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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF AG, Product Safety
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): beta-lonon R
- Test substance No.: 02/0449-1
- Physical state: liquid/colorless to yellowish
- Analytical purity: 97.8% (Analytical report 02L00370)
- Lot/batch No.: continuous production
- Date of production: October 9, 2002
- Stability under test conditions: 97.8% (proven by reanalysis Analytical report 03L00294)
- Storage condition of test material: Refrigerator, protected from light
- Other: The analyses of the test substance were carried out at the Analytical Department of BASF
Aktiengesellschaft, Ludwigshafen, Germany .

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats CrIGIxBrIHan:WI
- Source: Charles River, Sulzfeld, Germany
- Identification: ear tattoo
- Mean body weight at study initiation: males: 152g; females: 123.8g (weight variation of the animals did not exceed 20 percent of the mean weight of each sex)
- Age at study initiation: 42 ±1 days
- Housing: individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, Germany).
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet mouse/rat "GLP", supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): ad libitum, drinking water (from drinking bottles)
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- For each concentration, the test substance was weighed out and mixed with a small amount of food in a beaker.
- Subsequently, a premix was prepared in a mixer by adding an appropriate amount of food followed by mixing.
- Then corresponding amounts of food,
depending on dose group, were added to this premix in order to obtain the desired concentrations.
- Mixing was carried out for about 10 minutes in a laboratory mixer (details of the mixers used are retained with the raw data).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF AG .
- The stability of the test substance in the diet over a period of 49 days at room temperature was proven before the start of the study (Project No . : 01 Y0449/028010) .
- Homogeneity and concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and at the end of the administration period .
Duration of treatment / exposure:
3 months
Frequency of treatment:
continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 1000, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.(-> BASFAG30SO449/02045.Repeated dose toxicity: oral rat.28 d)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, check for signs of toxicity and mortality
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- following parameters were examined: behavior during "handling", fur,  skin, posture, salivation, respiration, activity/arousal level, tremors,  convulsions, abnormal movements, impairment of gait, lacrimation,  palpebral closure, exophthalmus, feces (appearance/consistency), urine  and pupil size


BODY WEIGHT: Yes
- Time schedule for examinations: prior to administration period, then weekly


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of the administration period the eyes of all animals, and on day 91 the eyes of the control and high dose animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning; in all surviving animals per test group and sex at the end of the administration period
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- Parameters examined: determined in blood with  EDTA-K3 as anticoagulant using a particle counter: leukocytes,  erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean  corpuscular hemoglobin, mean corpuscular hemoglobin concentration,  platelets, differential blood count, reticulocytes. Prothrombin time was  determined using a ball coagulometer


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning; in all surviving animals per test group and sex at the end of the administration period
- Animals fasted: Yes
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline  phosphatase, serum gamma-glutamyltransferase, sodium, potassium,  chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total  bilirubin, total protein, albumin, globulins, triglycerides, cholesterol,  magnesium.


URINALYSIS: Yes
- Time schedule: day 85
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

HORMONES:
- determined with  radioimmunoassys using commercially available test kits and a  gamma-counter
- parameters determined: total triiodothyronine (T3 ), total thyroxine (T4 ), thyroid-stimulating hormone (TSH)


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period starting at about 10 .00 a.m
- Dose groups that were examined: all animals
- Battery of functions tested (FOB): passive  observations without disturbing the animals, followed by removal from the  home cage, open field observations in a standard arena and sensorimotor  tests as well as reflex tests. 
- The findings were ranked according to the  degree of severity, if applicable.

-- Home cage observations: The animals were observed in their closed home  cages; any disturbing activities were avoided during these examinations  in order not to influence the behavior of the animals. Attention was paid  to posture, tremor, convulsions, abnormal movements, impairment of gait  and general observations.
-- Open field observations: The animals were transferred to a standard  arena (50 cm x 50 cm with sides of 25 cm high) and observed for at least  2 minutes. The following parameters were examined: behavior when removed  from cage, fur, skin, salivation, nose discharge, lacrimation, eyes /  pupil size, posture, palpebral closure, respiration, tremors,  convulsions, abnormal movements / stereotypics, impairment of gait,  activity/arousal level, feces, urine and number of rearings.
-- Sensorimotor tests/reflexes: The animals were removed from the open  field and subjected to following sensorimotor or reflex tests: approach  response, touch response, vision, pupillary reflex, pinna reflex,  audition ("startle response"), coordination of movements, behavior during, vocalization, pain  perception, grip strength of forelimbs, grip strength of hindlimbs,  landing foot-splay test

- Motor activity assessment: was measured on the same day as FOB was  performed. The measurement was performed in the dark using the  Multi-Varimex-System with 4 infrared beams per cage. During the  measurement the animals were kept in Polycarbonate cages with absorbent  material. The measurements started at about 2.00 p.m. and the number of  beam interrupts were counted over 12 intervals, each lasting 5 minutes.  The period of assessment for each animal started when the first beam was  interrupted by pushing the cage into the rack. Measurements ended exactly  60 minutes thereafter. 


