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Diss Factsheets

Administrative data

Description of key information

Repeated dose toxicity:
- oral: NOAEL 1 = 40 mg/kg bw/d  (OECD 408) for liver effects - indicative for peroxisomal proliferation
- oral: NOAEL 2 =  196 mg/kg bw/d (OECD 408) for hematological effects relevant for human hazard
- by inhalation: NOAEC systemic: 250 mg/m3 for effects in clinical chemistry parameter
- by inhalation: NOAEC local: 50 mg/m3 for several lesions in lung and nasal cavity

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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
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
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
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.
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.
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)
Critical effects observed:
not specified

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
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
196 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two feeding studies according to respective guidelines are available and considered sufficient for the evaluation of oral toxicity of the test item after repeated administration.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-05 to 2012-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
5-day inhalation study according to GLP and OECD/EU guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
7 September 2009
Deviations:
yes
Remarks:
only 5 day exposure
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
only 5 day exposure
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material: Palatinol 10-P
- Analytical purity: 99.4 corr. area-%
- Batch No.: B4504 v. 2.8.2012
- Expiry date: Not reported
- Appearance: Colourless, liquid
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; SandhoferWeg 7, 97633 Sulzfeld
- Age at study initiation: about 8 weeks
- Fasting period before study: no data
- Housing: Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- No. of animals per cage: up to 5 animals
- Diet: ad libitum, not during exposure
- Water: ad libitum, not during exposure
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30-70 %
- Air changes: 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12 from 06.00 a.m. - 06.00 p.m.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.4 - <= 2.2 µm
Remarks on MMAD:
MMAD / GSD:
50 mg/m3: 1.4 / 2.8 µm and 1.4 / 2.6 µm
250 mg/m3: 1.4 / 2.8 µm and 1.4 / 2.9 µm
1000 mg/m3: 2.2 / 2.1 µm and 1.6 / 2.6 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nümbrecht, Germany), Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)
- Exposure chamber volume: 90 L
- Method of holding animals in test chamber: The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamberto inhale the atmosphere.
- System of generating aerosols: For each concentration, a respective constant amount of the test substance was supplied to a two-component atomizer by means of a metering pump and sprayed with compressed air into a glass mixing stage. The so generated aerosol was mixed with conditioned air and passed into the inhalation system.
- Method of particle size determination: Gravimetrical Marple 298 cascade impactor measurements
- Treatment of exhaust air: The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air is lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the breathing zones ofthe animals.
- Temperature, humidity, pressure in air chamber:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated.

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentration was determined by gas chromatography of absorption samples.
- Samples taken from breathing zone: yes
- Sampling frequency: three samples per concentration and exposure

TEST ATMOSPHERE
- Particle size distribution:
Particle Size determinations of the test atmospheres was made with the TSI APS 3321. Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) was obtained directly by the piece of equipment used (TSI APS 3321).
- Frequency: two times during the exposure period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration was determined by gas chromatography of absorption samples.
Duration of treatment / exposure:
5 consecutive days
Frequency of treatment:
Once a day for 6 hours
Dose / conc.:
50 mg/m³ air
Dose / conc.:
250 mg/m³ air
Dose / conc.:
1 000 mg/m³ air
No. of animals per sex per dose:
10 male animals per concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of observation period following administration: one day
- Fasting period: animals were fasted over night before blood sampling
- Necropsy of survivors performed: yes
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until the day prior to gross necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 were examined.

ACUTE PHASE PROTEINS
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.3 were examined.

PALMITOYL CoA OXIDATION
- Tissue: liver
- How many animals: all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table No. 4
HISTOPATHOLOGY: Yes, see table No. 5
Other examinations:
not applicable
Statistics:
Body weights, body weight change: Dunnett's test
Clinical pathology examinations: Kruskal-Wallis test and Wilcoxon test
Weight of anesthetized animals and absolute and relative organ weight: Kruskal-Wallis test and Wilcoxon test
Clinical signs:
no effects observed
Description (incidence and severity):
During the whole study period, the animals showed no clinical signs and findings different from normal.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of all test groups were not statistically significant different from the control groups. However, the mean body weight change of test group 3 (1000 mg/m3) was slightly, though significantly lower than in the control.

