Alkaline earth metal and zinc hydroxy(substituted)acetate complexes and alkaline earth metal glyoxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 June 2012 - 7 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 June 2012 - 7 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals : Male and female animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing
- Basis for dose level selection : Dose levels were selected in consultation with the Sponsor

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Sprague Dawley:Crl:CD(SD) rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Italia S.p.A., Calco, Lecco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males & females: 6-8 weeks
- Weight at study initiation:
Males: 201 to 225 g for males and 151 to 175 g for females; The weight variation did not exceed ± 20 per cent of the mean weight.
- Fasting period before study:
no
- Housing:
Before mating: up to 5 of one sex to a cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: up to 5 animals / cage
Cage type: polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (pre-mating), polycarbonate cages measuring 42.5x26.6x18.5 cm (mating period and pregnant females)
Bedding: Nesting material was provided inside suitable bedding bags and changed at least twice a week
- Diet (e.g. ad libitum):
A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or diet.
- Water (e.g. ad libitum):
Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period:
An acclimatisation period of 20 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 2 °C
- Humidity (%):
55 ± 15 %
- Air changes (per hr):
15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):
artificial light for 12 hours each day
Actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved/suspended in the vehicle, purified water. The formulation was prepared daily (concentrations of 10, 30 and 100 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

- VEHICLE:
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified. The recovery of the test item from the vehicle was within the acceptance criteria.
The analytical method was validated in the range of 5 to 150 mg/mL.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The female was paired with the same male until positive identification occurred or 14 days had elapsed.
- Proof of pregnancy: A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
- After successful mating each pregnant female was caged (how): After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring approxiamtely 42.5x26.6x18.5 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and changed at least 3 times a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked during the pre-treatment period in the range of 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirm that the method was acceptable.

Final results for all levels were within the acceptability limits for concentration (90-110%) and homogeneity (CV<10%).
Final results for stability were within the acceptability limits for concentration (90-110%) and homogeneity (CV<10%).
Samples of the formulations prepared on Day 1 and during Week 4 were analysed to check the homogeneity and concentration. Results of the analyses were within the limits of acceptance for suspensions (90-110% for concentration and CV <10% for homogeneity).

Duration of treatment / exposure:

Males: minimum of 2 consecutive weeks prior to pairing and thereafter during pairing up to the day before necropsy.
Females: minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice.

Frequency of treatment:

Animals were dosed once a day, 7 days a week.

Details on study schedule:

Groups of 10 males and 10 females received the test item, by gavage at dosages of 100, 300 and 1000 mg/kg/day. A similarly constituted group of animals received the vehicle alone (purified water) and acted as a control. All doses were administered at a constant volume of 10 mL/kg body weight.
Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 4 consecutive weeks.
Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.
The following investigations were performed on all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter observation, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor
- Rationale for animal assignment (if not random): random

Positive control:

not required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Four animals were sacrificed for humane reasons at the end of gestation period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION
Food consumption was recorded at weekly intervals whenever possible, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
One day after the last treatment (i.e. on the day of necropsy).
- Animals fasted: Yes, animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
- How many animals: Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
- Parameters checked in table "Clinical chemistry parameters examined" were examined.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

See "Postmortem examinations (parental animals)"

Litter observations:

As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Parental animals killed for humane reasons and those that had completed the scheduled test period were killed with carbon dioxide.
Parental males:
The males were killed after the mating of all females, after at least 28 days of treatment period.
Parental females:
The females with live pups were killed on Day 4 post partum.
One female with total litter loss was killed one day after the occurrence of total litter loss. The females which had not given birth 25 days after the positive identification of mating were killed shortly after.

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 (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females:
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

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

Tissues fixed and preserved
Samples of all the tissues listed below were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid) from all animals.

Abnormalities
Coagulating glands
Epididymides
Ovaries
Penis
Prostate gland
Seminal vesicles
Testes
Uterus with cervix
Vagina

HISTOPATHOLOGY: Yes
The tissues required for histopathological examination are listed above. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. The examination was restricted to the following:
a) Tissues specified above from all animals in the control and high dose group killed at term.
b) Tissues specified above from all animals killed during the treatment period.
c) All abnormalities in all groups.

Postmortem examinations (offspring):

GROSS PATHOLOGY: Yes
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.

