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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: Guideline-Studie (GLP, QAU)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: EPA-TSCA Test Guideline "Functional Observational Battery", Fed. Reg. Vol. 50, No. 188, Sept. 27, 1985, pp. 39458 - 39461
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibutyl phthalate
EC Number:
201-557-4
EC Name:
Dibutyl phthalate
Cas Number:
84-74-2
Molecular formula:
C16H22O4
IUPAC Name:
dibutyl phthalate
Details on test material:
- Name of test material (as cited in study report): dibutyl phthalate (test substance number 89/449)
- Analytical purity: > 99% (GC)
- Lot/batch No.: P .66 A/89

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: male and female Wistar rats (Chbb = THOM, SPF) used were from Dr . Karl Thomae GmbH, Biberach/Riss, FRG, and clinically free from any signs of disease
- Age at study initiation: 35 days at supply
- Weight at study initiation: male animals, 186 (175 - 196) g; female animals, 144 (134 - 153) g
- Housing: during the study period the rats were housed singly in type DK III stainless steel wire mesh cages, supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area about 800 cm2)
- Diet (e.g. ad libitum): the feed used was ground KLIBA rat/mouse/hamster maintenance laboratory diet GLP 343 meal of KLINGENTALMÜHLE AG, CH-4303 Kaiseraugst
- Water (e.g. ad libitum): drinking water
- Acclimation period: 7-day

ENVIRONMENTAL CONDITIONS
The animals were accommodated in a fully air-conditioned room
- 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:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared by weighing in the test substance and mixing this thoroughly with a small amount of the feed. This was then mixed in a BOSCH household mixer. An appropriate amount of feed was then added to obtain the desired concentration, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer. Different amounts of DIBUTYL PHTHALATE and Bis-(2-ethylhexyl)- phthalate were added, depending on the test groups to the feed at the end of the adaptation period

DIET PREPARATION
- Rate of preparation of diet (frequency): the test substance preparations were prepared in up to 2-week intervals for reasons of the confirmed stability over 32 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical results verified the correctness of the test substance concentrations in the diet with two exceptions.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuously via food
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
ca. 30, 152, 752 mg/kg bw per day (ca 0, 27, 141, 688 mg/kg in males and 0, 33, 162, 813 mg/kg for females)
Basis:
other: calculated based on feed consumption (see Table 2)
Remarks:
Doses / Concentrations:
0, 400, 2000 and 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 (see Table 1)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the doses were chosen on the basis of the 28-day feeding study with test substance and positive control substance carried out at BIBRA, in which concentrations of 0.02, 0.05, 0.1, 0.5, 1.0 and 2.59 in the diet were used. According to preliminary results, the no effect level of test substance was at 0.1 % and of positive control substance is at 0.05%, which is possibly equivalent to a respective test substance intake of about 100 and 50 mg/kg body weight. Due to the above mentioned findings dose levels for test substance of 400, 2000 and 10000 ppm and for positive control substance 1400 ppm were chosen. The study was carried out from August 29, 1989 (day 0) to December 07, 1989 (last day of perfusion fixation).
- Rationale for selecting satellite groups: no satellite group used; at the end of the 3-month administration period all animals were sacrificed following a fasting period (withdrawal of feed for about 16 - 20 hours).
Positive control:
Bis-(2-ethylhexyl)- phthalate in a dosage of 1,400 ppm via the diet over 3 months as a positive control group in respect to the number and morphology of the liver peroxisomes

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for dead or moribund animals was carried out twice a day (Mondays to Fridays) or once a day (Saturdays, Sundays and on public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the animals were inspected for evident signs of toxicity twice a day (Mondays to Fridays) or once a day (Saturdays, Sundays and on public holidays). In addition, the animals were also subjected to an additional exact clinical examination once a week

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once a week on each occasion on the same day (Tuesday)

FOOD CONSUMPTION:
- 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 / No / No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the study and towards the end of the study the eyes were examined for any changes to the refracting media using a HEINE FOCALUX hand-held slit lamp.
- Dose groups that were examined: animals in test group 0(0 ppm) and in test group 3 (10000 ppm)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 45, 86 and 92/93
- Anaesthetic used for blood collection: No; blood taken from the retroorbital venous plexus in the morning
- Animals fasted: No
- How many animals: 10 animals per test group and sex
- Parameters examined: Hematological examinations (leukocytes, erythrocytes, hemoglobin, haematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets) and Clotting analyses (thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 45, 86 and 92/93
- Animals fasted: No
- How many animals: 10 animals per test group and sex
- Parameters examined: enzymes (alanine àminotransferase, aspartate aminotransferase, alkaline phosphatase, cyanide-insensitive palmitoyl-CoA-oxidation), blood chemistry (sodium, potassium, chloride, inorganicphosphate, calcium, urea, creatinine, glucose, totalbilirubin, totalprotein, albumin, globulins, triglycerides, cholesterol, magnesium) and hormones (total triiodothyronine [T3], total thyroxine [T 4])