The following significant body weight changes were observed:
- Test group 1 (50 mg/m³), from study day 0 to 4 (-1.9 g, p< 0.05)
- Test group 3 (1000 mg/m³) from study day 0 to 4 (-5.1 g, p< 0.01)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In rat of test group 3 (1000 mg/m³), relative eosinophil counts were higher compared to controls, but the values were within historical control ranges (relative eosinophil counts 1.12.8 %). Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of test group 3 (1000 mg/m3), globulin and total protein levels were decreased, whereas cholesterol levels were increased. These effects were considered test-substance related and adverse.

In the mentioned test group, total bilirubin levels were decreased. In the absence of an anemia this decrease was most probably due to an increased conjugation rate of bilirubin coupled with a greater excretion of bilirubin via the bile. This effect was regarded as treatment-related, but not adverse.

In the same test group calcium levels were lower compared to controls, but the mean was within the historical control range. In rats of test group 2 (300 mg/m3) potassium levels were low, but this decrease was not dose-dependent. Therefore, the lower calcium levels in rats of test group 3 as well as the lower potassium levels in test group 2 were regarded as incidental and not treatment-related.

In males of test groups 2 and 3 (250 and 1000 mg/m3), cyanide sensitive Palmitoyl CoA oxidation (Pal CoA) in liver tissue was higher compared to controls. However the mean in test group 2 was within the historical control range (4.30 – 7.69 mU/mg protein) and it was not accompanied with an alteration of any other liver parameter in serum. Therefore, the PalCoA in test group 2 (250 mg/m3) was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
A minimal to moderate granulomatous inflammation was observed in the lungs of two males in test group 2 (250 mg/m3) and in nine males of test group 3 (1000 mg/m3). showed very few to multiple granulomatous foci, often nearby the bronchi, bronchioli and terminal bronchioli. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells.

A minimal to moderate lympho-reticulocellular hyperplasia of the tracheobronchial and mediastinal lymph nodes was noted in six males of test group 3 (1000 mg/m³), that was regarded to be treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/m³:
- Increased absolute and relative weight of the lungs (+37%).
- Increased absolute and relative weight of the liver (+30%).

250 mg/m³:
- Increased absolute weight of the lungs (+9%).
- Increased relative weight of the lungs (+7%).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions reported in the following organs (control, group 1, group 2, group 3):
- Epididymides (3, 1, 1, 3)
- Liver (0, 0, 0, 1)
- Mediastinal lymph node (0, 0, 0, 1)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nasal cavity:
Single or few mucous cells occurred in the maxillary sinus (nasal cavity, level III) in all treated males. In the septal window (nasal cavity, level III), a minimal increased number of mucous cells was observed in four (out of 10) males in test group 3 (1000 mg/m³). Seven males in test group 3 (1000 mg/m³) showed a minimal increased number or hypertrophy of mucous cells in the nasopharyngeal duct (nasal cavity, level IV). The epithelium in the respective areas was regular and signs of inflammation or degenerative changes were completely missing in the whole nasal cavity. The occurrence, increase in number, and hypertrophy of mucous cells were regarded to be treatment-related.

Trachea:
A minimal to moderate multifocal hypertrophy or hyperplasia of the respiratory epithelium including mucous cells was seen in the trachea of eight males in test group 3 (1000 mg/m3). The occurrence of hypertrophy/ hyperplasia of the respiratory epithelium including mucous cells was considered to be treatment-related.

Lungs:
A minimal to moderate granulomatous inflammation was observed in the lungs of two males in test group 2 (250 mg/m3) and in nine males of test group 3 (1000 mg/m3). These males showed very few to multiple granulomatous foci, often nearby the bronchi, bronchioli and terminal bronchioli. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells. Partly, the alveolar walls were lined by large cuboidal cells with vacuolated cytoplasm and prominent nuclei (type II pneumocyte hyperplasia). Some of these males showed in addition areas with muco-purulent features.

