Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): DBPP
- Label information: DBPP: CP-850
- Description: colorless liquid

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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.