Registration Dossier

Administrative data

Description of key information

4 week repeated dose oral toxicity: NOAEL 1350 mg/kg bw/day, OECD 407, EU Method B.7 and Japanese guidelines (Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry), Fabreguettes 1996

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:
13 February 1996 to 04 April 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed to valid guidelines and the study was conducted under GLP conditions.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines: Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Stain: Crl CD (SD) BR
- Age at study initiation: approximately 6 weeks old.
- Weight at study initiation: mean body weight of 209 g (197 to 226 g) for the males and 176 g (156 to 200 g) for the females.
- Housing: The animals were individually housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metallic tray containing autoclaved sawdust was placed under each cage. Sawdust was changed once a week; trays, cages and racks were changed once every 4 weeks.
- Diet: Pelleted maintenance diet, ad libitum. Diet was distributed weekly. Feeders were changed once every 4 weeks.
- Water: Bottles containing tap water filtered using a 0.22 micron filter were provided ad libitum. Bottles were changed weekly.
- Acclimation period: 8-day acclimatisation period under the same conditions as the main test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (07:00 - 19:00)

IN-LIFE DATES: From: 13 February 1996 To: 04 April 1996
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified by reverse-osmosis
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was administered as a solution in the vehicle. The test material was dissolved in order to achieve concentrations of 30, 90 and 270 mg/mL, then homogenised using a magnetic stirrer.
The test material preparations were kept for up to 9 days, according to the stability data and were stored at +4 °C. The preparations were delivered each day to the animal room and maintained under continuous stirring during the dosing procedure.

ADMINISTRATION:
- Concentration in vehicle: A constant dosage-volume of 5 mL/kg/day was used.
The oral route was selected since it is a possible route of exposure in humans.
The test material or vehicle was administered by gavage using a glass syringe fitted with a metal tube. The quantity of the test material or vehicle administered to each animal was adjusted according to the most recently recorded body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
STABILITY
The stability of solutions (prepared with the batch used in this study) was demonstrated before the beginning of the treatment period.
Initially, samples of preparations at the lowest (30 mg/mL) and highest (270 mg/mL) concentrations used in the study were sampled in duplicate just after preparation, after 5 hours at room temperature and after 4 and 9 days of storage at +4 °C. These samples were stored frozen at -20 °C pending last sampling occasion (day 9), when all samples were assayed. As the highest dose was prepared at a concentration close to the limit of solubility of the test material in water (365 g/L at 25 °C, the first stability assay performed after the samples were frozen at -20 °C was difficult to interpret as the values for the highest dose were higher than nominal concentrations.
Therefore, further preparations at 30 mg/mL and 270 mg/mL were made and sampled in duplicate as described above and analysed immediately. This showed satisfactory results. Stability of preparations at 30 mg/mL and 270 mg/mL was therefore demonstrated for 9 days at +4 °C under the conditions of preparation previously mentioned.

CONCENTRATION
For each group, including the control, the preparations intended for use in weeks 1 and 4 were sampled for analysis. Throughout the study, a satisfactory concordance was found between obtained and nominal concentrations.

METHOD
An aliquot of the dosage form was diluted and analysed by Ultra-Violet spectrophotometry at 401 nm. Concentrations of the test material were estimated from a calibration curve prepared over a range from 0 to 50 µg/mL.
The solution was stirred with a magnetic stirrer. A 1 mL volume of solution was sampled in a flask tube using a Microlab 1000 Hamilton dilutor and diluted tenfold with water. After vortex mixing, serial dilutions were carried out with water in accordance with a target concentration of 0 µg/mL for group 1 and ranging from 0 to 50 µg/mL of the test material for other groups.

ASSAY
An aliquot of test solution was analysed by Ultra-violet spectrophotometry.
A calibration curve was obtained by linear regression analysis of Ultra-violet absorbance at 401 nm versus concentration of standard solutions for concentrations ranging from 0 to 50 µg/mL (four levels: 5, 10, 20 and 50 µg/mL).The concentrations were calculated using the following equation:
Y = aX + b
Where:
Y= absorbance of the test material
X = concentration of the test material (µg/mL)
a = slope
b = intercept
All results are expressed as mg/mL of the test material.

