Dimolybdenum trizinc nonaoxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species:

rat

Strain:

Wistar

Remarks:

RccHan™;WIST

Details on species / strain selection:

Standard for use in regulatory toxicology studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males 84 to 90 days and females 98 to 104 days
- Weight at study initiation: Males 319 to 355g and females 195 to 237g
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used (except during pairing). Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet provided ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, non restricted.
- Acclimation period: Six days before commencement of treatment (males) and 20 days before commencement of treatment (females).

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Between 20 to 24°C
- Humidity (%): Between 40 and 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hour light/dark cycle.

IN-LIFE DATES: From: To: 30 July 2019 (estrous cycle evaluation) to 13 October 2019 (last female necropsy).

Route of administration:

oral: gavage

Details on route of administration:

Standard for use in regulatory toxicology studies

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Starting with the lowest concentration, a sufficient amount of vehicle was added to the pre weighed test item to obtain a solution. Any large particles were crushed using a spatula and the solution was mixed with a magnetic stirrer. Once fully dissolved, the suspension was transferred to a measuring cylinder and made to the required volume using the vehicle. The suspension was transferred to a suitable container and stirred again using a magnetic stirrer. Formulations were transferred to their final containers using a syringe, whilst magnetically stirred. Since the formulation analysis method was not stability
indicating, formulations were prepared daily and administered within four hours of preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Purified water
- Concentration in vehicle: 0, 6, 12 and 25 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As no analytical method is available which is also stability indicating for this inorganic substance, the analytical method involved removing the purified water by evaporation and drying the residue formulations. Sample concentration were determined by weighing the final residue of the formulations remaining in the sample.

The formulations for Week 1 and the Final week of treatment were sampled, 1 × 10 mL (accurately weighed), from the middle of the formulation. For Week 1, Group 4 had out of specification results. The relative mean error (RME) was -28.4% from the nominal concentration and the difference from mean was ±5.41%.

Additional aliquots were taken as contingency samples for confirmation of results. The contingency results have been reported alongside the original results. The RME for Group 4 was within the acceptance criteria at +5.6% and the coefficient of variation was 37.21%.

Duration of treatment / exposure:

Males: Treated for 2 weeks prior to pairing , up to necropsy after a minimum of 5 consecutive weeks.
Females: Treated for 2 weeks prior to pairing , throughout pairing, gestation and until Day 13 of lactation.

Frequency of treatment:

Daily at roughly the same time each day

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males/females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this OECD 422 study (0, 30, 60 and 125 mg/kg/day) were based on the results of a 14-day preliminary study. In the preliminary study dose levels of 62.5, 250, 500 and 1000 mg/kg/day were investigated in male and female Han Wistar rats. Treatment at doses of 250, 500 and 1000 mg/kg/day were not tolerated, with all groups terminated early as a result of excessive body weight loss. Treatment at 62.5 mg/kg/day which showed no obvious effect on body weight or food consumption. Clinical signs of animals treated at 62.5 mg/kg/day did not indicate any systemic effect of treatment, and necropsy showed no abnormalities. Dose levels of 30, 60 and 125 mg/kg/day (follow the OECD guidelines with a regular interval) were considered suitable for this main investigation.
- Rationale for animal assignment (if not random): Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. Body weight of animals did not exceed +/- 20% of the mean for each sex.
- Fasting period before blood sampling for clinical biochemistry: Yes. Food witheld the night before.

Positive control:

