Tetraammonium decachloro-μ-oxodiruthenate(4-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Part of a combined repeated dose study (OECD 422) with reproductive and developmental toxicity screening.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September - 6 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Part of a combined repeated dose study (OECD 422) with reproductive and developmental toxicity screening.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September - 6 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Reason / purpose for cross-reference:

reference to same 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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for such studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: Males and females were aged 67 and 90 days, respectively, at first test material administration.
- Weight at study initiation: Males: 341.5 - 410.6 g at first test material administration. Females: 234.4 - 287.6 g at first test material administration.
- Fasting period before study: Not specified.
- Housing: Males and females kept in individual cages, except during mating period (see reproductive toxicity s
ection for further details).
- Diet (e.g. ad libitum): Standard commercial feed (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 (150 lux)/12

IN-LIFE DATES: From: 28 September 2016 To: 6 December 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were continuously stirred until the last animal of each group had been dosed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification specified (standard vehicle).
- Concentration in vehicle: No data
- Amount of vehicle (if gavage): Constant dose volume of 5 mL/kg bw/day/animal.
- Lot/batch no. (if required): Caesar and Loretz GmbH, Germany. Batch number 15296406.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each dose solution were analysed by ICP-OES for ruthenium content, at dates throughout the study period.

Duration of treatment / exposure:

Males and females were dosed from 2 weeks prior to mating and during the mating period. Males were further dosed after the mating period for a total treatment duration of 34 days. Females were dosed throughout gestation and at least up to and including day 13 post-partum (total of 40-55 days).

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

"Low dose"

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

"Mid dose"

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

"High dose"

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses selected on the basis of a 14-day range-finding study
- Rationale for animal assignment (if not random): Randomised based on body weight

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.
- Cage side observations included skin/fur, eyes, mucuous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure (to allow for within-subject comparisons) and weekly thereafter. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of dosing, weekly thereafter, and at termination. During gestation, females were weighed on days 0, 7, 14 and 20, and within 24 hours of parturition as well as on PND 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Recorded weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Monitored daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group, randomly selected from each group
- The following parameters were examined: Haemoglobin content (HGB), Erythrocytes (RBC), Leucocytes (WBC), Differential blood count (relative and absolute), Reticulocytes (Reti), Platelets (PCT), Haematocrit value (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), as well as Thromboplastin time (TPT) and Activated partial thromboplastin time (aPTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group, randomly selected from each group
- The following parameters were examined: Albumin, Globulin, Albumin/Globulin ratio, Bile acids, Bilirubin (total), Cholesterol (total), Creatinine, Glucose, Protein (total), Blood urea, Calcium, Chloride, Potassium, Sodium, Sodium/Potassium ratio, BUN/Creatinine ratio, Lactate dehydrogenase (LDH), Alanine, aminotransferase (ALAT), Alkaline phosphatase (aP), Aspartate aminotransferase (ASAT).

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: test day 43 or 50 (shortly before scheduled sacrifice); females: test day 65-71 (shortly before scheduled sacrifice). Screening was carried out two hours after dosing, and before any blood sampling.
- Dose groups that were examined: All (5/sex/group).
- Battery of functions tested: sensory activity (auditory, visual, proprioceptive stimuli); grip strength; motor activity.

IMMUNOLOGY: Not specified

OTHER: Thyroid hormone (T4) levels in serum were analysed for adult males (aside from those in high dose group) and PND 13 pups

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All tissues and organs were examined macroscopically.
The weight of the following organs was recorded before fixation (where applicable): adrenals, brain, heart, kidney (2), epididymis (2), liver, uterus (incl. cervix), ovary (2), spleen, testicle (2), thyroid, thymus, combined weight of prostate+seminal vesicles+coagulating glands. Paired organs were weighed individually and identified as left or right. Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY: Yes
The following organs/parts were fixed for microscopic examination: adrenal gland (2), bone, bone marrow (os femoris), brain, epididymis (2), eye with optic nerve (2), all gross lesions observed, heart, intestine, kidney and ureter (2), liver, lungs with bronchi and bronchioles, lymph node (1 cervical and 1 mesenteric), mammary gland, skeletal muscle, sciatic nerve, oesophagus, ovary and oviduct, pituitary, prostate/seminal vesicles/coagulating gland, spinal cord (3 sections), spleen, stomach, testicle (2), thyroid, thymus, tissue masses or tumours, tongue, trachea, urinary bladder, uterus (incl. cervix), vagina.

