Iron tris(2-ethylhexanoate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 13, 2018 to February 21 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 29, 2016

Deviations:

yes

Remarks:

Female in the weight range of 190-204 g and not 175-200 g, while males were in the range of 220-234 g, instead of 200-225 g. This deviation was not considered to have compromised the pur pose or conduct of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iron tris(2-ethylhexanoate)

IUPAC Name:

Iron tris(2-ethylhexanoate)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany, Sulzfeld, Germany
- Weight at study initiation: 220-234 g for males, 190-204 g for females
- Housing: from arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags.
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy) ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks before the start of treatment

DETAILS OF FOOD AND WATER QUALITY: Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was suspended in the vehicle.
The preparations were made daily (concentrations of 15.0, 30 and 60 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
The test item was weighed directly into a clear glass container by using disposable smartSpatula ® (Sigma-Aldrich Code number: Z740505). The container was subsequently filled to approximately 75% of vehicle and put into a water bath (set at 50°C). Every approximately ten minutes, the flasks were shaken manually until homogeneous distribution of the test item was visually observed. The preparations were then brought to room temperature (approximately 25°C) filled with the remaining amount of vehicle and shaken by hand again.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is insoluble in water. The vehicle was corn oil as in it the test item is stable and forms an homogeneous solution.
- Concentration in vehicle: 15.0, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1 male to 1 female
- Length of cohabitation: till proof of mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
The female was paired with the same male until positive identification occurred or 14 days had elapsed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment, analysis was performed in a separate study (IES Study No. 20180064) in order to validate the analytical method and the preparation procedure in the test item concentration range of 0.8 to 250 mg/mL and to verify the stability of the preparation at 28 hours at room temperature (20 ± 5 °C) and ambient light conditions and for 8 days at room temperature (20 ± 5 °C) in the dark at the concentrations of 0.8 and 250 mg/mL.
Samples of the preparations made during the current study (Week 1 and Week 4) were analysed to check the homogeneity and concentration. Analysis of test item preparations were carried out at the Test Site under the Delegated phase (IES Study No. 20180065). Samples were packed and transferred for analysis at room temperature (20 ± 5 °C) by a dedicated courier to the Principal Investigator of the Test Site.
Six replicates of approximately 10 mL were drawn from Groups 2/6 and 4/8 preparation (2 from the top, 2 from the middle and 2 from the bottom of the preparation), while 2 replicates approximately 10mL were drawn from Groups 1/5 and 3/7 preparations. Therefore, for each sampling date a total of 16 specimens were prepared for shipment.
For each specimen an additional back up sample of approximately 10 mL was retained at the test facility. Samples were labelled as follows: study number, sampling date and nominal concentration.
The 16 specimens were transported by a dedicated courier. Prior to shipment, the Principal Invest igator was notified by e-mail. Back up samples were maintained at -20 ± 5°C until positive results of the analysis were available and approved.
The recoveries for test samples from treatment Week 1 were found to be within the range of 86 % to 113 %. The % nominals determined for test samples from treatment Week 4 were within the range of 91 % to 104 %. This confirms the correct preparation of dose formulation samples at the test facility
and, since both sample sets included samples drawn from top, middle and bottom of dose group 2/6 (15 g Iron Tris(2 Ethylhexanoate)/L and 1.71 g Fe/L, respectively) and dose group 4/8 (60 g Iron Tris(2 Ethylhexanoate)/L and 6.84 g Fe/L, respectively) preparations, further confirms the homogeneous distribution of the test item in the selected corn oil vehicle.
All control samples (dose group 1/5) were found to be free of residues of Iron Tris(2 Ethylhexanoate) and iron, respectively. Thus, the concentrations in these samples were below the limit of detection (LOD) of 14.5 μg Iron Tris(2-Ethylhexanoate)/L and 1.65 μg Fe/L.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, during pairing with females until the day before necropsy, for a total of 32/33 days. Dose volumes were a djusted once per week for each animal according to the last recorded body weight.
Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum, for a total of 42 to 63 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Recovery groups (Groups 5 to 8)
Male animals were dosed once a day, 7 days a week, for a total of 28 days. Female animals were dosed once a day, 7 days a week, for a total of 63 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
No treatment was given during the recovery period.

