Registration Dossier

Administrative data

Description of key information

No adverse effects were observed in a 28-day gavage study in rats (OECD 407, GLP) at doses of up to 1000 mg/kg bw (NOTOX 2009b and c).

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:
2009-03-09-, 2009-05-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to OECD guideline and under GLP
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(December 2008)
Deviations:
yes
Remarks:
Humidity above 70 % (probably due to cleaning procedures in the room). Light periods with 1 hour fluctuations.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1987 according to the notification of November 21, 2003 by Ministry of Health, Labor and Welfare (No. 1121002), Ministry of Economy, Trade and Industry (No. 2) and Ministry of Environment (No. 031121002).
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. July 2000.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: all animals within ± 20 % of the sex mean
- Housing: 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cageenrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom)
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 22.3
- Humidity (%): 40 - 89. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room
Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethyl cellulose in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) in 1 % aqueous carboxymethyl cellulose were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle: the choice was based on trial formulations
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10 or 20 mL. For determination of accuracy, samples were taken at 50 % height or at 90 %, 50 % and 10 % height. The latter set of samples was also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50 % height and stored at room temperature for 6 hours.
Duration of treatment / exposure:
28 days treatment plus 14 days treatment-free recovery phase
Frequency of treatment:
once daily for 28 days
Remarks:
Doses / Concentrations:
0, 30, 300, and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected on the basis of a 5-day dose range finding study
- Post-exposure recovery period in recovery groups: 14 days
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations: mortality / viability; clinical signs (at least once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to start of treatment and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery phase
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: White blood cells; Differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils); Red blood cells; Reticulocytes; Red blood cell distribution width; Haemoglobin; Haematocrit; Mean corpuscular volume; Mean corpuscular haemoglobin; Mean corpuscular; haemoglobin concentration; Platelets; Prothrombin time; Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery phase
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: Alanine aminotransferase; Aspartate aminotransferase; Alkaline phosphatase; Total Protein; Albumin; Total Bilirubin; Urea; Creatinine; Glucose; Cholesterol; Sodium; Potassium; Chloride; Calcium; Inorganic Phosphate; Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Adrenal glands; (Aorta); Brain [cerebellum, mid-brain, cortex]; Caecum; Cervix; (Clitoral gland); Colon; Duodenum; Epididymides; Eyes (including optic nerve and harderian gland); (Female mammary gland area); Femur including joint; Heart; Ileum; Jejunum; Kidneys; (Larynx); (Lacrimal gland, exorbital); Liver; Lung, infused with formalin; Lymph nodes - mandibular, mesenteric; (Nasopharynx); (Oesophagus); Ovaries; (Pancreas); Peyer's patches [jejunum, ileum] if detectable; (Pituitary gland); (Preputial gland); Prostate gland; Rectum; (Salivary glands - mandibular, sublingual); Sciatic nerve; Seminal vesicles including coagulating gland; Skeletal muscle; (Skin); Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid [if detectable]; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; All gross lesions. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

HISTOPATHOLOGY: Yes. All tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals; uterus, cervix, vagina and ovaries of all female animals for determination of oestrus cycle; all tissues from animal no. 23 which was terminated in extremis; all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings. Histopathology was subjected to a peer review.
Other examinations:
Organ weights: Adrenal glands; Brain; Epididymides; Heart; Kidneys; Liver; Ovaries; Spleen; Testes; Thymus; Uterus (including cervix); Prostate; Seminal vesicles including coagulating glands; Thyroid including parathyroid.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
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:
not examined
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 AND MORTALITY. Animals at 300 and 1000 mg/kg/day showed yellow faeces during the treatment period and/or Day 1 of the recovery period. Since the test substance is a yellow pigment, this finding was considered to be related to staining properties of the test substance and of no toxicological significance.
Male no. 23 at 1000 mg/kg/day showed hunched posture, laboured respiration and piloerection prior to being killed in extremis. Female no. 52 at 1000 mg/kg/day showed hunched posture and/or piloerection between Days 13 and 15. The macro- and microscopic abnormalities in these animals were consistent with a gavage trauma and, therefore, these clinical signs were considered not to be related to toxic properties of the test substance. No mortality occurred that was considered to be related to the test substance. Male no. 23 at 1000 mg/kg/day was killed in extremis on Day 9. Based on the macro- and microscopic abnormalities observed in this animal, this death was considered a consequence of a gavage trauma and, therefore, not to be related to toxic properties of the test substance.