ESTROUS CYCLE DETERMINATION: Yes
- Time schedule: Vaginal smears for cycle determination were prepared in the morning and evaluated according to the timetable .

SPERM PARAMETERS: Yes
- Time schedule: Immediately after necropsy and organ weight determination the right testis and cauda epididymidis were taken from all male animals
- Parameters checked: Sperm motility, Sperm morphology, sperm head count (cauda epididymidis), sperm head count (testis)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Weight parameters from all animals sacrificed at scheduled dates were determined:  liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus,  spleen, brain, heart, thymus, prostate gland

HISTOPATHOLOGY: Yes
- left testis, left epididymis and both ovaries were  fixed in Bouin's solution. After fixation, the organs were embedded in  paraplast. 
- The following organs were fixed in 4% formaldehyde solution,  histopathologically processed and examined by light microscopy: All gross lesions, salivary glands (glandula mandibularis and glandula  sublingualis), esophagus, stomach (forestomach and glandular stomach),  duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, brain,  pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and  lumbar cord), eyes, adrenal glands, thyroid glands, parathyroid glands,  trachea, lungs, pharynx, larynx, nose (nasal cavities), aorta, heart,  bone marrow (femur), lymph nodes (mandibular and mesenteric), spleen,  thymus, kidneys, urinary bladder, oviducts/uterus/vagina, prostate gland,  seminal vesicles, female mammary gland, skin, skeletal muscle, sternum  with marrow, femur with knee joint, extraorbital lacrimal glands
Statistics:
Means and standard deviations of each test group were calculated for  several parameters. 
Further statistical analyses were performed.
- Dunnett test:  Food and water consumption, body weight change, food efficiency
- Kruskall-Wallis test:  Feces, rearing, grip strength length forelimbs, grip strength length  hindlimbs, landing foot-splay test, motor activity, clinical pathology,  pathological weight parameters (if p-value < = 0.05 Wilcoxon test was  additionally performed)
- Fishers exact test: Urinalysis, except volume, color, turbidity and specific gravity;  abnormal sperm > 4%
- Wilcoxon test: Total spermatids/g testis, total sperm/g cauda epi., % motility

Results and discussion

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
other: overall effects; urinalysis; organ weights; histopathology
Dose descriptor:
NOAEL
Effect level:
71.8 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: overall effects; urinalysis; organ weights; histopathology
Dose descriptor:
NOAEL
Effect level:
83 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: overall effects; urinalysis; organ weights; histopathology
Dose descriptor:
LOAEL
Effect level:
10 000 ppm
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
720 mg/kg bw/day (actual dose received)
Sex:
male
Dose descriptor:
LOAEL
Effect level:
801 mg/kg bw/day (actual dose received)
Sex:
female
Dose descriptor:
NOEL
Effect level:
100 ppm
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
8.2 mg/kg bw/day (actual dose received)
Sex:
female
Dose descriptor:
NOEL
Effect level:
7.1 mg/kg bw/day (actual dose received)
Sex:
male

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

ANALYSES

- Stability of the test substance: was demonstrated in the diet over a  period 

of 49 days at room temperature
- Concentration control: correctness of the concentrations were  confirmed. 

The recovery rates were within a range of 92.3% - 102.6% of  the target 

concentrations.

TEST SUBSTANCE INTAKE
 
Dose       Conc. (ppm)           Intake (mg/kg bw/d)
                                 Males      Females
Low               100             7.1          8.2
Mid              1000            71.8         83.0
High             10000           719.6        801.0

CLINICAL EXAMINATIONS

- Mortality: One female animal of the control group was sacrificed  moribund on 

day 42.

- Clinical signs: single findings (one moribund female in control group,  one 

male animal of the high dose group with alopecia) were assessed as  being 

incidental and not substance-related.

- Food consumption: Food consumption of male and female animals in the  high 

dose group was statistically significantly decreased on day 7  (-24.5%). As no 

further significant impairment was seen, this was  assessed as being incidental.

- Water consumption: no changes

- Body weight data: In high dose males, body weight (day 7) and body  weight 

change (day 7 and 14) was statistically significantly decreased.  These findings 

were connected with the above-mentioned impaired food  consumption and therefore 

assessed as being incidental.