URINALYSIS: Yes
- Time schedule for collection of urine: days 38 and 80
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters were examined: appearance, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the test substance administration, then on days 34, 59 and 90 on treatment
- Dose groups that were examined: all animals in test
- Battery of functions tested: functional observational battery (including general appearance, tremors, convulsions, piloerection, lacrimation/secretion of pigmented tears, salivation, pupil contraction/dilatation, diarrhea, vocalization, paresis, ataxia, body tone, posture, animal body (appearance), locomotor activity, respiration, urination, skin color, righting reflex, behavior, grip strength, papillary reflex, winking reflex, vision, audition, olfaction, sensitivity of the body surface, pain perception, tail pinch, toe pinch, visual placing response, miscellaneous (all other visible clinical signs).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; the animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The weight of the anesthetized animals as well as the weight of liver, kidneys, brain, adrenal glands and testes from a1l animals was determined. After weighing, parts of the liver have been transferred to the Laboratory of clinical chemistry and hematology for further investigations.
HISTOPATHOLOGY: Yes; subsequently to gross pathology, the following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, pituitary gland, thyroid glands, parathyroid glands, thymus, trachea, Lungs, heart, aorta, salivary glands (nandibular glands [gl. mandibularis] and sublingual glanils [gl. sublingualis]), liver, spleen, kidneys, adrenal glands, pancreas, ovaries, uterus/vagina, accessory genital organs (prostate and seminal vesicle), skin, esophagus, stomach (glandular and non glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric lymph nodes), female mammary gland, musculature, sciatic nerve, sternum, bone marrow (femur), eyes, femur with knee joint, spinal cord (cervical, mid thoracic, lumbar cord), extraorbital lacrimal glands. Testes of all animals were fixed in Bouin. After the organs have been fixed, processing, the examination by light microscopy and the evaluation of findings was performed.
Other examinations:
None
Statistics:
Statistical significances of the clinical data (body weight, grip strength fore- and hind limbs and hot plate values) were examined for statistical significance by analysis of variance (ANOVA) followed by DUNNETT's test. Clinical chemistry and hematology: in order to test if the results of the individual dose groups differed statistically significantly from the results of the control group, the means for the dose groups, excepting the differential blood count, were compared with those for the control group using the analysis of variance (ANOVA and DUNNETT's test. Urinalyses: the assessment to whether certain characteristics differed in degree in the control and test groups was carried out using the chi2 test in appropriate two-by two contingency tables. Gross- and histopathology: the statistical evaluation was carried out using the DIJNNETT test for a simultaneous comparison of several dose groups with a control group.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- The administration of test substance as addition to the diet in different dosages did not lead to any disturbances of the general state in any test animal.
- During the entire administration period no animal died prematurely.

BODY WEIGHT AND WEIGHT GAIN
In test group 3 (10000 ppm) the female rats showed reduced body weights up to 49% in comparison to the controls during the study period. A slight influence could also be seen in the males of test group 2 (2000 ppm). The body weights in this group were reduced up to 3% over the course of the study compared with the corresponding controls. In respect to no dose-response relationship and minimal degree, this was assessed as being a sign of biological variation and not substance-related.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- During the course of the study in the males and females no substance-induced influence in the feed consumption values could be detected. Only in the females of the 10000 ppm group an intermittent slight decrease of max. 3% and sporadically an increase was noted during the study period, but this was assessed as being a sign of biological variation and not substance-related.
- The approximate, mean daily test substance intake in mg/kg body weight for the complete administration period is presented in Table 2.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmological examinations carried out prior to the start of the study and at the end of the administration period revealed no substance-induced impairment of the refracting media.

HAEMATOLOGY AND CLINICAL CHEMISTRY
The following substance-induced changes:
- Test group 3 (10000 ppm): (a) decrease in triglycerides and triiodothyronine in both sexes; (b) increase in cyanide-insensitive palmitoyl-CoA-oxidation in both sexes; (c) decrease in red blood cells, hemoglobin and haematocrit in the males; (d) increase in glucose and albumin in the males
- Test group 2 (2000 ppm): (a) no substance-induced changes
- Test group 1 (400 ppm): (a) no substance-induced changes
Thus, under the test conditions given in this study, a dose without an effect is from the viewpoint of clinical chemistry and hematology for male and female rats between 2000 ppm and 10000 ppm.