Other examinations:

During a 14-day pre-exposure period, the oestrus cycles of 13 female animals per study group were monitored to yield at least 10 females having a normal oestrus cycle. Animals that fail to exhibit typical 4-5 day cycles were not included in the study.

Statistics:

Statistical analyses of the parametrical values, captured or calculated by Provantis (i.e. clinical signs, parental body weight, body weight gain, food consumption), were done by Provantis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The test item caused no changes in behaviour, the external appearance or the appearance of the faeces at any of the tested dose level. Salivation and breathing sounds were noted in all dose groups, with increasing incidences. These were not considered to be adverse since there was a lack of systemic toxicity (e.g. effects on body weight or food consumption) and were most likely due to a local (oesophagus/stomach tract) irritant nature of the test item. An increased water consumption and an increased respiratory rate were noted for one animal each at the mid dose level; these were considered to be incidental on the basis of a low incidence.

Mortality:

no mortality observed

Description (incidence):

No premature death was noted in the male or female rats of the control group and the low and the mid dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences in body weight and body weight gain were noted between the control group and the low and mid dose groups. Although there were some occasions where growth was lower in the mid dose group, there was no effect over the whole study period, and therefore these isolated differences were considered to be incidental to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For males, there were no test item-related changes in food consumption between the control and low dose groups. At the mid dose, a reduced food intake compared to the control group was noted in the first week of treatment. However, this normalised in the next week and this observation was considered to be not treatment-related. For low-dose females, a reduced food consumption was noted during the first week of the gestation period, normalising in the further course of the study and consequently was considered to be not of toxicological relevance. At the high dose, food consumption was markedly reduced during the first week of dosing and this likely contributed to the body weight loss for both sexes.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No influence on the water consumption (evaluated by visual appraisal) was noted for the male and female animals of the treatment groups.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increases in the absolute number of monocytes and neutrophils for the female animals of the mid dose group were considered to be unrelated to treatment as almost all values were within the range of the LPT background data and no difference was noted for the male animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted for the examined plasma levels of the biochemical parameters in male animals of the low and mid dose groups. For females, statistically significantly lower sodium concentrations were noted at these dose levels and a statistically significant higher LDH concentration was noted at the low dose. These observations were considered spontaneous since all values of the sodium concentration were within the range of the LPT background data and no dose response-relationship was noted for the LDH concentration.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

None of the examined mid dose females showed urination in contrast to the controls (3 of 5) or the animals of the low dose group (4 of 5). As no further abnormalities regarding the amount or appearance of the urine were observed, the reduced incidence of urination was considered to be spontaneous and not test item-related. There was no influence on fore- and hindlimb (males only) grip strength at the low and intermediate doses. Increased hindlimb grip strength (not significant) was noted for low-dose females, whilst a decrease in this parameterwas seen at the mid dose. As the differences showed no coherent dose response and all were within the LPT background data-range, these were considered to be unrelated to treatment. Slightly higher, but not statistically significant, differences in spontaneous mobility were noted for males at the low and mid doses; in contrast females showed a decrease in these values. These conflicting differences were considered to be not test item-related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted for the relative and absolute organ weights between control group and treated groups. Slight reductions for the absolute and relative weights of the left and right kidneys (statistically significant for the left kidney) were noted in the mid-dose animals, though these were within the background range and were considered to be not test item-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in the low dose group. At the mid dose, 3 males had with few to several haemorrhagic foci in the stomach and another male had a partly reddened stomach mucosa, while 3 females had haemorrhagic foci and/or ulcers. More sever signs of irritation were noted in the high dose animals (dosing was discontinued). These stomach changes resulted most likely from the irritant nature of the test item and therefore were considered to be not adverse.

The following isolated observations were considered as spontaneous and not as test item-related: a lung with several haemorrhagic foci and a discolouration of the superior part of the left lobe in a single low-dose animal; at the high dose, inflated lungs (agonal change), and inflated and reddened intestines as well as reduced spleen in two resptively animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted in the stomach in the low dose group. At the mid dose, erosion(s) and haemorrhages in the glandular mucosa. However, these changes were most likely due to the irritant nature of the test item and therefore, considered to be not adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Male thyroid hormone levels. The slight but statistically significant decrease that was noted for the mid dose group was considered to be spontaneous and therefore not test item related as all values were within the background range.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Critical effects observed:

not specified

Premature death and body weight losses (as well as various clinical signs) were reported for the high dose group (1000 mg/kg bw/day), leading to the termination of these animals after only 2 weeks of treatment.

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of tetraammonium-decachloro-µ-oxodiruthenate for at least 28 days, no clinical signs of toxicity or any adverse pathological or histopathological effects were observed at up to 300 mg/kg bw/day (considered the study NOAEL).

