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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July - 19 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July - 19 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Justification for study design:
To assess for treatment-related effects on reproduction parameters and developmental toxicity.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony. Males and females originated from different units to avoid subsequent brother/sister matings.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 10-11 weeks (12-13 weeks at mating).
- Weight at study initiation: Males: 351 g – 381 g, Females: 202 g - 268 g
- Fasting period before study: no data
- Housing: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption, from 500 ml bottle ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 24.5 (target range 22±3)
- Humidity (%): 36 – 70 (target range 30-70)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 July 2014 To: 19 September 2014
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose
Remarks:
1% in distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared for up to 14 days and kept in refrigerator pending dosage. Stability of the test item in the selected vehicle was assessed under the conditions employed on the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected by the sponsor. For the preparation of 2 L vehicle, 20 g Methylcellulose powder was added to approximately 1750 mL hot distilled water, mixed continuously, and gradually made up to 2000 mL with cold distilled water. The mixture was then stirred overnight.
- Concentration in vehicle: 0, 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1 (brother-sister matings were avoided)
- Length of cohabitation: Up to 12 days (until copulation, all pairs mated within this period)
- Proof of pregnancy: Vaginal smears were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were
examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation, referred to as Day 0 of pregnancy.
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed in Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken from each concentration. Each formulation used in the study was sampled and analyzed. One set of samples was collected for analysis and one set for a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the control solution to confirm the absence of test item.

Formulations prepared on Day 0 were sampled and sent to the test site for rapid formulation analysis to provide confirmation on formulation concentration and
homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.
Duration of treatment / exposure:
28 days for males (14 days pre-mating and 14 days mating/post-mating period) and satellite females; 52-54 days for females (14 days pre-mating, for up to 12 days of the mating period, through gestation (22-24 days) and up to and including the day before necropsy (at least 4 days post-partum dosing).
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 (5 additional females/group were included as satellites for toxicology endpoints)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of the repeated dose range finding study in the rat. The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.
- Rationale for animal assignment (if not random): All parental (P Generation) animals were sorted by computer according to body weight and divided in to weight ranges. An equal number of animals from each weight group were randomly assigned to each dose group to ensure that the mean group weights were similar. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for signs of morbidity and mortality; once a day for general clinical observations
- Cage side observations (general clinical) included pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and then weekly (in the morning) aside from week 1.
- Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also assessed. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice a week and at termination for all adult animals. In addition, parent females were weighed on gestation Days 0, 7, 10, 14, 17 and 20 and on postnatal Days (PND) 0 (within 24 hours after parturition) and PND4 (before termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable
- Food consumption was measured in parallel with body weight. Food efficiency was not calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OTHER: additional general toxicity observations included ophthalmoscopy, haematology, clinical chemistry, urinalysis and neurobehavioural exmination of parental animals
Oestrous cyclicity (parental animals):
Observed daily during the mating period, until a sperm positive vaginal smear or a vaginal plug was identified
Sperm parameters (parental animals):
Parameters examined in P0 male parental generation: testis and epididymis weight

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, runts, mortality, gross abnormalities, weight gain and behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups found dead (not cannibalised)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals one day after the last treatment
- Maternal animals: All surviving animals one day after the last treatment

GROSS NECROPSY
- Gross necropsy consisted of external examinations and assessment of the cranium, thoracic, and abdominal cavities as well as the appearance of the tissues and organs. The numbers of implantation sites and corpora lutea were recorded for main test females.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and the weight of the following organs from all adult animals (where appropriate) were determined: uterus (including cervix), ovaries, testes, epididymides, prostate, seminal vesicles with coagulating glands and brain. From all Main males and from all satellite females, the following organs were weighed in addition to the ones previously mentioned: heart, kidneys, liver, spleen and thymus, adrenals, thyroids with parathyroids. Paired organs were weighed together except testes and epididymides which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal. Relative organ weights (to body and brain weight) were calculated and reported.

The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, the testes with epididymides were retained in Bouin’s solution, and all other organs in 10% buffered formalin solution.

In addition, for all Main males and all satellite females and for all adults killed or found dead prior to scheduled termination, the following organs and tissues, or representative samples, were preserved: lungs with bronchi, skeletal muscle (quadriceps), adrenal gland, lymph node, small intestine, ovary, spinal cord (cervical, thoracic and lumbar levels), aorta, oviduct, brain, pancreas, spleen, epididymis, pituitary, sternum with marrow, eye with the optic nerve, prostate, stomach, oesophagus, salivary gland (including mandibular, sublingual and parotid glands), testis, femur with marrow, thymus, heart, thyroid with parathyroid gland, kidney, sciatic nerve, tongue, large intestine, seminal vesicle with coagulating gland, trachea, extraorbital lachrymal gland, urinary bladder, harderian gland, skin/subcutis and mammary gland (inguinal) area, uterus, liver and vagina.

