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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 March 2006 to 19 October 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study in agreement with OECD Guideline 408 and GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
Compliance statement that study was conducted in accordance with US, EU, Italian and OECD regulations on GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain specifics: Hsd: Sprague Dawley
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: males: 91-107 g, females: 84-105 g
- Housing: up to 5 of one sex to a cage, clear polycarbonate cages with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): commercially available laboratory rodent diet, ad libitum*
- Water (e.g. ad libitum): ad libitum* from water bottles
* except during week 13 of treatment, when samples of blood were withdrawn under anaesthesia of the first 10 surviving male and the first 10 surviving female animals from each group (food and water deprivation). At the same time interval, individual overnight (approximately 16 hours) urine samples were also collected from the same animals under the same conditions. Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 ml/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle, 0.5% carboxymethylcellulose in distilled water (CMC 0.5% w/v). The formulations were prepared daily. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Concentration in vehicle: 5, 20 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations prepared in weeks 1 and 13 of the study were also analysed to check the homogeneity and concentration. Chemical analyses were carried out by the Analytical Chemistry Department at RTC. All the analyses were within the accepted limit.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once a day, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
10 male and 10 female animals
The high dose group and the control group included 5 additional animals per sex sacrificed after 4 weeks of recovery
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on information from a preliminary study, in which the dose level of 1000 mg/kg/day was considered to be the NOAEL).
- Rationale for animal assignment (if not random): computerised stratified randomisation
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random):
a) Tissues from all main phase animals in the control and high dose groups killed at the end of the 13 weeks of treatment.
b) All abnormalities in all main phase groups.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Mortality: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after dosing and 1 hour after dosing. During the first week of treatment, an additional observation at 30 minutes after dosing was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
Once during week 12 of treatment and once during week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.

BODY WEIGHT: Yes
Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals assigned to the study were examined just prior to the commencement of treatment. In the first instance the eyes of all animals from high dose and control groups were re-examined during week 13 of treatment. Since no treatment-related changes were observed, no further examinations were performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes: isofluorane
- Animals fasted: No, but food and water deprivation during blood collection
- How many animals: the first 10 surviving male and the first 10 surviving female animals from each group
- Parameters examined:
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count (not performed as no signs of anaemia were seen)
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count
- Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: No, but food and water deprivation during blood collection
- How many animals: the first 10 surviving male and the first 10 surviving female animals from each group
- Parameters examined:
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride
(During week 4 of the recovery period, blood samples were taken from all surviving animals under identical conditions in order to re-evaluate Sodium, Potassium, Calcium and Phosphorus, parameters which showed possibly treatment-related changes at measurements performed during the treatment period.)

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined:
Appearance
Volume
Specific gravity
pH
Protein
Total reducing substances
Glucose
Ketones
Bilirubin
Urobilinogen
Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12 and week 4 of the recovery period
- Dose groups that were examined: all / controls and high dose recovery groups
- Battery of functions tested:
evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength
motor activity by an automated activity recording

OTHER:
- samples from liver and blood plasma from control and high dose group (1000 mg(kg bw) collected at the end of the exposure period
were analysed for the presence of the test item
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Continuous variables: analysis of variance.
Differences between each treated group and the control groups were assessed by Dunnett's test using a pooled error variance.
The homogeneity of the data was verified by Bartlett¿s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was
applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
One control male, one male receiving 50 and one male receiving 1000 mg/kg/day showed palpable masses in the right/left ventral region (mammary area) which were detected for the first time on days 80 and 74 of treatment and day 29 of recovery, respectively. Size and description of the mass
were confirmed every week until despatch to necropsy. No toxicological importance was attributed to these small masses, considered to be incidental in origin. In addition, abnormal colour of the faeces, the severity of which increased with the dose level, was observed in all cages of both male and female treated animals, starting from day 3 of treatment until the end of the treatment period. This sign disappeared after 3 days of the recovery.

HAEMATOLOGY
The statistically significant changes observed (decrease in mean corpuscular haemoglobin concentration in males and increase in females treated with 50 mg/kg/day, decrement of prothrombin time and monocytes percentage in males receiving 200 mg/kg/day) were considered to be incidental and with no toxicological significance.

CLINICAL CHEMISTRY
Changes in electrolytes (sodium, potassium and calcium) and phosphorus were observed in treated animals, mainly in males, during the dosing phase. In particular, statistically significant increases in sodium and decreases in phosphorus were seen in males receiving 50 and 1000 mg/kg/day while a statistically significant decrease in sodium was observed in 1000 mg/kg/day females; statistically significant decreases in phosphorus, calcium and potassium were seen in males receiving 200 mg/kg/day. The above mentioned changes were minimal and did not show a clear dose-relation and/or consistency between sexes, therefore they were not considered of toxicological significance.
Analyses on phosphorous, calcium, sodium and potassium were performed during the recovery phase. The changes observed during the treatment period in electrolytes and phosphorus, in animals receiving 1000 mg/kg/day, were seen to be recovered.

ORGAN WEIGHTS
Statistically significant increases in absolute and relative liver weights were observed in females receiving 1000 mg/kg/day, sacrificed at the end of the treatment period, when compared to controls. Absolute and relative liver weights were seen to be decreased (up to 15%) in this female treated group, at the end of the recovery period. The above mentioned changes and others, including statistically significant decreases in absolute and/or relative heart weights observed in males receiving 200 mg/kg/day (up to 10%), and increases in absolute and relative spleen weights (up to 13%) observed in females receiving 200 and 1000 mg/kg/day, were considered of no toxicological significance since they were minimal, not dose-related and/or not consistent between sexes and no corresponding histopathological findings were observed.