All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were 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 non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

The following reproductive indices were calculated:
Males
Copulatory Index (%) = no. of animals mated/no. of animals paired x 100
Fertility Index (%) = no. of males which induced pregnancy/no. of males pairedx 100

Females
Copulatory Index (%) = no. of animals mated/no. of animals paired x 100
Fertility Index (%) = no. of pregnant females/no. of females paired x 100

Males and females
Copulatory Interval = Mean number of days between pairing and mating

Females
Pre-birth loss was calculated as a percentage from the formula:
(No. of visible implantations - total litter size at birth )/No. of visible implantations x 100

Pup loss at birth was calculated as a percentage from the formula:
(Total litter size - live litter size)/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(Total litter size at birth - live litter size at Day 4)/Total litter size at birth x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Four high dose females (nos. 92510061, 92510067, 92510071 and 92510075) were sacrificed for humane reasons on Day 21/22 of gestation. At despatch to necropsy these animals had pale appearance, decreased activity, piloerection and/or were cold to touch. At necropsy examination they were all pregnant.
At post mortem examination, all females showed pale colour of the kidneys and reduced size of the spleen and thymus. In addition some females also showed multiple depressed dark areas in the glandular region of the stomach with associated yellow mucoid contents in the ileum and jejunum and/or distended duodenum with gas contents and/or pale colour of the liver.
Such changes were considered to be treatment-related.
Treatment-related changes were noted in the kidneys, spleen, thymus and stomach. The observed renal injury involved the nephrons, unit of the kidney, morphologically characterised by degeneration of some glomeruli (sclerosis) and the majority of renal tubules, sometimes associated with subacute inflammatory reactions. The renal tubules showed lined cells, in some instances basophilic with presence of exfoliated cells and/or amorphous material (crystal-like) in their lumen. Focal tubular necrosis in the papilla, associated with pelvic dilatation (hydronephrosis) and hyperplasia of the pelvic lining epithelium were also reported in female no. 92510075.
Moreover, mild to marked lymphoid depletion or red pulp depletion and atrophy were noted in the spleen and in the thymus and ulceration or erosion in the glandular mucosa of the stomach was also reported in the four unscheduled dead females. These changes were considered stress-related.
All females mated. Only one control female (no. 92510005) was sacrificed 26 days post coitum and found not pregnant at necropsy.
The number of females with live pups on Day 4 postpartum was: 8 in the control group, 10 in each of the low and mid-dose groups, 6 in the high dose group. Total litter loss was observed in a single female from the control group (no. 92510019).
Male animals did not show any signs of toxicological significance during the whole study. No significant clinical signs were seen in the female animals killed at the end of the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four females dosed at 1000 mg/kg/day were sacrificed for humane reasons on Day 21/22 of gestation due to the presence of a number of clinical signs (pale appearance, decreased activity, piloerection and/or cold to touch).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in body weight were observed in the treated animals when compared to controls. Reductions in body weight and body weight gain (these being also statistically significant) were observed in the treated males during the mating treatment period. These reductions, which were not dose-related, were not considered to be toxicologically significant. No significant changes were observed in the females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on food consumption were observed in the males. Slight and occasionally also significant at statistical analysis reductions were seen in the high dose females on Day 20 post coitum (-13%) and on Day 4 post partum (-19%).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Four high dose females (nos. 92510061, 92510067, 92510071 and 92510075) were sacrificed for humane reasons on Day 21/22 of gestation. At despatch to necropsy these animals had pale appearance, decreased activity, piloerection and/or were cold to touch.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were seen in selected organs/tissues evaluated in high dose males or females receiving the test substance, sacrificed at the end of treatment period, nor in the abnormalities detected at post mortem.
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.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrus cycle number before pairing was slightly reduced in the high dose females (2.2) when compared to controls (2.7).
The mean pre-coital interval and the number of copulation plugs were similar between control and treated groups.
All females mated. However, one female of the control group (no. 92510005) was found not pregnant.
A copulatory index of 100% was observed for both sexes from all groups. The fertility index in the treated groups (100%) was higher than in controls animals (90%). The number of females, with live pups on Day 4postpartum was: 8 in the control group, 10 in each of the low and mid-dose groups and 6 in the high dose group.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related differences were noted in implantation number, pre-birth loss data and gestation length between control and treated groups.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: systemic toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility and reproduction paramters

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Remarks on result:

not measured/tested

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs that could be related to treatment were reported at the examination of pups during the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant differences in pup loss were observed among the surviving treated dams and the controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Four females from the high dose group were sacrificed for humane reasons at the end of gestation period.
No significant differences in total litter size, live litter size, mean pup weights and pup loss were observed among the surviving treated dams and the controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant differences between control and treated groups were noted at necropsy in the decedent pups.
No significant abnormalities were recorded in the pups sacrificed on Day 4 post partum.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios:
No treatment-related effects were seen on sex ratios.
Males percentage at birth appeared to be slightly reduced in the high dose when compared to other groups, but this reduction was considered to be not toxicologically relevant.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility and reproduction parameters

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Remarks on result:

not measured/tested

Reproductive effects observed:

no

Conclusions:

Treatment-related effects indicating systemic toxicity were observed in some pregnant females dosed at 1000 mg/kg/day.
No changes were observed in males at any dose and in females dosed at 100 and 300 mg/kg/day.
No adverse effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.
The dose level of 300 mg/kg/day is considered to be the NOEL for systemic toxicity, while 1000 mg/kg/day is the NOAEL for fertility and reproduction parameters.

