Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity tests are available for the oral, inhalation and dermal route. Adverse effects were observed via the three routes of exposure. The main effects were observed on the skin, respiratory tract, plasma cholinesterase, kidney, bladder and the liver. These adverse effects did not justify a classification according to the CLP criteria.

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:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP stuy according to EPA pesticides guideline. No urinalysis, no neurobehavioural examination. Doses separated by relatively large intervals (5-10).
Qualifier:
according to
Guideline:
other: EPA Pesticides Assessment Guidelines, Subdivision F, Section 158.82-1
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley, CD-Crl: CD (SD)BR
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: 43 days old
- Weight at study initiation: males: 177.9-218.7g; females: 140.8-167.2g.
- Fasting period before study: no data
- Housing: individually, in suspended stainless-steel cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- 6°F
- Humidity (%): 50+/-20%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle

IN-LIFE DATES: From: March 13, 1985 To: March 18, 1985
Route of administration:
oral: feed
Vehicle:
other: feed
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material required for each sex of each DBPP-treated group was weighed on an Arbor electronic balance and added to approximately 200 grams of feed. This combination was mixed for approximately 2 minutes (or until a homogeneous mix consistency was achieved) in a Waring blender as a premix.

DIET PREPARATION
The premix for each level was added to the additional amount of feed required for that mix. The compound/diet formulation was then mixed in a Hobart mixer at a rate of one minute per kilogram.
- Rate of preparation of diet (frequency): weekly
- Diet preparation schedule: diets were prepared one week from being offered to the test animals in order to allow for the completion of concentration analyses prior to administration.
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 200g samples from the control and the DBPP diets for each sex/group were taken. One of each replicate for weeks 1, 2, 3, 4, 9 and 12 was submitted to Hazleton Biotechnologies Corporation for concentration analysis. Dietary mixtures were found to be stable and homogenous by analyses conducted on diets mixed for a concurrent study in rats using the same test material and the same target dose levels.

Analysis principle: DBPP is extracted from the feed by shaking with methylene chloride. The extract is diluted and injected on a gas chromatograph (GC) equipped with a nitrogen-phosphorous detector (NPD). Quantitation is done by linear regression analysis on the total area of three peaks of DBPP.
Duration of treatment / exposure:
91 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
5, 50 and 250 mg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
15 animals per sex per dose (120 animals in total, control animals included)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data available
- Rationale for animal assignment (if not random): random, but selecting the random assignment that produced homogeneity of both body weight variances and means.
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at initiation and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: at initiation and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Mean food consumption and standard deviations calculated per sex per dose group per week.
Total food consumption and standard deviations calculated per sex per dose group
Mean compound consumption calculated per sex per dose group per week.

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and at termination
- Dose groups that were examined: all
- Examination method: indirect ophthalmoscope with 1% Mydriacyl to dilate the pupils.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 5 and 13
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all
- Parameters that were examined: Hematocrit (HCT), Hemoglobin (HGB), Erythrocyte count (RBC), Leukocyte count (WBC), Differential Leukocyte count, Platelet count (PLATELET), Erythrocyte morphology. Reticulocyte count was not deemed necessary.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5 and 13
- Animals fasted: Yes, except for acetyl-cholinesterase determinations
- How many animals: all
- Parameters that were examined: Calcium, Phosphorus (IN PHOS), Chloride, Sodium, Potassium, Glucose, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea nitrogen (BUN), Albumin, Globulin, Total protein, Creatinine, Total bilirubin, Total cholesterol.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Rats that died or were sacrificed in extremis during the study were necropsied, and gross observations were recorded. After 13 weeks of DBPP-treatment, all surviving rats were killed by exsanguination following sodium pentobarbital anesthesia and necropsied. The terminal body weight of each rat was recorded. Terminal necropsies included examination of: the external surface; all orifices; cranial cavity; carcass; external surface of the brain; nasal cavity and paranasal sinuses; thoracic, abdominal and pelvic cavities and their viscera; and the cervical tissues and organs.

ORGAN WEIGHTS:
After careful dissection and trimming of fat and other contiguous tissue in a uniform manner, the following organs from each rat sacrificed after 13 weeks were weighed, and organ/body weight ratios and organ/brain weight ratios were determined: brain, kidneys, liver, testes with epididymides.

TISSUE PRESERVATION:
The following tissues from each animal were preserved in 10% neutral buffered formalin: lesions; brain; pituitary; thyroid with parathyroids; thymus; lung; trachea; heart; bone marrow (femur); salivary glands (mandibular); liver; spleen; kidneys; adrenals; pancreas; testes with epididymides; ovaries; uterus; aorta (thoracic); esophagus; stomach; duodenum, jejunum, ileum; colon, cecum, rectum; urinary bladder; mesenteric lymph node; sciatic nerve; prostate; seminal vesicles; spinal cord (thoracic).
The following tissues were preserved in 10% neutral buffered formalin for possible future examination: sternum with bone marrow; mammary gland; thigh musculature; eyes; femur (including articular surface); cervical spinal cord; lumbar spinal cord; extra orbital lacrimal glands.

HISTOPATHOLOGY: Yes
Except for the tissues preserved for possible future examination, tissues from each rat were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
- Tissues from all control and high-dose rats.
- Tissues from the low- and mid-dose rats which died or were sacrificed in extremis
- All gross lesions
- Lung, liver kidneys and urinary bladder from the low- and mid-dose rats.
Statistics:
Statistical analysis base on decision tree presented in study report.
Levene's test of homogeneity of variances + anova.
Terpstra-Jonckheere nonparametric test for trend and Dunnett's control vs treatment comparisons where relevant.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased body weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased food consumption
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
observed abnormalities considered related to orbital sinus bleeding trauma
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
decreased erythorcyte count, hemoglobin and hematocrit values
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
plasma, red blood cell and brain cholinesterase inhibitions
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
elevated liver weights
Gross pathological findings:
no effects observed
Description (incidence and severity):
observed eye abnormalities considered related to orbital sinus bleeding trauma
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
lesions in the liver and the urinary bladder
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
controversial interpretation by two independent pathologists with regard to neoplastic effect in one control male
Details on results:
CLINICAL SIGNS AND MORTALITY
Five rats died or were sacrificed in extremis during the study:
- 1 group 4 male was sacrificed following getting his nose caught in the cage wire
- 1 group 1 female, 1 group 3 female and 1 group 4 female were found dead following orbital sinus bleeding during week 13
- 1 grouo 2 female was found dead following orbital sinus bleeding during week 5
All signs noted were not unlike those findings generally seen in rats from this laboratory or were considered as associated with orbital sinus bleeding.