Frequency of treatment:

Each day for the periods described above (duration of treatment/exposure)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups
10 males + 10 females for the control and for each tested dose

Recovery groups
5 males + 5 females for the control and for each tested dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the dose range finding test, where signs of toxicity were seen at 320 mg/kg bw, the highest dosage selected for the final test was 300 mg/kg bw

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once daily during the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations
Main groups
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD Consumption
- Time schedule for examinations: the weight of food consumed by each cage of males and females were recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.
Recovery groups
The weight of food consumed by each cage of rats were recorded at weekly intervals following allocation.

HAEMATOLOGY: Yes
Blood collection from pups on Days 4 and 14 post partum
Blood sampleswere withdrawn under light ether anaesthesia fromthe heart (by intracardiac puncture).
On Day 4 post partum, approximately 0.2mL of blood samples were taken from at least two pups (1 sample/sex, when possible) per litter.
On Day 14 post partum, approximately 0.5mL of blood samples were taken from at least two pups (1 sample/sex, when possible) per litter.
As a part of the necropsy procedure, approximately 0.8mL of blood was taken from all parental males and females. Parameters checked in the "Any other information on materials...." field of the IUCLID dossier were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
see HAEMATOLOGY
- Parameters checked in the "Any other information on materials...." field of the IUCLID dossier were

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
see DETAILD CLINICAL OBSERVATIONS
- Battery of functions tested: sensory activity / grip strength / motor activity.
examined.

Oestrous cyclicity (parental animals):

Oestrus cycle was monitored by vaginal smears in all females for at least 1 week before start of dosing in order to exclude a replace from the study females with irregular cycle. Vaginal smears were also taken from Day 1 of treatment, up to positive identification of
mating.
The vaginal smear data was examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before dispatch to necropsy.

Sperm parameters (parental animals):

Parameters examined in male parental generations
Examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation was done taking into account the tubular stages of the spermatogenic cycle.
Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952. The PAS-stained sections were used to identify the permatogenic
stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and to the integrity of the various cell types within the different stages; regular layering in
the germinal epithelium was noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 5 males and 3 females as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in the offsprings daily for a litter: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, blood collection and thyroid hormone determination

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females (Main groups)
All females were examined also for the following:
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals)
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium
sulphide to reveal evidence of implantation.

Organ weights (All groups)
From all animals completing the scheduled test period, the organs indicated were dissected free of fat and weighed. The ratios of organ weight to body weight was calculated for each animal.

Postmortem examinations (offspring):

Pups at Day 4 post partum
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination and internal examination of gonads in order to detect abnormalities (ie presence of undescendent testes). Sex was determined during the internal examination.

Pups at Day 14 post partum
All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and internal examination of gonads in order to detect abnormalities (i.e. presence of undescendent testes). All pups with abnormalities were retained in a 10% neutral buffered formalin.

Nipples retention at Day 14 post partum
No nipples/areolae were observed in male pups.

Statistics:

Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

Copulation index, fertility index, precoital interval, preimplantation loss and prenatal loss were calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed among main and recovery females administered the highest dose of 300 mg/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Very slight reductions in body weight, only occasionally significant at statistical analysis, were observed in the high dose females of the recovery group (Group 8, dosed at 300mg/kg/day) in the last 3 weeks of treatment period. Reductions gradually recovered during the recovery
period.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slight decrease of erythrocytes was recorded in males dosed at 150 and 300mg/kg/day. Due to the minimal severity, this change was considered to be not adverse.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Subcutaneous masses were observed in one female dosed at 75mg/kg/day and in 2 females dosed at 300mg/kg/day. Two of these were recognised at histopathological examination as inflammation or adenocarcinoma of the mammary gland, respectively, while the third one
was unremarkable.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Subcutaneous masses were observed in one female dosed at 75mg/kg/day and in 2 females dosed at 300mg/kg/day. Two of these were recognised at histopathological examination as inflammation or adenocarcinoma of the mammary gland, respectively, while the third one
was unremarkable.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone determination
An increase of thyroid stimulating hormone was recorded in one animal receiving 75 mg/kg/day (no. X0990034), with no related changes of the other thyroid hormones. Due to the absence of dose-relation, this finding was considered to be incidental.