Alveolar histiocytosis, characterized by focal accumulation of some histiocytes (alveolar macrophages) in few alveoli, frequently occurs spontaneously. This type of alveolar histiocytosis was observed in four control males as well as in three males of test group 1 (50 mg/m³). In these males, one or few foci of alveolar histiocytosis occurred in one or two lobes of the lungs (minimal, grade 1) and is therefore interpreted as spontaneous lesion. However, in test groups 2 (250 mg/m³) and 3 (1000 mg/m³) all males were affected and the alveolar histiocytosis was often seen in the same areas as the inflammation, distributed over the whole lung. The occurrence of granulomatous inflammation and the increased incidence and severity of alveolar histiocytosis in males of test groups 2 (250 mg/m³) and 3 (1000 mg/m³) correlated very well with the increased lung weights in these test groups and was related to treatment.

In the epithelium of bronchi, bronchioles, and/ or terminal bronchioles, a concentration-related minimal to moderate hypertrophy/ hyperplasia, partly accompanied by an increase of mucous cells, was observed in all males of test groups 2 (250 mg/m³) and 3 (1000 mg/m3). The occurrence of the epithelial hypertrophy/ hyperplasia in bronchi, bronchioles, and/ or terminal bronchioles was regarded to be treatment-related.

Liver:
A slight diffuse hepatocellular hypertrophy was observed in all males of test group 3 (1000 mg/m³). The diffuse hepatocellular hypertrophy correlated with the increased absolute and relative liver weights in this test group and was regarded to be treatment-related.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEC
Remarks:
(systemic toxicity)
Effect level:
250 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEC
Remarks:
(local effects)
Effect level:
50 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Respiratory irritation
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
250 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
A 5-day inhalation toxicity study conducted according to GLP regulations is available and considered sufficient for evaluation of systemic toxicity after repeated administration.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-05 to 2012-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
5-day inhalation study according to GLP and OECD/EU guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
7 September 2009
Deviations:
yes
Remarks:
only 5 day exposure
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
only 5 day exposure
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material: Palatinol 10-P
- Analytical purity: 99.4 corr. area-%
- Batch No.: B4504 v. 2.8.2012
- Expiry date: Not reported
- Appearance: Colourless, liquid
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; SandhoferWeg 7, 97633 Sulzfeld
- Age at study initiation: about 8 weeks
- Fasting period before study: no data
- Housing: Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- No. of animals per cage: up to 5 animals
- Diet: ad libitum, not during exposure
- Water: ad libitum, not during exposure
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30-70 %
- Air changes: 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12 from 06.00 a.m. - 06.00 p.m.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.4 - <= 2.2 µm
Remarks on MMAD:
MMAD / GSD:
50 mg/m3: 1.4 / 2.8 µm and 1.4 / 2.6 µm
250 mg/m3: 1.4 / 2.8 µm and 1.4 / 2.9 µm
1000 mg/m3: 2.2 / 2.1 µm and 1.6 / 2.6 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nümbrecht, Germany), Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)
- Exposure chamber volume: 90 L
- Method of holding animals in test chamber: The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamberto inhale the atmosphere.
- System of generating aerosols: For each concentration, a respective constant amount of the test substance was supplied to a two-component atomizer by means of a metering pump and sprayed with compressed air into a glass mixing stage. The so generated aerosol was mixed with conditioned air and passed into the inhalation system.
- Method of particle size determination: Gravimetrical Marple 298 cascade impactor measurements
- Treatment of exhaust air: The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air is lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the breathing zones ofthe animals.
- Temperature, humidity, pressure in air chamber:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated.

