Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Oct. 25, 2004 to July 19, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted: 21st September 1998
Deviations:
no
GLP compliance:
yes
Remarks:
according to OECD and Swiss principles of GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-amino-4-hydroxyethylaminoanisole sulfate
- TSIN: A084, WR 20381
- Substance type: Pure active substance
- Physical state: Pale grey powder
- Storage condition of test material: Away from direct sunlight at room temperature (15-25°C)
- Stability of the test substance: Stable under storage conditions
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 57
- Expiration date of the lot/batch: July 2005
- Purity test date: 03.08.2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (15-25°C) in the original container away from direct sunlight
Stability: The substance on storage in dryness and darkness to be stable  July 2005.
Stability in solution: The stability over a total period of seven days was tested by HPLC. The test stock solutions (approx. 5 weight%) were stored at room temperature and in the absence of light.
Water solution: the results (t = 0h: 100.0%; 6h: 93.8%; 2d: 95.1%; 7d: 79.7%) confirm a low degradation (G2000/003)
DMSO solution: the results (t = 0h: 100.0%; 6h: 98.8%; 2d: 92.0%; 7d: 80.5%) confirm a low degradation (G2000/003)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetix stirrer and used at room temperature (15-25°C), protected from light (i.e. aluminium foil). Homogeneity of the test item in the vehicle was maintened during the daily administration period using a magnetic stirrer.
- Final dilution of a dissolved solid, stock liquid or gel: 1.5/ 2.0/20.0 mg/ml in vehicle

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar (HanBrl, SPF bred)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Fullinsdorf/Switzerland.
- Age at study initiation: 6 weeks upon delivery
- Weight at study initiation: Males: 134.8-159.0 g (mean weight - 146.5 g); Females: 114.0-132.8 g (mean weight - 124.2 g).
- Housing: The animals were housed in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding.
- Diet: Pelleted standard Provimi Kliba 3433 rat maintenance diet; ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water (from Itingen); ad libitum. Results of bacteriological assay, chemical and contaminant analyses of representative samples were recorded.
- None of the contaminants analyzed in the water and diet was considered to have been present in the concentration which would have affected the validity of the results.
- Acclimation period: 7 days (under laboratory conditions after health examinations).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Relative humidity: 30 - 70%.
- Air changes: Air-conditioned room with 10-15 air changes per hour.
- Photoperiod: 12-hour fluorescent light/12-hour dark cycle with music during the light period.
IN-LIFE DATES: From: Nov. 01, 2004 To: Feb. 18, 2005

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Rationale : Accidental oral ingestion is a possible route of human exposure
Vehicle:
water
Remarks:
bidistilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared daily. Test substance was weighed into a glass beaker on a tared mettler balance and the vehicle was added. The mixtures were prepared using a magnetic stirrer and used at room temperature (15 - 25°C), protected from light (i.e. in aluminum foil).
- Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE: Bidistilled water
- Concentration in vehicle: 1.5, 5 and 20 mg/mL for 15, 50 and 200 mg/kg bw, respectively.
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONS: The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the sponsor.
- Concentration, homogeneity and stability (after 4 hours) of the dose formulations were determined in samples taken after start of the experiment.
- Concentration and homogeneity of the dose formulations were determined in samples taken monthly during treatment.
The results of homogeneity and concentration analyses showed that the formulation procedure resulted in adequate dose formulations. The mean concentrations of the test samples were 88.3 to 101.4%, 90.4 to 96.6% and 83.4 to 100.0% of the nominal values for the 15, 50 and 200 mg/kg bw, respectively, confirming proper dosing for the entire study period.
The details on the analytical verification of doses are provided in the study report.
Duration of treatment / exposure:
108/109 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 animals/sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were provided by the sponsor.
- Rationale for animal assignment: Randomization was performed by a computer-generated random algorithm.