URINALYSIS
No adverse effects observed.

NEUROBEHAVIOUR
In none of the 4 tests performed during the study the dose groups differed from the untreated control, regarding neurofunction.

ORGAN WEIGHTS (Table 3)
- The absolute and relative liver weights of the close group 3 females were statistically significantly increased. The relative liver weights of the dose group 3 males were also statistically significantly increased compared to the control. The absolute liver weights of the males of dose group 3 also tended to be higher than these of the males of dose groups 1 and 2 and these of the control. The increased liver weights of close group 3 are interpreted as being related to the test substance.
- The kidney weights (absolute and relative) of the males of dose group 1 were statistically slightly significantly decreased, the absolute and relative kidney weights of the females of dose group 3, however, were statistically slightly significantly increased. The kidney weight changes were regarded as incidental. Moreover, there were no substance-induced histomorphological kidney changes either.

GROSS PATHOLOGY
Macroscopy revealed no substance-induced changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Light microscopy revealed after HE staining no hepatocellular changes correlating with the increased liver weights. If the assessment of the fatty deposits is based on detectable lipid vacuoles in hepatocytes, there are differences between the control and dose groups 1 and 2 on the one hand and the dose group 3 on the other. While all animals of the control had at least a few or slight fatty deposits they were not found in 6 males and 5 females of dose group 3, on the basis of a uniform comparable magnification chosen for evaluation. The higher grades of fatty infiltration, as they were noted in some animals of the control and dose groups 1 and 2, were wholly absent. However, this does not explain the liver weight increase observed. In conjunction with statistically slightly significantly changed clinicochemical parameters (decreased triglyceride values in the females and also as the trend in the males of close group 3) this can be assessed as an indication of a substance-induced influence on the lipid metabolism, which is based on a hypolipidemic mechanism. A morphological equivalent for this assumption would be the detection of peroxisome proliferation in the hepatocytes. While in the HE stain characteristic changes or indications of peroxisome proliferation were not yet detectable in dose group 3 at the chosen dose of 10000 ppm, a multifocal peroxisome proliferation was successfully detected in the same dose by cytochemical presentation of the catalase with diaminobenzidine in 1 male and 1 female animal of the study 9950449/89021. All other histomorphological findings were assessed as being of incidental or spontaneous origin.

The substance-induced histopathological effects were increases in the absolute and relative liver weights, which remained however without a morphological correlate and decreased or missing lipid deposition in the hepatocytes, all observed in male and female animals of dose group 3 (10000 ppm). This findings correlates with the multifocal peroxisome proliferation in the liver diagnosed at the same dose level in 1 male and 1 female of the study 9950449/89021 after cytochemical presentation of the catalase with diaminobenzidine. Thus, under the given study conditions and from the viewpoint of pathology the "no adverse effect level" is at a dose between 2000 and 10000 ppm test substance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
152 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
752 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: LOAEL based on liver effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Test substance intake

Test group

Concentration (ppm)

Mean daily test substance intake in mg/kg body weight

Male animals

Female animals

1

400

27

33

2

2000

141

162

3

10000

688

816

 

Table 3: mean absolute/relative organ weights at necropsy (only organs showing some significant differences from control values shown)

Organ

Weight parameter

Sex

Mean values in indicated groups and dose levels

0 (0 mg/kg)

1 (30 mg/kg)

2 (152 mg/kg)

3 (752 mg/kg)

 

 

Liver

Absolute (g)

Female

7.30±0.46

7.44±0.73

7.75±0.74

8.44±0.98*

male

16.15±0.52

16.32±2.68

16.15±1.80

18.45±2.03

Relative to body weight (%)

Female

2.80±0.15

2.91±0.22

2.96±0.22

3.34±0.15*

male

3.45±0.32

3.45±0.34

3.54±0.24

3.85±0.25*

 

Kidneys

Absolute (g)

Female

1.83±0.09

1.86±0.12

1.94±0.15

1.99±0.13*

male

3.19±0.09

2.94±0.29*

3.12±0.13

3.42±0.25

Relative to body weight (%)

Female

0.71±0.04

0.73±0.06

0.74±0.05

0.80±0.09*

male

0.68±0.04

0.63±0.03*

0.69±0.05

0.72±0.05

 

Applicant's summary and conclusion