BODY WEIGHT AND WEIGHT GAIN. No toxicologically relevant changes in body weight and body weight gain were observed.
Female no. 52 at 1000 mg/kg/day showed body weight loss in the second week of treatment, but body weight gain similar to control animals during continuing treatment. The macro- and microscopic abnormalities in this animal were consistent with a gavage trauma and, therefore, the body weight loss was considered not to be related to toxic properties of the test substance.

FOOD CONSUMPTION AND COMPOUND INTAKE. Food consumption before or after allowance for body weight was similar between treated and
control animals.

HAEMATOLOGY. No toxicologically relevant changes occurred in haematological parameters of treated rats.
Female no. 52 at 1000 mg/kg/day showed the following changes in haematology parameters compared to other females: higher relative neutrophil counts, relative reticulocyte counts and red blood cell distribution width (RDW) and lower white blood cell counts (WBC), relative lymphocyte counts, red blood cell counts and haemoglobin levels. The macro- and microscopic abnormalities in this animal were consistent with a gavage trauma and, therefore, these changes in haematology parameters were considered not to be related to toxic properties of the test substance.
Minor statistically significant differences when compared to control animals included lower relative basophil counts in males at 30 mg/kg/day at the end of treatment, higher relative lymphocyte counts and lower relative neutrophil counts in males at 1000 mg/kg/day at the end of recovery and higher relative monocyte counts in females at 1000 mg/kg/day at the end of recovery. These changes were considered of no toxicological significance as they remained within the range considered normal for rats of this age and strain, occurred in the absence of a treatment-related distribution and/or occurred only at the end of the recovery period.

CLINICAL CHEMISTRY. No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Bile acid levels of two males (nos. 27 and 30) at 1000 mg/kg/day were higher at the end of treatment, but returned to levels comparable to control males at the end of recovery. In the absence of any corroborative findings in these animals and normal bile acid levels in other animals of this dose group, this finding was considered of no toxicological significance.
Minor statistically significant differences when compared to control animals included lower creatinine levels in females at 300 mg/kg/day at the end of treatment, higher chloride and inorganic phosphate levels in males at 1000 mg/kg/day at the end of recovery and lower albumin levels in females at 1000 mg/kg/day at the end of recovery. These changes were considered of no toxicological significance as they remained within the range considered normal for rats of this age and strain, occurred in the absence of a treatment-related distribution and/or occurred only at the end of the recovery period.

NEUROBEHAVIOUR. No toxicologically relevant findings in hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
One male (no. 19) at 300 mg/kg/day and one male (no. 25) at 1000 mg/kg/day showed abnormalities in pupillary reflex of the left eye, which coincided with exophthalmos and opacity for the male at 1000 mg/kg/day. In the absence of findings in the right eyes of both animals and in the absence of findings in other treated animals, these findings were considered incidental and not related to treatment with the test substance.
The variation in motor activity did not indicate a relation with treatment.


