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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:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study. No international guideline followed but well documented.
Justification for type of information:
This information is in the dossier in support of the RA of the key study.
Cross-reference
Reason / purpose:
read-across source
Remarks:
the substance used as RA is considered worse case for DBPP, no recalculations were done.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed to or a minimum of 28 exposure days (5 days/week (except holidays) during an approximate 6-week period) or to
a minimum of 62 exposure days (5 days/week (except holidays) during an approximate 14-week period). An esposure period was 6 h. Rats were assigned to 4 groups: a control, a low, a mid and a high dose group.
Mortality, clinical signs, body weights, eyes, hematological and serum biochemistry parameters, gross and microscopic pathology were examined.
GLP compliance:
yes
Remarks:
Conducted in general comformance with the Environmental Protection Agency GLP Standards with the following exception: Test substance characterization and stability data are available but were not developed under the Standards cited above
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name: SKYDROL 500B-4
= formulation of Tributyl phosphate (19.8%; mono-constituent); DBPP (40-70%; multi-constituent); butyl diphenyl phosphate (10-30%; mono-constituent); 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate (≤ 10%)
- Lot No.: QC-35001
- Specific gravity: 1.0564
- Description at receipt: clear purple fire resistant hydraulic fluid
- Source: Monsanto Chemical Company

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory (Portage, Michigan)
- Age at study initiation: 60 days
- Weight at study initiation: males: 318 g, females: 196 g
- Housing: suspended individual stainless steel wire mesh cages
- Diet: ad libitum (except during the exposure period), Purina laboratory certified Rodent Chow
- Water: ad libitum (except during the exposure period), Sodium zeolite conditioned tap water (St. Louis City, MO)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.4
- Humidity (%): 35-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1984-06-19 To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: - mid level: MMAD: 2.56-3.55 μm, GSD: 1.83-2.55 μm
- high level: MMAD: 3.06-3.60 μm, GSD: 1.82-2.00 μm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 10 m3 New York University-style stainless steel chambers with a pyrimidal top and bottom
- System of generating particulates/aerosols:
Low level test atmosphere generation system: test material was metered from a Harvard apparatus syringe drive pump using a capillary restrictor to a Laskin-style nebulizer which generated the test atmosphere. The concentration of the test material in the inhalation chamber was controlled by regulating the pressure on the syringe pump system, and consequently, the flowrate of the test material into the nebulizer.
Mid and high level test atmosphere generation sytems: test material was metered from a pressurized tank using a capillary restrictor to a Laskin-style nebulizer which generated the test atmosphere. The concentration of the test material in the inhalation chamber was controlled by regulating the pressure in the tank headspace, and consequently, the flowrate of the test material into the nebulizer.
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination: Andersen cascade impactor. A sample was drawn for 10 min at a flowrate of approximately 1 CFM. The mass of material collected on each stage was determined gravimetrically and was used to determine mass median aerodynamic diameter (MMAD), geometric standard deviation, and % of particles < 10 microns

TEST ATMOSPHERE
- Brief description of analytical method used: Liquid Chromatography
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Atmospheric analytical sampling: 4 per exposure from each chamber, except the control level, which was sampled during the first study week and, thereafter, every 2 weeks.
- Uniformity of atmosphere analysis: atmospheric concentrations were measured twice during the study period (week 1 and week 13) from 5 specified locations in each chamber to demonstrate the uniformity of distribution of the test material atmosphere.
- Sampling method: test atmosphere was drawn at a known rate through a single glass impinge containing propanol-2
Duration of treatment / exposure:
Period 1 animals: minimum of 28 exposure days, 5 days/week (except holidays) during an approximate 6-week period
Period 2 animals: minimum of 62 exposure days, 5 days/week (except holidays) during an approximate 14-week period
Frequency of treatment:
5 days per week (except holidays)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5.3, 100, 300 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
16, 133, 483 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: checks for mortality and moribundity: preceding each exposure and on non-exposure days

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations between the 2nd and 5th h of each exposure, immediately following each exposure (normal work days only), a thorough examination weekly for gross signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 2nd to last study week
- Dose groups that were examined: control and high level exposure group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 10/sex/group on week 6 (period 1), 15/sex/group at terminal sacrifice (period 2)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters checked: total erythrocyte count (RBC), total leukocyte count (WBC), platelet count, hematocrit (HCT), level of hemoglobin (Hgb), red cell indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC)], leukocyte differential, reticulocyte count, plasma and red cell cholinesterase

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 10/sex/group on week 6 (period 1), 15/sex/group at terminal sacrifice (period 2)
- Animals fasted: No data
- Parameters checked: albumin, total protein, blood urea nitrogen (BUN), total bilirubin, glucose, glutamic pyruvic-transaminase (D-GPT/ALT), alkaline phosphatase, glutamic oxaloacetate-transaminase (D-GOT/AST), globulin, phosphorous, creatinine, calcium, chloride, sodium, potassium