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentration was determined by gas chromatography of absorption samples.
- Samples taken from breathing zone: yes
- Sampling frequency: three samples per concentration and exposure

TEST ATMOSPHERE
- Particle size distribution:
Particle Size determinations of the test atmospheres was made with the TSI APS 3321. Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) was obtained directly by the piece of equipment used (TSI APS 3321).
- Frequency: two times during the exposure period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration was determined by gas chromatography of absorption samples.
Duration of treatment / exposure:
5 consecutive days
Frequency of treatment:
Once a day for 6 hours
Dose / conc.:
50 mg/m³ air
Dose / conc.:
250 mg/m³ air
Dose / conc.:
1 000 mg/m³ air
No. of animals per sex per dose:
10 male animals per concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of observation period following administration: one day
- Fasting period: animals were fasted over night before blood sampling
- Necropsy of survivors performed: yes
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until the day prior to gross necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 were examined.

ACUTE PHASE PROTEINS
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.3 were examined.

PALMITOYL CoA OXIDATION
- Tissue: liver
- How many animals: all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table No. 4
HISTOPATHOLOGY: Yes, see table No. 5
Other examinations:
not applicable
Statistics:
Body weights, body weight change: Dunnett's test
Clinical pathology examinations: Kruskal-Wallis test and Wilcoxon test
Weight of anesthetized animals and absolute and relative organ weight: Kruskal-Wallis test and Wilcoxon test
Clinical signs:
no effects observed
Description (incidence and severity):
During the whole study period, the animals showed no clinical signs and findings different from normal.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of all test groups were not statistically significant different from the control groups. However, the mean body weight change of test group 3 (1000 mg/m3) was slightly, though significantly lower than in the control.

The following significant body weight changes were observed:
- Test group 1 (50 mg/m³), from study day 0 to 4 (-1.9 g, p< 0.05)
- Test group 3 (1000 mg/m³) from study day 0 to 4 (-5.1 g, p< 0.01)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In rat of test group 3 (1000 mg/m³), relative eosinophil counts were higher compared to controls, but the values were within historical control ranges (relative eosinophil counts 1.12.8 %). Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of test group 3 (1000 mg/m3), globulin and total protein levels were decreased, whereas cholesterol levels were increased. These effects were considered test-substance related and adverse.

In the mentioned test group, total bilirubin levels were decreased. In the absence of an anemia this decrease was most probably due to an increased conjugation rate of bilirubin coupled with a greater excretion of bilirubin via the bile. This effect was regarded as treatment-related, but not adverse.

In the same test group calcium levels were lower compared to controls, but the mean was within the historical control range. In rats of test group 2 (300 mg/m3) potassium levels were low, but this decrease was not dose-dependent. Therefore, the lower calcium levels in rats of test group 3 as well as the lower potassium levels in test group 2 were regarded as incidental and not treatment-related.

In males of test groups 2 and 3 (250 and 1000 mg/m3), cyanide sensitive Palmitoyl CoA oxidation (Pal CoA) in liver tissue was higher compared to controls. However the mean in test group 2 was within the historical control range (4.30 – 7.69 mU/mg protein) and it was not accompanied with an alteration of any other liver parameter in serum. Therefore, the PalCoA in test group 2 (250 mg/m3) was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
A minimal to moderate granulomatous inflammation was observed in the lungs of two males in test group 2 (250 mg/m3) and in nine males of test group 3 (1000 mg/m3). showed very few to multiple granulomatous foci, often nearby the bronchi, bronchioli and terminal bronchioli. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells.

A minimal to moderate lympho-reticulocellular hyperplasia of the tracheobronchial and mediastinal lymph nodes was noted in six males of test group 3 (1000 mg/m³), that was regarded to be treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/m³:
- Increased absolute and relative weight of the lungs (+37%).
- Increased absolute and relative weight of the liver (+30%).