Oestrous cycle
Absence or reduced oestrous cycle number were seen in the females of mid- and high dose groups (dosed at 150 and 300mg/kg/day) during the pre-mating phase of the study.

Copuylatory index
copulatory indices were 100% for controls, low and mid-dose groups (dosed at 75 and 150mg/kg/day), 90% for high dose animals (dosed at 300mg/kg/day).

Fertility index
The fertility indices were 100% for controls and low dose animals (dosed at 75mg/kg/day) and 90% for animals dosed at 150 and 300mg/kg/day.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicity recorded

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pallors,cold to touch and small appearance were in general the clinical signs noted in control and treated pups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant differences in mean pup loss were observed among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum.

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

no change in thyroid weight. No other organs weighed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Autolysed abdominal organs was observed in pups found dead at birth and in pups which died during the lactation period.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sex ratio
A dose-related reduction of the percentage of males at birth, statistically significant in the high dose group (receiving 300mg/kg/day) was observed in comparison to controls.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicity recorded

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

1. Clinical signs

Main and Recovery groups

No treatment-related clinical signs were observed in the male from the main phase and in those from the recovery phase, during treatment and recovery periods.

Salivation was observed in 8/10 main phase females dosed at 300mg/kg/day during mating phase and post partum phases and in 4/5 recovery females, dosed at the same dose level, during treatment period. In addition, pioloerection, pallor and vaginal discharge were seen in a single female from the low dose group (no. X0990039, dosed at 75mg/kg/day) from Day 1 to Day 4 post partum.

No clinical signs were observed during recovery period.

A subcutaneous mass was observed in one female dosed at 75mg/kg/day (no. X0990045) in the right ventral area of the neck (data not tabulated). This was recognised at histopathological examination as acute, multifocal inflammation of the mammary gland.

A second subcutaneous mass was observed in one female dosed at 300mg/kg/day (no. X0990063) in the left dorsal area (data not tabulated). This was recognised at histopathological examination as an adenocarcinoma of the mammary gland.

A third subcutaneous mass was observed in a second female dosed at 300mg/kg/day (no. X0990079) in the right ventral area of the neck (data not tabulated). This was recognised at histopathological examination as unremarkable.

2. Clinical observations (Functional Observation Battery Tests)

Main and Recovery groups

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

3.Motor activity, grip strength and sensory reactivity to stimuli

Main and Recovery groups

A slight statistically significant increase in motor activity was observed in the females of the mid- and high dose recovery groups (dosed at 150 and 300mg/kg/day) when compared to controls. No other changes in motor activity were observed during treatment of recovery periods in either males of females. Due to the episodical nature of this change and to the low magnitude of the measured values (below the mean historical control value), this increase was condidered to be of no toxicological significance.

No changes of neurotoxicological significance were observed in grip strength, landing foot splay or other sensory reactivity parameters evaluated.

4. Body weight

Main and Recovery groups

Very slight reductions in body weight (not greater than 10%), only occasionally significant at statistical analysis, were observed in the high dose females of the recovery group (Group 8, dosed at 300mg/kg/day) in the last 3 weeks of treatment period. Reductions gradually recovered during the recovery period.

No significant changes in body weight and body weight gain were observed in the treated male animals and in the main group females, when compared to controls during treatment or recovery periods.

5. Food consumption

Main and Recovery groups

No changes of toxicological significance were observed in food consumption during the study in the males and in the females from the main phase groups.

Slight reductions in food consumption (from 7% to 20%) were seen in the recovery low and high dose females (Groups 6 and 8, dosed at 75 and 300mg/kg/day) during the treatment period. These reductions were no longer present during the recovery period. Due to the absence of dose relation and to the low magnitude, this change was considered of no toxicological relevance and, therefore, not adverse.

6. Haematology

Dosing phase (main phase animals)

Slight decrease of erythrocytes was recorded in males dosed at 150 and 300mg/kg/day (7% and 6% below controls, respectively). Due to the minimal severity, this change was considered to be not adverse.