Executive summary:

A study according to OECD Guideline 421 and GLP was performed to investigate the repeated dose toxicity of the test item when administered orally to male and female Sprague Dawley rats. Groups of 10 males and 10 females received the test item, by gavage at dosages of 100, 300 and 1000 mg/kg/day. A similarly constituted group of animals received the vehicle alone (purified water) and acted as a control. All doses were administered at a constant volume of 10 mL/kg body weight.

Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 4 consecutive weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.

The following investigations were performed on all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter observation, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Mortality and fate of females

Four high dose females were sacrificed for humane reasons on Day 21/22 of gestation. At despatch to necropsy these animals had pale appearance, decreased activity, piloerection and/or were cold to touch. At necropsy examination they were all pregnant. Pale colour of the kidneys, reduced size of the spleen and thymus, multiple depressed dark areas in the glandular region of the stomach with associated yellow mucoid contents in the ileum and jejunum and/or distended duodenum with gas contents and/or pale colour of the liver were also observed.

Histopathological examination revealed treatment-related changes in the kidneys (nephrons, unit of the kidney, morphologically characterised by degeneration of some glomeruli and the majority of renal tubules, sometimes associated with subacute inflammatory reactions), spleen and thymus (lymphoid depletion or red pulp depletion and atrophy) and stomach (ulceration or erosion in the glandular mucosa). Only one control female was found not pregnant at necropsy.

The number of females with live pups on Day 4 post partum was: 8 in the control group, 10 in each of the low and mid-dose groups, 6 in the high dose group.

Clinical signs

No signs of toxicological significance were observed in animals from the control, low and mid-dose groups and in the surviving female animals from the high dose group.

Body weight and body weight gain

No changes of toxicological significance were observed in body weight and body weight gain in treated males and females when compared to controls.

Food consumption

No effects on food consumption were observed in the males and in low and mid-dose females. Reduced food consumption was seen in the high dose females during the post coitum period.

Oestrus cycle, mating performance and reproductive parameters

Mean oestrus cycle was slightly reduced in the females dosed at 1000 mg/kg/day (approximately 2 in the 15 day pre-mating period) when compared to controls (approximately 3).

No significant differences between groups were observed in pre-coital interval and copulation plugs, as well as in the reproductive parameters (copulatory index and fertility index).

Implantation, pre-birth loss data and gestation length

No treatment-related differences were noted in implantation number, pre-birth loss data and gestation length between control and treated groups.

Litter data and sex ratios

Litter data and sex ratios were unaffected by treatment.

Pre-weaning clinical signs of pups

Clinical signs were comparable between treated and control groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum No treatment-related effects were seen.

Organ weights

No toxicologically relevant changes were observed in organ weights.

Macroscopic observations

No relevant changes were detected at post mortem examination in treated animals killed at end of treatment period, when compared with controls.

Microscopic observations

No treatment-related changes were seen in selected organs/tissues evaluated in high dose males or females receiving the test item, sacrificed at the end of treatment period, or in the abnormalities detected at post mortem.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Sprague Dawley:Crl:CD(SD) rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Lecco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males & females: 6-8 weeks
- Weight at study initiation:
Males: 201 to 225 g for males and 151 to 175 g for females; The weight variation did not exceed ± 20 per cent of the mean weight.
- Fasting period before study: no
- Housing:
Before mating: up to 5 of one sex to a cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: up to 5 animals / cage
Cage type: polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (pre-mating), polycarbonate cages measuring 42.5x26.6x18.5 cm (mating period and pregnant females)
Bedding: Nesting material was provided inside suitable bedding bags and changed at least twice a week
- Diet (e.g. ad libitum):
A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or diet.
- Water (e.g. ad libitum):
Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period:
An acclimatisation period of 20 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 2 °C
- Humidity (%):
55 ± 15 %
- Air changes (per hr):
15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):
artificial light for 12 hours each day
Actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved/suspended in the vehicle, purified water. The formulation was prepared daily (concentrations of 10, 30 and 100 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified. The recovery of the test item from the vehicle was within the acceptance criteria.
The analytical method was validated in the range of 5 to 150 mg/mL.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked during the pre-treatment period in the range of 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirm that the method was acceptable.

