Strontium 3-hydroxy-4-[(1-sulfonato-2-naphthyl)azo]-2- naphthoate 5/2 hydrate

Endpoint summary

Description of key information

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for R507-2 of 1000 mg/kg/day was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2007 - 08 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Crl:Wl(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: All animals within ± 20 % of the sex mean
- Fasting period before study: Not specified
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in Mill type; height 15 cm.) with sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage­ enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). No cage-enrichment was provided during overnight activity monitoring.
Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7-21.9 °C
- Humidity (%): 26-81 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: 11 December 2007 to 07 January 2008:

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

propylene glycol

Details on oral exposure:

Frequency: Once daily, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing, and were homogenised to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation; 10 %). Formulations were stable for at least 5 hours over the entire concentration range when stored at room temperature.

Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector: Dual A Absorbance Detector
Column: Waters Symmetry C18, 150 mm x 3 mm i.d., dp = 5 µm (Waters)
Column temperature: 40 °C ± 5 °C
Injection volume : 10 µL
Mobile phase: 30/70 (v/v) ACN/ 30 mM NH4Ac in water
Flow: 0.3 mL/min
UV detection: 518 nm

Stock and spiking solutions
Stock solutions of the test substance were prepared in DMSO at concentrations of
554 - 988 mg/L. In order to dissolve the test substance completely, the stock solutions were placed in a water bath at approximately 50 °C for a maximum duration of 60 minutes.

Calibration solutions
Calibration solutions in the concentration range 0.050 - 10 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) DMSO/water.

Procedural recovery samples
Approximately 500 mg blank vehicle was spiked with the test substance at a target concentration of 9 or 200 mg/g.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 95 % and 97 %).

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation of 0.37 % and 0.92 %, respectively).

Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of -1.9 % and -0.46 %, respectively. Based on this, the formulations were found to be stable during storage at room temperature for at least 5 hours.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

not specified

Details on study design:

- Dose selection rationale: Based on the results of a 5-day range finding study (NOTOX Project 486325), the dose levels for this 28-day oral gavage study were selected to be 0, 50, 150 and 1000 mg/kg/day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, At least once daily, detailed clinical observations were made in all animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded.
All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade O = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
-Time schedule for examinations: Weekly


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: ) immediately prior to scheduled post mortem examination at the end of the treatment, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (identity)-iso-flurane anaesthesia
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
-Time schedule for collection of blood:immediately prior to scheduled post mortem examination at the end of the treatment, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (identity)-iso-flurane anaesthesia
- Animals fasted: Yes
- How many animals:
All animals
- Parameters checked in table [No.1] were examined.


URINALYSIS: Not specified


NEUROBEHAVIOURAL EXAMINATION: During week 4 of treatment, the following tests were performed on all animals of the first cage of each main group alocation:
Hearing ability, pupillary reflex, static righting reflex and grip strength.
Motor activity test (recording period:12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical services, Debenham, stowmarket England).


IMMUNOLOGY: Not specified


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- all gross lesions. For groups 2 and 3, macroscopic abnormalities consisting of red discolouration of the contents of (parts of) the gastro-intestinal tract were not further processed for microscopic examination. Histological examination of the gastro-intestinal tract of group 4 animals showing similar macroscopic abnormalities showed no treatment-related morphological changes of (parts of) the gastro-intestinal tract. It could therefore be reasonably assumed that no correlating treatment related morphological changes of (parts of) the gastro­ intestinal tract would be visible at histological examination.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Red faeces was noted among all groups treated with the test substance.

Incidental findings that were noted included rales, chromodacryorrhoea, a dark right eye. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted among control animals.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant changes in body weights and body weight gain were noted.

The statistically significant lower body weight gain of females at 1000 mg/kg/day at the end of the treatment period was of a very slight nature (i.e. within the normal range), and was therefore considered to be of no toxicological significance.

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 specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats. The statistically significant higher red blood cell and relative basophil counts in females at 1000 mg/kg/day were considered to be of no toxicological significance. These changes occurred in the absence of supportive morphological or haematological changes and/or remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

Any statistically significant changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and were of a very slight nature (i.e. remained within the range considered normal for rats of this age and strain). These changes included lower inorganic phosphate levels in males at 50 mg/kg/day, while in females at 1000 mg/kg/day these changes consisted of higher total bilirubin and calcium levels and lower chloride levels.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant changes were noted in organ weights and organ to body weight ratios.

Any statistically significant changes in organ weights and organ to body weight ratios were considered not to be a sign of toxicity, as a dose-related trend was absent, and means remained within the range considered normal for rats of this age and strain. These changes included lower thymus weights in males at 50 mg/kg/day and higher, lower thymus to body weight ratio in males at 1000 mg/kg/day, and lower epididymides weight in males at 50 mg/kg/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Reddish contents of the gastro-intestinal tract were noted in 1/5 males at 50mg/kg/day, 3/5 males and females at 150 mg/kg/day (one additional male showing reddish contents of the stomach only) and in all animals at 10000 mg/kg/day. Other necropsy findings were considered to be unrelated to treatment with the test substance since they occured in the absence of a treatment- related ditribution, and are occasionally seen among rats used in these types of studies. These findings including red discolouration of the mandibular lymph node, exophthalmus of the right eye, fluid in the uterus, and a reddish focus on the liver.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

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:

behaviour (functional findings)

body weight and weight gain

clinical signs

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Conclusions:

Wistar rats were treated with R507-2 for 28 consecutive days by daily oral gavage administration at dose levels up to 1000 mg/kg/day.

The red faeces noted among all groups treated with the test substance was confirmed macroscopically by a dose-related incidence of reddish contents of the gastro-intestinal tract/stomach. No histopathological correlates in the gastro-intestinal tract were noted. These findings were considered to be due to passive staining properties of the test substance (a red powder) and to be of no toxicological significance.

No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for R507-2 of 1000 mg/kg/day was established.

Executive summary:

Title

Repeated dose 28-day oral toxicity study with R507-2 by daily gavage in the rat.

 

Guidelines

The study was based on the following guidelines.

-      EC Directive 96/54/EEC, 8.7 Repeated Dose (28 days) Toxicity (oral), 1996.

-      OECD407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995.

-      OPPTS870.3050,Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

 

Rationale for dose levels

Based on the results of a 5-day range finding study (NOTOX Project 486325), the dose levels for this 28-day oral gavage study were selected to be 0, 50, 150  and 1000 mg/kg/day.

 

Study outline

The test substance was administered daily for 28 days by oral gavage administration to SPF­ bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

 

Evaluated parameters

Chemical analyses of formulations in propylene glycol were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

Results

 

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours.

 

No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

 

Conclusion

 

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for R507-2 of 1000 mg/kg/day was established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

Based on the study report for Repeat oral dose toxicity no adverse effects were observed. The No Observed Adverse Effect Level (NOAEL) for R507-2 is 1000 mg/kg/day.

Therefore the substance is not classified for specific target organ toxicity after repeated exposure (STOT RE category 1 or 2) according to Regulation EC No 1272/2008.