Not a guideline requirement

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. Detailed observations were also performed to establish and confirm a pattern of signs in association with dosing. Males were observed daily during the first week and twice weekly thereafter. Females were observed daily during the first week, twice during the second week, on Days 0, 7, 14 and 20 of gestation and Days 1, 6 and 12 of the lactation phase.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”. After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity.
Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded in males before dosing on the day treatment commenced and weekly thereafter including at necropsy. In females bodyweights were recorded prior to treatment, weekly before pairing, Days 0, 7, 14 and 20 after mating and on Days 1, 4, 7 and 13 of lactation including at necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded weekly from the day treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Day 15-22), but recommenced for males from Day 22. For females after mating food consumption was recorded on Days 0-7, 7-14, 14-20 after mating and on Days 1-4, 4-7 and 7-13 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: The 5 lowest numbered males per group and the first five lactating females with surviving litters per group.
- Parameters: Hematocrit (L/L), Hemoglobin concentration, Erythrocyte count, Absolute reticulocyte count, Mean cell hemoglobin, Mean cell hemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, lymphocytes, eosinophils, basophils, monocytes and large unstained cells, platelet count. Prothrombin time and activated partial thromboplastin time were also assessed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Yes
- How many animals: The 5 lowest numbered males per group and the first five lactating females with surviving litters per group.
- Parameters: Alkaline phosphatase, Alanine aminotransferase, Alanine aminotransferase, Aspartate aminotransferase, Total bilirubin, bile acids, urea, Creatinine, glucose, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein and albumin. Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 5 of treatment (males) and lactation days 7-9 (females)
- Dose groups that were examined: Five males and females. per group.
- Battery of functions tested: Sensory reactivity and grip strength, approach response, pinna reflex, auditory startle response, tail pinch response, grip strength and motor activity.

IMMUNOLOGY: Yes
- Time schedule for examinations: At termination for all F0 adult females with a surviving litter. Day 4 of age for F1 offspring (where possible) and Day 13 of age.
- How many animals: All adult males and F0 females with a surviving litter.
- Dose groups that were examined: All
- Parameters: Samples from offspring on Day 13 of age and adult males were assessed for levels of thyroxine (T4). There was no effect of treatment following thyroxine analyses, therefore, TSH and Day 4 of age T4 samples were not analyzed.

Sacrifice and pathology:

GROSS PATHOLOGY: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. See table 1 in 'any other information on materials incl. methods' for full list.

HISTOPATHOLOGY: Tissues were routinely preserved in 10% Neutral Buffered Formalin except for the testes (initially in modified Davidson's fluid) and the eyes (in Davidson's fluid). Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. In addition to the lists in table 1, the five lowest numbered males and the first five lactating females with a surviving litter in Groups 2 and 3 at scheduled termination were examined. This is as a result of histopathological changes observed in the high dose requiring further examination.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.

The following sequence of statistical tests was used for body weight, food consumption, hematology, blood chemistry and organ weight data:

A parametric analysis (e.g. Williams' test, Dunnett's test) was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. A non-parametric analysis (e.g. Kruskal-Wallis' test, Wilcoxon rank sum test, Shirley's test) was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 2 of treatment, one female which received 30 mg/kg/day was observed having dry rales. There were no clinical signs observed during the treatment period prior to pairing, during gestation or during lactation that were considered related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male which received 60 mg/kg/day was terminated for welfare reasons on Day 3 of treatment due to signs of high pitched, dry rales. There were no findings seen at macroscopic evaluation. This death was considered not to be related to treatment as no dose dependency was noted and it occurred early on in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain for males which received 125 mg/kg/day during treatment was lower than that of Controls (64% of Control). This was due to low body weight gain up to Day 22 of treatment; body weight gain for these animals thereafter (Days 22-36 of treatment) was similar to that of Controls. Body weight gain for males which received 30 or 60 mg/kg/day were unaffected by treatment.

Body weight gain for females which received 125 mg/kg/day was similar to that of Controls during Days 1-8 of treatment and decreased during Days 8-15 of treatment (14% of Control). Bodyweight gains for females which received 30 or 60 mg/kg/day were unaffected before pairing.

Bodyweight gains for females which received 30, 60 or 125 mg/kg/day during gestation and lactation were unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for males which received 125 mg/kg/day were slightly low when compared to Controls for Days 1 to 15 of treatment. Food consumption values for these animals were comparable to Controls for the remainder of the treatment period.

Overall food consumption for males which received 30 or 60 mg/kg/day were unaffected during treatment; females which received 30, 60 or 125 mg/kg/day were also unaffected during treatment, gestation and lactation.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology investigations at scheduled termination for treated males revealed statistically significantly lower hematocrit and erythrocyte counts at all dose levels when compared to Controls, however no dose-response was apparent. Hemoglobin concentrations for males which received 125 mg/kg/day were also statistically significantly lower than controls.

All other differences from Control for males and females, including those which attained statistical significance, were minor, confined to one sex or showed no dose-related response and therefore, considered unrelated to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry investigations at scheduled termination revealed statistically significantly higher bile acid levels for males which received 125 mg/kg/day when compared to Controls; creatinine levels in males were also statistically significantly higher than Controls at this dose level.