- Food efficiency: In high dose males, food efficiency was statistically  

significantly decreased on day 7 and 28. Due to the isolated occurrence  and in 

connection with the above-mentioned impaired food consumption,  this finding was 

assessed as being incidental.

- Ophthalmoscopy: No substance-related effects were obtained. 

- Functional observational battery and motor activity measurement: All  findings 

were assessed as being incidental, as they occurred in single  animals, only, or 

were equally distributed between treated groups and  controls.

- Estrous cycle determination: No substance-related effects were  obtained. 

CLINICAL PATHOLOGY

- Hematology: At the end of the administration period, prothrombin times  were 

significantly shortened in the high dose females. No  treatment-related effects 

were seen in the other hematological parameters  of both sexes.

- Clinical chemistry: Enzyme examinations revealed marked increases in  

gamma-glutamyltransferase activities in the serum of the high dose  animals of 

either sex after 3 months of test substance administration. No  treatment-related 

changes were found in the other enzyme measurements.
Blood chemistry investigations showed increased calcium, total protein,  albumin, 

globulins and cholesterol concentrations in the serum of the  high dose male and 

female animals. However, the increase in albumin in  the females of the high dose 

group was not statistically significantly  different to the concurrent control, 

but was seen as a tendency towards  higher values. Dose-dependent, statistically 

significantly increased  inorganic phosphate levels were measured in the serum of 

all treated  males. Although the increases in inorganic phosphate were statistically  

significantly different to the control value and showed a dose-response relationship, 

these isolated findings were assessed as being fortuitous  since similar increases 

were not observed in the females. The differences  were attributed to a low 

control group value and not to the test compound  administered. No toxicologically 

relevant changes were observed in the  other blood chemistry parameters.

- Thyroid hormone measurements: Slightly but significantly decreased  thyroxine 

(T4) levels (-19%) were found in the high dose males. A  relationship to 

treatment could not be excluded. The significant changes  in the low and mid dose 

females were not regarded as treatment related as  they occurred without a dose 

relationship.

Table: Thyroid hormone determinations

                T3 (nM)         T4 (nM)        TSH (µg/l
Dose Groups   M      F        M       F       M       F
Control      1.31   1.49    55.2    30.7     7.1     5.8
Low          1.34   1.74    54.0    42.4**   7.6     5.0
Mid          1.36   1.44    60.6    37.3*    8.0     5.8
High         1.6    1.63    44.9**  33.1     7.2     5.9

* p < = 0.05, ** p < = 0.01

- Urinalyses: At the end of the study, an increased amount of ketones  were 

excreted by the high dose males and females and in the urine  specimens of the 

high dose females increased urobilinogen levels were  measured. Microscopic 

examination of the urine sediments revealed  increased numbers of degenerated 

transitional epithelial cells in the mid  and high dose males as well as in the 

high dose females. Higher number of  granular and epithelial cell casts was also 

observed in the urine  specimens of the mid and high dose males. The test 

compound did not  affect the other urine parameters.

- Sperm analysis: There were no treatment-related effects on sperm  parameters.

PATHOLOGY

- Absolute organ weights: When compared to control group, the mean  absolute 

weights of following organs were significantly increased:
        
Group             L                M              H
Liver       na (m,f)          na (m)      +65.3% (m)
                          +10.2% (f)      +52.5% (f) 
Kidneys     na (m,f)        na (m,f)      +22.7% (m)
                                              na (f)                   
Testes      na                na          +11.9%        

L, M, H = low, mid, high dose
m, f = males, females
na = no abnormalities        

All other mean absolute weight parameters did not show significant  differences 

when compared to the control group.

- Relative organ weights (related to terminal body weight): 
When compared to control group 0, the mean relative weights of following  organs 

were significantly decreased or increased:
        
Group                 L             M              H
Liver          na (m,f)     +9.1% (m)     +73.4% (m)
                            +7.6% (f)     +54.8% (f)
Kidneys        na (m,f)      na (m,f)     +28.0% (m) 
                                          +10.6% (f)
Testes         na            na           +17.5%        
Epididymides   na            na           +11.0%
Adrenal glands na (m,f)     -11.1% (f)      na (m,f)


The decrease of the mean relative adrenal weight in females of the mid  dose group 

was considered incidental. 
All other mean relative weight parameters did not show significant  differences 

when compared to the control groups.

- Gross lesions: All gross lesions occurred singly. In two high dose  males, a 

unilateral pelvic dilation of the kidneys was observed.