Final results for all levels were within the acceptability limits for concentration (90-110%) and homogeneity (CV<10%).
Final results for stability were within the acceptability limits for concentration (90-110%) and homogeneity (CV<10%).
Samples of the formulations prepared on Day 1 and during Week 4 were analysed to check the homogeneity and concentration. Results of the analyses were within the limits of acceptance for suspensions (90-110% for concentration and CV <10% for homogeneity).

Duration of treatment / exposure:

Males: minimum of 2 consecutive weeks prior to pairing and thereafter during pairing up to the day before necropsy.
Females: minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice.

Frequency of treatment:

Animals were dosed once a day, 7 days a week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor
- Rationale for animal assignment (if not random): random

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Four animals were sacrificed for humane reasons at the end of gestation period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION
Food consumption was recorded at weekly intervals whenever possible, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
One day after the last treatment (i.e. on the day of necropsy).
- Animals fasted: Yes, animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
- How many animals: Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
- Parameters checked in table "Clinical chemistry parameters examined" were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Reproductive parameters. See IUCLID chapter 7.8.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Parental animals killed for humane reasons and those that had completed the scheduled test period were killed with carbon dioxide.
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.
Parental males:
The males were killed after the mating of all females, after at least 28 days of treatment period.
Parental females:
The females with live pups were killed on Day 4 post partum.
One female with total litter loss was killed one day after the occurrence of total litter loss. The females which had not given birth 25 days after the positive identification of mating were killed shortly after.

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 (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females:
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.
Pups:
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed and examined for external abnormalities and sex confirmation by
gonadal inspection.

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

Tissues fixed and preserved
Samples of all the tissues listed below were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid) from all animals.

Abnormalities
Coagulating glands
Epididymides
Ovaries
Penis
Prostate gland
Seminal vesicles
Testes
Uterus with cervix
Vagina

HISTOPATHOLOGY: Yes
The tissues required for histopathological examination are listed above. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. The examination was restricted to the following:
a) Tissues specified above from all animals in the control and high dose group killed at term.
b) Tissues specified above from all animals killed during the treatment period.
c) All abnormalities in all groups.

Other examinations:

Reproductive parameters (see IUCLID chapter 7.8)

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were 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 non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Four high dose females (nos. 92510061, 92510067, 92510071 and 92510075) were sacrificed for humane reasons on Day 21/22 of gestation. At despatch to necropsy these animals had pale appearance, decreased activity, piloerection and/or were cold to touch. At necropsy examination they were all pregnant.
At post mortem examination, all females showed pale colour of the kidneys and reduced size of the spleen and thymus. In addition some females also showed multiple depressed dark areas in the glandular region of the stomach with associated yellow mucoid contents in the ileum and jejunum and/or distended duodenum with gas contents and/or pale colour of the liver.
Such changes were considered to be treatment-related.
Treatment-related changes were noted in the kidneys, spleen, thymus and stomach. The observed renal injury involved the nephrons, unit of the kidney, morphologically characterised by degeneration of some glomeruli (sclerosis) and the majority of renal tubules, sometimes associated with subacute inflammatory reactions. The renal tubules showed lined cells, in some instances basophilic with presence of exfoliated cells and/or amorphous material (crystal-like) in their lumen. Focal tubular necrosis in the papilla, associated with pelvic dilatation (hydronephrosis) and hyperplasia of the pelvic lining epithelium were also reported in female no. 92510075.
Moreover, mild to marked lymphoid depletion or red pulp depletion and atrophy were noted in the spleen and in the thymus and ulceration or erosion in the glandular mucosa of the stomach was also reported in the four unscheduled dead females. These changes were considered stress-related.
All females mated. Only one control female (no. 92510005) was sacrificed 26 days post coitum and found not pregnant at necropsy.
The number of females with live pups on Day 4 postpartum was: 8 in the control group, 10 in each of the low and mid-dose groups, 6 in the high dose group. Total litter loss was observed in a single female from the control group (no. 92510019).
Male animals did not show any signs of toxicological significance during the whole study. No significant clinical signs were seen in the female animals killed at the end of the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four females dosed at 1000 mg/kg/day were sacrificed for humane reasons on Day 21/22 of gestation due to the presence of a number of clinical signs (pale appearance, decreased activity, piloerection and/or cold to touch).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in body weight were observed in the treated animals when compared to controls. Reductions in body weight and body weight gain (these being also statistically significant) were observed in the treated males during the mating treatment period. These reductions, which were not dose-related, were not considered to be toxicologically significant. No significant changes were observed in the females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on food consumption were observed in the males. Slight and occasionally also significant at statistical analysis reductions were seen in the high dose females on Day 20 post coitum (-13%) and on Day 4 post partum (-19%).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Four high dose females (nos. 92510061, 92510067, 92510071 and 92510075) were sacrificed for humane reasons on Day 21/22 of gestation. At despatch to necropsy these animals had pale appearance, decreased activity, piloerection and/or were cold to touch.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Terminal body weight as well as absolute and relative weight of testes, epididymides and ovaries were similar between groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations:
No relevant changes were detected at post mortem examination in treated animals, when compared with controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were seen in selected organs/tissues evaluated in high dose males or females receiving the test substance, sacrificed at the end of treatment period, nor in the abnormalities detected at post mortem.
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.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Reproductive parameters: see IUCLID chapter 7.8