For the adult animals, detailed histological examination was performed with a light microscope as follows:
- on the retained organs in the control and high dose groups (Subgroup A males and satellite females)
- any animals found dead or euthanized pre-terminally in all groups
- all macroscopic findings (abnormalities) on the retained reproductive organs of all animals of the control and high dose group

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries included the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on PND4.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examination for abnormalities

HISTOPATHOLOGY / ORGAN WEIGTHS
Not examined
Statistics:
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations. The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2
test was performed as feasible.
Reproductive indices:
Male/female mating and fertility indices, gestation index
Offspring viability indices:
Number of viable pups per litter was measured on PND0 and 4; survival index, pre-implantation, intrauterine and total mortality were also measured
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection, pale skin colour, moderately decreased activity, laboured respiration from moderate to severe, hunched back and brown liquid around the vulva was found in one female, which was found dead on PND4.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One low-dose female was diagnosed with prolapse of the uterus, and was euthanized for animal welfare reasons on gestation day 21. This was considered to be an incidental finding and unrelated to the test item. One high-dose female was found dead on PND4. This was considered as incidental, correlated with necropsy and histopathology findings.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse effects noted on the body weight and growth values at any of the administered doses. A statistically significant reduction in growth was noted in high-dose males from Day 3-7 and from Day 7-10, though overall growth (Days 0-27) aligned with the control group. A statistically significant growth reduction was noted in low-dose males from Day 7-10; no dose response was noted and this difference was therefore considered not to be treatment-related. There were no statistically significant growth differences for main test females. For the satellite females, all test groups showed reduced growth from Day 3-7 but there was no dose response and no difference thereafter.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences were observed in the mid- or low-dose groups only and were considered incidental to treatment. Statistically significant increases in the eosinophil percent (EOS%) group mean value were measured in high-dose males, though the individual results were within the historical control range of the laboratory. Therefore, this difference, which occurred in isolation, was considered to be of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant reductions in phosphorus serum concentration values were measured in males from the low- and high-dose groups, though these were ascribed to a higher control mean and not a test item related effect. Similarly, a significantly lower serum urea concentration in the low- and high-dose group females was ascribed to the high control mean. The control values for sodium and chloride serum concentration in females were also outside the historical control range. With the exception of serum urea concentration, the concurrent control values were not representative of the historical control values of the laboratory. Therefore, assessment of the individual results was done, and values were compared with the historical control data set. No test item-related effects were observed and so the statistically significant differences are considered to have no toxicological relevance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total distance travelled was comparable to the control for both sexes in the high dose group.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There was no evidence of test item-related histological findings in the found dead or preterminally euthanized animals. In the found dead high-dose female, congestion/haemorrhage in the adrenals, lymphocytic apoptosis and increased mixed mononuclear cellularity in the mandibular lymph nodes, necrotizing, mural inflammation in the stomach were observed and correlated with macroscopic findings. Necrotizing abscess in the uterine horns, myeloid hyperplasia in the femur and sternum bone marrow, agonal congestion/hemorrhages in the lungs proteinaceus cast in the right kidney, decreased zymogen granules in pancreas, and decreased cellularity in the thymus were noted as well. In the preterminally euthanized low-dose female, mixed cellular inflammation and necrotizing abscess in the uterine cervix, and lymphoid atrophy and extramedullary hematopoeisis in the spleen could be detected.

At termination, there was no evidence of test item-related histological findings in high-dose animals or macroscopic observations from all groups in the reproductive organs. Histopathological evaluation of the male gonads, testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in control and high-dose males. Histological structure of the follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar in both control and high-dose females. Focal/multifocal, mononuclear cell infiltrate in the prostate, the diffuse/multifocal tubular degeneration/atrophy and diffuse tubular dilatation in the testes, the reduced sperm content/debris material, or spermatocoele in the epididymis and the extramedullary hematopoeisis in the spleen were noted. Based on low incidence and/or severity and/or distribution in control and treated animals, these changes were considered as incidental or regarded as a common background.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 4 days of pairing (cohabitation) in the majority of the animals, except one female from the control group
(11 days), one female from the low dose group (7 days) and one female from the high dose group (12 days).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Focal/multifocal, mononuclear cell infiltrate in the prostate, the diffuse/multifocal tubular degeneration/atrophy and diffuse tubular dilatation in the testes, the reduced sperm content/debris material, or spermatocoele in the epididymis were noted. Based on low incidence and/or severity and/or distribution in control and treated animals, these changes were considered as incidental or regarded as a common background.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on reproductive parameters.