- Histopathology:

Liver: A minimal to slight central hypertrophy of hepatocytes was noted  in three 

mid dose males and in all high dose males and females. In  addition, two high 

dose males showed a peripheral hypertrophy of  hepatocytes. In the cytoplasm of 

these hypertrophic hepatocytes,  myelinoid figures were noted. The myelinoid 

figures most likely represent  arrays of concentric membranes (smooth endoplasmic 

reticulum).

Kidneys: Eosinophilic droplets were observed in some of the tubular  epithelial 

cells in most of males. These eosinophilic droplets stained  positive with 

Mallory-Heidenhain. The immunohistochemical examination of  eosinophilic droplets 

with a specific antibody revealed  alpha2u-globuline. The amount of this protein in tubular epithelial cells  was higher in

 mid and high dose males than in controls. Chronic  nephropathy was observed in 

a high incidence with higher degrees of  severity in males of the high dose group. 

The macroscopically diagnosed  unilateral pelvic dilation of the kidneys in two 

high dose males was  confirmed histopathologically. The occurrence of this 

finding was  considered incidental.

Thyroid gland: A flaky appearance of the colloid ("altered colloid") was  noted 

in most untreated and treated males and females. The gradings of  this finding, 

however, were increased in high dose males.

Stomach: In the region of the limiting ridge - the border between  forestomach 

and glandular stomach - the epithelium showed prominent rete  packs (diagnosed 

as "acanthosis") in two high dose males and in one mid  and one high dose female. 

The occurrence of this finding was considered  incidental.

Testes: The absolute and relative weights of testes were significantly increased 

in high dose males. Because there were no histopathological  correlates for the 

increased weights, a substance-related effect seemed  unlikely.

Epididymides: The relative weight of epididymides was significantly  increased 

in high dose males. The absolute weight of epididymides did not  show significant 

differences and there were no histopathological  correlates for the increased 

weight. Therefore, this finding was  considered due to the slightly (-4.2%) but 

not significantly decreased  terminal body weight in these animals.

All other findings noted were either single observations or they were  biologically 

equally distributed between control and treatment groups.  All of them were 

considered to be incidental or spontaneous in origin and  without any relation 

to treatment.

In summary, the following substance-related findings were obtained:

10,000 ppm (719.6 mg/kg bw/d in males ; 801.0 mg/kg bw/d in females)

• Increased y-glutamyltransferase, calcium, total protein, albumin,

globulins, cholesterol, urinary ketones and urinary transitional epithelial

cells in both sexes

• Increased urinary casts in males

• Shortened prothrombin times and increased urinary urobilinogen in females

• Decreased thyroxine in males

• Significant increase of the absolute and relative liver weights in males and females;

• Significant increase of the absolute and relative kidney weights in males and

of the relative kidney weight in females;

• Central hypertrophy of hepatocytes in the liver of all males and females;

• Peripheral hypertrophy of hepatocytes in the liver of two males;

• High incidence of chronic nephropathy in males

• Higher amounts of alpha2u-globuline in tubular epithelial cells of the kidneys in males

• Higher degrees of severity of altered colloid in the thyroid gland of males

and high incidence with higher gradings of altered colloid in females

1,000 ppm (71.8 mg/kg bw/d in males ; 83.0 mg/kg bw/d in females)

• Increased urinary casts and urinary transitional epithelial cells in males

• Significant increase of the relative liver weight in males and of the absolute

and relative liver weights in females

• Central hypertrophy of hepatocytes in the liver of 3 males

• Higher amounts of alpha2u-globuline in tubular epithelial cells of the kidneys

in males

100 ppm (7.1 mg/kg bw/d in males ; 8.2 mg/kg bw/d in females)

• No substance-related findings obtained

Applicant's summary and conclusion

Conclusions:
The administration of beta-Ionone over a period of 3 months at dietary concentrations of 100, 1000 and 10 000 ppm lead to signs of general systemic toxicity. Target organs were liver, kidneys and thyroid glands. Regarding estrous cycle determination and sperm evaluation, no influence on the reproduction system was obtained. Moreover, no signs of neurotoxicity were observed during functional observational battery as well as measurement of motor activity performed towards the end of the administration period.

Thus, the no-observed-effect-level (NOEL) under the conditions of the present study was 100 ppm for both sexes (about 7 and 8 mg/kg bw/d for males and females) based on adaptive liver effects in both sexes and minor urine findings in males at 1000 ppm which correspond to a dosage of 72 and 83 mg/kg bw/day for males and females (no-observed-adverse-effect-level, NOAEL). The lowest-observed-adverse-effect-level (LOAEL) was found at 10 000 ppm (720 and 801 mg/kg bw/day for males and females) due to liver, kidney and thyroid findings in both sexes.