250 mg/m³:
- Increased absolute weight of the lungs (+9%).
- Increased relative weight of the lungs (+7%).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions reported in the following organs (control, group 1, group 2, group 3):
- Epididymides (3, 1, 1, 3)
- Liver (0, 0, 0, 1)
- Mediastinal lymph node (0, 0, 0, 1)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nasal cavity:
Single or few mucous cells occurred in the maxillary sinus (nasal cavity, level III) in all treated males. In the septal window (nasal cavity, level III), a minimal increased number of mucous cells was observed in four (out of 10) males in test group 3 (1000 mg/m³). Seven males in test group 3 (1000 mg/m³) showed a minimal increased number or hypertrophy of mucous cells in the nasopharyngeal duct (nasal cavity, level IV). The epithelium in the respective areas was regular and signs of inflammation or degenerative changes were completely missing in the whole nasal cavity. The occurrence, increase in number, and hypertrophy of mucous cells were regarded to be treatment-related.

Trachea:
A minimal to moderate multifocal hypertrophy or hyperplasia of the respiratory epithelium including mucous cells was seen in the trachea of eight males in test group 3 (1000 mg/m3). The occurrence of hypertrophy/ hyperplasia of the respiratory epithelium including mucous cells was considered to be treatment-related.

Lungs:
A minimal to moderate granulomatous inflammation was observed in the lungs of two males in test group 2 (250 mg/m3) and in nine males of test group 3 (1000 mg/m3). These males showed very few to multiple granulomatous foci, often nearby the bronchi, bronchioli and terminal bronchioli. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells. Partly, the alveolar walls were lined by large cuboidal cells with vacuolated cytoplasm and prominent nuclei (type II pneumocyte hyperplasia). Some of these males showed in addition areas with muco-purulent features.

Alveolar histiocytosis, characterized by focal accumulation of some histiocytes (alveolar macrophages) in few alveoli, frequently occurs spontaneously. This type of alveolar histiocytosis was observed in four control males as well as in three males of test group 1 (50 mg/m³). In these males, one or few foci of alveolar histiocytosis occurred in one or two lobes of the lungs (minimal, grade 1) and is therefore interpreted as spontaneous lesion. However, in test groups 2 (250 mg/m³) and 3 (1000 mg/m³) all males were affected and the alveolar histiocytosis was often seen in the same areas as the inflammation, distributed over the whole lung. The occurrence of granulomatous inflammation and the increased incidence and severity of alveolar histiocytosis in males of test groups 2 (250 mg/m³) and 3 (1000 mg/m³) correlated very well with the increased lung weights in these test groups and was related to treatment.

In the epithelium of bronchi, bronchioles, and/ or terminal bronchioles, a concentration-related minimal to moderate hypertrophy/ hyperplasia, partly accompanied by an increase of mucous cells, was observed in all males of test groups 2 (250 mg/m³) and 3 (1000 mg/m3). The occurrence of the epithelial hypertrophy/ hyperplasia in bronchi, bronchioles, and/ or terminal bronchioles was regarded to be treatment-related.

Liver:
A slight diffuse hepatocellular hypertrophy was observed in all males of test group 3 (1000 mg/m³). The diffuse hepatocellular hypertrophy correlated with the increased absolute and relative liver weights in this test group and was regarded to be treatment-related.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEC
Remarks:
(systemic toxicity)
Effect level:
250 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEC
Remarks:
(local effects)
Effect level:
50 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Respiratory irritation
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
50 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
A 5-day inhalation toxicity study conducted according to GLP regulations is available and considered sufficient for evaluation of local toxicity after repeated administration.