BODY WEIGHT AND WEIGHT GAIN
There was a treatment-related decrease in body weight and food consumption for the group 3 and 4 males and the group 4 females. The decreased body weights became most evident beginning at approx. week 7 for males. For the females, the difference was apparent for the duration of the study but became progressively more ponounced beginning with week 5.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In most instances the weekly food consumption values for groups 3 and 4 males and group 4 females were less than respective control values and tended to follow the same pattern noted for body weight.
The average compound consumption for the 13-week study period in all groups was within 86% of the target dosage.

OPHTHALMOSCOPIC EXAMINATION
There were no compound-related opthalmologic abnormalities noted during the study. Some of the findings noted in the right eye at week 13, i.e. total cataracts, retinochoroidal degeneration and phthisis bulbi, may be related to orbital sinus bleeding trauma.

HAEMATOLOGY
Decreased erythrocyte count, hemogolobin and hematocrit values occurred for the group 4 males and females at weeks 5 and 13. All of the changes for week 13 and the male hematocrit value for week 5 were significantly less than respective control values.
Slight changes in erythrocyte morphologic findings were noted in several treated groups. An increased incidence and/or severity grade of acanthocytosis was noted in the groups 3 and 4 males at 5 and 13 weeks. A similar change was noted in the females at week 5 only. A slight increase in the grade of polychormasia occurred in the groups 3 and 4 males at week 13. In addition, the mean total cholesterol values for the group 4 males at week 5 and 13 were significantly elevated when compared to respective control values.

CLINICAL CHEMISTRY
Plasma, red blood count cell and brain cholinesterase inhibitions were seen as compound-related effects. The plasma cholinesterase inhibition involved the group 3 females and group 4 males at females at weeks 6 and 14. The red blood cell inhibition involved the group 4 males and females at weeks 6 and 14. With the exception of the red blood cell cholinesterase values for the group 4 males, all of the changes were statistically significant. The mean brain cholinesterase value for the group 4 females was significantly less than the control female value.

Numerous instances of significantly changed values occurred in other clinical chemistry parameters; however, these were considered to be of little biological significance. All remaining hematology and clinical chemistry values were within acceptable laboratory limits and comparable to control values.

ORGAN WEIGHTS
Changes in organ weight data resulting from treatment with the test substance at the hig-dose (group 4) level were elevated absolute liver weights and liver weights relative to terminal body weights and brain weights. The absolute liver weight and the liver/brain ratio values for the males, and the liver/body weight ratios for both males and females were significantly increased. The liver/body weight ratio increases were further enhanced by decreased terminal body weights for the group 4 males and females (significant for the females).
The group 4 females mean kidney weight was significantly decreased and the mean brain/body weight ratio for the same group was significantly increased when compared to respective female control values. These changes were considered as incidental in nature. No other treatment-related changes were seen in any of the remaining organ weight data.

GROSS PATHOLOGY
With the exception of the eyes, all gross pathology findings were not unlike those routinely seen in rats from this laboratory. The majority of the eye problems were considered related to orbital sinus bleeding trauma.

HISTOPATHOLOGY:
Compound-related lesions were present in the liver and urinary bladder. The liver in the high-dose males had decreased hepatocytic vacualation (probably representing glycogen storage) and the liver in high-dose males and females had increased fatty change.
A peer review of urinary bladders was conducted by a second pathologist without knowledge of dose level or the original pathologist's findings. This review was due to the lack of a no effect level reported by the original study pathologist based on trace to minimal levels of mononuclear cell infiltrate in the submucose immediately below and adjacent to the mucosal basement membrane and sometimes extending into the mucosal epithelium. In order to define the distribution of mono-nuclear cell infiltrate, the original study pathologist subdivided the location of the infiltrate into into epithelial/perivascular. The original pathologist also used a six-step grading system for the mononuclear cell infiltrate.
The reviewing pathologist used a five-step grading system and did not subdivide the distribution of the mononuclear infiltrate. The finding was designated as submucosal inflammation and is described in this summary.

Results of the peer review confirm the presence of treatment-related epithelial hyperplasia and submucosal inflammation in rats of both sexes in the high-dose group. No effect levels were apparent for both findings at 5 mg/kg for males and 50 mg/kg for females. When the original pathologist's grade of trace is included within the minimal grade, the findings also support this conclusion for the inflammation/mononuclear cell infiltrate. The reviewing pathologist's findings do show a slight increase in the incidence of inflammation and hyperplasia in the 50 mg/kg females versus controls. However, the severity of these findings (minimal/slight) was comparable in control and treated females. The slight increase in incidence was considered insufficient to substantiate an effect at the 50 mg/kg level.

The columnar metaplasia of the transitional epithelium noted by the original pathologist as part of the hyperplasia was not subdivided from hyperplasia by the reviewing pathologist but was discussed in his report as pleomorphism.

Control male n° B37467 had urinary calculi and gross thickened walls of the urinary bladder. The epithelial proliferation was diagnosed as severe epithelial hyperplasia by the original pathologist, and as severe epithelial hyperplasia which also exhibited a transitional cell carcinoma by the reviewing pathologist. This is a controversial lesion, the diagnosis of which does not affect the interpretation of this study.
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment-related effects on body weights, food consumption, clinical pathology results (females) and histopathology results (males) were observed.
Critical effects observed:
not specified
Conclusions:
In conclusion, biologically significant findings of epithelial hyperplasia and submucosal inflammation of the urinary bladder both appear to be limited to groups 3 and 4 males and group 4 females. Decreased hepatocytic vacuolation and increased fatty change of questionnable biological significance were present in the liver of group 4 males and both group 4 males and females, respectively.
Executive summary:

To evaluate the subchronic toxicity of DBPP, Sprague-Dawley rats were exposed daily to diets containing target dose levels of 5, 50 and 250 mg of DBPP per kilogram of body weight for 91 days. Survival, clinical signs, body weights, food consumption values, ophthalmoscopic examination results, clinical pathology data, terminal body weights, absolute and relative organ weights and gross and microscopic pathology were evaluated for compound effects.