EXPERIMENTAL CONDITIONS
- Spectrophotometer: Shimadzu UV-260 (Roucaire) Double-beam
- Cell: Quartz cell
- Path length: 1 cm
- Blank: water
- Wavelength: 401 nm

In addition, an UV spectrum from each group diluted to 50 µg/mL was recorded and compared with the spectrum of an analytical standard solution at 50 µg/mL to verify the structure of the test material. The UV spectrum from each group was found to tally with the analytical standard.

VALIDATION
- Specificity: Specificity of the analytical method was demonstrated since no absorbance was measured in water.
- Linearity: A satisfactory linearity was obtained in the range of 0 to 50 µg/mL (4 levels in duplicate; 5, 10, 20 and 50 µg/mL) for the test material. The coefficient of determination was 0.9998.
- Repeatability: Replicate analyses (n = 10) of a solution containing 20 µg/mL of the test material was correct since the coefficient of variation obtained was 0.6 %.
Duration of treatment / exposure:
4 consecutive weeks. At the end of the treatment period, the first five animals of the control and high dose-level groups were kept for a 2-week reversibility period.
Frequency of treatment:
Animals were administered the test material once a day, at approximately the same time, 7 days a week over a period of 29 days.
Remarks:
Doses / Concentrations:
150, 450 and 1350 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
The 150 and 450 mg/kg/day dose groups both consisted of 5 animals per sex per dose; 10 animals per sex per dose were exposed in the top dose group, 1350 mg/kg/day, and the control.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were determined following the results of a preliminary 7 day toxicity study performed in the same species. The administration of the test material at 150, 450 or 1350 mg/kg/day by gavage induced yellow coloured faeces in animals given 150 or 450 mg/kg/day and orange coloured faeces in animals given 1350 mg/kg/day. Yellow coloured extremities were noted in some animals given 1350 mg/kg/day. These findings were considered to be due to the staining properties of the test material. No clinical signs or macroscopic findings of toxicological significance were noted in any treated groups. Consequently, the same dose-levels were used for the 4 week toxicity study.
- Rationale for animal assignment: The required number of animals was selected according to body weight and clinical condition, and allocated by sex to the groups according to a computerised randomisation.
- Rationale for route selection: The oral route was selected since it is a possible route of exposure in humans.
Observations and examinations performed and frequency:
MORTALITY AND CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice a day (including weekends and public holidays) for mortality or signs of morbidity during the treatment period and at least once a day during the reversibility period.
Clinical signs were observed at least once a day for each animal, at approximately the same time of day.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded for each animal once before allocation of the animals into groups, on the first day of treatment, and then once a week until the end of the study, including the reversibility period.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each animal was recorded once a week, over a 7-day period, until the end of the study, including the reversibility period.
Food intake per animal and per day was calculated using the amount of food given and left in each food hopper.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from the orbital sinus of the animals (before the daily treatment). Haematology parameters were determined on day 28 after blood was collected into tubes containing EDTA. Coagulation parameters were determined on blood that had been collected into tubes containing sodium citrate.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia.
- Animals fasted: Yes, for at least 14 hours.
- How many animals: 5 per sex per dose.
- Haematology parameters examined: Erythrocytes (RBC), Haemoglobin (HB), Mean Cell Volume (MCV), Packed Cell Volume (PCV), Mean Cell Haemoglobin Concentration (MCHC), Mean Cell Haemoglobin (MCH), Thrombocytes (PLAT), Leucocytes (WBC), Differential white cell count with cell morphology - neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L) and monocytes (M).
- Coagulation parameters examined: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the orbital sinus of the animals whilst under light ether anaesthesia (before the daily treatment) into tubes containing lithium heparinate. Blood chemistry parameters were determined on day 28.
- Animals fasted: Yes, for at least 14 hours.
- How many animals: 5 per sex per dose.
- Parameters examined: Sodium (Na+), Potassium (K+), Chloride (CI), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total Bilirubin (TOT.BIL), Total Proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT) and Gamma glutamyl transferase (GGT).