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS AND MICROSCOPIC PATHOLOGY: Yes
Organs weighed: adrenals, brain, heart, kidneys, liver, spleen, testes with epididymides
Tissues retained: aorta, adrenals, bone and bone marrow, brain, colon, esophagus, eyes, heart, ileum, kidneys, lesions or abnormal masses, liver, lung with mainstem bronchi, lymph nodes (thymic and mesenteric), mammary gland, nasal passages, nerve (sciatic), ovaries, pancreas, prostate, pituitary, salivary gland, (sub-mandibular), skeletal muscle, skin, spinal cord, spleen, stomach, tested with epididymides, thymus, thyroid/parathyroid, trachea, uterus (with cervix), urinary bladder
Statistics:
The group differences in inlife body weights, hematology, and serum chemistry values were analyzed statistically by the use of Dunnett's test for comparing multiple treatments with a control.
Terminal body weights and absolute organ weights were analyzed for group differences by analysis of variance and Dunnett's test. Organ to body weight ratios were statistically tested for group differences by the Mann-Whitney test with the Bonferroni inequality procedure.
The incidence of microscopic lesions were analyzed by the use of the Fisher's exact test with the Bonferroni inequality procedure.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
All animals survived the scheduled exposures. No notable observations were seen during exposure. Notable observations in the mid and high exposure level animals included red/pink nasal discharge, salivation, loss of hair, and red ocular, discharge. The observation of red ocular discharge was only noted twice, once in mid level females and once in high level females. The only observations noted in the control and low level animals were red/pink nasal discharge and loss of hair, which were insignificant. Additional observations noted on weekly weigh days were red/brown perinasal encrustation, salivation, and focal and/or general loss of hair.

BODY WEIGHT AND WEIGHT GAIN
Significant (p ≤ 0.05) weight differences occurred in females of the high exposure level at different times thoughout the study, especially during the final 3 weeks.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmic examination of the control and high level animals showed no ocular changes which could be attributed to test material exposure.

HAEMATOLOGY
Marginal decreases in erythrocyte parameters (RBC, HGB, HCT) in both males and females of the high exposure level occurred in both period 1 and 2. The only statistically significant (p≤0.01) RBC, HGB and HCT changes were in the high level females from period 2.

CLINICAL CHEMISTRY
The changes in serum chemistry parameters, that were apparent, were moderate decreases in plasma cholinesterase levels in the high level females from both period 1 and 2 and lesser (not statistically significant) decreases in the mid level females from both periods.
Creatinine values were marginally lower in all exposure level males in period 2, however, this was apparently due to a slightly increased mean control value as compared to the historical control mean of 0.6. All other changes in chemistry parameters were apparently unrelated to the test material.

ORGAN WEIGHTS
The only changes apparently test related in organ weights were increases in both the absolute and relative hepatic weights in the high exposure animals.

GROSS PATHOLOGY
No gross necropsy observations of importance or that could be related to test material exposure were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically hepatocellular vacuolization of mild severity was present either randomly or in centrilobular regions in the livers of males (10/15, 5/15, 1/15 and 0/15 affected in high, mid, low and control groups, respectively). Centrilobular hepatocellular hypertrophy of mild severity occurred in 10/15 females from the high exposure level. These were the only lesions observed microscopically which were considered to have been related to test material exposure.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
5.3 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects
Dose descriptor:
LOAEC
Effect level:
100 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: red/pink nasal discharge, microscopically hepatocellular vacuolization (centrilobular) in males

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The “no-effect” level reported in the study is 100 mg/m3. In our opinion, however, the observed effects at this dose cannot be neglected, and this dose level should be considered as the LOAEC instead of the "no-effect" level.
Executive summary:

4 groups of 25 male and 25 female Sprague/Dawley rats per group were exposed to mean analytical concentrations of 0, 5.3, 100 or 300 mg SKYDROL 500B-4 per m3 in air in 10 m3 inhalation chambers. For period 1 animals a minimum of 28 6 h exposures were conducted over an approximate 6 -week period. For Period 2 animals a minimum of 62 6 h exposures were conducted over an approximate 14 -weel period. Period 1 animals (10/sex/group) were sacrificed and used for hematology and serum biochemical analyses only. All animals survived the scheduled exposures. Red/pink nasal discharge, salivation, loss of hair, and red ocular discharge were notable observations in the mid and high exposure level animals. An insignificant incidence of observations were noted in the control and low level animals. Additional observations noted on weekly weigh days were red/brown perinasal encrustation, salivation, and loss of hair. During the study and especially in the final 3 weeks, high exposure level females weighed significantly less than controls. Marginal decreases in erythrocytes (RBC, HGB, HCT) in both males and females of the high exposure level occurred, However, the only significant changes were in the high level females from period 2. Moderate decreases in plasma cholinesterase levels in the high level females were considered test exposure related. All other changes in chemistry parameters were slight and/or sporadic and no correlation to test material exposure could be made. No gross necropsy observations of importance were noted. Both the absolute and relative hepatic weights in the high exposure animals were increased. Microscopic findings related to test material exposure were hepatocellular vacuolization of mild severity in the livers of the high level males and centrilobular hepatocellular hypertrophy of mild severity in the livers of the high level females.

The "no-effect" level reported in the study is 100 mg/m3. However, based on the observed effects at this dose that cannot be neglected, this dose level should be considered as the LOAEC instead of the "no-effect" level.