At 250 mg/kg, the high-dose level, dietary exposure to DBPP resulted in derpessed weekly body weights and/or growth rates, as well as decreased weekly food consumption in males and females and significantly lower total food consumption in female rats. Inhibition of plasma, erythrocyte and brain cholinesterase levels (females only for the brain cholinesterase) was determined in this group of rats, with depression of the plasma cholinesterase being most pronounced. Hematology effects noted included depressed erythrocyte counts, hematocrits and hemoglobin levels, as well as some slight changes in erythrocyte morphology. Total cholesterol was increased in males only. Decreased terminal body weights and increased absolute and relative liver weights were also noted as treatment effects. Histopathologic changes were seen in the liver and urinary bladder of males and females exposed to DBPP at this level. Because the initial histopathology evaluation did not establish a no-effect level with regard to changes in the urinary bladder, a second pathologist reviewed the bladder slides without prior knowledge of treatment or of the original pathologist's findings. There was a consensus by both pathologists, and a conclustion by the coordinating pathologist, that treatment related epithelial hyperplasia and submucosal inflammation were present in the bladders of males and females of the high-dose group. The liver findings were characterized by decreased hepatocytic vacuolization in males and increased fatty change in males and females. These liver findings are considered of questionnable biological significance.

At 50 mg/kg, the mid-dose level, some depression of weekly body weights and total food consumption was noted. Plasma cholinesterase levels were inhibited in female rats only. The histomorphological changes in the urinary bladder of epithelial hyperplasia and submucosal inflammation were considered treatment effects in males at this level. The coordinating pathologist drew the conclusion that in females the incidence and severity of these bladder changes were not sufficient to substantiate a treatment effect. Hepatocytic vacuolization in the liver was observed in males, but again was of questionnable biological significance.

No effects of exposure to DBPP were seen at the 5 mg/kg level. The review of urinary bladders on a blind basis established no clear difference in incidence or severity of urinary bladder findings between this low-dose group and controls.

In conclusion, treatment-related effects on body weights, food consumption, clinical pathology results, organ weights and histopathology results were seen in males and females exposed at 250 mg/kg. Treatment-effects on body weights, food consumption, clinical pathology results (females) and histopathology results (males) were observed at 50 mg/kg. There were no treatment-related effects at the 5 mg/kg level.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Three 90-day studies are available and the results are consistent with each other. The selected study is a 90-day study, GLP, key study, providing NOAEL and higher doses resulting in adverse effects.
System:
nervous system
Organ:
other: no specific target organ identified

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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:
The test substance for this study was SKYDROL 500B-4 which is a 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%). This test substance contains a considerable % of DBPP (multi-constituent), and additional TBP. TBP is considered a worse compound compared to DBPP based on its classification and therefor read-across to this formulation is deemed appropriate and conservative.
Reason / purpose:
read-across source
Remarks:
the substance used as RA is considered worse case for DBPP, no recalculations were done.
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
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:
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)
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
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.
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.
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is GLP compliant and of good quality (Klimisch score = 2). The selected study is the only available study.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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:
The test substance for this study was SKYDROL 500B-4 which is a 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%). This test substance contains a considerable % of DBPP (multi-constituent), and additional TBP. TBP is considered a worse compound compared to DBPP based on its classification and therefor read-across to this formulation is deemed appropriate and conservative.
Reason / purpose:
read-across source
Remarks:
the substance used as RA is considered worse case for DBPP, no recalculations were done.
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
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:
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)
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
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.
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.
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5.3 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is GLP compliant and of good quality (Klimisch score = 2). The selected study is the only available study.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline mentioned, but test protocol is of good quality and described in detail. Study performed according to US FDA GLP principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was applied to the shaved backs of 4 male and 4 female rabbits at dosage levels of 10, 100 and 100 mg/kg/day, 5 days a week for 3 weeks. The two control-groups, each composed of 4 male and 4 female rabbits, received distilled water at a dosage level of 2 mL/kg/day on the same regimen as the treated rabbits. The rabbits were observed daily. Individual body weights were recorded weekly. Hematological and biochemical studies and urinalyses were conducted once in the pretest period and at 3 weeks of study. At necropsy the cholinesterase activity of the brain was determined for all of the rabbits.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: H.A.R.E. Rabbits for Research, Hewitt, New Jersey.
- Age at study initiation: no data
- Weight at study initiation: 1960-2760 grams
- Fasting period before study: no data
- Housing: individually in hanging wire-mesh cages
- Diet (e.g. ad libitum): Purina Rabbit Chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks.

To ensure that the rabbits were free from desease, during the conditioning period the drinking water contained 0.1% Triple Sulfa for the first 4 days and 0.02% Triple Sulfa for the remainder of the conditioning period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, but no data on actual temperature-range.
- Humidity (%): controlled, but no data on actual humidity-range.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): controlled, but no data on actual light cycle.
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: dorsal
- % coverage: approximately 10% of the body surface
- Time intervals for shavings or clipplings: as necessary

REMOVAL OF TEST SUBSTANCE
- Washing (if done): tepid tap water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): individual daily doses were based upon the body weights obtained weekly.
- Constant volume or concentration used: constant dose

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: Ejay Saf-T Shield #412, W.A. Butler Company, Columbus, Ohio.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
5 days/week, 6 h per day.
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
analytical per unit area
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
analytical per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
analytical per unit area
No. of animals per sex per dose:
4 males and 4 females
Control animals:
yes
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): computer-generated random table
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (7d/w)
- Cage side observations: mortality, moribundity and overt signs of toxicity.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily following 6h exposure period and again before the next application

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during pre-test period and at 3 weeks of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: control animals and animals at the 100 and 1000 mg/kg/d dosage levels
- Parameters checked: hemoglobin, hematocrit, erythrocyte count, leucocyte count (total and differential)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during pre-test period and at 3 weeks of study
- Animals fasted: No data
- How many animals: control animals and animals at the 100 and 1000 mg/kg/d dosage levels
- Parameters checked: blood urea nitrogen (BUN), fasting blood glucos, activities of serum alkaline phosphatase, serum glutamic oxalacetic transaminase (SGOT) and cholinesterase activity for the erythrocytes and plasma

URINALYSIS: Yes
- Time schedule for collection of urine: no data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: determination of volume, specific gravity, color and appearance, pH and qualitative tests for albumin, glucose, occult blood and bilirubin

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After 21 days of compound administration, all surviving animals were sacrificed by intravenous overdosing of sodium pentobarbital and necropsied. At necropsy, organs and tissues were examined for gross abnormalities and tissues were collected in buffered neutral 10% formalin. The spleen, liver, adrenals, kidneys, ovaries/testes and brain were weighed fresh. The pituitary was weighed after fixation.
All rabbits that died during the study period were necropsied and tissues were collected and weighed as above.