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, CD rats (10/sex/group) were orally administered tetraammonium-decachloro-µ-oxodiruthenate by stomach tube (gavage) at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were dosed for 34 days (14 days pre-mating, as well as during the mating and post-mating periods). Females were dosed for 14 days pre-mating, through mating, gestation (around 22 days) and up to post-natal day 13 (40-55 days in total).

Premature death and body weight losses (as well as various clinical signs) were reported for the high dose group (1000 mg/kg bw/day), leading to the termination of these animals after only 2 weeks of treatment. No test-item related mortality, clinical signs of toxicity, neurological observations, or changes in the body weight, food consumption, haematology or clinical chemistry parameters were observed in the remaining two dose groups. There were no adverse treatment-related changes in organ weights, or following macroscopic examination and histopathology for the adult animals of either sex, aside from some local effects on the stomach at the mid dose (also observed in prematurely terminated animals at the high dose). On this basis, a study NOAEL of 300 mg/kg bw/day was established.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 (incl. QA statement)

Limit test:

no

Justification for study design:

Not applicable

Test material

Test material information

Constituent 1

Reference substance name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

EC Number:

286-924-7

EC Name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

Cas Number:

85392-65-0

Molecular formula:

Cl10ORu2.4H4N

IUPAC Name:

tetraammonium decachloro-μ-oxodiruthenate(4-)

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for such studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: Males and females were aged 67 and 90 days, respectively, at first test material administration.
- Weight at study initiation: Males: 341.5 - 410.6 g at first test material administration. Females: 234.4 - 287.6 g at first test material administration.
- Fasting period before study: Not specified.
- Housing: Males and females kept in individual cages, except during mating period (see reproductive toxicity s
ection for further details).
- Diet (e.g. ad libitum): Standard commercial feed (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 (150 lux)/12

IN-LIFE DATES: From: 28 September 2016 To: 6 December 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were continuously stirred until the last animal of each group had been dosed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification specified (standard vehicle).
- Concentration in vehicle: No data
- Amount of vehicle (if gavage): Constant dose volume of 5 mL/kg bw/day/animal.
- Lot/batch no. (if required): Caesar and Loretz GmbH, Germany. Batch number 15296406.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed (all males successfully mated).
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each dose solution were analysed by ICP-OES for ruthenium content, at dates throughout the study period.

Duration of treatment / exposure:

Males and females were dosed from 2 weeks prior to mating and during the mating period. Males were further dosed after the mating period for a total treatment duration of 34 days. Females were dosed throughout gestation and at least up to and including day 13 post-partum (total of 40-55 days).

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 81 days (males) or 104 days (females)

F1 animals killed at postnatal day 13

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

"Low dose"

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

"Mid dose"

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

"High dose"

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses selected on the basis of a 14-day range-finding study
- Rationale for animal assignment (if not random): Randomised based on body weight

Positive control:

Not included

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.
- Cage side observations included skin/fur, eyes, mucuous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure (to allow for within-subject comparisons) and weekly thereafter. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of dosing, weekly thereafter, and at termination.
During gestation, females were weighed on days 0, 7, 14 and 20, and within 24 hours of parturition as well as on PND 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Recorded weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Monitored daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group, randomly selected from each group
- The following parameters were examined: Haemoglobin content (HGB), Erythrocytes (RBC), Leucocytes (WBC), Differential blood count (relative and absolute), Reticulocytes (Reti), Platelets (PCT), Haematocrit value (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), as well as Thromboplastin time (TPT) and Activated partial thromboplastin time (aPTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group, randomly selected from each group
- The following parameters were examined: Albumin, Globulin, Albumin/Globulin ratio, Bile acids, Bilirubin (total), Cholesterol (total), Creatinine, Glucose, Protein (total), Blood urea, Calcium, Chloride, Potassium, Sodium, Sodium/Potassium ratio, BUN/Creatinine ratio, Lactate dehydrogenase (LDH), Alanine, aminotransferase (ALAT), Alkaline phosphatase (aP), Aspartate aminotransferase (ASAT).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: test day 43 or 50 (shortly before scheduled sacrifice); females: test day 65-71 (shortly before scheduled sacrifice). Screening was carried out two hours after dosing, and before any blood sampling.
- Dose groups that were examined: All (5/sex/group).
- Battery of functions tested: sensory activity (auditory, visual, proprioceptive stimuli); grip strength; motor activity.

