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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-Oct-1979 to 18-Jan-1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
(incomplete exposure schedule; no ophthalmology examination; some details missing from report)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Santicizer 160 (butyl benzyl phthalate)
- Substance type: no data
- Physical state: no data
- Analytical purity: 99.3%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: DR-7783, Drum No. 3
- Expiration date of the lot/batch: no data
- Stability under test conditions: "no evidence of significant decomposition of Santicizer 160 during the study"
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Portage, MI
- Age at 2 days prior to study initiation: 47-51 days
- Weight at 2 days prior to study initiation: males 228-252 g , females 160-186 g
- Fasting period before study: no data
- Housing: individually in stainless steel mesh cages
- Diet (e.g. ad libitum): Ralston Purina Rodent Chow 5002, ad libitum (except during 6-hour exposure periods)
- Water (e.g. ad libitum): tap water via an automatic watering system (except during 6-hour exposure periods)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 1
- Humidity (%): 35-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 18-Oct-1979 To: 18-Jan-1980

Administration / exposure

Route of administration:
other: inhalation: aerosol and vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 0.05 mg/l, 16 measurements, range 1.852 +- 1.939 µm to 3.282 +- 2.000 µm
0.2 mg/l, 17 measurements, range 2.399 +- 2.091 µm to 3.692 +- 2.095 µm
0.8 mg/l, 18 measurements, range 2.482 +- 1.819 µm to 3.315 +- 1.939 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: four 10 m3 Rochester-style inhalation chambers
- Method of holding animals in test chamber: animals placed in chambers which each had a stainless steel top, bottom and 3 sides and a glass door
- Source and rate of air: central ventilation supply, ~9.8 chamber volumes/hour
- Method of conditioning air: no data
- System of generating particulates/aerosols: Laskin-style nebulizer, 1 for each of the low- and mid-level exposure generations systems, 3 in the high-level exposure generation system; each positioned in side of vertical particle-size separator connected to air inlet of inhalation chamber
- Temperature, humidity, pressure in air chamber:
- mean temperatures in control, low, mid and high concentration chambers respectively, 23.0, 21.8, 24.4 and 24.3 ºC; temperature measured >=4 times/day in each chamber
- humidity - no data
- pressure - no data
- Air flow rate: constant air flow rate in all chambers throughout the study, 1644 litres/min
- Air change rate: no data
- Method of particle size determination: particle-size separators used to prevent most of the large, non-respirable aerosol particles from entering the chamber; non-viable, nine-stage Anderson impactor; concentrations of particles <10 µm and >10 µm determined
- Treatment of exhaust air: chambers were vented through the bottom cone

TEST ATMOSPHERE
- Brief description of analytical method used: atmospheres drawn through Bendix 7207 impinger containing n-nonane; sampled 4 times/exposure day; concentration of Santicizer 160 determined by gas chromatography;
- Samples taken from breathing zone: yes
- Other:
- Concentration of test material in inhalation chamber controlled by regulating pressure in tank headspace and, consequently, flow rate of test material into nebulizer
- Nominal concentration calculated as net amount of test material entering air inlet of inhalation chamber (i.e. amount delivered to nebulizer minus amount recovered in bottom of separator) per unit time, divided by total airflow through chamber per unit time

VEHICLE
- None
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
10 litres test chamber atmosphere drawn at ~1 litre/min through Bendix 7207 impinger containing 15 ml n-nonane; concentration of Santicizer 160 determined by gas chromatography; sampled 4 times/exposure day; additional samples of test atmospheres obtained at 5 different locations in each chamber during pretest period to demonstrate uniformity of distribution of Santicizer 160
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
target: 5 days/week for 13 weeks (actual: total of 59 exposure days)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.05, 0.2 and 0.8 mg/l
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.051, 0.218 and 0.789 mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
25
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): "Animals of each sex were randomly assigned via a computer program on the basis of body weight to either treatment or control groups. This randomization precluded significant differences among group mean body weights or variability of individual body weights within groups."
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, prior to and following exposure, or at similar times on non-exposure days
- Cage side observations included: no data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 7 and week 13
- Anaesthetic used for blood collection: no data, but blood obtained from periorbital sinus of the eye, so presumably yes
- Animals fasted: Yes
- How many animals: 10/sex per group
- Parameters examined:
- red blood cell count (RBC)
- white blood cell count (WBC)
- haemoglobin (Hgb)
- haematocrit (Hct)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
- mean corpuscular haemoglobin (MCH)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 7 and week 13
- Animals fasted: Yes
- How many animals: 10/sex per group
- Parameters examined:
- alkaline phosphatase (ALP)
- bilirubin
- blood urea nitrogen (BUN)
- glucose
- total protein (TP)
- serum glutamic pyruvic transaminase (SGPT)