HISTOPATHOLOGY: Yes
The following tissues from all rabbits in the controls and 1000 mg/kg/day groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin and examined microscopically: skin (treated and untreated), regional lymph node, spleen, pancreas, stomach, duodenum, colon, mesenteric lymph node, liver, gallbladder, adrenals, kidneys, urinary bladder, ovaries/testes, nerve, muscle, bone/marrow, thymus, heart, lung, thyroid, parathyroid, eyes, brain, pituitary.
Sections of the regional lymph node and skin (treated and untreated) were microscopically examined from all rabbits in the 10 and 100 mg/kg/day dosage levels.
Statistics:
All statistical analyses compared the treatment groups with the control groups, by sex. Body weights (21d), hematological, biochemical and urinalysis parameters (21d) and absolute and relative organ weights (terminal) were compared by analysis of variance (one-way classification).
Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances), described by Steel and Torrie and employing Dunnett's multiple comparison tables, were used to judge significance of differences.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
erythema, edema, fissuring, blanching
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
some minor effects observed, but no dose-response relationship
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
decreased mean cholinesterae activity at 1000 mg/kg/d level
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
at skin of application site, for all 3 dosage levels
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at skin of application site and regional lymph nodes, for all 3 dosage levels
Details on results:
CLINICAL SIGNS AND MORTALITY
No pharmacodynamic signs considered to be related to the test substance were observed. Two male rabbits receiving 100 mg/kg/d died during the study. One of them was found dead on day 3. Previous observations included anorexia, cyanosis and possible fecal impaction. For the other rabbit anorexia was noted on day 5 which, by day 11, was accompanied by dehydration, diarrhea, ataxia and prostration. This rabbit was sacrificed in extremis on day 12.
One rabbit at the 10 mg/kg/d dosage level had diarrhea on and after day 17, which was accompanied by a distended abdomen and swollen anogenital area for the last 3 days of study. Another rabbit at the 1000 mg/kg/d level also had diarrhea the last 2 days of the study. For a few of the control rabbits, nasal and ocular discharges were noted occasionally. During the last 5 days of the study a female control group I rabbit was described as having its "entire body curved to the right".

DERMAL IRRITATION
No dermal irritation was observed for any of the rabbits in either of the control groups during the study.
At the 10 mg/kg/d dosage level marked erythema was observed for 2 of the 4 abraded rabbits and moderate edema was noted for 2 abraded and 2 unabraded rabbits. By contrast, moderate desquamation noted in week 3 was observed for 2 unbaraded rabbits but the moderate fissuring appeared on an abraded rabbit. Erytheme was accompanied by blanching for 2 abraded rabbits for 1 day only but persisted for several days for one unabraded rabbit.
At the 100 mg/kd/d dosage level the erythema became definitely marked for 2 abraded and 1 unabraded rabbit. The only blanching observed was for the rabbit that was sacrificed in extremis (see section clinical signs and mortality). For this rabbit severe atonia and moderate coriaceousness were noted for study days 10 and 11. The degree of dermal irritation probably is related to the pharmacotoxic signs observed for this rabbit. The marked edema was noted for an unabraded rabbit, but the edema became moderate-to-marked for the other surviving rabbits. Atonia became moderate for 2 abraded and 2 unabraded surviving rabbits. Moderate desquamation was noted for an unabraded rabbit. For the surviving rabbits, moderate coriaceousness was observed for 2 abraded and 1 unabraded rabbit and moderate fissuring was noted for 3 abraded and 2 unabraded rabbits.
At the 1000 mg/kg/d dosage level, the erythema became marked for an abraded rabbit in week 1 and for 2 unabraded rabbits in week 3. At some time during the study, usually in week 2, the erythema was accompanied by blanching for all the rabbits. Moderate edema was observed for 3 abraded and 2 unabraded rabbits. Similarly moderate atonia and desquamation were noted for 4 abraded and 3 unabraded rabbits. For all the abraded and 3 unabraded rabbits the coriaceousness became moderate at some time during the study. Similarly moderate fissuring was noted for 1 abraded and 2 unabraded rabbits.

BODY WEIGHT AND WEIGHT GAIN
Between weeks 2 and 3 of the study period, loss of body weight was observed for a female rabbit of control group I, for 2 male rabbits at the 10 mg/kg/d dosage level, for 1 male rabbit at the 100 mg/kg/d dosage level and for 1 male and 3 female rabbits at the 1000 mg/kg/d dosage level. There was, however, only 1 statistical difference between the body weights of the control and treated groups. This difference was in comparison with control group I mean.

HAEMATOLOGY
No changes considered to be related to the test substance were seen in the hematological studies. All of the hematological values were within the range usually observed for New Zealand White rabbits in the test laboratory. This indicates that the statistically significant differences between the male rabbits of control group I and those at the 100 mg/kg/d dosage level for neutrophil, lymphocyte and basophil counts were not biologically significant.

CLINICAL CHEMISTRY
With the exception of one male and one female rabbit in control group I the blood urea nitrogen concentration increased over the 3 weeks study for all the rabbits. However, at the end of the study none of these values were unusually high. High values for alkaline phosphates were found for at least one rabbit in each of the control and test groups. The high alkaline phosphatase values were most evident in control group I. Similarly high values for serum glutamic pyruvic transaminase were found for individual rabbits in each of the groups. One male rabbit of the control group I also had a moderately high value for serum glutamic oxalacetic transaminase.
For both the male and the female rabbits treated at the 1000 mg/kg/d level, the mean cholinesterase activity of the plasma was lower than that of either control group; the differences were statistically significant. Similarly, the differences between the mean cholinesterase activity for the cells of these treated female rabbits and the female rabbits of control group II were statistically significant and may be biologically significant. However, no physiological signs of decreased cholinesterase activity was observed.

URINALYSIS
Trace amounts of occult blood in the urine were noted for some of the rabbits in each of the control and test groups at the termination of the study.

ORGAN WEIGHTS
Statistical variations occurred when sex-group mean organ weights of the treated groups were compared with the corresponding weights in the control groups. Variations were seen in spleen (10 mg/kg/d group), kidney (0, 10 and 1000 mg/kg/d group), pituitary (0 mg/kg/d group) and brain (100 mg/kg/d group) weihts of female animals of indicated dosage levels when compared to Control group I and/or Control group II. In the absence of compound-related morpholigic alterations in the above organs, these weight variations were not considered of toxicologic significance.