URINALYSIS: Yes
- Time schedule for collection of urine: For urine collection, the animals were deprived of food and placed in metabolism cages for an overnight period of at least 14 hours. The urine was collected into a tube containing thymol crystals over this period.
- Metabolism cages used for collection of urine: Yes, for at least 14 hours.
- Animals fasted: Yes, for at least 14 hours.
- Parameters examined: Volume (VOLUME), pH (pH), Specific gravity (SP.GRAV), Proteins (PROT), Glucose (GLUC), Ketones (CETO), Blood (BLOOD), Appearance (APP) and Colour (COLOUR).
Sacrifice and pathology:
SACRIFICE
On completion of the treatment period, after at least 14 hours of fasting, all animals of groups 2 and 3 and the last five animals of each sex in groups 1 and 4 were asphyxiated using carbon dioxide and killed by exsanguination.
On completion of the reversibility period, after at least 14 hours fasting, the remaining five animals of the control and high dose group were killed under the same conditions.

ORGAN WEIGHTS
For all animals killed at the end of the treatment or reversibility period, the body weight was recorded before necropsy and the organs specified in Table 1 were weighed wet as soon as possible after dissection. Paired organs were weighed separately (except for thyroids with parathyroids).

GROSS PATHOLOGY: Yes
A complete macroscopic examination was performed on all animals. All gross observations were recorded individually.
For all animals, all the macroscopic lesions and the tissues specified in Table 1 were preserved in 10 % buffered formalin (except for the eyes which were fixed in Davidson's fixative).

HISTOPATHOLOGY: Yes
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at approximately 4 microns in thickness and stained with haematoxylin-eosin.
Microscopic examination was performed on:
- All macroscopic lesions and tissues listed in Table 1 for animals of the control and high dose-level groups (1350 mg/kg/day) killed at the end of the treatment period.
- All macroscopic lesions for animals of the low and intermediate dose-level groups (150 and 450 mg/kg/day).
- All macroscopic lesions for all animals of control and high dose-level groups (1350 mg/kg/day) killed at the end of the reversibility period.
Statistics:
The following sequence was used for the statistical analysis of body weight, food consumption, haematology, blood biochemistry, urinalysis and organ weight data:

1) First, data was tested for normality of distribution using Kolmogorov-Lilliefore's test.

1a) Data found to have a normal distribution was then assessed according to the following scheme, dependant on the number of groups:
> Assessment of homogeneity of variance between groups of 3 or more = Bartlett’s test.
Data found to be heterogeneous using the Bartlett’s test was then tested according to Dunn’s test.
Data found to be homogeneous using the Bartlett’s test was then tested according to Dunnett’s test.
> Assessment of homogeneity of variance between groups of 2 =Fisher’s test.
Data found to be heterogeneous using Fisher’s test was then tested according to Mann-Whitney’s test.
Data found to be homogeneous using Fisher’s test was then tested according to Dunnett’s test.

1b) Data found not to have a normal distribution using Kolmogorov-Lilliefore's test was then treated as follows:
Logarithmic transformation of values (except for organ weights), then re-tested for normality of data according to Kolmogorov-Lilliefore's test. Data found to have a normal distribution then followed the scheme laid out above (under 1a). Failure to normalise the distribution resulted in the following:
> Comparison of treated and control groups using original (untransformed values, including organ weights) using tested using Dunn’s test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No unscheduled deaths occurred throughout the study.
Ptyalism was noted in 1/5 males and females given 450 mg/kg/day and in 7/10 males and 8/10 females given 1350 mg/kg/day. Since this sign was occasionally noted, generally in week 1, it was considered to be of no toxicological importance.
The following observations were noted during the treatment period, which were likely to be related to the elimination of the test material or metabolites:
- Yellow or reddish orange coloured faeces in all animals given 450 or 1350 mg/kg/day, respectively, from week 1;
- Yellow coloured extremities in 1/5 females given 450 mg/kg/day and in 4/10 males and 9/10 females given 1350 mg/kg/day from weeks 2 or 3.
These signs were considered to be an indirect proof of the absorption of the test material.
During the reversibility period, the coloured faeces disappeared in the animals of the high dose level group, and the only sign observed was a yellow coloured tail in 4/5 treated females.

BODY WEIGHT AND WEIGHT GAIN
The mean body weight gain was considered to be similar in control and treated animals during the treatment and reversibility periods.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean food consumption was considered to be similar in control and treated animals during the treatment and reversibility periods.