URINALYSIS: Yes
- Time schedule for collection of urine: week 7 and week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
- volume
- specific gravity
- colour
- appearance
- pH
- haemoglobin
- ketone
- glucose
- presence of blood
- total protein

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
INTERIM AND TERMINAL SACRIFICE:
- 10 pre-selected, fasted animals/sex per group were sacrificed for the respective interim or terminal necropsy after urine and blood collections
- remaining 5 fasted animals/sex per group were sacrificed for terminal necropsy

GROSS PATHOLOGY: Yes, for all animals
- adrenals*, aorta, bone marrow (femur), brain*, eye, heart*, intestine (large), intestine (small, one level), kidney*, liver*, mesenteric lymph node, muscle, nasal turbinate, ovary, pancreas, pituitary*, prostate, sciatic nerve, spinal cord (one section), spleen, stomach, testis*, thyroids, trachea, urinary bladder, uterus, any lesion or abnormal mass
- organs weighed indicated by *
- pituitary weighed at terminal sacrifice based on observation of an abnormality at interim sacrifice

HISTOPATHOLOGY: Yes
- all tissues taken at gross pathology retained in 10% buffered formalin (eyes retained in 2% glutaraldehyde in 10% buffered formalin) and examined microscopically
Other examinations:
none
Statistics:
Body weight, organ weights, haematology, serum chemistry and urine volume: 2-tailed Dunnett's test
Organ weight relative to body weight: Mann Whitney test with Bonferoni Inequality Modification

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
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:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality
At 0.218 and 0.789 mg/l, urine-stained fur and chromodacryorrhea were seen in both sexes, which the investigators considered to be treatment-related. Piloerection and alopecia were also observed, but these effects were sporadic and inconsistent, and were not considered as reliable indicators of toxicity of the test compound

BODY WEIGHT AND WEIGHT GAIN
No effects on body weight that were considered to be biologically significant

FOOD CONSUMPTION
Not measured

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
Not performed

HAEMATOLOGY
No effects that were considered to be biologically significant

CLINICAL CHEMISTRY
At 0.789 mg/l, statistically significant, marked decrease in serum glucose at week 13 in males (not seen at 7 weeks; no dose response)

URINALYSIS
No effects

NEUROBEHAVIOUR
Not measured

ORGAN WEIGHTS
At 0.789 mg/l, increased relative liver and kidney weights in both sexes at 7 and/or 13 weeks
At 0.218 mg/l, increased kidney weight in males at 7 (but not 13) weeks
At 0.051 mg/ml, increased pituitary weight in females at 13 weeks - not considered treatment-related since only seen in lowest exposure group

GROSS PATHOLOGY
No effects, including liver and kidney

HISTOPATHOLOGY: NON-NEOPLASTIC
No effects, including liver and kidney

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No effects

OTHER FINDINGS
None

Effect levels

Dose descriptor:
NOAEC
Effect level:
0.218 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Uniform distribution of vapour and aerosol within exposure chambers throughout the study; >90% of particles were <10 µm; some vapour existed at the high and mid exposure levels.

Applicant's summary and conclusion

Conclusions:
In a reliable study, a no-observed-adverse-effect-concentration (NOAEC) of 0.218 mg/l was established for rats exposed to benzyl butyl phthalate by inhalation for 6 hours/day on 5 days/week for 13 weeks. Increased relative liver and kidney weights were observed at the higher test concentration of 0.789 mg/l, although there were no accompanying histopathological findings. The study was performed in compliance with GLP.
Executive summary:
Groups of 25 male and 25 female Sprague-Dawley rats were exposed by whole-body inhalation to benzyl butyl phthalate at concentrations of 0.051, 0.218 or 0.789 mg/l (analytical) for 6 hours/day on 5 days/week. A control group was sham-exposed. An interim sacrifice was performed on 10/sex per group after 7 weeks of exposure and the remaining animals were sacrificed after 13 weeks of exposure. Evaluation included clinical observation, body weight, haematology, clinical chemistry, urinalysis, organ weights and macroscopic and microscopic examination of a range of organs and tissues.

Urine-stained fur and chromodacryorrhea were observed in both sexes in the mid- and high-exposure groups. Although the investigators described these findings as "seemingly compound and dose-related", the EU RAR made no mention of chromodacryorrhea but stated that urine-stained fur, piloerection and alopecia "were not considered as reliable indicators of the test compound". Increased relative liver and kidney weights were seen at the top dose, and males in this group had a marked decrease in serum glucose at terminal sacrifice, leading the EU RAR to set a no-observed-adverse-effect concentration (NOAEC) of 0.218 mg/l.

In a reliable study, an NOAEC of 0.218 mg/l was established for rats exposed to benzyl butyl phthalate by inhalation for 6 hours/day on 5 days/week for 13 weeks. Increased relative liver and kidney weights were observed at the higher test concentration of 0.789 mg/l, although there were no accompanying histopathological findings. The study was performed in compliance with GLP.