There were no differences between the control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 4 days of pairing (cohabitation) in the majority of the animals, except one female from the control group
(11 days), one female from the low dose group (7 days) and one female from the high dose group (12 days).
There was no effect of treatment noted during gestation, parturition or the post-partal period. The mean duration of pregnancy was comparable in the control and treated groups. All parturitions were normal. The number of corpora lutea and number of implantation sites was comparable to the control mean in all dose groups.

There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, post-natal or total mortality values (litter mean and %) at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse general systemic or reproductive effects seen at highest tested dose
Critical effects observed:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The number of viable pups on PND 0 and 4 was comparable to the control values. The incidence of mortality during 4 postnatal days was negligible
Body weight and weight changes:
no effects observed
Description (incidence and severity):
When evaluated on per litter basis, the mean body weight and body weight gain in the F1 generation evaluated on PND 0 and 4 were similar in all treated groups and the values were comparable to controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally on PND 4.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The sex ratios did not differ significantly between the control and treatment groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No abnormal behaviour of the pups was noted.
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental effects seen at highest tested dose
Critical effects observed:
not specified
Reproductive effects observed:
no
Conclusions:
In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of diammonium sodium hexakis(nitrito-N)rhodate for at least 28 days, no treatment-related adverse effects on any measured reproductive or fertility parameters were observed. The systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day).
Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, Wistar rats (12/sex/group in the main test) were orally administered diammonium sodium hexakis(nitrito-N)rhodate by stomach tube (gavage) at doses of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day. Males were dosed for 28 days (14 days pre-mating and 14 days through the mating and post-mating periods). Main test females were dosed for 14 days pre-mating, through mating (up to 12 days), gestation (22-24 days) and up to post-partum day 5 (around 7 weeks in total).

There were no reported changes to reproductive parameters (including number of corpora lutea, implantation sites, number of pups, fertility and gestation indices, reproductive performance as well as pre- and post-implantation losses).

The systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Diammonium sodium hexakis(nitrito-N)rhodate
- Substance type: no data
- Physical state: solid (pale yellow/green powder)
- Analytical purity: assumed 100% for experimental suspension formulation
- Impurities (identity and concentrations): traces of platinum (0.01%), ruthenium (0.073%), iridium (0.044%), silver (0.016%); traces of other metals (iron, nickel, bismuth, copper, tellurium, chromium, magnesium) documented in the study report's certificate of analysis
- Composition of test material, percentage of components: Rhodium 16.63%; sodium 3.88%
- Purity test date: 01 April 2014
- Lot/batch No.:RHL 653
- Expiration date of the lot/batch: 03 June 2015. The expiry date of the test article was based on two years from the production date of the material. As far as the manufacturer is aware, no degradation/aging is expected in any long term time frame however there is no analytical data available to confirm this.
- Stability under test conditions: test article stability in the vehicle was not determined in this study; fresh preparations were routinely employed.
- Storage condition of test material: 15-25 degrees C, protected from light

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony. Males and females originated from different units to avoid subsequent brother/sister matings.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 10-11 weeks (12-13 weeks at mating).
- Weight at study initiation: Males: 351 g – 381 g, Females: 202 g - 268 g
- Fasting period before study: no data
- Housing: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- Diet (e.g. ad libitum): Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption, from 500 ml bottle ad libitum.
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd. Food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 24.5 (target range 22±3)
- Humidity (%): 36 – 70 (target range 30-70)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 July 2014 To: 19 September 2014

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Test item or vehicle control-treated animals were administered the dosing formulations daily by oral gavage, using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.
Vehicle:
methylcellulose
Remarks:
1% in distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared for up to 14 days and kept in refrigerator pending dosage. Stability of the test item in the selected vehicle was assessed under the conditions employed on the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected by the sponsor. For the preparation of 2 L vehicle, 20 g Methylcellulose powder was added to approximately 1750 mL hot distilled water, mixed continuously, and gradually made up to 2000 mL with cold distilled water. The mixture was then stirred overnight.
- Concentration in vehicle: 0, 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed in Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken from each concentration. Each formulation used in the study was sampled and analyzed. One set of samples was collected for analysis and one set for a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the control solution to confirm the absence of test item.