HAEMATOLOGY
No test material-related changes were noted at the end of the treatment period.
The only differences from controls (including: leucocyte count, mean cell volume and mean cell haemoglobin concentration in females) were not dose-related, and all the individual values were within the range of the testing facility’s historical background data. Consequently, they were considered to be without relationship to the treatment with the test material.

CLINICAL CHEMISTRY
No test material-related changes were noted at the end of the treatment period.
The only differences from controls (including sodium and glucose levels, alkaline phosphatase activity) were slight and not dose-related. Therefore, they were considered to be without relationship to the treatment with the test material.

URINALYSIS
No test material-related changes were noted at the end of the treatment period.

ORGAN WEIGHTS
At the end of the treatment and reversibility periods: Some minor differences in organ weights were noted between individuals within the same group and between groups. However, there were no major findings that were considered to be treatment related.

GROSS PATHOLOGY
At the end of the treatment period: Yellowish colouration of extremities and hair was found in 1/5 males and all females given 1350 mg/k/day. Orange deposit on the caecum mucosa was noted in 2/5 males and orange or yellowish deposit on the stomach mucosa in 1/5 males from the same group.
These discolourations were considered to be due to the physical properties of the test material (dyeing substance).
The few other macroscopic findings encountered are all commonly recorded findings in the rat of this strain and age. None were considered to be treatment-related.

At the end of the reversibility period: Reversibility was not found on completion of the study, as extremities and/or hair were yellow in 4/5 females given 1350 mg/kg/day.
In addition, yellowish areas measuring approximately 0.5 cm long x up to 0.5 cm wide were noted in the left lobe of the lung in 2/5 females given 1350 mg/kg/day. The few other macroscopic findings noted are those commonly recorded in the untreated laboratory rat of this strain and age. None were considered to be treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
- At the end of the treatment period: Unilateral, focal, minimal degeneration of seminiferous tubules was found in 1/5 males given 1350 mg/kg/day. As this finding can be found spontaneously in the untreated laboratory rat, this was considered to be of no toxicological importance.
The few other microscopic findings noted are all commonly recorded spontaneous changes in the untreated laboratory rat of this strain and age and none was considered to be of toxicological importance.

- At the end of the reversibility period: Very few macrophages overloaded with yellowish pigment were found in some alveoli of the lungs of 2/5 treated females which showed yellowish areas in the lungs at macroscopic examination. This was probably due to aspiration of test material, as a consequence of regurgitation or misdosing.
Moderate degeneration of seminiferous tubules together with moderate oligospermia were noted in 1/5 treated males. As this can be found spontaneously in the untreated laboratory rat of this strain and age, this was considered to be without relationship to the treatment with the test material.
The few other microscopic findings noted are all commonly recorded spontaneous changes in the untreated laboratory rat of this strain and age and thus, they were considered to be of no toxicological importance.
Dose descriptor:
NOAEL
Effect level:
1 350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the absence of mortality or overt signs of toxicity in animals tested up to the highest dose level.
Critical effects observed:
not specified
Conclusions:
Under the conditions of the test, the NOAEL was determined to be 1350 mg/kg/day in male and female rats.
Executive summary:

The potential toxicity of the test material was assessed in a 4 week repeated dose toxicity study with a 2 week reversibility period in the rat. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 407 and EU Method B.7, and three Japanese guidelines (Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry).

Two groups of five male and five female Sprague-Dawley rats received the test material daily by gavage at 150 and 450 mg/kg/day, and one group of 10 males and 10 females was given 1350 mg/kg/day. An additional group of 10 males and 10 females received the vehicle alone (purified water) under the same conditions.

The animals were checked daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Haematological and blood biochemical investigations and urinalysis were performed at the end of the treatment period (week 4). On completion of the 4 week treatment period, the first five animals of each sex in the control and high dose-level group (1350 mg/kg/day) were kept for a 2-week reversibility period.

At the end of the treatment or reversibility periods, the designated animals were killed and submitted to a complete macroscopic examination. Designated organs were weighed. A microscopic examination was performed on selected tissues for animals of the control and high dose-level groups killed at the end of the treatment period, macroscopic lesions were microscopically examined for the animals of the low and intermediate dose-level groups and for the animals of the control and high dose-level groups killed at the end of the reversibility period.

