Nickel, 5-[2-(2-hydroxy-3,7-disulfo-1-naphthalenyl)diazenyl]-1H-1,2,4-triazole-3-carboxylate sodium complexes

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health and Welfare (MHW) Guidelines 1986 for a 28-day repeat dose oral toxicity study as required by the Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (MITI) amended 1986.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-510-2

EC Name:

-

Cas Number:

738587-10-5

Molecular formula:

C26H14N10Ni2O18S4 as free acid

IUPAC Name:

dinickel(2+) sodium 3-carboxy-5-[(1E)-2-(7-oxido-2,6-disulfonaphthalen-1-yl)diazen-1-yl]-1H-1,2,4-triazol-1-ide 3-carboxy-5-[(1E)-2-(7-oxido-2-sulfo-6-sulfonatonaphthalen-1-yl)diazen-1-yl]-1H-1,2,4-triazol-1-ide

Details on test material:

ponsor's identification: S190700
Description : Dar brown powder
Purity : 82.8%
Batch number : NBZ3341/70
Date received : 22 October 2002
Storage conditions: room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for seven days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted into the study. At the start of treatment the males weighed 139 to 188g, the females weighed 119 to 160g, and were approximately five to six weeks old. The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water.

A pelleted diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Northants, UK) was used. A certificate of analysis of the batch of diet used is given in Appendix 15. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK).

The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity controls were set to achieve target
values of 21 ± 2°C and 55 ± 15% respectively. Occasional deviations from these targets were
considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using random letter tables and the
group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear marking system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method of administration:
Gavage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation and were analysed for concentration of test substance at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:
other: adjusted for purity of 82.8% w/w

No. of animals per sex per dose:

Male: 5 animals at 15 mg/kg bw/day
Male: 5 animals at 250 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day

Dosed as active (a correction factor for 82.8% purity applied)
Female: 5 animals at 15 mg/kg bw/day
Female: 5 animals at 250 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Positive control:

None

Examinations

Observations and examinations performed and frequency:

All animals were examined for overt Signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends and on public holidays). All observations were recorded.

Sacrifice and pathology:

On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone (Rhone Merieux, Dagenham, Essex, UK) followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen,Testes, Thymus

Histopathology

Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:

Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain
Caecum
Colon
Duodenum
Epididymides
Eyes
Gross lesions
Heart
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spleen
Stomach
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Muscle (skeletal)
Testes
Thymus
Thyroid/parathyroid
Trachea
Urinary bladder
Uterns

All tissues were despatched to Precision Histology International, One Eyed Lane, Weybread, Diss, Norfolk, UK for processing. The tissues from 1000 mg/kg/bw/day and the non-recovery group were prepared as paraffin blocks, sectioned at nominal thickness of 5flm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 250 mg/kg/day dose groups animals.

Since there were indications of treatment-related gastric changes, examination was subsequently extended to include similarly prepared sections of stomach from all animals in the remaining
groups.

Other examinations:

Behavioural Assessments

Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait
Tremors
Twitches
Convulsions
Bizarre/ Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

Functional Performance Tests

MOTOR ACTIVITY: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).

FORELIMB/HINDLIMB GRIP STRENGTH: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

SENSORY REACTIVITY: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:

Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were confined to red staining detected on the cage tray liners of either sex treated with 1000 mg/kg/day from Day 2 onwards together with associated observations of a generalised red staining of the fur.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment-related findings were confined to red staining detected on the cage tray liners of either sex treated with 1000 mg/kg/day from Day 2 onwards together with associated observations of a generalised red staining of the fur.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Animals treated with 250 or 1000 mg/kg/day showed red urine.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Various treatment related effects seen in the stomach at 250 and 1000 mg/kg/bw/day

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical observations:
Mortality. There were no deaths during the study. Clinical red staining detected on the cage tray liners of either sex treated with 1000 mg/kg/day from Day 2 onwards together with associated observations of a generalised red staining of fur. There was one instance of red stained fur among animals treated with 250 mg/kg/day. These findings resolved in recovery 1000 mg/kg/day animals within a few days of cessation fo treatment. Such changes are often seen when a highly coloured test material is administrated by gavage and may result from the presence of test material and/or its metabolite(s) in the urine.

Laboratory findings:
Functional Observations.

Behavioural Assessment. No treatment-related findings were observed.


Functional Performance Tests. No treatment-related effects were detected.


Sensory Reactivity Assessments. No treatment-related effects were detected.

Effects in organs:
Bodyweight. No adverse effect on bodyweight development was detected.


Food Consumption. There was no adverse effect on food onsumption during the study. Food efficiency in test animals was comparable with that seen in controls.


Water Consumption. Daily visual inspection of water bottles revealed no overt intergroup differences.


Haematology. No treatment-related effects were detected.


Blood Chemistry. No treatment-related effects were detected.


Urinalysis: There were no toxicologically significant changes detected but animals treated with 250 or 1000 mg/kg/day showed red urine.


Organ Weights. No treatment-related effects were detected.


Necropsy. No macroscopic abnormalities were observed.


Histopathology. The following treatment-related changes were observed:

Stomach: Agglomeration of secretion and mucous cell hyperplasia were observed in realtion to treatment in the gastric mucosa of rats of either sex dosed at 1000 mg/kg/day. Agglomeration of secretion was also seen for two male rats and for one female rat, with associated mucous
cell hyperplasia, receiving 250 mg/kg/day of the test material, but since these conditions do occasionally occur considered to be present only at the high dose level. Acanthosis and hyperkeratosis of the limiting ridge were also observed among male rats dosed at 1000 mg/kg/day. There
were some indication of sligh regression of the conditions among recovery 1000 mg/kg/day animals compared with controls following an additional fourteen days without treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

2

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Classified as: Not classified

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test material and complies with the following regulatory guidelines:

i) Commission Directive 96/54/EC (Method B7).
ii)  The Japanese Ministry of Health and Welfare (MHW) Guidelines 1986 for a twenty-eight day repeat dose oral toxicity study as required by the Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (MITI) amended 1986.
iii)  The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 28 July 1995).

Methods

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 250 and 1000 mg/kg/day (incorporating a correction factor for 82.8% purity). A control group of five males and five females was dosed with vehicle alone (distilled water). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Conclusion

Oral administration of the test material, S190700, to rats, by gavage, for a period of twenty-eight consecutive days resulted in treatment-related changes in either sex at a dose level of 1000 mg/kg/day. There was no convincing evidence of treatment observed in either sex treated with 250 mg/kg/day or 15 mg/kg/day and for this reason the "No Observed Effect Level" (NOEL) was considered to be 250 mg/kg/day.

 

The effects detected at 1000 mg/kg/day, were confined to gastric changes considered to be attributable to the irritant nature of the test material formulation and were considered not to represent a true systemic effect of treatment.