GROSS PATHOLOGY
Gross patiologic lesions observed at necropsy which were considered related to topical application of the test substance were limited to the skin of the application site. These changes, which were thickening, fissuring, scabbing, crusting, erythema and hemorrhage, occurred at all 3 dosage levels. Thickening and fissuring were the most commonly observed lesions and the degree of thickening was somewhat dose-dependent. All rabbits from the 3 experimental groups had compound-related lesions at the application sites. Gross pathologic lesions other than those at the applications site were those which commonly occur spontaneously in untreated rabbits. They were not considered significant with respect to the outcome of the study.

HISTOPATHOLOGY
Compound-related changes were limited to the skin of application site and the regional lymph nodes draining the application site. The skin changes consisted of very slight-to-moderate acanthosis, very slight-to-moderate hyperkeratosis, dermal edema, parakeratosis and occasional ulceration of vesicle formation. Hyperkeratosis and acanthosis were the most commonly observed conditions, occurring in almost all rabbits from the 3 experimental groups. Dermal edema also was seen in most rabbits from the 100 and 1000 mg/kg/d groups. Accumulation of necrotic debris on the epidermal surface was seen in numerous rabbits from the 3 experimental groups. The severity of these skin lesions did not appear to be dose dependent.
Compound-related findings in the regional lymph nodes consisted of an increased incidence of lymphoid hyperplasia at the 10, 100 and 1000 mg/kg/d levels and occurrence of reticuloendothelial hyperplasia in 2 rabbits from the 1000 mg/kg/d group.
Most rabbits, control and treated, had a very slight to moderate, multifocal dermal inflammatory cell infiltrate. This condition occurred in the untreated skin site as well as in the application site. Other microscopic findings in these rabbits were generally parasitic lesions and mild inflammatory conditions. They were not considered of significance with respect to the outcome of the study.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This NOAEL is based on systemic effects
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This LOAEL is based on systemic effects
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No NOAEL could be identified in this test for local effects
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This LOAEL is based on local effects
Critical effects observed:
not specified
Executive summary:

The test substance was applied to the shaved backs of 4 female and 4 male rabbits at dosage levels of 10, 100 and 1000 mg/kg/d, 5 days a week for 3 weeks. The 2 control groups, each composed of 4 male and 4 female rabbits, received distilled water at a dosage level of 2 mL/kg/d on the same regimen as the treated rabbits. The rabbits were observed daily. Individual body weights were recorded weekly. Hematological and biochemical studies and urinalysis were conducted once in the pretest period and at 3 weeks of study. At necropsy the cholinesterase activitiy of the brain was determined for all rabbits.

One male rabbit was found dead on day 3, and one male rabbit was sacrificed in extremis on day 12 of the study period. Each of these rabbits were in the 100 mg/kg/d dose group. No changes considered to be related to the compound were seen in general appearance, body weights, hematological studies or urinalysis.

At each dosage level, the test substance elicited erythema, edema, atonia, desquamation, coriaceousness and fissuring on all the rabbits. For 3 or more rabbits at each dosage level the erythema became marked and the edema became moderate. Blanching accompanied the erythema for 3 and 8 rabbits in the 10 and 1000 mg/kg/d dosage levels, respectively. The coriaceousness remained slight at the 10 mg/kg/d dosage levels but became moderate for 3 and 6 rabbits at the 100 and 1000 mg/kg/d dosage levels, respectively. At each dosage level the atonia, desquamation and fissuring became moderate for 1or more rabbits. The number of rabbits on which these signs of dermal irritation were noted increased with the dosage level.

For both male and female treated rabbits the mean cholinesterase activity of the plasma was, with the exception of the 100 mg/kg/d females in control period, less than that of the control group at the initiation as well as termination of the study. The differences between the mean cholinesterase activitiy for the brain of the female rabbits treated at the 1000 mg/kg/d dosage level and Control group II was statistically significant. These differences may be biologically significant, however, no physiological signs of decreased cholinesterase activity were observed.

All rabbits which were sacrificed at termination or which died during the study period were necropsied. All rabbits from the 3 experimental groups had compound-related changes in the skin of the application sites. Thinkening and fissuring were the most comonly observed lesions. Other compound-related findings included scabbing, crusting, erythema and hemorrhage.

Microscopically, compound-related lesions were limited to the skin of the application site where most rabbits from the 3 experimental groups had acanthosis and hyperkeratosis. Most rabbits from the 100 and 1000 mg/kg/d groups had dermal edema. Accumulation of necrotic debris was observed in several rabbits from each experimental group. Parakeratosis, ulceration and vesicle formation occurred in occasional rabbits from one or more experimental groups. Increased incidence of lymphoid hyperplasia in the regional lymph nodes in the 3 experimental groups and reticuloendothelial hyperplasia of the regional lymph nodes in 2 rabbits from the 1000 mg/kg/d group were also considered compound-related.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The study is GLP compliant and of good quality (Klimisch score = 2). The selected study is the only available study.
System:
nervous system
Organ:
other: no specific target organ identified

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline mentioned, but test protocol is of good quality and described in detail. Study performed according to US FDA GLP principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was applied to the shaved backs of 4 male and 4 female rabbits at dosage levels of 10, 100 and 100 mg/kg/day, 5 days a week for 3 weeks. The two control-groups, each composed of 4 male and 4 female rabbits, received distilled water at a dosage level of 2 mL/kg/day on the same regimen as the treated rabbits. The rabbits were observed daily. Individual body weights were recorded weekly. Hematological and biochemical studies and urinalyses were conducted once in the pretest period and at 3 weeks of study. At necropsy the cholinesterase activity of the brain was determined for all of the rabbits.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: H.A.R.E. Rabbits for Research, Hewitt, New Jersey.
- Age at study initiation: no data
- Weight at study initiation: 1960-2760 grams
- Fasting period before study: no data
- Housing: individually in hanging wire-mesh cages
- Diet (e.g. ad libitum): Purina Rabbit Chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks.