Plasma glucose levels were statistically significantly lower when compared to Controls for both males and females which received 125 mg/kg/day and males which received 60 mg/kg/day.

Females which received 125 mg/kg/day showed statistically significantly higher levels of plasma urea when compared to Controls.

All other differences from Control for males and females, including those which attained statistical significance, were minor, confined to one sex or showed no dose-related response and therefore, considered to represent normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were observed for sensory reactivity and grip strength assessment during Week 5 of treatment for males and Day 7-9 of lactation for females at all dose levels. The mean forelimb grip strength for males and females at 125 mg/kg/day was slightly lower than in Controls; with differences attaining statistical significance; however both values were within the historical control data ranges so no effect of treatment is inferred.

Motor activity was considered to be unaffected by treatment.

Activity (high and low beam) was statistically significantly low in males which received 30, 60 or 125 mg/kg/day at the 48 minute period, and for males which received 125 mg/kg/day at the 24 minute period. Overall total scores for both low and high beam at all dose levels were low when compared to Controls, however these did not attain statistical significance, and there was no consistent difference over time at 125 kg/kg/day so no effect of treatment is inferred.

Immunological findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. Consequently, there was no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The evaluation of organ weights performed after five weeks of treatment in males revealed, slightly high absolute and adjusted kidney weights and slightly low absolute liver weights with no effect on adjusted liver weight at 125 mg/kg/day. Adjusted spleen weights were high in males at all dose levels which were treated with the substance.

Organ weights for females which received 125 mg/kg/day at Day 13 of lactation revealed slightly high absolute and adjusted adrenal and kidney weights. Females which received 30 or 60 mg/kg/day revealed no effect on organ weights at Day 13 of lactation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three males which received 125 mg/kg/day showed pale kidneys with one male also showing marked enlargement.

In the stomach, dark areas observed macroscopically in some treated animals, correlated with the microscopic finding of minimal agonal hemorrhage/congestion, and is not related to treatment.

There were no other test item related changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with the test substance were seen in the kidney (moderate to marked tubular degeneration/regeneration and tubular dilatation) in males which received 125 mg/kg/day and in the stomach (eosinophilic globules in surface/mucus neck cells) in males and females which received 125 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

General systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, were evaluated with by oral gavage administration; concentrations of 30, 60 or 125 mg/kg/day were generally well-tolerated when administered daily for at least five weeks in males, and in females for two weeks before pairing, throughout pairing and gestation to Day 13 of lactation. One male which received 60 mg/kg/day was terminated for welfare reasons on Day 3 of treatment due to signs of high pitched, dry rales. Macroscopic examination revealed no findings and this death was considered unrelated to treatment. The clinical condition of the adults remained good throughout the period of treatment.

Effects on body weight performance and food consumption (comprising low mean weight gain and reduced food consumption) were evident during the treatment period for males and females which received 125 mg/kg/day. Overall group mean body weight gain for males which received 125 mg/kg/day were low during the first three weeks of treatment (Days 1 to 22) and for females at the same dose level during the second week of treatment (Days 8 to 15). In addition, food consumption was lower than Control for males which received 125 mg/kg/day for the first two weeks of treatment (Days 1 to 15). Following five weeks of treatment, the terminal body weights for males which received 125 mg/kg/day were slightly low when compared to Controls.

Sensory reactivity, grip strength and motor activity investigations, performed in Week 5 or Day 7-9 of lactation, were unaffected by treatment. There was no effect on serum T4 concentrations in adult males and females treated at 30, 60 or 125 mg/kg/day, nor in the F1 offspring.

Macroscopic and microscopic examination after five weeks of treatment revealed findings in the kidney of males which received 125 mg/kg/day. Pale kidneys were recorded in three males and marked enlargement of the kidneys in one male was observed at 125 mg/kg/day; mean absolute and adjusted kidney weights were increased when compared to Controls. These findings correlated with increased creatinine concentrations in the plasma, as well as tubular degeneration/regeneration and tubular dilation seen at histopathological examination which were considered adverse changes. One female which received 125 mg/kg/day was recorded as having abnormal color of the adrenals and one other female had a dilated pelvis of the kidney at macroscopic examination; high absolute and adjusted kidney weights were recorded, and increased urea concentrations in the plasma. However, there were no related microscopic findings in the kidneys or adrenals for females which received 125 mg/kg/day so these findings were of no toxicological significance and of uncertain relationship to treatment.

