Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (GLP, QUA)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
For clinical and neurofunctional examinations and pathology OECD Guideline No. 407 adopted 27 .07.95 was followed additionally
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): di-n-butylphthalate (test substance number 98/486-1)
- Physical state: liquid / colorless, homogeneous
- Analytical purity: 99.8% prior to the study, 99.8% after the in-life phase of the study (reanalysis)
- Lot/batch No.: P.151 A/98
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar rats (Chbb:THOM (SPF)) were supplied by Boehringer Ingelheim Pharma KG
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: male animals 281.2 (277.9 - 284.7) g; female animals 195.5 (192.5 - 197.1) g
- Fasting period before study: none; the animals did not have access to water or feed during the exposure
- Housing: during the period when the rats were not exposed they were housed singly in makrolon-wire cages (type MD III, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2)). Underneath the cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding, supplied by SSNIFF, Soest, FRG). The motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm2) and bedding.
- Diet (e.g. ad libitum): the animals were maintained on rat/mouse/hamster laboratory diet, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: 9 to 15 days (see Table 1); in order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on 2 days before start of exposure (preflow period).

ENVIRONMENTAL CONDITIONS
The animals were kept in fully air-conditioned rooms
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Mean MMADs between 1.5 and 1.9 µm with GSDs around 2 were measured in the test groups (MMAD µm / GSD of 1.9 /1.9, 1.6 /1.9, 1.5 /1.9 and 1.5 /2.0, respectively for the 0, 1, 5, 50 and 500 mg/m3 dose groups). The mean mass fractions of particles below 3 µm aerodynamic size ranged between 78 and 84%. Thus the aerosols were highly respirable for rats.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the inhalation atmosphere was maintained inside aerodynamic exposure systems (INA
60, volume V~ 90 l, BASF AG) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets (head-nose exposure system s)
- Method of holding animals in test chamber: the rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol
- Source and rate of air: see Table 2
- Method of conditioning air: conditioned supply air is filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C. Compressed air is filtered air pressurized to about 6 bar
- System of generating particulates/aerosols: for each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air in the mixing stage, mixed with conditioned dilution air and passed via the cyclonic separator and the dilution tube into the inhalation system. The desired concentrations in test groups 1-4 were achieved by substituting appropriate amounts of aerosol (exhaust air 1) by conditioned supply air (supply air 3)
- Temperature, humidity, pressure in air chamber:
- Air flow rate: see Table 2
- Air change rate: see Table 2
- Method of particle size determination: the calculation of the particle size distribution was carried out on the basis of mathematical methods for evaluating particle measurements