Effect levels

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: systemic toxicity

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Treatment-related effects indicating systemic toxicity were observed in some pregnant females dosed at 1000 mg/kg/day.
No changes were observed in males at any dose and in females dosed at 100 and 300 mg/kg/day.
No adverse effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.
The dose level of 300 mg/kg/day is considered to be the NOEL for systemic toxicity, while 1000 mg/kg/day is the NOAEL for fertility and reproduction parameters.

Executive summary:

A study according to OECD Guideline 422 and GLP was performed to investigate the repeated dose toxicity of the test item when administered orally to male and female Sprague Dawley rats. Groups of 10 males and 10 females received the test item, by gavage at dosages of 100, 300 and 1000 mg/kg/day. A similarly constituted group of animals received the vehicle alone (purified water) and acted as a control. All doses were administered at a constant volume of 10 mL/kg body weight.

Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 4 consecutive weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.

The following investigations were performed on all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter observation, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Mortality and fate of females

Four high dose females were sacrificed for humane reasons on Day 21/22 of gestation. At despatch to necropsy these animals had pale appearance, decreased activity, piloerection and/or were cold to touch. At necropsy examination they were all pregnant. Pale colour of the kidneys, reduced size of the spleen and thymus, multiple depressed dark areas in the glandular region of the stomach with associated yellow mucoid contents in the ileum and jejunum and/or distended duodenum with gas contents and/or pale colour of the liver were also observed.

Histopathological examination revealed treatment-related changes in the kidneys (nephrons, unit of the kidney, morphologically characterised by degeneration of some glomeruli and the majority of renal tubules, sometimes associated with subacute inflammatory reactions), spleen and thymus (lymphoid depletion or red pulp depletion and atrophy) and stomach (ulceration or erosion in the glandular mucosa). Only one control female was found not pregnant at necropsy.

The number of females with live pups on Day 4 post partum was: 8 in the control group, 10 in each of the low and mid-dose groups, 6 in the high dose group.

Clinical signs

No signs of toxicological significance were observed in animals from the control, low and mid-dose groups and in the surviving female animals from the high dose group.

Body weight and body weight gain

No changes of toxicological significance were observed in body weight and body weight gain in treated males and females when compared to controls.

Food consumption

No effects on food consumption were observed in the males and in low and mid-dose females. Reduced food consumption was seen in the high dose females during the post coitum period.

Oestrus cycle, mating performance and reproductive parameters

Mean oestrus cycle was slightly reduced in the females dosed at 1000 mg/kg/day (approximately 2 in the 15 day pre-mating period) when compared to controls (approximately 3).

No significant differences between groups were observed in pre-coital interval and copulation plugs, as well as in the reproductive parameters (copulatory index and fertility index).

Implantation, pre-birth loss data and gestation length

No treatment-related differences were noted in implantation number, pre-birth loss data and gestation length between control and treated groups.

Litter data and sex ratios

Litter data and sex ratios were unaffected by treatment.

Pre-weaning clinical signs of pups

Clinical signs were comparable between treated and control groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum No treatment-related effects were seen.

Organ weights

No toxicologically relevant changes were observed in organ weights.

Macroscopic observations

No relevant changes were detected at post mortem examination in treated animals killed at end of treatment period, when compared with controls.

Microscopic observations

No treatment-related changes were seen in selected organs/tissues evaluated in high dose males or females receiving the test item, sacrificed at the end of treatment period, or in the abnormalities detected at post mortem.

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.