Under the conditions of the test, there were no signs of toxicologically relevant effects in any of the animals exposed to the test material up to the highest dose of 1350 mg/kg/day. The only signs noted in animals of the intermediate and high dose-level groups during the treatment period were related to the dyeing properties of the test material. Some findings of discolouration persisted in animals given 1350 mg/kg/day during the reversibility period. Therefore the NOAEL was determined to be 1350 mg/kg/day in male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 350 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality is high. Both the key and supporting studies were performed under GLP conditions to standardised guidelines. The reports are conclusive and reported to a sufficient standard for classification. In accordance with the principles for assessing data quality defined by Klimisch et al. (1997), these studies were both assigned a reliability score of 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two studies have been provided to address repeated dose oral toxicity; a 4 week repeated dose study and the preliminary 7 day range finding study.

The potential toxicity of the test material in the rat was assessed in a preliminary 7 day study in order to define the dosing range for a subsequent 4 week repeated dose toxicity study using the same species. The study was performed under GLP conditions and in accordance with the principles of the standardised guidelines OECD 407 and EU Method B.7.

Three groups of 3 male and 3 female Sprague Dawley rats received the test material daily by oral gavage, at dose levels of 150, 450 and 1350 mg/kg/day for 7 days. An additional group of animals received the vehicle alone (purified water) under the same conditions. The animals were checked daily for clinical signs and mortality. Food consumption and body weight were recorded twice a week. At the end of the treatment period, the animals were killed and submitted to a complete macroscopic examination.

No unscheduled deaths occurred during the study. No clinical signs of toxicological significance were observed during the study. Yellowish or orange-coloured faeces noted in treated animals were considered to be related to the elimination of the test material or its metabolites. The food consumption and the body weight gain were similar in the control and treated animals during the study. With the exception of yellow colouration of the hair and extremities seen in some animals which was considered to be due to the staining properties of the test material, no findings of toxicological significance were noted at necropsy.

Under the conditions of the test, there were no treatment related mortalities or overt signs of toxicity. Therefore the NOAEL can be said to be greater than the highest dose tested (> 1350 mg/kg/day).

The administration of the test material to rats was well-tolerated at 150, 450 and 1350 mg/kg/day. Consequently, the same dose-levels were proposed for the 4-week study.

The potential toxicity of the test material was assessed in a 4 week repeated dose toxicity study with a 2 week reversibility period in the rat. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 407 and EU Method B.7, and three Japanese guidelines (Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry).

Two groups of five male and five female Sprague-Dawley rats received the test material daily by gavage at 150 and 450 mg/kg/day, and one group of 10 males and 10 females was given 1350 mg/kg/day. An additional group of 10 males and 10 females received the vehicle alone (purified water) under the same conditions.

The animals were checked daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Haematological and blood biochemical investigations and urinalysis were performed at the end of the treatment period (week 4). On completion of the 4 week treatment period, the first five animals of each sex in the control and high dose-level group (1350 mg/kg/day) were kept for a 2-week reversibility period.

At the end of the treatment or reversibility periods, the designated animals were killed and submitted to a complete macroscopic examination. Designated organs were weighed. A microscopic examination was performed on selected tissues for animals of the control and high dose-level groups killed at the end of the treatment period, macroscopic lesions were microscopically examined for the animals of the low and intermediate dose-level groups and for the animals of the control and high dose-level groups killed at the end of the reversibility period.

Under the conditions of the test, there were no signs of toxicologically relevant effects in any of the animals exposed to the test material up to the highest dose of 1350 mg/kg/day. The only signs noted in animals of the intermediate and high dose-level groups during the treatment period were related to the dyeing properties of the test material. Some findings of discolouration persisted in animals given 1350 mg/kg/day during the reversibility period. Therefore the NOAEL was determined to be 1350 mg/kg/day in male and female rats.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
There were two studies available, a preliminary 7 day toxicity study and the subsequent 4 week repeated dose toxicity study. The 4 week study was selected as key; the purpose of the preliminary study was to define the dosing range for the 4 week study in which the animals were exposed for a longer period of time and where a more extensive set of parameters were investigated.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the test material does not require classification for specific organ toxicity, repeated dose. The only signs noted in animals were related to the dyeing properties of the test material and were considered to be an indirect proof of the absorption of the test material.