TEST ATMOSPHERE
- Brief description of analytical method used: sampling (Sampling velocity: 1.25 m/s; flow rate of sampling: 1 l/min, test group 0; flow rate of sampling: 3 l/min, test groups 1-4; sample volumes:100 l for test group 0 & 1, 20 l for test group 2 and 5 l for test group 3 &4). The sample volumes were adjusted to achieve comparable amounts of the test substance in the samples of the different test groups. The desired amounts refer to the calibration of the analytical method (sampling site: immediately adjacent to the animals' noses; sampling frequency: as a rule, 2 samples per concentration and exposure). After the final sampling of each exposure day the content of the last absorption vessel was transferred to a 50 ml graduated flask and analyzed separately, to check the absorbing efficiency of sampling. Samples were analyzed by gas chromatograph.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control. Daily means were calculated based on, as a rule, 2 measured samples per concentration and one measured sample in the control group. From these daily mean values, mean concentrations and standard deviations for the entire study were derived. The constancy of concentrations in each inhalation system (except test group 0) was continuously monitored by means of a scattered light photometers. The aerosol generation effectiveness was as expected. For aerosol inhalation studies the effectiveness usually lies in the range between 10 and 30% (here 12.2, 9.7, 14.8 and 22.6%, respectively for the 1, 5, 50 and 500 mg/m3 dose groups). Loss of concentration occurs in the devices for particle size selection and in the tubing. Real time surveillance of the inhalation atmospheres with scattered light photometers proved the constancy of each concentration throughout the daily exposures.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours on each workday over a time period of 28 days (exposure period). The number of exposures was 20 .
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1.2±0.29, 5.6±0.87, 49.3±4.23 and 509±23.1 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 1, 5, 50 and 500 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 9.7, 57.4, 333.4 and 2253 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
other: concurrent control group was exposed to clean air
Details on study design:
- Dose selection rationale: in the di-n-butyl Phthalate Risk Assessment (Draft of Sept, 1997), it is shown that the acute inhalation toxicity of di-n-butyl Phthalate is low (LC50 rat 4h >15 mg/l). Additionally, irritation of the respiratory tract was observed in cats and mice at exposure levels of 1 or 0.25 mg/l respectively.
Three repeated exposure inhalation studies with limited scopes of examination describe changes in cytochrome P450 in the lung, leukocyte counts, blood levels of liver enzymes, blood protein fractions and other clinico-pathology parameters as well as changes in organ weights.
Clear effect levels were reported to be 80 mg/m3 for 5-day exposure and 50 mg/m3 for 6-month exposure. A Low Observed Adverse Effect Concentration (LOAEC) in a 90-day study was 1 mg/m3. The Risk Assessment concludes 0.1 mg/m3 to represent a NOAEC although the validity of all available studies is at least doubtful.
The database on oral studies in rats allows to establish a robust systemic NOAEL for subchronic oral exposures at 150 mg/kg. The oral NOEL for peroxisome proliferation (which is not taken as relevant for the risk assessment in humans) is about 20 mg/kg. Considering that di-n-butyl Phthalate is readily absorbed in the digestive system and assuming that this will be the case after inhalation, too, concentrations corresponding to the oral NO(A)ELs can be calculated. Exposure of rats for 6 hours daily to concentrations of 625 or 80 mg/m3 would lead to the respective daily doses.
Taking into account the above mentioned considerations the target concentrations are set to: 0, 1, 5, 50 and 500 mg/m3.
If there are appreciable differences in systemic toxicity depending on route of exposure the high concentration should exert some adverse effects. Distinct local effects in the respiratory tract are expected. Exposure to the high intermediate concentration should be the systemic NOAEC but may still cause local effects in the respiratory tract. Either the low intermediate or the low concentration is expected to represent the NO(A)EC under the condition of a 28-day inhalation study.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: an observation of the general state of health of the animals as well as a check for dead or moribund animals was performed twice a day on working days or once a day on weekends or public holidays, respectively. Open field observations were carried out on study days -1, 7, 14 and 21. The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes.
- Cage side observations included: behaviour when removed from cage, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupillary size

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical examinations of the test animals were carried out on workdays at least 3 times on exposure days and, as a rule, once during the preflow period and the post-exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was determined at the start of the preflow, at the start of the exposure period and then, as a rule, once a week. As a rule, the animals were weighed at the same time of the day.
Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the start of exposure. Group means were derived from the individual differences.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was determined weekly and calculated as mean water consumption in grams per animal and day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the exposure period (day -1 (females) or -2 (males)) the eyes of all animals, and at the end of the study (day 26) the eyes of the animals of test group 0 (control group) and of test group 4 (high concentration) were examined with an ophthalmoscope for any changes in the refracting media.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29, before necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters examined: leukocytes, erythrocytes, hemoglobin, haematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count; furthermore, differential blood smears were prepared and stained according to Wright without being evaluated .

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29, before necropsy
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, 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: day 26
- 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: Yes
- Time schedule for examinations: a functional observation battery (FOB) was carried out after the end of the exposure period on study day 28. Motor activity (MA) was measured on the same day as FOB was performed.
- Dose groups that were examined:
- Battery of functions tested: FOB (functional observation battery; home cage observations, open field observations, sensorimotor tests/reflexes); motor activity measurements.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; the animals were sacrificed by exsanguination via the abdominal aorta and vena cava under anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The following weight determinations were performed from all animals with scheduled kill: anesthetized animals, lungs, liver, kidneys, adrenal glands, testes, epididymides, ovaries, brain, thymus, heart, spleen
HISTOPATHOLOGY: Yes; the following organs or tissues were fixed in 4% neutral buffered formaldehyde or in Bouin's solution: gross lesions, brain, pituitary gland, thyroid glands with parathyroid, glands, thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenal glands, testes/ovaries (Bouin's solution), uterus/vagina, accessory genital organs (epididymides,
prostate gland, seminal vesicles), stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, mandibular, mediastinal and mesenteric lymph nodes, sciatic nerve, bone marrow (femur), eyes, spinal cord (cervical, thoracic and lumbar cord), head, larynx. 2 parts of the liver dedicated for routine investigation were fixed in Carnoy's solution. After the organs were fixed, histotechnic processing and examination by light microscopy were performed.
Other examinations:
not applicable
Statistics:
- Body weight, body weight change: parametric one-way analysis using the F-test (ANOVA) (two- sided). If the resulting p-value was equal or less 0.05, a comparison of each group with the control group using the DUNNETT's test (two-sided) was performed for the hypothesis of equal means.
- Feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two-sided) for the equal medians.
- Clinical pathology parameters, except differential blood count: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two-sided) for the equal medians.
- Urinalysis, except volume, color, turbidity and specific gravity: pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions sciences.
- Weight parameters at necropsy: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- During the preflow period and the post-exposure observation days the animals showed no clinical signs and findings different from normal.
During the exposure period the animals of the control group, low concentration (1 mg/m3), low intermediate concentration (5 mg/m3) and the high intermediate concentration (50 mg/m3) showed no clinical signs and findings different from normal, while the animals of the high concentration (500 mg/m3) showed red crust formation at the snouts after daily exposure in a maximum number of 4 animals and a maximum duration of the symptom from study day 13 to day 27.
- No deaths were recorded throughout the study.