In addition, male no. X0990068 (300mg/kg/day) showed moderate neutrophilia. Compared with mean control data, the increment was 4.1 fold. Due to the minimal incidence, this finding was considered to be unrelated to treatment. The statistically significant difference of reticulocytes recorded between controls and females dosed at 150mg/kg/day (39% above controls) was not dose-related, therefore it was considered to be incidental.

Recovery phase (recovery phase animals)

No changes were recorded.

Coagulation

Dosing phase (main phase animals)

No changes were recorded.

Recovery phase (recovery phase animals)

The statistically significant difference of prothrombin time recorded between controls and females dosed at 75mg/kg/day (4% above controls) was not dose-related, therefore it was considered to be incidental.

7. Clinical chemistry

Dosing phase (main phase animals)

One male dosed at 150mg/kg/day (no. X0990050) showed an increase of aspartate aminotransferase (2.8 fold compared with controls). Due to the absence of dose-relation, this change was considered to be unrelated to treatment.

Recovery phase (recovery phase animals)

The statistically significant differences recorded between control and treated animals (protein, globulin, chloride and potassium in males, chloride and bile acids in females) were not observed during the Dosing Phase and/or not dose-related, therefore they were considered to be unrelated to treatment.

8. Thyroid hormone determination

Parental males

No changes were recorded. An increase of thyroid stimulating hormone was recorded in one animal receiving 75mg/kg/day (no. X0990034), with no related changes of the other thyroid hormones. Due to the absence of dose-relation, this finding was considered to be incidental.

Pups - Day 14 post partum

No changes were recorded.

9. Terminal body weight and organ weights

Final and recovery sacrifice

No relevant changes were observed on terminal body, absolute and relative organ weights of treated animals that completed the treatment or recovery period, when compared to controls.

10. Macroscopic observations

Final and recovery sacrifice

No remarkable changes were noted at post mortem examination in treated animals at the end of treatment and recovery period, when compared with controls.

The sporadic changes such as adhesions to lungs and sternum in one mid-dose male (no.X099048), subcutaneous mass observed in one mid- and high dose female (no. X0990045 and X0990063, respectively), pelvic dilatation of kidneys or dark and/or red areas in the stomach of control and/or treated animals of both sexes of treatment and/or recovery period, were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

11. Microscopic observations

No treatment-related changes were noted in animals sacrificed at the end of the treatment period.

The sporadic lesions such as mucosal erosion of non glandular region of the stomach (sometimes associated in treated females with pigmentation - test item like) or pelvic dilatation of kidneys reported in control and/or treated animals of both sexes, chronic inflammation of prostate in one high dose male (no. X0990068) or M-Adenocarcinoma in the mammary glands of one high dose female (no. X0990063) correlated to the subcutaneous mass observed during necropsy were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age. In particular, acute inflammation of lungs associated with necrosis, fibrosis and presence of foreign material (test item like), correlated to adhesions to lungs, sternum and thymus.

12. Oestrous cycle, reproductive parameters, pairing combination and mating performance

No treatment-related anomalies were noted in the pre-coital interval of the treated females, when compared to controls. Absence or reduced oestrous cycles number were seen in the females of mid- and high dose groups (dosed at 150 and 300 mg/kg/day) during the pre-mating phase of the study. The number of copulation plugs was similar between control and treated groups.

One female from the high dose group (no. X0990067, dosed at 300mg/kg/day) did not mate. One female from the mid-dose group (no. X0990043, dosed at 150 mg/kg/day) was found not pregnant, while 1 female from the high dose group (no. X0990069, dosed at 300 mg/kg/day) showed unilateral implantation and total litter loss on Day 1 post partum.

One female dosed at 75mg/kg/day (no. X0990025) and one from the control group (no. X0990001) conceived after Day 5 of mating (Days 14 and 7, respectively).

Males and females

The copulatory indices were 100% for controls, low and mid-dose groups (dosed at 75 and 150mg/kg/day), 90% for high dose animals (dosed at 300mg/kg/day).