ORGAN WEIGHTS. No toxicologically relevant changes occurred in organ weights and organ to body weight ratios
of treated rats.
Female no. 52 at 1000 mg/kg/day showed higher thymus and kidney weights and thymus and kidney to body weight ratios at the end of treatment. The macro- and microscopic abnormalities in this animal were consistent with a gavage trauma and, therefore, these changes in organ weights were considered not to be related to toxic properties of the test substance.
Several minor differences in organ weights and organ to body weight ratios achieved statistical significance when compared to control animals. These changes included higher seminal vesicle weights and seminal vesicles to body weight ratios in males at 30 and 300 mg/kg/day at the end of treatment and in males at 1000 mg/kg/day at the end of recovery, higher spleen weights and/or spleen to body weight ratios in females at all dose levels at the end of treatment, higher liver to body weight ratio in females at 1000 mg/kg/day at the end of treatment and higher thyroid weight and higher thyroid to body weight ratio in females at 1000 mg/kg/day at the end of recovery. These changes were considered of no toxicological significance as they remained within the range considered normal for rats of this age and strain and occurred in the absence of supportive microscopic findings in these organs. Furthermore, they occurred in the absence of a treatment-related distribution, were considered to have arisen as a result of slightly high or low control values and/or occurred only at the end of the recovery period.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY. Greenish content in the gastrointestinal tract was noted for the majority of animals at 300 and 1000 mg/kg/day at the end of treatment, but not at the end of recovery. Since the test substance is a yellow pigment, this finding was considered to be related to staining properties of the test substance and of no toxicological significance.
Male no. 23 at 1000 mg/kg/day showed yellowish discolouration of the glandular mucosa of the stomach, yellowish watery-cloudy fluid in the thoracic cavity, pale discolouration of enlarged kidneys, a gelatinous thymus and several tissues which were grown together: pericardium with thymus, left lobe of lungs with right medial lobe of lungs and pleura with lungs. Female no. 52 at 1000 mg/kg/day showed yellowish discolouration of the esophagus at the level of the thymus, pale discolouration of kidneys, isolated yellowish hard nodules on the thymus and many yellowish hard nodules on the diaphragm which was grown together with the lungs. Together with the microscopical findings, the macroscopical abnormalities in these animals were consistent with a gavage trauma and, therefore, considered not to be related to toxic properties of the test substance.
Other incidental findings among control females and treated animals included alopecia, a sore, scabs, exophthalmus and reddish discolouration of the left eye, isolated red foci on the thymus, reddish discolouration of the mandibular lymph nodes and/or reduced size of seminal vesicles. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
No macroscopical abnormalities were noted for control males, females at 30 mg/kg/day and recovery males at 1000 mg/kg/day.

HISTOPATHOLOGY. There were no microscopic findings recorded which could be attributed to toxic properties of the test substance.
The greenish contents of the gastrointestinal tract, considered due to pigment nature of the test substance, were not associated with any pathological alterations in animals at 1000 mg/kg/day.
There was no evidence of alterations in estrus cycle between the control and treated females. The microscopic alterations recorded for male no. 23 and female no. 52 at 1000 mg/kg/day were consistent with a gavage trauma and, therefore, considered not to be related to toxic properties of the test substance. These alterations included tubular dilatation, granular casts, tubular basophilia and yellow brown crystals in the kidneys of both animals at 1000 mg/kg/day and osteonecrosis in the trabecular bone of the femur and tibia of male no. 23.
The remainder of the recorded microscopic findings were within the normal range of background
alterations encountered in Wistar rats of this age and strain.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at any dose level.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at any dose level.
Critical effects observed:
not specified
Conclusions:
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) and a No Observed Effect Level (NOEL) 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

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

Repeated dose toxicity was assessed in a subacute gavage study in rats following OECD guideline 407 and GLP (NOTOX 2009b). Based on the results of a 5-day range finding study (NOTOX 2009c), the dose levels were selected to be 0, 30, 300 and 1000 mg/kg/day. The test substance, formulated in 1% aquaous carboxymethyl cellulose, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.

Animals at 300 and 1000 mg/kg/day showed yellow faeces and/or green contents of the gastrointestinal tract. Based on the absence of any supportive pathological alterations in animals at 1000 mg/kg/day and the test substance being a yellow pigment, these findings were considered to be related to staining properties of the test substance and 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). The NOEL for subacute oral toxicity in rats is 1000 mg/kg bw.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No serious irreversible effects were observed at dose levels of less than 150 mg/kg bw upon subacute exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats. As a result the substance is not considered to be classified for repeated dose toxicity in Category 2 under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.