BODY WEIGHT AND WEIGHT GAIN
- The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
- The body weight change was statistically significantly decreased in the female animals: (1) low concentration ( 1 mg/m') on the study day 14 (about 38%); (2) low intermediate concentration (5 mg/m3) on the study day 7 (about 63%) and day 14 (about 42%); (3) high intermediate concentration (50 mg/m') on the study day 7 (about 74%), day 14 (about 62%), day 21 (about 45%) and day 28 (about 34%); (4) high concentration (500 mg/m3) on the study day 7 (about 64%), day 14 (about 46%), day 21 (about 31 %) and day 28 (about 29%).
These findings are considered to be incidental and not substance related, because they only occurred in one sex, did not show a consistent concentration time response relation, were not related to the food consumption data and did not lead to impairment of body weight development.
- There were no significant weight changes in males or females at necropsy.

FOOD CONSUMPTION
The food consumption was statistically significantly decreased in the female animals: (1) low concentration ( 1 mg/m3) on the study day 14 (about 15%) and day 28 (about 10%); (2) low intermediate concentration (5 mg/m3) on the study day 14 (about 11 % ); (3) high intermediate concentration (50 mg/m3) on the study day 14 (about 11 %) and day 28 (about 9%); (4) high concentration (500 mg/m3) on the study day 7 (about 10%), day 14 (about 10%) and day 28 (about 9%).
These findings are considered to be incidental and not substance related, because they only occurred in one sex, did not show a consistent concentration time response relation and did not lead to impairment of body weight development.

FOOD EFFICIENCY
The food efficiency of the animals were not statistically significantly different from the control group 0(except in the female animals of the low intermediate concentration (5 mg/m3), the high intermediate concentration (50 mg/m3) and the high concentration (500 mg/m3) on the study day 7, were it was statistically significantly decreased (about 60-72'%)).
These findings are considered to be incidental and not substance related. They are calculated from body weight and food consumption data displaying changes, which were already identified as incidental because they only occurred in one sex and did not show a consistent concentration time response relation.

WATER CONSUMPTION
The water consumption of the animals were not statistically significantly different from the control group 0(except in the female animals of the low concentration (1 mg/m3) and the low intermediate concentration (5 mg/m3) on the study day 8, where it was statistically significantly decreased (about 17-18%), which are considered incidental, because of the lack of concentration time response relation)

OPHTHALMOSCOPIC EXAMINATION
The ophthalmologic examinations did not show any impairment of the refracting media. Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of the test groups and the control group without any concentration-response relationship. These findings are regularly observed in rats of this strain and age and are therefore not assessed to be abnormal findings.
HAEMATOLOGY
There were no treatment-related changes in the hematology parameters measured.

CLINICAL CHEMISTRY
Compound-related differences in clinical chemistry parameters were not evident at any concentration in either males or females. The only one statistically significant intergroup difference in the results of clinical pathology testing was decrease in sodium in the high dose females). This deviation is marginal and inconsistent, when compared with the other sex. Accordingly, this finding is considered to be of no toxicological significance.

URINALYSIS
No treatment-related changes were found in the urinalyses of either sex.

