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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
Study period:
From 12 Sep 1994 To 21 Dec 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-propylheptyl) phthalate
EC Number:
258-469-4
EC Name:
Bis(2-propylheptyl) phthalate
Cas Number:
53306-54-0
Molecular formula:
C28H46O4
IUPAC Name:
1,2-bis(2-propylheptyl) benzene-1,2-dicarboxylate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- Identification: ear tattoo
- Age at study initiation: 38 days on arrival
- Weight at study initiation: mean 184 (176 - 194) g for the males, mean 149 (140 - 163) g for the females
- Fasting period before study: about 16 - 20 hours
- Housing: single, DK III stainless steel wire mesh cages (Becker, Castrop-Rauxel, Germany); floor area about 800 cm2
- Diet: commercial diet (ground Kliba laboratory diet rat/mouse/hamster 343 meal, Klingenthalmuehle AG, Kaiseraugst, Switzerland); ad libitum
- Water: drinking water ad libitum; ad libitum
- Acclimation period: 7 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:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently, a premix was prepared in a BOSCH household mixer by adding an appropriate amount of food and mixing for about 3 minutes. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): 343 meal, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland.
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the diet at room temperature was proven for 4, 8 and 32 days. The homogeneous distribution of the test substance in the diet was checked in samples of the high and low concentration drawn at the beginning of the study. These analyses served also as concentration controls for low and high concentration. Further concentration control analyses were performed in samples drawn at the start of the administration period (mid concentration) and towards the end of the administration period (all concentrations).
Duration of treatment / exposure:
3 months
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Remarks:
Nominal in diet
Dose / conc.:
2 500 ppm
Remarks:
Nominal in diet
Dose / conc.:
15 000 ppm
Remarks:
Nominal in diet
Dose / conc.:
39 mg/kg bw/day (actual dose received)
Remarks:
Mean actual dose received
Dose / conc.:
196 mg/kg bw/day (actual dose received)
Remarks:
Mean actual dose received
Dose / conc.:
1 266 mg/kg bw/day (actual dose received)
Remarks:
Mean actual dose received
Dose / conc.:
36 mg/kg bw/day (actual dose received)
Remarks:
Actual dose received (males)
Dose / conc.:
181 mg/kg bw/day (actual dose received)
Remarks:
Actual dose received (males)
Dose / conc.:
1 187 mg/kg bw/day (actual dose received)
Remarks:
Actual dose received (males)
Dose / conc.:
42 mg/kg bw/day (actual dose received)
Remarks:
Actual dose received (females)
Dose / conc.:
211 mg/kg bw/day (actual dose received)
Remarks:
Actual dose received (females)
Dose / conc.:
1 344 mg/kg bw/day (actual dose received)
Remarks:
Actual dose received (females)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The doses were chosen an the basis of a previous investigation with 5 animals/sex/group were dosed with concentrations of 0, 1000, 10000 and 20000 ppm (Administration in the diet for two weeks; BASF AG, Project No. 10C0110/94019)
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for overt clinical signs or mortality was made twice a day from Mondays to Fridays and once a day on Saturdays, Sundays and public holidays.
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Three days prior to the start of the administration period and 91 days after start of the administration period
- Dose groups that were examined: the animals in the highest dose group and in the control group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3 days prior and at the end of administration period; in the morning
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3 days prior and at the end of administration period; in the morning
- Animals fasted: No
- How many animals: 10/sex/group
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, cyanide-insensitive palmitoyl-CoA-oxidation, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