TREATMENT: Animals (15 animals/sex/dose group) were treated according to following dosing schedule.
Group 1: Control (Bidistilled water)
Group 2 (Low dose level): 15 mg/kg bw/day
Group 3 (Mid dose level): 50 mg/kg bw/day
Group 4 (High dose level): 200 mg/kg bw/day
10 animals/sex/dose group were used in toxicity testing after 13 weeks treatment period (referred as Allocation A) while 5 animals /sex/dose group were used in hormone testing after 13 weeks treatment period (referred as Allocation B)

Examinations

Observations and examinations performed and frequency:
MORTALITY/VIABILITY: Yes
- Time schedule: Twice daily.

GENERAL CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before the commencement of administration of test substance and once daily during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of administration and once every week (1-14 weeks) thereafter.
- The animals were observed in their home cages, outside their home cages in a standard arena and in the hand.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during pretest, treatment and before necropsy. Body weights were recorded using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION: Yes,
- Food consumption recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At pretest and Week 13, after application of mydriatic solution to both eyes of the animals and using a Microflex 2 Opthamoscope.
- Dose groups that were examined: All animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 16 weeks (For Allocation A).
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia).
- Animals fasted: Yes, animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals.
- Parameters examined: Erthrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, platelet (thrombocyte) count, reticulocytes count, reticulocytes maturity index, methemoglobin, Heinz bodies (to be completely assessed only if changes in methemoglobin are noted), total leukocyte count, differential leukocyte count, coagulation, thromboplastin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 16 weeks (For Allocation A)
- Animals fasted: Yes, animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals.
- Parameters examined: Glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus (inorganic), protein (total), protein (electrophoresis) and albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18-hour fasting period into a specimen vial.
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: Yes, animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: Volume (18 hours), specific gravity (relative density), color, appearance, pH, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes, leukocytes and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Dose groups that were examined: All animals
- Battery of functions tested: Grip strength and locomotor activity.
- Grip Strength: Grip strength of the fore and hind limb were measured using a push-pull strain gauge (Mecmesin,AFG 25N). Each measurement was repeated thrice; the mean was calculated and recorded.
- Locomotor activity: Animals were monitored for a 60-minute period, during the 14th week of treatment. Locomotor (decreased or increased) activity was measured quantitatively with AMS FohrMedical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System and the total activity of this time period was recorded.

HORMONE ANALYSIS (Allocation B): Blood samples were drawn from the retro-orbital plexus prior to scheduled necropsy, placed on ice and were deep frozen at approximately -80°C. Total and free (T3 and T4) and TSH were analyzed in five aliquots of 300 µL serum.
Sacrifice and pathology:
NECROSCOPY: All the animals (allocation A and B) were weighed and necropsied after 16 weeks. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
- Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution :
Adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain (4 levels), cecum, colon, duodenum, epididymides (fixed in Bouin's solution), esophagus, exorbital lacrimal glands, eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), heart, ileum (with Peyer's patches), jejunum (with Peyer's patches), kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), mammary gland area, nasal cavity (turbinates), ovaries, pancreas, pituitary gland, prostate gland (including coagulating glands), rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, mid thoracic, lumbar), spleen, stomach, testes (fixed in Bouin's solution), thymus, thyroid (including parathyroid gland, if possible), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina and gross lesions.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS: The following organ weights were taken from all animals necropsied at termination of treatment: brain, heart, liver, thyroids/parathyroids, thymus, kidneys, adrenals, uterus, spleen, testes, epididymides and ovaries.
- The organ to terminal body weight ratios as well as organ to brain weight ratios was determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOPATHOLOGY: Yes
Histopathology: All organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin. Slides of all organs and tissues were collected at scheduled sacrifice of the animals of control and high-dose groups.
Allocation B animals: Thyroid glands of animals were examined by study pathologist. The morphologic changes observed in high dose groups resulted in examination of thyroid glands of mid and low dose groups.
Statistics:
a) The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, and organ weights and ratios:
-The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
-Fisher's exact-test was applied to the macroscopic findings.