To ensure that the rabbits were free from desease, during the conditioning period the drinking water contained 0.1% Triple Sulfa for the first 4 days and 0.02% Triple Sulfa for the remainder of the conditioning period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, but no data on actual temperature-range.
- Humidity (%): controlled, but no data on actual humidity-range.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): controlled, but no data on actual light cycle.
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: dorsal
- % coverage: approximately 10% of the body surface
- Time intervals for shavings or clipplings: as necessary

REMOVAL OF TEST SUBSTANCE
- Washing (if done): tepid tap water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): individual daily doses were based upon the body weights obtained weekly.
- Constant volume or concentration used: constant dose

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: Ejay Saf-T Shield #412, W.A. Butler Company, Columbus, Ohio.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
5 days/week, 6 h per day.
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
analytical per unit area
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
analytical per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
analytical per unit area
No. of animals per sex per dose:
4 males and 4 females
Control animals:
yes
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): computer-generated random table
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (7d/w)
- Cage side observations: mortality, moribundity and overt signs of toxicity.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily following 6h exposure period and again before the next application

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during pre-test period and at 3 weeks of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: control animals and animals at the 100 and 1000 mg/kg/d dosage levels
- Parameters checked: hemoglobin, hematocrit, erythrocyte count, leucocyte count (total and differential)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during pre-test period and at 3 weeks of study
- Animals fasted: No data
- How many animals: control animals and animals at the 100 and 1000 mg/kg/d dosage levels
- Parameters checked: blood urea nitrogen (BUN), fasting blood glucos, activities of serum alkaline phosphatase, serum glutamic oxalacetic transaminase (SGOT) and cholinesterase activity for the erythrocytes and plasma

URINALYSIS: Yes
- Time schedule for collection of urine: no data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: determination of volume, specific gravity, color and appearance, pH and qualitative tests for albumin, glucose, occult blood and bilirubin

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After 21 days of compound administration, all surviving animals were sacrificed by intravenous overdosing of sodium pentobarbital and necropsied. At necropsy, organs and tissues were examined for gross abnormalities and tissues were collected in buffered neutral 10% formalin. The spleen, liver, adrenals, kidneys, ovaries/testes and brain were weighed fresh. The pituitary was weighed after fixation.
All rabbits that died during the study period were necropsied and tissues were collected and weighed as above.

HISTOPATHOLOGY: Yes
The following tissues from all rabbits in the controls and 1000 mg/kg/day groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin and examined microscopically: skin (treated and untreated), regional lymph node, spleen, pancreas, stomach, duodenum, colon, mesenteric lymph node, liver, gallbladder, adrenals, kidneys, urinary bladder, ovaries/testes, nerve, muscle, bone/marrow, thymus, heart, lung, thyroid, parathyroid, eyes, brain, pituitary.
Sections of the regional lymph node and skin (treated and untreated) were microscopically examined from all rabbits in the 10 and 100 mg/kg/day dosage levels.
Statistics:
All statistical analyses compared the treatment groups with the control groups, by sex. Body weights (21d), hematological, biochemical and urinalysis parameters (21d) and absolute and relative organ weights (terminal) were compared by analysis of variance (one-way classification).
Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances), described by Steel and Torrie and employing Dunnett's multiple comparison tables, were used to judge significance of differences.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
erythema, edema, fissuring, blanching
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
some minor effects observed, but no dose-response relationship
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
decreased mean cholinesterae activity at 1000 mg/kg/d level
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
at skin of application site, for all 3 dosage levels
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at skin of application site and regional lymph nodes, for all 3 dosage levels
Details on results:
CLINICAL SIGNS AND MORTALITY
No pharmacodynamic signs considered to be related to the test substance were observed. Two male rabbits receiving 100 mg/kg/d died during the study. One of them was found dead on day 3. Previous observations included anorexia, cyanosis and possible fecal impaction. For the other rabbit anorexia was noted on day 5 which, by day 11, was accompanied by dehydration, diarrhea, ataxia and prostration. This rabbit was sacrificed in extremis on day 12.
One rabbit at the 10 mg/kg/d dosage level had diarrhea on and after day 17, which was accompanied by a distended abdomen and swollen anogenital area for the last 3 days of study. Another rabbit at the 1000 mg/kg/d level also had diarrhea the last 2 days of the study. For a few of the control rabbits, nasal and ocular discharges were noted occasionally. During the last 5 days of the study a female control group I rabbit was described as having its "entire body curved to the right".

DERMAL IRRITATION
No dermal irritation was observed for any of the rabbits in either of the control groups during the study.
At the 10 mg/kg/d dosage level marked erythema was observed for 2 of the 4 abraded rabbits and moderate edema was noted for 2 abraded and 2 unabraded rabbits. By contrast, moderate desquamation noted in week 3 was observed for 2 unbaraded rabbits but the moderate fissuring appeared on an abraded rabbit. Erytheme was accompanied by blanching for 2 abraded rabbits for 1 day only but persisted for several days for one unabraded rabbit.
At the 100 mg/kd/d dosage level the erythema became definitely marked for 2 abraded and 1 unabraded rabbit. The only blanching observed was for the rabbit that was sacrificed in extremis (see section clinical signs and mortality). For this rabbit severe atonia and moderate coriaceousness were noted for study days 10 and 11. The degree of dermal irritation probably is related to the pharmacotoxic signs observed for this rabbit. The marked edema was noted for an unabraded rabbit, but the edema became moderate-to-marked for the other surviving rabbits. Atonia became moderate for 2 abraded and 2 unabraded surviving rabbits. Moderate desquamation was noted for an unabraded rabbit. For the surviving rabbits, moderate coriaceousness was observed for 2 abraded and 1 unabraded rabbit and moderate fissuring was noted for 3 abraded and 2 unabraded rabbits.
At the 1000 mg/kg/d dosage level, the erythema became marked for an abraded rabbit in week 1 and for 2 unabraded rabbits in week 3. At some time during the study, usually in week 2, the erythema was accompanied by blanching for all the rabbits. Moderate edema was observed for 3 abraded and 2 unabraded rabbits. Similarly moderate atonia and desquamation were noted for 4 abraded and 3 unabraded rabbits. For all the abraded and 3 unabraded rabbits the coriaceousness became moderate at some time during the study. Similarly moderate fissuring was noted for 1 abraded and 2 unabraded rabbits.

BODY WEIGHT AND WEIGHT GAIN
Between weeks 2 and 3 of the study period, loss of body weight was observed for a female rabbit of control group I, for 2 male rabbits at the 10 mg/kg/d dosage level, for 1 male rabbit at the 100 mg/kg/d dosage level and for 1 male and 3 female rabbits at the 1000 mg/kg/d dosage level. There was, however, only 1 statistical difference between the body weights of the control and treated groups. This difference was in comparison with control group I mean.