Formulations prepared on Day 0 were sampled and sent to the test site for rapid formulation analysis to provide confirmation on formulation concentration and
homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.
Duration of treatment / exposure:
28 days for males (14 days pre-mating and 14 days mating/post-mating period); 52-54 days for females (14 days pre-mating, for up to 12 days of the mating period, through gestation (22-24 days) and up to and including the day before necropsy (at least 4 days post-partum dosing).
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 (5 additional females/group were included as satellites for toxicology endpoints)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of the repeated dose range finding study in the rat. The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.
- Rationale for animal assignment (if not random): All parental (P Generation) animals were sorted by computer according to body weight and divided in to weight ranges. An equal number of animals from each weight group were randomly assigned to each dose group to ensure that the mean group weights were similar. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately. Five males of each dose group were allocated to each of subgroups A and B (these were subject to slightly different examinations, see 'Examinations' section below).
- Rationale for selecting satellite groups: for toxicology endpoints
- Post-exposure recovery period in satellite groups: none (treated for 28 days and necropsied on Day 28)
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for signs of morbidity and mortality; once a day for general clinical observations
- Cage side observations (general clinical observations) included pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and then weekly (in the morning) aside from week 1.
- Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also assessed. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice a week and at termination for all adult animals. In addition, parent females were weighed on gestation Days 0, 7, 10, 14, 17 and 20 and on postnatal Days (PND) 0 (within 24 hours after parturition) and PND4 (before termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

FOOD EFFICIENCY:
- Food consumption was measured in parallel with body weight. Food efficiency was not calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
Conducted for all males and Satellite females before treatment commenced and for the Control group and High dose group males and Satellite females, during Week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy on day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: 5 males (subgroup B) and satellite females
- See attached file for a full list of the evaluated parameters.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy on day 28
- Animals fasted: Yes (overnight)
- How many animals: 5 males (subgroup B) and satellite females
- See attached file for a full list of the evaluated parameters.

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to necropsy on day 28
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (overnight)
- See attached file for a full list of the evaluated parameters.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once
- Dose groups that were examined: Performed during the last exposure week on subgroup A males and on the satellite females
- Battery of functions tested: Functional observational battery (FOB including quantitative assessment of grip strength (manual and instrumental), and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessment of motor activity were measured, while a manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive) was also conducted.

IMMUNOLOGY: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal irrespective of the date of death. The external appearance was then examined and the cranium, thoracic and abdominal cavities were opened. The appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes
The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, the testes with epididymides were retained in Bouin’s solution, and all other organs in 10% buffered formalin solution.

In addition, for all Main males and all satellite females and for all adults killed or found dead prior to scheduled termination, the following organs and tissues, or representative samples, were preserved: lungs with bronchi, skeletal muscle (quadriceps), adrenal gland, lymph node, small intestine, ovary, spinal cord (cervical, thoracic and lumbar levels), aorta, oviduct, brain, pancreas, spleen, epididymis, pituitary, sternum with marrow, eye with the optic nerve, prostate, stomach, oesophagus, salivary gland (including mandibular, sublingual and parotid glands), testis, femur with marrow, thymus, heart, thyroid with parathyroid gland, kidney, sciatic nerve, tongue, large intestine, seminal vesicle with coagulating gland, trachea, extraorbital lachrymal gland, urinary bladder, harderian gland, skin/subcutis and mammary gland (inguinal) area, uterus, liver and vagina.

For the adult animals, detailed histological examination witha light microscope was performed as follows:
- on the retained organs in the control and high dose groups (Subgroup A males and Toxicology satellite females)
- any animals found dead or euthanized pre-terminally in all groups
- all macroscopic findings (abnormalities) on the retained reproductive organs of all animals of the control and high dose group

