Tetraammonium decachloro-μ-oxodiruthenate(4-)

Endpoint summary

Description of key information

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of tetraammonium-decachloro-mu-oxodiruthenate for at least 34 days, no clinical signs of toxicity or any adverse pathological or histopathological effects were observed at up to 300 mg/kg bw/day (Hansen, 2017).

 

No repeated dose toxicity studies by the inhalation or dermal route were identified, or are required.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Overall, good-quality database which meets REACH Standard Information Requirements.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

No relevant human data were identified.

 

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-mu-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. Tetraammonium-decachloro-mu-oxodiruthenate did not result in test item related mortality, clinical signs of toxicity, neurological observations, or changes in the body weight, food consumption, haematology or clinical chemistry parameters at up to 300 mg/kg bw/day during the treatment period. 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 (Hansen, 2017).

 

According to REACH Annex VIII (EC 1907/2006), repeated dose toxicity studies only need to be conducted on one species taking into consideration the most appropriate route of administration regarding human exposure. The compound is not expected to reach the lungs in appreciable quantities (based on respiratory tract deposition modelling data). Thus, inhalation will not be a significant route of exposure. Similarly, skin contact during production and/or use is expected to be negligible. As the oral route of exposure is considered the most appropriate, repeated dose toxicity studies were not carried out for the dermal or inhalation routes.

Justification for classification or non-classification

In a reliable repeated dose toxicity study (combined with a reproductive/developmental screening assay) involving gavage administration of tetraammonium-decachloro-mu-oxodiruthenate to rats for at least 34 days, no adverse systemic effects were seen at up to 300 mg/kg bw/day. As such, classification of this substance as STOT-RE is not required, according to EU CLP criteria (EC 1272/2008). The observed effects on the stomach are likely the result of local irritancy.