b) The following statistical methods were used for statistical analysis of clinical laboratory data:
-Quantitative data was analyzed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to Bartlett. Alternatively, if the variances were considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<=0.05).
-Ordinal data such as urine sediment were analyzed using the Kruskal-Wallis test. If this test was significant (p<=0.05), comparisons were made between the control group and each of the treatment groups using Dunn's test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
CLINICAL SIGNS (DAILY AND WEEKLY)
- No toxicologically relevant clinical signs were observed in the test animals.
Mortality:
no mortality observed
Description (incidence):
MORTALITY
- All animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
BODY WEIGHT AND WEIGHT GAIN
- The mean body weights and the mean body weight gain of the test substance treated females were comparable to the controls throughout the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
FOOD CONSUMPTION
- No test substance related changes in the mean daily food consumption were noted at any time point during the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
OPHTHALMOSCOPIC EXAMINATION
- No ocular changes of toxicological relevance were seen after 14 weeks of treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
HAEMATOLOGY
- Slight anaemia with a compensatory reticulocytosis was seen in both sexes of 200 mg/kg bw dose group, evident as lower red blood cell counts, lower haemoglobin, elevated methaemoglobin, lower haematocrit levels and elevated reticulocyte counts and reticulocyte maturity indices. Similar effects but of less severity as described at 200 mg/kg bw were noted for the 50 mg/kg bw dose groups, but predominately in females only. Indication of a slight anaemia with a compensatory reticulocytosis was noted in females only.
- The methemoglobin level noted in the blood of females treated with 50 and 200 mg/kg bw/day was higher than that of the control females. Although Heinz bodies were not seen, this finding was considered to be a slight test substance related change.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
CLINICAL CHEMISTRY
- Reduced creatinine, elevated triglyceride elevated sodium and chloride concentrations were noted in 200 mg/kg bw dose group. Elevated plasma sodium and chloride levels were seen more distinctly in females than in males. These electrolytes were clearly elevated in females treated with 50 mg/kg bw/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
URINALYSIS
- Yellow/brown to brown to black discoloration of the urine was observed with increasing dose levels in test substance treated males and females. This was considered likely to be excreted test substance. Evaluation of urine sediment did not reveal test substance-related changes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
NEUROBEHAVIOUR
- No treatment related differences were observed in the mean fore and hind limb grip strength values and the mean locomotor activity at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
ORGAN WEIGHTS AND ORGAN WEIGHT RATIO:
- Marginally elevated thyroid-to-brain weight ratios were noted in both sexes treated with 200 mg/kg bw/day, whereas females treated with 200 mg/kg bw/day showed elevated mean absolute and relative weights in liver, kidney and spleen. These differences were considered to be test substance related. All other organ weights and ratios were unaffected.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
GROSS PATHOLOGY
- Dark red or black discoloration of the thyroid was recorded at necropsy in most animals treated with 50 and 200 mg/kg bw/day, which was assessed as a test substance related gross lesion. All other macroscopic findings recorded were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age in oral toxicity studies and were considered incidental, reflecting the usual individual variability.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
HISTOPATHOLOGY
- Histopathological changes were recorded in thyroid (follicular cell enlargement due to storage of brown fine-granular pigment in both sexes), pituitary (slight hypertrophy of chromophobic cells in males) and kidneys (pigment storage, tubulus swelling with necrosis of tubulus cells and basal membrane thickening) at 50 and 200 mg/kg bw/day. Increased mean grade of extra medullary hemopoiesis in combination with slightly increased mean grade of hemosiderin storage in animals treated with 200 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
HORMONE ANALYSIS (Allocation B)
- No test substance related changes in the thyroid hormones (TSH, free and total T3 and T4) were observed at any dose level after 15 weeks of treatment.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
2-amino-4-hydroethoxyethylamino-anisole sulfate when administered by oral gavage daily for 108-109 days to rats at dose levels of 0, 15, 50 and 200 mg/kg bw/day, revealed a no observed adverse effect level (NOAEL) at 15 mg/kg bw/day based on hematology, clinical biochemistry, gross pathology and histopathology. Considering the effects observed at 50 and 200 mg/kg bw/day: anaemia and morphological and histological changes in the thyroid, the kidneys and the pituitary, and according to CLP criteria the substanec should be classified as STOT RE Categorie 2, H373: May cause damage to organs (blood, thyroid, kidneys, pituitary) through prolonged or repeated exposure by oral route.
Executive summary:

The repeated dose oral toxicity study of 2-amino-4-hydroxyethylaminoanisole sulfate was performed by following the OECD Guideline 408(Repeated Dose 90-Day Oral Toxicity in Rodents).