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries included the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Other examinations:
Organ weights: At the time of termination, body weight and the weight of the following organs from all adult animals (where appropriate) were determined: uterus (including cervix), ovaries testes, epididymides, prostate, seminal vesicles with coagulating glands and brain. From all Main males and from all satellite females, the following organs were weighed in addition to the ones previously mentioned: heart, kidneys, liver, spleen and thymus, adrenals, thyroids with parathyroids. Paired organs were weighed together except testes and epididymides which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal. Relative organ weights (to body and brain weight) were calculated and reported.
Statistics:
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations. The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection, pale skin colour, moderately decreased activity, laboured respiration from moderate to severe, hunched back and brown liquid around the vulva was found in one female, which was found dead on PND4.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One low-dose female was diagnosed with prolapse of the uterus, and was euthanized for animal welfare reasons on gestation day 21. This was considered to be an incidental finding and unrelated to the test item. One high-dose female was found dead on PND 4. This was considered as incidental, correlated with necropsy and histopathology findings.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse effects noted on the body weight and growth values at any of the administered doses. A statistically significant reduction in growth was noted in high-dose males from Day 3-7 and from Day 7-10, though overall growth (Days 0-27) aligned with the control group. A statistically significant growth reduction was noted in low-dose males from Day 7-10; no dose response was noted and this difference was therefore considered not to be treatment-related. There were no statistically significant growth differences for main test females. For the satellite females, all test groups showed reduced growth from Day 3-7 but there was no dose response and no difference thereafter.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences were observed in the mid- or low-dose groups only and were considered incidental to treatment. Statistically significant increases in the eosinophil percent (EOS%) group mean value were measured in high-dose males, though the individual results were within the historical control range of the laboratory. Therefore, this difference, which occurred in isolation, was considered to be of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant reductions in phosphorus serum concentration values were measured in males from the low- and high-dose groups, though these were ascribed to a higher control mean and not a test item related effect. Similarly, a significantly lower serum urea concentration in the low- and high-dose group females was ascribed to the high control mean. The control values for sodium and chloride serum concentration in females were also outside the historical control range. With the exception of serum urea concentration, the concurrent control values were not representative of the historical control values of the laboratory. Therefore, assessment of the individual results was done, and values were compared with the historical control data set. No test item-related effects were observed and so the statistically significant differences are considered to have no toxicological relevance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on organ weights. Occasional statistically significant differences were not dose-related and therefore considered incidental and of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the high-dose female which died during the study, red material could be seen on the fur of the perinasal/periorbital region, the adrenals were enlarged and dark red, and the mandibular lymph nodes were enlarged. In the non-glandular mucosa of the stomach multifocal dark red discoloration was detected at necropsy. No macroscopic changes were noted at necropsy in the low-dose female which was preterminally euthanized due to prolapse of the uterus.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total distance travelled was comparable to the control for both sexes in the high dose group.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was no evidence of test item-related histological findings in the found dead or preterminally euthanized animals. In the high-dose female found dead, congestion/haemorrhage in the adrenals, lymphocytic apoptosis and increased mixed mononuclear cellularity in the mandibular lymph nodes, necrotizing, mural inflammation in the stomach were observed and correlated with macroscopic findings. Necrotizing abscess in the uterine horns, myeloid hyperplasia in the femur and sternum bone marrow, agonal congestion/hemorrhages in the lungs proteinaceus cast in the right kidney, decreased zymogen granules in pancreas, and decreased cellularity in the thymus were noted as well. In the preterminally euthanized low-dose female, mixed cellular inflammation and necrotizing abscess in the uterine cervix, and lymphoid atrophy and extramedullary hematopoeisis in the spleen could be detected.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects were noted on general toxicology parameters

Target system / organ toxicity

Critical effects observed:
not specified

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 diammonium sodium hexakis(nitrito-N)rhodate for at least 28 days, no treatment-related adverse effects were observed at up to the highest tested dose (1000 mg/kg bw/day).
Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, Wistar rats (12/sex/group in the main test) were orally administered diammonium sodium hexakis(nitrito-N)rhodate by stomach tube (gavage) at doses of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day. Males were dosed for 28 days (14 days pre-mating and 14 days through the mating and post-mating periods). Main test females were dosed for 14 days pre-mating, through mating (up to 12 days), gestation (22-24 days) and up to post-partum day 5 (around 7 weeks in total). Additional females (5/group), assigned to satellite groups for toxicology endpoints, were treated for 28 days.

Daily gavage administration of diammonium sodium hexakis(nitrito-N)rhodate to rats did not result in test item related mortality, clinical signs of toxicity, neurological (including FOB) or ophthalmoscopy observations, or changes in the body weight, food consumption, haematology, clinical chemistry or urinalysis parameters at dose levels of up to 1000 mg/kg bw/day. There were no adverse treatment-related changes in organ weights, or following macroscopic and microscopic examination of a wide range of tissues and organs for the adult animals of either sex.

On this basis, a study NOAEL of 1000 mg/kg bw/day was established.