HAEMATOLOGY
No changes considered to be related to the test substance were seen in the hematological studies. All of the hematological values were within the range usually observed for New Zealand White rabbits in the test laboratory. This indicates that the statistically significant differences between the male rabbits of control group I and those at the 100 mg/kg/d dosage level for neutrophil, lymphocyte and basophil counts were not biologically significant.

CLINICAL CHEMISTRY
With the exception of one male and one female rabbit in control group I the blood urea nitrogen concentration increased over the 3 weeks study for all the rabbits. However, at the end of the study none of these values were unusually high. High values for alkaline phosphates were found for at least one rabbit in each of the control and test groups. The high alkaline phosphatase values were most evident in control group I. Similarly high values for serum glutamic pyruvic transaminase were found for individual rabbits in each of the groups. One male rabbit of the control group I also had a moderately high value for serum glutamic oxalacetic transaminase.
For both the male and the female rabbits treated at the 1000 mg/kg/d level, the mean cholinesterase activity of the plasma was lower than that of either control group; the differences were statistically significant. Similarly, the differences between the mean cholinesterase activity for the cells of these treated female rabbits and the female rabbits of control group II were statistically significant and may be biologically significant. However, no physiological signs of decreased cholinesterase activity was observed.

URINALYSIS
Trace amounts of occult blood in the urine were noted for some of the rabbits in each of the control and test groups at the termination of the study.

ORGAN WEIGHTS
Statistical variations occurred when sex-group mean organ weights of the treated groups were compared with the corresponding weights in the control groups. Variations were seen in spleen (10 mg/kg/d group), kidney (0, 10 and 1000 mg/kg/d group), pituitary (0 mg/kg/d group) and brain (100 mg/kg/d group) weihts of female animals of indicated dosage levels when compared to Control group I and/or Control group II. In the absence of compound-related morpholigic alterations in the above organs, these weight variations were not considered of toxicologic significance.

GROSS PATHOLOGY
Gross patiologic lesions observed at necropsy which were considered related to topical application of the test substance were limited to the skin of the application site. These changes, which were thickening, fissuring, scabbing, crusting, erythema and hemorrhage, occurred at all 3 dosage levels. Thickening and fissuring were the most commonly observed lesions and the degree of thickening was somewhat dose-dependent. All rabbits from the 3 experimental groups had compound-related lesions at the application sites. Gross pathologic lesions other than those at the applications site were those which commonly occur spontaneously in untreated rabbits. They were not considered significant with respect to the outcome of the study.

HISTOPATHOLOGY
Compound-related changes were limited to the skin of application site and the regional lymph nodes draining the application site. The skin changes consisted of very slight-to-moderate acanthosis, very slight-to-moderate hyperkeratosis, dermal edema, parakeratosis and occasional ulceration of vesicle formation. Hyperkeratosis and acanthosis were the most commonly observed conditions, occurring in almost all rabbits from the 3 experimental groups. Dermal edema also was seen in most rabbits from the 100 and 1000 mg/kg/d groups. Accumulation of necrotic debris on the epidermal surface was seen in numerous rabbits from the 3 experimental groups. The severity of these skin lesions did not appear to be dose dependent.
Compound-related findings in the regional lymph nodes consisted of an increased incidence of lymphoid hyperplasia at the 10, 100 and 1000 mg/kg/d levels and occurrence of reticuloendothelial hyperplasia in 2 rabbits from the 1000 mg/kg/d group.
Most rabbits, control and treated, had a very slight to moderate, multifocal dermal inflammatory cell infiltrate. This condition occurred in the untreated skin site as well as in the application site. Other microscopic findings in these rabbits were generally parasitic lesions and mild inflammatory conditions. They were not considered of significance with respect to the outcome of the study.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This NOAEL is based on systemic effects
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This LOAEL is based on systemic effects
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No NOAEL could be identified in this test for local effects
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This LOAEL is based on local effects
Critical effects observed:
not specified
Executive summary:

The test substance was applied to the shaved backs of 4 female and 4 male rabbits at dosage levels of 10, 100 and 1000 mg/kg/d, 5 days a week for 3 weeks. The 2 control groups, each composed of 4 male and 4 female rabbits, received distilled water at a dosage level of 2 mL/kg/d on the same regimen as the treated rabbits. The rabbits were observed daily. Individual body weights were recorded weekly. Hematological and biochemical studies and urinalysis were conducted once in the pretest period and at 3 weeks of study. At necropsy the cholinesterase activitiy of the brain was determined for all rabbits.

One male rabbit was found dead on day 3, and one male rabbit was sacrificed in extremis on day 12 of the study period. Each of these rabbits were in the 100 mg/kg/d dose group. No changes considered to be related to the compound were seen in general appearance, body weights, hematological studies or urinalysis.

At each dosage level, the test substance elicited erythema, edema, atonia, desquamation, coriaceousness and fissuring on all the rabbits. For 3 or more rabbits at each dosage level the erythema became marked and the edema became moderate. Blanching accompanied the erythema for 3 and 8 rabbits in the 10 and 1000 mg/kg/d dosage levels, respectively. The coriaceousness remained slight at the 10 mg/kg/d dosage levels but became moderate for 3 and 6 rabbits at the 100 and 1000 mg/kg/d dosage levels, respectively. At each dosage level the atonia, desquamation and fissuring became moderate for 1or more rabbits. The number of rabbits on which these signs of dermal irritation were noted increased with the dosage level.

For both male and female treated rabbits the mean cholinesterase activity of the plasma was, with the exception of the 100 mg/kg/d females in control period, less than that of the control group at the initiation as well as termination of the study. The differences between the mean cholinesterase activitiy for the brain of the female rabbits treated at the 1000 mg/kg/d dosage level and Control group II was statistically significant. These differences may be biologically significant, however, no physiological signs of decreased cholinesterase activity were observed.

All rabbits which were sacrificed at termination or which died during the study period were necropsied. All rabbits from the 3 experimental groups had compound-related changes in the skin of the application sites. Thinkening and fissuring were the most comonly observed lesions. Other compound-related findings included scabbing, crusting, erythema and hemorrhage.