NEUROBEHAVIOUR
1) Open field observations: the open field observations from the animals did not reveal any abnormalities in the test group.
2) Functional observational battery
- Home cage observations: no abnormalities were detected in any of the test groups when the animals were observed in their home cages.
- Open field observations: no abnormalities were detected in any of the test groups when the animals were observed.
- Sensorimotor tests/Reflexes: minor findings were observed but do not show changes in the substance exposed groups as compared to control.
- Quantitative observations (Feces, Rearing, Grip strength, Landing foot-splay test): the rearing was statistically significantly increased in males of the high intermediate concentration (50 mg/m3) when compared to controls.
This finding is judged to be incidental, because it only occurred in the male animals, a clear concentration response is lacking and no further indication on clinical abnormalities were detected in the other test groups during multiple open field observations and motor activity measurement .
No other statistically or biologically relevant findings were observed.
3) Motor activity: there were no statistically significant deviations from the control group 0 in overall motor activity (summation of all intervals). Comparing the single intervals with the control group, the only statistically significant deviations seen was the increased values were seen in the female animals of the low concentration (1 mg/m3), the low intermediate concentration (5 mg/m3) and the high intermediate concentration (50 mg/m3) at interval 10. This observation was due to a high value in the control group and is therefore judged to be incidental and not substance related. No other abnormalities were detected.

ORGAN WEIGHTS (Table 3)
- The absolute lung weights were significantly increased in males of group 2 (+18.4%; p≤0.05) and in group 3 (+11.1%; p≤0.05). The lung weights in males of group 4 were also elevated (+9.1%) though this was not significant. As there is no dose-response relationship, no significant change in the relative weights and no corresponding histopathological finding, this is regarded as incidental.
- The absolute but not relative weights of the testes were decreased in all treatment groups though this was not significant in group 4. But again, there was no dose response-relationship, no change in the relative weights, no corresponding histopathological finding that is different from the controls, and the values lie within the range of historical controls (mean: 3.19 g). In fact, the mean of 3.418 g in the present control group is even higher than the maximum value of historical controls (maximum: 3.364 g). Therefore, this is also regarded as incidental.
- The heart weight of females in group 1 was slightly decreased (-12.8%; p <_ 0.05), but this was incidental.
- The other mean absolute weight parameters of the other groups did not show significant differences when compared with the control group.

GROSS PATHOLOGY
The only macroscopic lesion was a focus in the liver of a top concentration female which histologically came out to be focal fatty infiltration. A relation to treatment can be excluded.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment related findings were: (1) crust formation at the snouts of high concentration animals (500 mg/m3) after daily exposure (2) concentration dependent hyperpiasia of mucous cells in some sites of levels II to IV of the nasal cavity and (3) concentration dependent squamoid metaplasia in level I of the larynx. Neither in the nose nor in the larynx there were any signs of toxic changes, like inflammatory cell infiltration, cell exfoliation or necrosis.
It is assumed that the treatment-related findings represent unspecific adaptation reactions toward the aerosol load imposed on the epithelia of the upper respiratory tract. They are not considered to represent specific adverse effects of the test substance.

HISTORICAL CONTROL DATA (if applicable)
See above

OTHER FINDINGS
There was no indication of an affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune system by the test article, neither when looking on the weight parameters determined, nor when reflecting the gross lesions or microscopic findings in the organs of the nervous system (brain, spinal cord, sciatic nerve), the reproductive system (testes, ovaries, prostate gland, uterus), or the immunocompetent organs (spleen, thymus, mediastinal and mandibular lymph nodes, Peyer's patches of the jejunum). The inhalation of the test article thus did not cause substance-related adverse effects.

Effect levels

Dose descriptor:
NOAEC
Effect level:
>= 500 mg/m³ air
Sex:
male/female
Basis for effect level:
other: the inhalation of the test article up to this dose level did not cause substance-related systemic adverse effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3: Table: absolute and relative body weights at necropsy

Organ

Test groups

0 (0 mg/m3)

1 (1.2 mg/m3)

2 (5.6 mg/m3)

3 (49.3 mg/m3)

4 (509 mg/m3)

Testes

Mean absolute weight (g)

3.418

3.02*

3.056*

3.1*

3.168n.s.

Percent of control (%)

100

-11.6

-10.6

-9.3

-7.3

Lungs

Mean absolute weight (g; in males/females)

1.118

1.174

1.324*

1.242*

1.22

Percent of control (%; in males/females)

1.004

0.922

1.022

0.964

1.068

Heart

Mean absolute weight (g; in males/females)

1.158

1.162

1.116

1.09

1.162

Percent of control (%; in males/females)

0.924

0.806*

0.936

0.884

0.924

*: significant difference compared to the control conditions; n.s. not significant

 

Applicant's summary and conclusion