Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is old, but well reported and included the majority of endpoints typical of a guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1975

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Repeated doses of test article applied to skin of rabbits for 28 days. Various parameters were monitored/measured throughout the study. Gross necropsy and histopathology performed at scheduled sacrifice. No guideline was available at the time of the study's conduct.
GLP compliance:
no
Remarks:
performed prior to GLPs
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
FM PHT4 (micronized) was used as test article.

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
12/12 M/F rabbits were used on the study. The rabbits were housed individually in metal metabolism cages and maintained in a temperature and humidity controlled room. Purina Rabbit Chow and water were available ad libitum.

Administration / exposure

Type of coverage:
open
Vehicle:
other: saline
Details on exposure:
The test article was applied dermally at 0, 50, 500 or 5000 mg/kg/d, 5 days/week for 4 weeks. Three male and three female rabbits were used at each dose level. The control rabbits were treated with saline only at a volume of 12 ml on the same regimen as treated animals.

The dorsal skin of each rabbit was shaved with electric clippers as necessary during the study. The shin of one-half of the rabbits was abraded twice a week. The compound was mixed with a small amount (maximum of 12 ml) of saline to form a paste. The was spread over the skin with a glass rod. The rabbits were held in wooden stocks during the 6 hour test article administration period after which the backs were washed with tepid tap water and the rabbits were returned to their individual cages. Individual daily doses were based upont the body weights obtained weekly.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 500, 5000 mg/kg/d
Basis:
nominal per unit body weight
No. of animals per sex per dose:
3M/3F per dose.
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
The rabbits were observed daily for changes in general behavior and apppearance. Observations for dermal irritation were done prior to and following the 6 hr test article administration period. Individual body weights were recorded weekly. Once in the control group and on study day 14 and 26, blood and urine samples were collected from all rabbits.
Sacrifice and pathology:
All rabbits were sacrificed following 28 d of treatment. Selected tissues (approximately 28) in the control and 500 mg/kg/d groups were examined by histopathology (paraffin embeddded, sectioned, stained with hematoxylin and eosin). Skin, liver, kidney and bone marrow from the 50 mg/kg/d group were also examined histologically.
Other examinations:
Hematology: hemoglobin, hematorcrit, total RBC, total and differential WBC. Serum chemistries: glucose, BUN, SGOT, SGPT, SAP. Urinalysis: volume, pH, specific gravity, color and appearance, qualatative tests for albumin, glucose, bilirubin and occult blood. Bromine analysis: in the 50 and 500 mg/kg/d groups liver, fat, kidney, skin and blood were analyzed by neutron activation analysis.
Statistics:
Statistics were run on bromide levels: one way ANOVA with Dunnet's post test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
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 specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
Very slight to slight and occasionally moderate erythema was noted in control rabbits and in rabbits at the 50 mg/kg/d dose. Very slight to moderate erythema was noted for rabbits at the 500 and 5000 mg/kg/d doses. Moderate desquamation was noted for 3 of the rabbits at the 5000 mg/kg/d dose. One rabbit at the 5000 mg/kg/d dose showed marked ataxia, unable to lift head or right itself and dyspnea and was sacrificed in extremis. All of the rabbits at the 5000 mg/kg/d dose died or were sacrificed in extremis between days 10 and 26 of the study.

Body weights were similar in control, 50 and 500 mg/kg/d groups. Rabbits at the 5000 mg/kg/d dose lost weight prior to death. Organ weights among groups appeared comparable.

At day 14, 1 rabbit in the high dose group had a moderate increase in BUN. At day 26, the hematology, serum chemistry and urinalysis results in the one surviving rabbit at the high dose were: neutrophilia with lymphopenia, nucleated erythrocytes, marked increase in glucose and BUN, and albumiand glucose in the urine.

Deaths or declining condition necessitating early sacrifice of all rabbits at the high dose were considered compound-related. At necropsy, several rabbits in the high dose group had pale livers, accentuated liver lobulation and gastric irritation. These effects observed at necropsy may have been compound-related. No gross lesions were observed in the low and mid-dose groups. Microscopically, the only lesion seen in animals in the low and mid-dose groups considered compound related was a very slight hyperkeratosis of the application site in one rabbit in the mid-dose group.