In addition, microscopic examination revealed eosinophilic globules in the surface/mucus cells of the stomach in both males and females which received 125 mg/kg/day. Eosinophilic globules occur infrequently in rodents as a spontaneous lesion or associated with other lesions. They may be considered as a precursor of hyaline eosinophilic crystals and are usually located in mucus neck cells of the glandular mucosae near the limiting ridge. This finding is considered to be non-adverse (Nolte et al 2016). There were no macroscopic or microscopic correlates for the non-dose related decreased hematocrit and erythrocyte counts, decreased hemoglobin concentrations, decreased plasma glucose levels and increased bile acid levels in males which received 125 mg/kg/day, and decreased plasma glucose levels in females which received 60 or 125 mg/kg/day when compared to Controls. These changes were therefore considered non-adverse and of uncertain relationship to treatment.

The high adjusted spleen weights in all groups of treated males, slightly low absolute liver weights as well as slightly high absolute and adjusted adrenal weights in females which received 125 mg/kg/day were not associated with any macroscopic or microscopic changes and, therefore, considered non-adverse. There were no treatment-related macroscopic or microscopic findings for males and females at 30 or 60 mg/kg/day. In addition, the substance did not show any evidence of reproductive/developmental effects or endocrine disruption at any dose level investigated.

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

125 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

General systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, were evaluated with by oral gavage administration; concentrations of 30, 60 or 125 mg/kg/day were generally well-tolerated when administered daily for at least five weeks in males, and in females for two weeks before pairing, throughout pairing and gestation to Day 13 of lactation.

Based on the histopathological finding of tubular degeneration/regeneration in the kidneys of male Han Wistar rats at 125 mg/kg/day, it is concluded that the kidney is a target organ for toxicity and 60 mg/kg/day is the No-Observed-Adverse-Effect (NOAEL) for general systemic toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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

Test material information

Constituent 1

Reference substance name:

Dimolybdenum trizinc nonaoxide

EC Number:

245-322-4

EC Name:

Dimolybdenum trizinc nonaoxide

Cas Number:

22914-58-5

Molecular formula:

Mo2O9Zn3

IUPAC Name:

dimolybdenum trizinc nonaoxide

impurity 1

Reference substance name:

Molybdenum zinc tetraoxide

EC Number:

237-377-8

EC Name:

Molybdenum zinc tetraoxide

Cas Number:

13767-32-3

Molecular formula:

MoO4Zn

IUPAC Name:

molybdenum zinc oxide

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan™;WIST

Details on species / strain selection:

Standard for use in regulatory toxicology studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males 84 to 90 days and females 98 to 104 days
- Weight at study initiation: Males 319 to 355g and females 195 to 237g
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used (except during pairing). Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet provided ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, non restricted.
- Acclimation period: Six days before commencement of treatment (males) and 20 days before commencement of treatment (females).

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Between 20 to 24°C
- Humidity (%): Between 40 and 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hour light/dark cycle.

IN-LIFE DATES: From: To: 30 July 2019 (estrous cycle evaluation) to 13 October 2019 (last female necropsy).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Starting with the lowest concentration, a sufficient amount of vehicle was added to the pre weighed test item to obtain a solution. Any large particles were crushed using a spatula and the solution was mixed with a magnetic stirrer. Once fully dissolved, the suspension was transferred to a measuring cylinder and made to the required volume using the vehicle. The suspension was transferred to a suitable container and stirred again using a magnetic stirrer. Formulations were transferred to their final containers using a syringe, whilst magnetically stirred. Since the formulation analysis method was not stability indicating, formulations were prepared daily and administered within four hours of preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Purified water
- Concentration in vehicle: 0, 6, 12 and 25 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear. Day 0 of gestation counted as day evidence of mating was detected.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: None

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As per repeated dose entry.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after a minimum of five weeks of treatment.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

Daily via oral gavage

Details on study schedule:

- Age at mating of the mated animals in the study: Males 84 to 90 days and females 98 to 104 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