GROSS PATHOLOGY
Instances of dark/red or pink granular material were reported at post mortem examination, in the gastro-intestinal tract of animals receiving 1000 mg/kg/day and killed at the end of the treatment period. This abnormal content probably represented residues of the test item in the gastro-intestinal
tract.
No other relevant change was observed in treated animals killed at the end of treatment or at completion of recovery period, when compared with controls.

OTHER FINDINGS
Liver and blood plasma samples of male and female rats of the 1000 mg/kg bw/day group were below quantifiable limit concentrations of 1.5 ug/g dried liver and 0.4 / 0.6 ppm dried blood plasma.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Highest dose level was considered to be the NOAEL

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item (Pigment Red 122) when given by oral administration (gavage) to rats for 13 consecutive weeks at dosages of 0, 50, 200 or 1000 mg/kg bw/day, for 7 days/week and recovery from any treatment-related effects over a recovery period of 4 weeks, have been investigated. No toxicological alterations were observed during the in vivo phase at the following investigations: pre- and post-dose observations, clinical signs, neurotoxicity assessment, sensory reaction to stimuli, motor activity, body weight, food consumption, ophthalmoscopy and clinical pathology investigations. Necropsy gross observations and histopathological examination were performed after sacrifice of animals at the end of treatment and recovery periods, and no toxicologically relevant changes were observed. On the basis of these results, the high dose level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL). Liver and blood plasma samples of male and female rats of the 1000 mg/kg bw/day group were below quantifiable limit concentrations of 1.5 ug/g dried liver and 0.4 / 0.6 ppm dried blood plasma, indicating that the test item is not or only to a negligible extent bioavailable.
Executive summary:

Study design

The toxicity of the test item in Sprague Dawley rats after daily oral administration (by gavage) for 13 consecutive weeks and recovery from any treatment-related effects during a recovery period of 4 weeks, have been investigated.

The treatment groups and dose levels were as follows:

Group number  Treatment (mg/kg/day)+   Number of animals   
 Main phase  Recovery phase
 1   0  10 males and 10 females 5 males and 5 females 
 2  50  10 males and 10 females  
 3  200 10 males and 10 females   
 4  1000 10 males and 10 females  5 males and 5 females 

+: in terms of test item as supplied

The following investigations were performed during the in vivo phase: pre- and post-dose observations, clinical signs, neurotoxicity assessment, motor activity, body weight, food consumption, ophthalmoscopy and clinical pathology investigations.

Necropsy gross observations and histopathological examination were performed after sacrifice of animals at the end of treatment or recovery periods.

Mortality

No deaths occurred during the study.

Pre- and post-dose observations

No signs of reaction to treatment were recorded during the study.

Clinical signs, neurotoxicity assessments, sensory reaction to stimuli, motor activity and observation of cage trays

No relevant clinical signs were recorded during the dosing phase including the observation of animals in an open arena. Neurotoxicity tests and motor activity measurements performed at the end of treatment and recovery did not show changes attributable to the test item. In addition, abnormal colour of the faeces, the severity of which increased with the dose level, was observed in all cages of treated animals, starting from day 3 of treatment until the end of the treatment period. This sign disappeared after 3 days of the recovery period (data not tabulated).

Body weight

Body weight was not affected by treatment.

Food consumption

No differences were noted in mean food consumption in treated groups when compared to the control group.

Ophthalmoscopy

No significant lesions were observed in the control and high dose group at the examination performed during week 13 of administration.

Haematology

Following 13 weeks of treatment no toxicologically significant changes were observed in the haematological parameters.

Clinical chemistry

No alterations of toxicological significance were seen in the clinical chemistry parameters.

Urinalysis

No changes of toxicological significance were observed during the dosing phase.

Terminal body weight and organ weights

No relevant changes were observed in terminal body weight and in absolute and relative organ weights among treated animals, when compared with controls.

Macroscopic observations

No macroscopic finding was described that could be considered correlated with the administration of the test item, apart from instances of dark/red or pink granular material seen in the gastro-intestinal tract of animals receiving 1000 mg/kg/day and killed at the end of the treatment period. This abnormal content probably represented residues of the test item in the gastro-intestinal tract.

Microscopic observations

The histopathological examination did not reveal any evident differences in the incidence of the findings observed in treated and control animals, which could be considered treatmentrelated.

Conclusion

The toxicity of the test item when given by oral administration (gavage) to rats for 13 consecutive weeks at dosages of 50, 200 or 1000 mg/kg/day, and recovery from any treatment-related effects over a recovery period of 4 weeks, have been investigated. No toxicologically relevant changes were observed during the in vivo phase or at the post mortem examinations.

On the basis of these results, it could be concluded that the No Observed Adverse Effect Level (NOAEL) in this study was 1000 mg/kg/day.

Liver and blood plasma samples of male and female rats of the 1000 mg/kg bw/day group collected at the end of the exposure period were below quantifiable limit concentrations of 1.5 ug/g dried liver and 0.4 / 0.6 ppm dried blood plasma.