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
mortality
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Mortality in the 5000 mg/kg/d group.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Lack of effect on body weights, organ weights, hematology, urinalysis, serum chemistries. Slight effects on skin at site of application of test article.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

After treatment for 28 days, bromine levels measured in skin (50, 500 mg/kg/d) and blood (500 mg/kg/d) were statististically significantly higher when compared to the control group. In skin, mean measured bromine levels in the control, 50 and 500 mg/kg/d groups were 39.6, 712.0 and 726.5 ppm, respectively. Similarly, mean measured bromine levels in blood were 8.7, 10.0 and 13.3 ppm. No statistical differences were observed between the control and 50 or 500 mg/kg/d groups in bromine levels measured in the liver, fat or kidney. For the purposes of statistical comparison, meausrements from male and female rabbits in each dose group were combined (n=6/group).

Applicant's summary and conclusion

Conclusions:
The NOEL for mortality was 500 mg/kg/d. The NOAEL for systemic toxicity was 500 mg/kg/d.
Executive summary:

The test article was applied dermally at 0, 50, 500 or 5000 mg/kg/d, 5 days/week for 4 weeks. Three male and three female rabbits were used at each dose level. The control rabbits were treated with saline only at a volume of 12 ml on the same regimen as treated animals. The rabbits were observed daily for changes in general behavior and apppearance. Observations for dermal irritation were done prior to and following the 6 hr test article administration period. Individual body weights were recorded weekly. Once in the control group and on study day 14 and 26, blood and urine samples were collected from all rabbits. All rabbits were sacrificed following 28 d of treatment. Selected tissues (approximately 28) in the control and 500 mg/kg/d groups were examined by histopathology (paraffin embeddded, sectioned, stained with hematoxylin and eosin). Skin, liver, kidney and bone marrow from the 50 mg/kg/d group were also examined histologically. All rabbits were sacrificed following 28 d of treatment. Selected tissues (approximately 28) in the control and 500 mg/kg/d groups were examined by histopathology (paraffin embeddded, sectioned, stained with hematoxylin and eosin). Skin, liver, kidney and bone marrow from the 50 mg/kg/d group were also examined histologically.

Very slight to slight and occasionally moderate erythema was noted in control rabbits and in rabbits at the 50 mg/kg/d dose. Very slight to moderate erythema was noted for rabbits at the 500 and 5000 mg/kg/d doses. Moderate desquamation was noted for 3 of the rabbits at the 5000 mg/kg/d dose.

One rabbit at the 5000 mg/kg/d dose showed marked ataxia, unable to lift head or right itself and dyspnea and was sacrificed in extremis. All of the rabbits at the 5000 mg/kg/d dose died or were sacrificed in extremis between days 10 and 26 of the study. Body weights were similar in control, 50 and 500 mg/kg/d groups. Rabbits at the 5000 mg/kg/d dose lost weight prior to death. Organ weights among groups appeared comparable. At day 14, 1 rabbit in the high dose group had a moderate increase in BUN. At day 26, the hematology, serum chemistry and urinalysis results in the one surviving rabbit at the high dose were: neutrophilia with lymphopenia, nucleated erythrocytes, marked increase in glucose and BUN, and albumin in the urine.

Deaths or declining condition necessitating early sacrifice of all rabbits at the high dose were considered compound-related. At necropsy, several rabbits in the high dose group had pale livers, accentuated liver lobulation and gastric irritation. These effects observed at necropsy may have been compound-related. No gross lesions were observed in the low and mid-dose groups. Microscopically, the only lesion seen in animals in the low and mid-dose groups considered compound related was a very slight hyperkeratosis of the application site in one rabbit in the mid-dose group.

The NOEL for mortality was 500 mg/kg/d. The NOAEL for systemic toxicity was 500 mg/kg/d.