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

Oral

Repeated dose toxicity of di-(2-propylheptyl) phthalate was evaluated in a study performed under GLP according to guidelines OECD 408 and EU Method B.26 (BASF, 1995). Groups of 10 male and 10 female Wistar rats received di-(2-propylheptyl) phthalate dietary concentrations of 0, 500, 2500, and 15000 ppm in the diet over a period of 3 months. At all doses, no deaths occurred, no clinical findings were found and ophthalmoscopy revealed no treatment-related changes. While there were slightly decreased body weights and retarded body weight gain in male and female rats at 15000 ppm, no effect on food consumption was noted. No effects were found at lower dietary levels. Clinical pathology including hematology and urinalysis showed an increase in alkaline phosphatase, cyanide-insensitive palmitoyl-CoA-oxidation, albumin and urinary volume in both sexes of the 15000 ppm dose groups. Also at the high concentration, findings included an increase in platelets in males and creatinine in females, a decrease in hemoglobin, mean corpuscular hemoglobin and chloride in both sexes, decreases in hematocrit and triglycerides in males and decrease in glucose in females. At 2500 ppm only an increase in cyanide-insensitive PCoA-oxidation in females was noted and 500 ppm evoked no treatment-related changes. Although the gross pathology revealed no treatment-related findings at any dose level, the liver weights (absolute and/or relative) were increased at 2500 and 15000 ppm but not at 500 ppm, which corresponds to the histopathologically observed liver cell hypertrophy at 2500 and 15000 ppm. The increased PCoA, liver weight, and liver hypertrophy are indicative of peroxisomal proliferation in the liver. While at 500 ppm no treatment-related changes were recorded by histopathology, further minor findings in the anterior part of the pituitary gland in male rats and in the thyroid glands were observed in both sexes at 15000 ppm and 2500 ppm. Virtually no effects were noted at the organs relevant for reproductive function.

Changes in the thyroid and pituitary gland are secondary to the changes observed in the liver: peroxisome proliferation and parallel induced xenobiotic metabolism, which results in an increased elimination of T3/T4 from rat serum by increased glucuronidation. This mode of action has been extensively studied with Perchlorate and Phenobarbital for example (Meek et al., 2003 Critical Reviews in Toxicology, 33(6):591–653; Lewandowski et al., 2004 Regulatory Toxicology and Pharmacology 39, 348–362; McClain et al., 1989 Toxicology And Applied Pharmacology 99,216 -228; Fisher et al., 2013 Joural of Environmental Science and Health, Part C, 30:81 -105) and is therefore well understood. It is not relevant to humans as rodents are more sensitive to thyroid effects (summarised in Meek et al., 2003 Critical Reviews in Toxicology). Accordingly Bhat et al. (2014), Regulatory Toxicology and Pharmacology 70, 65 -74, who published the comprehensive oral risk assessment undertaken by the US EPA chaired Health Advisory board of NSF International, reduced the interspecies extrapolation factor for derivation of the oral reference dose (RfD) to 1 applying the US EPA guidance. The lack of human relevance of these thyroid effects is also reflected in the opinion of the German MAK Commission (2015). Furthermore, in humans, activation of peroxisome proliferator-activated receptor α (PPARα) does not lead to increased relative liver weights, oxidative enzyme induction or other responses typically associated with sustained PPARα activation observed in wild-type mice (Corton et al., 2018 Arch Toxicol. 92(1): 83–119). The weight of evidence supports the conclusion that adverse effects related to a PPARα MOA is either “not relevant” or “unlikely to be relevant” in humans (Felter et al., 2018 Regulatory Toxicology and Pharmacology 92, 1 -7).

Thus, the No Observed Adverse Effect Level (NOAEL) was found to be 500 ppm (39 mg/kg bw/d) for peroxisomal proliferation in liver of male and female Wistar rats, but the NOAEL for hazards relevant to human is 2500 ppm (196 mg/kg bw/d) for hematological effects.

 

In another subchronic study reported in letters submitted to the U.S. EPA, 12 male and 12 female Alpk:APfSD rats per dose were administered di-(2-propylheptyl) phthalate in the diet at concentrations of 500, 5000 and 12000 ppm corresponding to 0, 40, 420 or 1,000 mg/kg bw/d for a period of 14 weeks (Union Carbide, 1997, 1998). Two additional groups received the diet containing 0 or 12000 ppm and were held for 4 weeks without further treatment. As results, decreases in body weight gain and food consumption were observed dose dependent at 5000 ppm in males and at 12000 ppm in both sexes. An increased liver weight with concurrent increases in peroxisome enzyme levels were noted in all treatment groups. The observed hematological changes consisted of decreased hemoglobin levels. Although the sperm velocity was affected at 5000 and 12000 ppm, but no other of the selected sperm parameters as viability, total sperm, static count, percent motile, motile count, total sperm concentration and sperm/g tissue were affected. Moreover, there were no effects at any dose level on the development stages in epididymal sperm. Histology revealed lesions in the zona glomerulosa of the adrenal glands (only minimal at 500 ppm, slight at 5000 ppm and moderate at 12000 ppm) and findings indicative of peroxisome proliferation were noted in the livers at 5000 and 12000 ppm. Thus, based on this study the No Observed Adverse Effect Level (NOAEL) was 500 ppm (40 mg/kg/day) for effects on hematological parameters and liver.