OTHER: Thyroid hormone (T4) levels in serum were analysed for adult males (aside from those in high dose group) and PND 13 pups

Oestrous cyclicity (parental animals):

During a 14-day pre-exposure period, the oestrus cycles of 13 female animals per study group were monitored to yield at least 10 females having a normal oestrus cycle. Animals that fail to exhibit typical 4-5 day cycles were not included in the study.

Sperm parameters (parental animals):

Detailed histopathologic examination were performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of the the control and mid dose groups.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (4-5/sex/litter as nearly as possible); excess pups were killed and blood samples for T4 hormone level determination were taken on PND 4 .

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnormalities, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead. External reproductive genitals were examined for signs of altered development.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed one day after the last treatment
- Maternal animals: All surviving animals were sacrificed on PND 14 (one day after the last treatment).

GROSS NECROPSY
All tissues and organs were examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weight of the following organs was recorded before fixation (where applicable): adrenals, brain, heart, kidney (2), epididymis (2), liver, uterus (incl. cervix), ovary (2), spleen, testicle (2), thyroid, thymus, combined weight of prostate+seminal vesicles+coagulating glands. Paired organs were weighed individually and identified as left or right.

The following organs/parts were fixed for microscopic examination: adrenal gland (2), bone, bone marrow (os femoris), brain, epididymis (2), eye with optic nerve (2), all gross lesions observed, heart, intestine, kidney and ureter (2), liver, lungs with bronchi and bronchioles, lymph node (1 cervical and 1 mesenteric), mammary gland, skeletal muscle, sciatic nerve, oesophagus, ovary and oviduct, pituitary, prostate/seminal vesicles/coagulating gland, spinal cord (3 sections), spleen, stomach, testicle (2), thyroid, thymus, tissue masses or tumours, tongue, trachea, urinary bladder, uterus (incl. cervix), vagina.

Postmortem examinations (offspring):

SACRIFICE
- All F1 animals were sacrificed on PND 13

GROSS NECROPSY
- Dead pups and pups sacrified at day 13 post-partum were carefully examined externally for gross abnormalities. External reproductive genitals were examined for signs of altered development. The thyroid from 1 male and 1 female pup/litter was weighed and fixed for examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid glands were weighed and examined microscopically.

Thyroid hormone T4 levels in serum were also measured.

Statistics:

Statistical analyses of the parametrical values, captured or calculated by Provantis (i.e. clinical signs, parental body weight, body weight gain, food consumption), were done by Provantis.

Reproductive indices:

Female fertility index and gestation index

Offspring viability indices:

Birth index, Live birth index, Viability index (pre and post cull), Pre-implantation loss, Post-implantation loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The test item caused no changes in behaviour, the external appearance or the appearance of the faeces at any of the tested dose level. Salivation and breathing sounds were noted in all dose groups, with increasing incidences. These were not considered to be adverse since there was a lack of systemic toxicity (e.g. effects on body weight or food consumption) and were most likely due to a local (oesophagus/stomach tract) irritant nature of the test item. An increased water consumption and an increased respiratory rate were noted for one animal each at the mid dose level; these were considered to be incidental on the basis of a low incidence.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No premature death was noted in the male or female rats of the control group and the low and the mid dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences in body weight and body weight gain were noted between the control group and the low and mid dose groups. Although there were some occasions where growth was lower in the mid dose group, there was no effect over the whole study period, and therefore these isolated differences were considered to be incidental to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For males, there were no test item-related changes in food consumption between the control and low dose groups. At the mid dose, a reduced food intake compared to the control group was noted in the first week of treatment. However, this normalised in the next week and this observation was consider ed to be not treatment-related. For low-dose females, a reduced food consumption was noted during the first week of the gestation period, normalising in the further course of the study and consequently was considered to be not of toxicological relevance. At the high dose, food consumption was markedly reduced during the first week of dosing and this likely contributed to the body weight loss for
both sexes.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No influence on the water consumption (evaluated by visual appraisal) was noted for the male and female animals of the treatment groups.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increases in the absolute number of monocytes and neutrophils for the female animals of the mid dose group were considered to be unrelated to treatment as almost all values were within the range of the LPT background data and no difference was noted for the male animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted for the examined plasma levels of the biochemical parameters in male animals of the low and mid dose groups. For females, statistically significantly lower sodium concentrations were noted at these dose levels and a statistically significant higher LDH concentration was noted at the low dose. These observations were considered spontaneous since all values of the sodium concentration were within the range of the LPT background data and no dose response-relationship was noted for the LDH concentration.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