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

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The statistical evaluation and calculation of the data were carried out on the computer systems of the Department of Toxicology of BASF AG:
For the parameters body weight and body weight change a parametric one-way analysis of variance was done via the F-test (ANOVA). If the resulting p-values were equal or less than 0.05, a comparison of each dose group with the control group was carried out. These comparisons were performed simultaneously via DUNNETT's test for the hypothesis of equal means. If the results of this test were significant, labels (* for p < 0.05, ** for p < 0.01) were printed together with the group means in the tables. Both tests were performed two-sided.
For all clinical chemistry and hematology parameters, excepting the differential blood count, a parametric one-way analysis of variance was done via the F-test (ANOVA). If the resulting p-value was equal or less than 0.05, a comparison of each dose group with the control group was carried out. These comparisons were performed simultaneously via DUNNETT's test for the hypothesis of equal means. If the results of this test were significant, labels (* for p < 0.05, ** for p < 0.01) were printed together with the group means in the tables. Both tests were performed two-sided.
With the exception of volume, color, turbidity, pH and specific gravity the scale for the urine parameters is divided into 4 sections (0, 1, 2, 3). For the parameter "Nitrite" only a division in two sections (0, 1) is made. The parameters were sorted into 2 classes. This was done for the statistical analysis. A pairwise comparison of each dose group with the control was carried out using FISHER's exact test for the hypothesis of equal proportions. If the results of this test are significant, labels (* for p < 0.05, ** for p < 0.01) were printed in the tables.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 15000 ppm: the body weight values of the high dose group were slightly decreased in comparison to the control values. At the end of the administration period values of body weight were 6% below controls in males and 8% below controls in females. Although statistically significant only in females on days 63 to 91, the effects in both sexes were assessed as being substance-related. Corresponding body weight change values at the end of the administration period were 11% below controls in males and 17% below controls in females. Statistical significance was obtained for males on day 7 and for females on several days from day 42 onwards. This was assessed as being substance-related.
- 2500 ppm: no statistically or biologically relevant changes
- 500 ppm: no statistically or biologically relevant changes
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 15000 ppm: at the end of the administration period statistically significantly decreased hemoglobin concentrations were found in the peripheral blood of the high dose animals of either sex. Furthermore, in the high dose males statistically significantly decreased hematocrit values (-5.1%) and increased platelet counts were observed (+20%). In the high dose females mean corpuscular hemoglobin (MCH) was statistically significantly decreased (-5.2%); in the males only a trend towards reduced mean corpuscular hemoglobin was seen.
- 2500 ppm: no statistically or biologically relevant changes
- 500 ppm: no statistically or biologically relevant changes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 1500 ppm: the blood chemistry examinations revealed statistically significantly decreased chloride concentrations (-2.5% and -3%) and increased albumin levels in the high dose males and females, respectively. However, in the females of the highest dose group only a trend towards increased albumin concentrations was seen. Furthermore, a statistically significant decrease in triglycerides was found in the high dose males (-43%); in the sera of the females statistically significantly increased creatinine concentrations (+9%) and decreased glucose levels (-12.5%) were detected. After 3 month, alkaline phosphatase activities were statistically significantly increased in the sera of the high dose males and females. Moreover, a marked increase in cyanide-insensitive palmitoyl-CoA-oxidation was detected in the liver of the high dose animals of either sex (+824% in male, +680% in females). No other statistically or biologically relevant changes were noted.
- 2500 ppm: statistically significant increases in magnesium (9%) and liver cyanide-insensitive palmitoyl-Coenzyme A-oxidation (47%) in females and a statistically significant increase in albumin (6%) in males. No other statistically or biologically relevant changes were noted.
- 500 ppm: no statistically or biologically relevant changes
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 15000 ppm: Males and females of the highest dose group produced slightly greater volumes of urine than the corresponding controls. No other treatment-related changes were seen in the urinalytical parameters examined.
- 2500 ppm: no statistically or biologically relevant changes
- 500 ppm: no statistically or biologically relevant changes
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 15000 ppm: statistically significant increases in absolute (51.1% M, 59.4% F) and relative (64.2% M, 73.6% F) liver weights; a statistically significant decrease in absolute adrenal weight (15.5%) was observed in males, along with increases in relative kidney (14% M, 10% F) and relative brain (10% M, 8% F) weights
- 2500 ppm: increase in absolute liver weight in females and relative liver weight in males
- 500 ppm: no treatment-related weight changes
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 15000 ppm: liver cell hypertrophy due to peroxisome proliferation in both sexes, increase of basophilic (thyreotrophic cells in the anterior part of the pituitary gland in male rats (8/10), hypertrophy of the follicular epithelium of the thyroid glands in both sexes
- 2500 ppm: liver cell hypertrophy in one male, increase of basophilic (thyreotrophic cells in the anterior part of the pituitary gland in male rats
(3/10), hypertrophy of the follicular epithelium of the thyroid glands in 8 males and 4 females
- 500 ppm: no treatment-related changes
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were some additional statistically significant inter-group differences in the results of clinical pathology testing. These deviations are marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Effect levels

open allclose all
Key result
Dose descriptor:
other: NOAEL disregarding peroxisomal proliferation
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
other: NOAEL disregarding peroxisomal proliferation
Effect level:
196 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver weight (peroxisomal proliferation)
Key result
Dose descriptor:
NOAEL
Effect level:
39 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver weight (peroxisomal proliferation)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mean body weight (g):