Male and female Wistar (HanBrl, SPF bred) rats obtained from RCC Ltd, Laboratory Animal Services, CH-4414, Fullinsdorf/ Switzerland were used in the study. The body weight range of male and female rats was 134.8-159.0 g (mean weight - 146.5 g) and 114.0-132.8 g (mean weight - 124.2 g), respectively. Animals were housed in groups of five in Makrolon type-4 cagesandmaintained under controlled environmental conditions (temperature: 22 ± 3°C, humidity: 30-70%, 10-15 air changes/ hand12 h fluorescent light /12 h dark cycle with music during the light period).The study consisted of 3 test treatment groups and 1 control group containing 15 animals/sex/dose (10 animals/sex/dose group were used in toxicity testing after 13 week treatment period (referred as Allocation A) while 5 animals /sex/dose group were used in hormone testing after 13 week treatment period (referred as Allocation B)).Test substance formulations were prepared freshly daily in bidistilled water. The animals of different groups were treated as follows:

Group 1: Vehicle Control (Bidistilled water)

Group 2 (low dose group): 15 mg/kg bw/day

Group 3 (mid dose group): 50 mg/kg bw/day

Group 4 (high dose group): 200 mg/kg bw/day

The following adverse effects were noted at the different dose levels:

200 mg/kg bw/day

Indication of a slight anaemia with a compensatory reticulocytosis was seen in both sexes, evident as lower red blood cell counts, lower haemoglobin, elevated methaemoglobin, lower haematocrit levels and elevated reticulolycte counts and reticulocyte maturity indices. The effects on clinical biochemistry parameters (reduced creatinine, elevated triglyceride sodium and chloride concentrations), and urinalysis (slight proteinuria and increased

bilirubin and nitrite) observed in both sexes may indicate changes in the liver metabolism and, to a lesser extent, changes in the kidneys. Marginally elevated thyroid-to-brain weight ratios in both sexes and elevated mean absolute and relative liver, kidney and spleen weights in females were recorded. Histopathological and morphological test item-related changes were recorded in thyroid (follicular cell enlargement due to storage of brown fine-granular pigment, both sexes), pituitary (slight hypertrophy of chromophobic cells in males), kidneys (pigment storage, tubulus swelling with necrosis of tubulus cells and basal membrane thickening) and spleen

(increased mean grade of extra medullary haemopoiesis, both sexes).

50 mg/kg bw/day

Similar effects but of less severity as described at 200 mg/kg bw/day were noted for the mid dose groups, but predominately in females only. Indication of a slight anaemia with a compensatory reticulocytosis as described for the high dose was noted in females only. Elevated plasma sodium and chloride levels in females were also seen. Histopathological and morphological changes similar to those of the high dose group were observed in thyroid (follicular cell enlargement due to storage of brown fine-granular pigment, both sexes), pituitary (slight hypertrophy of chromophobic cell in males) and kidneys (pigment storage, tubular swelling with necrosis of tubular cells and basal membrane thickening).

15 mg/kg bw/day

No adverse effects were noted

Based on the adverse effects noted at 50 mg/kg bw/day, evident as slight anaemia and morphological and histological changes in the thyroid, the kidneys and the pituitary, a “no observed adverse effect level” (NOAEL) of 15 mg/kg bw/day of 2-amino-4 - hydroethoxyethylamino-anisole sulfate was derived from this subchronic oral toxicity study in rats.

This repeated dose toxicity study is classified as acceptable, and satisfies the guideline requirements of OECD 408 method.