None of the examined mid dose females showed urination in contrast to the controls (3 of 5) or the animals of the low dose group (4 of 5). As no further abnormalities regarding the amount or appearance of the urine were observed, the reduced incidence of urination was considered to be spontaneous and not test item-related. There was no influence on fore- and hindlimb (males only) grip strength

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted in the stomach in the low dose group. At the mid dose, erosion(s) and haemorrhages in the glandular mucosa. However, these changes were most likely due to the irritant nature of the test item and therefore, considered to be not adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Male thyroid hormone levels. The slight but statistically significant decrease that was noted for the mid dose group was considered to be spontaneous and therefore not test item related as all values were within the background range.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related influences on the oestrus cycles were noted between the control group and the treatment groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Histopathological examination performed on one testicle and one epididymis from the mid-dose group (with special emphasis on the qualitative stages of spermatogenesis and histopathology of the interstitial testicular structure) did not reveal any test item-related effects.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for the female fertility index, gestation index, pre-coital time, gestation length, number of implantation sites, pups born (alive and dead), live born pups, number of stillbirths, birth index, live birth index and percentages of the post-implantation loss at the low and mid doses.

Details on results (P0)

Premature death and body weight losses (as well as various clinical signs) were reported for the high dose group (1000 mg/kg bw/day), leading to the termination of these animals after only 2 weeks of treatment.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

Fertility and reproductive parameters

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted between the survival index of the pups from the dams of the control group and the pups from the dams treated at the low and mid doses. In the control group, one dam was noted with 3 stillborn pups and one further pup was cannibalized during the lactation period. At the low dose, no dam was noted with stillbirths and 3 dams with 1 dead pup each (2 pups were cannibalized and 1 was found dead). In the mid dose group, 2 dams had 1 stillbirth each and no further dead pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related difference was noted between the mean body weight of the control pups and those from treated dams on lactation days 1, 4 or 13. A statistically significant increase in body weight was noted for the male pups and for the male and female pups combined of the low dose group on lactation day 13. As no dose-response relationship was present, the increased pup body weight was considered to be spontaneous and hence not test item-related.

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

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid gland weights were unaffected aside from at the mid dose, where a statistically significant increase was noted for weight of the right glands. As this was only a slight increase and none of the individual data was above the highest value of the of the concurrent control group and no difference was noted for the weight of the left thyroid glands, this difference was considered to be spontaneous and not test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross abnormalities were noted during the macroscopic external and internal examination of the control pups and the pups from the dams treated at the low and mid doses.

Histopathological findings:

no effects observed

Description (incidence and severity):

No test item-related microscopic changes were noted for the thyroid gland(s) of the selected pups from the dams treated at the low and mid doses.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The of the pups of the dose groups on LD 13 was evaluated and the results were compared with the control group.
No test item-related differences in the level of the thyroid hormone T4 in the blood were noted for the pups of the treated dams. Statistically significantly decreased T4 levels were noted for the male pups of the low dose group and for male and female pups combined in the low and high dose groups. However, all values for the T4 levels were within the range of the LPT background data for male and female pups combined and thus considered to be not test item-related.

AGD was statistically significantly increased in males from the low dose group, but was considered to reflect the slightly higher body weights. No nipples were noted in any male animal of the test groups. A statistically significant increased AGD was noted for the female pups at the mid dose. As all individual values were still inside the range of LPT background data, this increase is considered as spontaneous and not as test item-related.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Pre- and post-natal development of pups

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at mid dose (high dose parental animals were prematurely terminated)

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of tetraammonium-decachloro-µ-oxodiruthenate for at least 34 days, no effects on any measured reproductive or fertility parameters, microscopic changes in the reproductive organs, or developmental effects, were observed at up to 300 mg/kg bw/day (the study NOAEL).

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, CD rats (10/sex/group) were orally administered tetraammonium-decachloro-µ-oxodiruthenate by stomach tube (gavage) at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were dosed for 34 days (14 days pre-mating, as well as during the mating and post-mating periods). Females were dosed for 14 days pre-mating, through mating, gestation (around 22 days) and up to post-natal day 13 (40-55 days in total).

Premature death and body weight losses (as well as various clinical signs) were reported for the high dose group (1000 mg/kg bw/day), leading to the termination of these animals after only 2 weeks of treatment. There were no reported changes to reproductive parameters (including number of implantation sites, number of pups, fertility and gestation indices, reproductive performance as well as pre- and post-implantation losses). Further, no treatment-related microscopic changes were observed in the reproductive organs. There were no adverse effects on the F1 offspring viability, growth or developmental effects. The study NOAEL was 300 mg/kg bw/day.