Microscopically, compound-related lesions were limited to the skin of the application site where most rabbits from the 3 experimental groups had acanthosis and hyperkeratosis. Most rabbits from the 100 and 1000 mg/kg/d groups had dermal edema. Accumulation of necrotic debris was observed in several rabbits from each experimental group. Parakeratosis, ulceration and vesicle formation occurred in occasional rabbits from one or more experimental groups. Increased incidence of lymphoid hyperplasia in the regional lymph nodes in the 3 experimental groups and reticuloendothelial hyperplasia of the regional lymph nodes in 2 rabbits from the 1000 mg/kg/d group were also considered compound-related.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
0.144 mg/cm²
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The study is GLP compliant and of good quality (Klimisch score = 2). The selected study is the only available study.

Additional information

Oral repeated dose toxicity was evaluated in 3 study reports (Snyder, 1986 (key study); Jessup, 1980 (supporting study); Jessup, 1983 (supporting study)). All three study reports were 90-day repeated dose studies.

 In the key study (Snyder, 1986), the test compound was administered in the diet of Sprague-Dawley rats at dose levels of5, 50 and 250 mg/kg bw/day. Treatment-related effects on body weights, food consumption, clinical pathology, organ weights and histopathology results were seen in males and females exposed at 250 mg/kg bw/day. Treatment-effects on body weights, food consumption, clinical pathology results (females) and histopathology results (males) were observed at 50 mg/kg bw/day. There were no treatment related effects at 5 mg/kgbw/day.

In the oldest supporting study (Jessup, 1980), the test compound was administered in the diet of rats at dose levels of 50, 150 and 500 mg/kg bw/day. Kidneys, livers, ovaries and urinary bladders developed lesions in response to compound administration. The kidneys were the only organs affected at the 50 mg/kg bw/day level with the presence of a yellowish-brown pigment in the epithelium of the renal tubules. This compound-induced change at the lowest dose level precludes the establishment of a NOAEL for DBPP in this study.

In the other supporting study (Jessup, 1983), rats were fed with a control and with a dosage level of 5 mg/kg bw/day. The test substance was fed in the diet and the concentration was adjusted weekly based upon the most recent body weights and food consumption values. Observations addressed clinical signs and mortality, body weights and weight gain, food consumption and compound intake, haematology and clinical chemistry, gross pathology and histopathology. There were no significant findings in any of the criteria evaluated for treatment with the test compound and as a consequence the dose level of 5 mg/kg bw/day is equal to the NOAEL.

The results of the abovementioned studies are consistent with each other. We conclude a NOAEL of 5 mg/kg bw/day for oral repeated dose toxicity.

For repeated-dose toxicity via the inhalation route, read-across to Skydrol 500B-4 (for details on composition see tabel in section 1.1 of the CSR) was applied.

Four 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/m3. 15 animals of each group were exposed for 3 months, the other 10 animals for approximately 6 weeks. Red/pink nasal discharge and salivation were notable observations in the mid and high exposure level animals. 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. Both the absolute and relative hepatic weights in the high exposure animals were increased. 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 was observed in 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. Consequently the NOAEC is 5.3 mg/m3.

Dermal repeated dose toxicity was evaluated in one study (Laveglia, 1986).

DBPP was applied to the shaved backs of four male and four female New Zealand White rabbits at dosage levels of 10, 100 and 1000 mg/kg bw/day, 5 days a week for 3 weeks. The back (approximately 10% of the body surface) of each rabbit was clipped as necessary during the study and the skin of 2 rabbits of each sex group was abraded twice weekly by producing a shallow incision (not sufficiently deep to cause bleeding) by a scalpel blade.

At each dose level, the test compound elicited erythema, edema, atonia, desquamation, coriaceousness and fissuring on all the rabbits. For both the male and female rabbits treated at the 1000 mg/kg bw/day level, the mean cholinesterase activity of the plasma was statistically significant lower than that of either control group. These differences may be biologically significant, however, no physiological signs of decreased cholinesterase activity were observed. Increased incidence of lymphoid hyperplasia in the regional lymph nodes in the 3 experimental groups and reticuloendothelial hyperplasia of the regional lymph nodes in 2 rabbits from the 1000 mg/kg bw/day group also were considered compound-related. The observed effects in the lymph nodes are considered adaptive responses.

A local NOAEL could not be derived due to the harmful effects of the test substance on the skin at all test concentrations. The systemic NOAEL was 100 mg/kg bw/day.

Justification for classification or non-classification

Repeated dose toxicity: oral

Of the concentrations tested in the evaluated study reports for oral repeated dose toxicity, 50 mg/kg bw/day is the only dose with treatment-related effects that fits in the guidance value range for a STOT Category 2 classification (CLP Annex I, Table 3.9.3). 

In the key study (Snyder, 1986), 50 mg/kg bw/day causes treatment related effects on body weights and food consumption in males, clinical pathology result in females (plasma cholinesterase) and histopathology results in males (epithelial hyperplasia and submucosal inflammation in the urinary bladder).

Small changes in body weight gain and food consumption do not justify classification. It is possible that DBPP influences the palatability of the feed. The other significant effects occurred only in males or females.

In the oldest supporting study (Jessup, 1980), there was a presence of a yellowish-brown pigment in the epithelium of the renal tubules at the 50 mg/kg bw/day. But there was a decrease of incidence with this pigment with increasing dosage and it did not occur in the females at this dosage level. 

We conclude that we cannot ascribe effects to a dose level of 50 mg/kg bw/day that support a classification for specific target organ toxicity following repeated exposure for DBPP via the oral route.

Repeated dose toxicity: inhalation

Adverse effects in the 90-day inhalation repeated dose study were observed at concentrations of 100 and 300 mg/m3/6 h/day (= respectively 0.1 and 0.3 mg/L/6 h/day).

According to the CLP Regulation (Annex I, Table 3.9.3), significant observed toxic effects between 0.02 and 0.2 mg/L/6 h/day (mist) result in a STOT RE Cat. 2 classification.

The adverse effects observed at 100 mg/m3/6 h/day were red/pink nasal discharge, salivation and microscopically hepatocellular vacuolization of mild severity in males only. Since these effects do not indicate significant toxicity, the test substance is not classified as STOT RE via the inhalation route.

Repeated dose toxicity: dermal

In the dermal repeated dose study, 1000 mg/kg bw/day was the lowest concentration at which a systemic adverse effect was observed. The observed effect at this dose was a statistically significant decrease in the mean cholinesterase activity. This effect can be biologically significant but no physiological signs of decreased cholinesterase activity were observed.

Regarding the high dose level that is required to result in the decreased cholinesterase activity and the absence of physiological signs, this substance will not be classified for specific target organ toxicity STOT RE via the dermal route.