Day 0 ppm 500 ppm 2500 ppm 15000 ppm
Males 7 183.5 185.2 183.2 185.6
28 336.6 337 338.7 327.2
56 412.5 413.7 410.4 388.8
91 447 458.3 449.7 420.7
Females 0 148.7 149.9 151.9 147.3
28 212.4 218.6 222.5 209
56 249.6 254.2 258.2 236.9
91 269.5 273.5 283.8 248.0*

*p<0.05 (Dunnett's tests and ANOVA)

Mean food consumption (g):

Day 0 ppm 500 ppm 2500 pppm 15000 ppm
Males 7 26.4 25.7 25.9 21.6
28 27.4 27.5 27.1 29.2
56 26.6 26.8 26.1 28
91 23 23.3 22.5 24.1
Females 0 19.6 19.6 20.5 17.6
28 19.3 19.9 20.4 20.7
56 19.6 19.5 19.6 19.9
91 23 23.3 22.5 24.1

Clinical chemistry, hematology and urinalysis:

0 ppm 500 ppm 2500 pppm 15000 ppm
Males HGB (mM/L) 9.6 9.6 9.8 9.1**
HCT (L/L) 0.451 0.452 0.464 0.428*
MCH (FM) 1.11 1.09 1.09 1.08
PLT (Giga/l) 728 763 770 877**
ALP (Mykat/L) 5.42 5.47 5.52 10.28**
Pal CoA (Mu/mg P) 6.03 6 9.07 55.75**
Cl (mM/L) 104.7 103.9 103.4 102.0*
Crea (MyM/L) 55 53.2 55.9 54.5
Gluc (mM/L) 7.48 7.79 7.91 7.78
Alb (G/L) 33.4 33.38 35.41* 37.52**
Trig (G/L) 2.75 3.19 2.25 1.56**
Urine ml 4.7 5 4.2 7.2
Females HGB (mM/L) 9.6 9.7 9.5 9.1**
HCT (L/L) 0.436 0.44 0.439 0.424
MCH (FM) 1.13 1.15 1.14 1.10*
PLT (Giga/l) 794 759 692 805
ALP (Mykat/L) 4.04 4.32 4.87 5.10*
Pal CoA (Mu/mg P) 5.07 5.8 7.46* 39.71**
Cl (mM/L) 106.3 106 104.9 102.8**
Crea (MyM/L) 54.2 56.2 57.9 59.1*
Gluc (mM/L) 8.38 7.92 8.29 7.33*
Alb (G/L) 36.07 37.28 37.2 38.93
Trig (G/L) 1.58 1.9 2.71 1.23
Urine ml 3.4 2.6 3.6 4.8

*p<0.05; **p<0.01 (Dunnett's test and ANOVA)

Liver weight:

0 ppm 500 ppm 2500 pppm 15000 ppm
Males Absolute (g) 13.01 14.29 14.76 19.66**
Relative (%) 3.08 3.29 3.47 5.05
Females Absolute (g) 7.76 7.97 9.28* 12.38**
Relative (%) 3.1 3.16 3.47 5.39**

*p<0.05; **p<0.01 (Dunnett's test and ANOVA)

Histopathological incidences:

0 ppm 500 ppm 2500 pppm 15000 ppm
Males Liver: diffuse hypertrophy 0/10 0/10 1/10 10/10
Pituitary: increased basophilic cells 0/10 0/10 3/10 8/10
Thyroid: hypertrophy 1/10 2/10 8/10 10/10
Females Liver diffuse hypertrophy 0/10 0/10 0/10 10/10
Pituitary: increased basophilic cells 0/10 0/10 0/10 0/10
Thyroid: hypertrophy 0/10 0/10 4/10 10/10

Applicant's summary and conclusion