The fertility indices were 100% for controls and low dose animals (dosed at 75mg/kg/day) and 90% for animals dosed at 150 and 300mg/kg/day.

13. Implantation, pre-natal loss data and gestation length of females

Gestation periods were similar in treated groups and controls. All dams gave birth between Days 21 and 23 post coitum.

Corpora lutea, implantations and pre-implantation loss, total litter size and pre-natal loss (percentage) did not show dose-related or treatment-related differences.

14. Litter data of females and sex ratio of pups

No treatment-related nor significant differences in total litter size, live litter size, mean pup loss (percentage), mean pup weights (percentage) and sex ratio of pups were observed among the treated and control dams at birth and on Days 1, 4, 13 and 14 post partum.

A dose-related reduction of the percentage of males at birth, statistically significant in the high dose group (receiving 300mg/kg/day) was observed in comparison to controls. This change was considered to be of no toxicological relevance.

15. Clinical signs of pups

Pallors,cold to touch and small appearance were in general the clinical signs noted in control and treated pups.

16. Anogenital distance and nipple count

No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups. No nipples were observed in male pups, when examined on Day 13 post partum.

17. Necropsy findings in decedent pups

Autolysed abdominal organs was observed in pups found dead at birth and in pups which died during the lactation period. No abnormalities were observed in pups from all groups killed on Day 14 post partum.

Applicant's summary and conclusion

Conclusions:

NOAEL >= 300 mg/kg bw/day

Executive summary:

Method

The toxic effects on Sprague Dawley rats after repeated dosing by oral route with the test item, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition

and early lactation of the offspring were investigated. The vehicle was corn oil. All doses (0, 75, 150 and 300 mg/kg/day) were administered at a constant volume of 5mL/kg body weight.

Males of the main groups were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 32/33 days. Males of the recovery group were treated for a total of 28 days. Females of the main groups were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 63 days. Females of the recovery groups were treated for a total of 63 days.

The following investigations were performed in all groups (main and recovery): body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five animals/sex/main group randomly selected and in all recovery

animals), post mortem macroscopic observations, organ weights. In addition, for main phase groups only (parental animals), oestrous cycle evaluation for parental females (1 week before dosing, during pre-mating and mating phases, prior to necropsy), mating performance, thyroid hormone determination (parental main phase males) and litter data were performed. Clinical signs, anogenital distance, external and internal examination and thyroid weight at necropsy were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.

Routine histopathological examination was performed only in main control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in five males.

Results

- Repeated oral administration of the tested substance to rats by gavage at the dose levels of 75, 150 and 300 mg/kg bw/day did not cause any mortality.

- Fertility and developmental part of the study

Oestrous cycle, reproductive parameters, pairing combination and mating performance: no treatment-related anomalies were noted in the pre-coital interval of the treated females. Absence or reduced oestrous cycles number were seen in the females of mid- and high dose groups (dosed at 150 and 300 mg/kg/day) during the pre-mating phase of the study. The number of copulation plugs was similar between control and treated groups.

The copulatory indices were 100% for controls, low and mid-dose groups (dosed at 75 and 150 mg/kg/day), 90% for high dose animals (dosed at 300 mg/kg/day).

The fertility indices were 100% for controls and low dose animals (dosed at 75 mg/kg/day) and 90% for animals dosed at 150 and 300 mg/kg/day.

Implantation, pre-birth loss data and gestation length of females: no significant differences were observed for these parameters between the treated groups and the controls. All pregnant dams gave birth between Days 21 and 23 post coitum.

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups: no significant differences in total litter size, live litter size, mean pup loss and mean pup weights were observed among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum.

A dose-related reduction of the percentage of males at birth, statistically significant in the high dose group (receiving 300 mg/kg/day) was observed in comparison to controls.

Clinical signs of pups: there were no compound-related effects.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum: necropsy did not reveal any treatment-related effect.

Conclusion

No effects which could be considered adverse were observed on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring at any of the dose levels investigated.

Therefore, the NOAEL (No Observed Adverse Effect Level) for fertility and reproduction parameters was considered to be 300 mg/kg/day for males and females.