125 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this OECD 422 study (0, 30, 60 and 125 mg/kg/day) were based on the results of a 14-day preliminary study. In the preliminary study dose levels of 62.5, 250, 500 and 1000 mg/kg/day were investigated in male and female Han Wistar rats. Treatment at doses of 250, 500 and 1000 mg/kg/day were not tolerated, with all groups terminated early as a result of excessive body weight loss. Treatment at 62.5 mg/kg/day which showed no obvious effect on body weight or food consumption. Clinical signs of animals treated at 62.5 mg/kg/day did not indicate any systemic effect of treatment, and necropsy showed no abnormalities. Dose levels of 30, 60 and 125 mg/kg/day (follow the OECD guidelines with a regular interval) were considered suitable for this main investigation.

- Rationale for animal assignment (if not random): Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. Body weight of animals did not exceed +/- 20% of the mean for each sex.

- Fasting period before blood sampling for clinical biochemistry: Yes. Food witheld the night before.

Positive control:

Not a guideline requirement

Examinations

Parental animals: Observations and examinations:

See repeated dose study for details on parental animal observations and examinations.

Oestrous cyclicity (parental animals):

Smears were taken for 15 days before pairing using cotton swabs (dry smears). Wet smears were taken for 14 days before treatment, after pairing until mating and for 4 days before scheduled termination (nominally Days 10-13 of lactation).

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Clinical observations (examined approximately 24 hours after birth and then daily), litter size (daily), sex ratio (Days 1, 4, 7, 11 and 13), individual bodyweights (Days 1, 4, 7, 11 and 13), ano-genital distance (day 1) and nipple/areolae count (Day 13 in males).

GROSS EXAMINATION OF DEAD PUPS:
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.

Postmortem examinations (parental animals):

See repeated dose study for details on parental animal observations and examinations.

Postmortem examinations (offspring):

GROSS EXAMINATION OF DEAD PUPS:
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.

Reproductive indices:

Pre-coital interval, percentage mating, conception rate, fertility index, gestation length and index, litter size and post-implantation survival index,

Offspring viability indices:

Survival index, live birth index, viability index, lactation index and sex ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

See repeated dose entry for paternal data.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See repeated dose entry for paternal data.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles were considered unaffected by treatment when compared with controls. All females showed regular 4 or 5 day cycles prior to treatment and there was no effect of treatment of estrous cycle regularity. All females mated within 1-4 days with the exception of one Control female which was observed to be acyclic during pairing. At termination, all females were observed to be in diestrus.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-coital interval, mating performance, gestation length, gestation index and fertility were considered unaffected by treatment with the substance, when compared to Controls.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No effects observed on reproduction parameters. NOAEL is set as the high dose.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs observed amongst offspring following parental administration of the test substance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of parental treatment of the mean number of offspring survival indices.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of male and female offspring on Day 1 of age and subsequent body weight gain until Day 13 of age was similar to that of the Control in all treated groups. Minor intergroup differences were considered to reflect the marginally higher litter sizes in the 30 or 125 mg/kg/day groups.

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):

no effects observed

Description (incidence and severity):

There was no effect of treatment on ano-genital distance in the male and female offspring at any dose level investigated.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in male offspring of parents treated with the test substance.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to treatment.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

The estrous cycle investigations, during the pre-mating period was unaffected, and all animals with litters were in di-estrus at termination, indicating that estrous cycles had ceased during lactation. Pre-coital interval, mating performance, gestation length and gestation index were unaffected by treatment. The litter size, sex ratio, survival, ano-genital distance and growth of the offspring (F1) were unaffected by treatment, the males did not develop nipples and there were no parental treatment related macroscopic findings in the offspring. There was no effect on serum T4 concentrations in adult males and females treated at 30, 60 or 125 mg/kg/day, nor in the F1 offspring.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No effects observed relating to offspring. NOAEL is set as the high dose.

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

General systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, were evaluated with by oral gavage administration; concentrations of 30, 60 or 125 mg/kg/day were generally well-tolerated when administered daily for at least five weeks in males, and in females for two weeks before pairing, throughout pairing and gestation to Day 13 of lactation.

As administration of the substance at doses up to and including 125 mg/kg/day did not affect the reproductive performance of male and females, or the development of their offspring, it was concluded that the NOAEL for reproductive/developmental toxicity was the high dose of 125 mg/kg/day.