 

Conclusion

Since no adverse effects relevant for human health were observed in the guideline compliant 90-day feeding study (BASF 1995) up to a dose level of 2500 ppm the NOAEL of 500 ppm assessed in a second non-guideline compliant subchronic feeding study (Union Carbide, 1997, 1998) was considered to overestimate human health hazard of the test substance. Therefore the NOAEL of 2500 ppm (196 mg/kg bw/day) was considered to be the relevant No Observed Adverse Effect Level for human health.

 

By inhalation

In an inhalation study (BASF SE, 2013) ten male Wistar rats per test group were head-nose exposed to respirable liquid aerosol for 6 hours per day at target concentrations of 50, 250 and 1000 mg/m3. The dew point, which designates the transition from the vapour phase to an aerosol, is 0.0007 mg/m3 in the case of di(2-propylheptyl) phthalate and thus far below the lowest test concentration. Aerosols were therefore present at all exposure concentrations.

Inhalation exposure to the test substance did not lead to any clinical signs of toxicity or impaired body weight development. Hematology and acute phase proteins in blood did not change toxicologically relevantly when compared with controls. At 1000 mg/m3, globulin and total protein levels in blood were decreased, whereas blood cholesterol was increased. These effects were considered to be substance-related and adverse. Thus, the no observed adverse concentration (NOAEC) for systemic toxicity was 250 mg/m3 for the test substance under the current test condition.

Regarding local effects, the following observations can be seen as sequels of the overloading of the lungs and macrophages with the oily, insoluble material: foam cell infiltrations in the terminal bronchioles and slight to moderate alveolar histiocytosis, combined with a slight to moderate lymphoreticular reaction in the mediastinal lymph nodes. In addition, there was slight to moderate hypertrophy in the terminal bronchial epithelium and in the trachea. These effects, were observed dose dependently at 1000 and 250 mg/m3, but not at 50 mg/m3. Therefore, the local no observed adverse effect concentration (NOAEC) for the test substance was 50 mg/m3 under the current study conditions.

Independent of this, exposure to the test substance aerosol produced goblet cell hyperplasia, the increased presence of goblet cells in plane of section III of the nasal conchae, in all concentration groups and in all exposed animals, but not in the controls. As summarised by MAK (2015), the increased presence of goblet cells is a physiological reaction to an overload phenomenon as a reaction to the impaction of the oily particles and does not represent a substance-specific adverse effect, but is an adaptive process, resulting in proliferative effects. The increased differentiation of stem cells in the basal epithelial layer to form goblet cells (instead of cylinder cells) is a physiological process that serves to produce more mucus as a reaction to the aerosol impaction, which is reversible over a long period of time (Renne et al. 2009, Toxicol Pathol 37: 5S–73S; Rogers 1994, Eur Respir J 7: 1690–1706). However, it is not known how such findings develop over longer exposure periods.

Justification for classification or non-classification

In the 90 -day study in rats, a human-relevant LOAEL above 1000 mg/kg bw/d was identified. Due to the very low vapour pressure of the test substance, inhalation of vapours is not considered relevant. Repeated inhalaltion of aerosols for 5 days led to changes in blood chemistry. Because of the nature of the effects and the fact that they only occured at the highest dose of 1000 mg/m³/d (>1000mg/kg bw/d after oral exposure), no classification according to GHS criteria is required.