted in this study. Body weight gain was reduced in the 1000 mg/kg dose group from day 13 -17 of pregnancy. After correction for the uterus weight, body weight gain remained low on day 21 of pregnancy. Food consumption in the 1000 mg//g group was reduced to a statistically significant level during days 9 to 13 and 13 to 17 of pregnancy. Food consumption was back to normal from days 17 to 21. Post-mortem examinations of females of the treated groups (50, 200 and 1000 mg/kg/day), did not reveal any test substance- related effects.

REPRODUCTION PARAMETERS: treatment of pregnant females with FeEDDHMANa did not induce any adverse effect on the reproduction parameters recorded in this study.

FOETAL DATA: external, skeletal and visceral examinations of foetuses of dams treated at 50, 200 or 1000 mg/kg/day did not reveal any adverse

effect of the test substance. Sex ratios were comparable in treated and control groups. A slight reduction in mean foetal weights on an individual basis was seen in animals of the high dose group.

CONCLUSION: oral dosing of mated female Wistar rats with FeEDDHMANa at 50, 200 and 1000 mg/kg body weight revealed a slight reduction in maternal body weight gain and food consumption and in one pregnant female signs of ill health, towards the end of the treatment period, in the high dose group only. The effect on food consumption had disappeared by the end of the study. No adverse effects were recorded on any of the reproduction or foetal parameters recorded in this study. Visceral and skeletal examinations of foetuses did not indicate any teratogenic potential of FeEDDHMANa. From the results of this study, the No-Observed-Effect-Level (NOEL) based on maternal toxicity was 200 mg/kg body weight/day. The No-Observed-Adverse-Effect- Level (NOAEL) based on embryo-foetal parameters was 1000 mg/kg body weight/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Well performed study and results corroborated by those from the one-generation study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a developmental toxicity study (Reijnders, 1996) EDDHMA-Fe was administered once daily to groups of mated female rats by oral gavage at 0, 50, 200 or 1000 mg/kg bw/day from day 6 through day 16 of gestation. Control group females received the vehicle only. All dams were sacrificed on day 21 of the gestation period and foetuses removed for examination. In dams, there were no treatment-related clinical signs or incidences of mortality. The body weight gain and food intake was reduced at 1000 mg/kg bw/day. No adverse effects on pregnancy and no embryo-/foetotoxic effects were observed. There was no indication of teratogenic potential. On the basis of these results, the NOAEL was 200 mg/kg bw/day for maternal toxicity and 1000 mg/kg bw/day for develomental toxicity and teratogenicity. Although the duration of treatment was short, the results of this study are corroborated by the results of the one-generation study. The effects seen in fetuses in the one-generation study, consisting of mortality or reduced viability were related to the poor physical condition of the mothers that had been treated for a much longer period.

The NOAEL in a developmental study with the structurally related substance EDDHA-Fe resulted in a NOAEL of 500 mg/kg bw (highest level tested).


Justification for selection of Effect on developmental toxicity: via oral route:
Relatively well performed and reported GLP study

Justification for classification or non-classification

The test item EDDHMA-Fe caused no developmental toxicity and no teratogenicity in the rat in a key developmental toxicity study according to test guideline OECD 414. Effects on reproductive performance/fertility were assessed using a one generation toxicity study according to test guideline OECD 415. Only a slight decrease of the fertility and conception indices were seen in the high dose group of 750 mglkg bw. However, the fertility and conception indices in the control group were also low. In the absence of concomitant histopathological findings, this slight change in reproductive performance was considered to be secondary to the poor clinical condition of the animals at this dose level. Similarly, developmental effects were seen in this study at dose levels at pronounced maternal toxicity only.

Accordingly, EDDHMA-Fe is not subject to classification for toxicity to reproduction according to Regulation 1272/2008/EC.

Additional information