Registration Dossier

Toxicological information

Repeated dose toxicity: dermal

Currently viewing:

Some information in this page has been claimed confidential.

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: K2 since geing used for read-across
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose:
read-across source
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Based on presence of mildly increased liver enzymes at 1000 mg/kg/day.
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
Only male rats were tested. Clinical Pathology – some parameters for haematology and serum chemistry were not examined. Pathology parameters – some tissues were not collected or weighed.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
yes
Remarks:
Only male rats were tested. Clinical Pathology – some parameters for haematology and serum chemistry were not examined. Pathology parameters – some tissues were not collected, weighed, or given histopathology examination.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: not reported as such

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR rats
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 56 days old, approximately
- Weight at study initiation: Male Rats – 238.9-305.3 grams
- Fasting period before study: None
- Housing: Stainless steel, wire-mesh cages suspended above cageboards, individually.
- Diet (e.g. ad libitum): Ad libitum, except when fasted
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Quarantined for 6 days of 14-day pretest period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: deionized water for control treatment only
Details on exposure:
All of the test substance administered was assumed to be available for absorption. For the purposes of this study, all toxic effects were reported as a function of the administered dosage.

TEST SITE
- Area of exposure: The area to be treated, the skin from the back and trunk of each rat, was marked on the back of each rat with a water-insoluble marker. On each day of treatment, the rats wore plastic collars during the exposure period to prevent ingestion of the test substance and disruption of the wrappings.
- % coverage: The approximate total body surface area covered for the 0, 10, 100, and 1000 mg/kg/day groups was 1.2%, 0.1%, 0.3%, and 1.5%, respectively. The volume of neat test substance or vehicle was not sufficient to cover 10% of the total body surface area. The test substance was applied evenly and as thinly as possible to the test site.
- Type of wrap if used: The test substance was covered with a 2-ply porous gauze dressing followed by successive layers of stretch gauze (no more than 8 layers) and self-adhesive bandage.
- Time intervals for shavings or clippings: On the day prior to the first treatment, the fur of each rat was closely shaved to expose the skin from the back and trunk. The animals were reshaved as needed during the study to facilitate the evaluation of dermal effects. The entire area that was originally shaved (including the untreated control skin) was reshaved. The animals were shaved only after an evaluation.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the exposure period, the collars and wrappings were removed and the test site was wiped with a wet paper towel and washed with warm tap water. The test site of each rat was then gently patted dry and the rat was returned to its cage. Control animals received the same washing technique as the treated animals.
- Time after start of exposure: The exposure period was approximately 6 hours.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Groups of 10 male rats were treated at dosages of 0, 10, 100, or 1000 mg of test substance per kg body weight. The amount of neat test substance needed to treat each rat was based on the most recently determined body weight measurement and the test substance density of 1150 mg/mL. Each day of dosing, the control rats received the same volume of deionized water as the high-dose treatment group.

VEHICLE
Deionized water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Daily for approximately 6 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 100, 1000 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A range-finding study was conducted to aid in dosage selection for the 28-day dermal study. Groups of 2 male rats received dermal applications of test substance at dosages of 10, 60, 500, or 1000 mg/kg for 6 hours for 4 consecutive days. No dermal irritation was observed, and no test substance-related clinical signs or body weight losses occurred. Based on the lack of toxicity observed in the range finding study, dosages of 0, 10, 100, and 1000 mg/kg/day were selected for the 28-day study.
- Rationale for animal assignment (if not random): Forty rats were selected for study use on the bases of adequate body weight gain, freedom from clinical signs of disease or injury, and a body weight within ± 20% of the mean. The selected rats were divided by computerized, stratified randomization into 4 groups of 10 rats, so that there were no statistically significant differences among group body weight means.
- Rationale for selecting satellite groups: Total Fluorine and Perfluorooctanoic Acid Level Evaluations were conducted on the first 5 rats in each group.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

GENERAL CAREFUL CLINICAL OBSERVATIONS
- Time schedule: At each weighing

DERMAL IRRITATION
- Time schedule: After removal of the test substance. The Draize Scale was used to score skin irritation. Adjacent areas of untreated skin were used for comparison.

BODY WEIGHT: Yes
- Time schedule for examinations: At 3-4 day intervals

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Once per week
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: Yes
- Time schedule for examinations: Once per week

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: Test day 29
- Anaesthetic used for blood collection: Yes, Carbon dioxide anesthesia
- Animals fasted: Yes
- How many animals: All rats
- Parameters in Table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: Test day 29.
- Animals fasted: Yes
- How many animals: All rats.
- Parameters in Table 2 were examined.

URINALYSIS: Yes.
- Time schedule for collection of urine: Test day 29.
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: Yes
- How many animals: All rats.
- Parameters in Table 3 were examined.

TOTAL BLOOD FLUORINE EVALUATIONS
- Time schedule for collection of blood: test days -4, 3, 9, and 21, approximately one hour after removal of the test substance.
- How many animals: First 5 rats in each group.
Blood was collected into EDTA tubes from the orbital sinus of the rats. The blood was refrigerated.
The total fluorine content of the day 21 blood samples was determined; the remaining samples were not analyzed.
- Time schedule for collection of blood: At study termination at necropsy.
- How many animals: All rats.
Additional blood was collected from the vena cava of all rats into a tube containing EDTA. Plasma was prepared and stored frozen at 80°C to 20°C.
Sacrifice and pathology:
SACRIFICE: Carbon dioxide asphyxiation and exsanguination.
GROSS PATHOLOGY: Yes. (see Table 4 for tissues collected).
ORGAN WEIGHTS: Yes. (see Table 4 for tissues weighed).
HISTOPATHOLOGY: Yes. (see Table 4).
Statistics:
See Table 5.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical signs resulting in systemic toxicity occurred. Red ocular discharge was observed in all rats during the study. Red nasal discharge and swollen face were observed in rats in all dose groups. These clinical signs are considered to be due to the wrapping/collar application procedure. Superficial wounds and hair loss were present in both treated and control animals and were not considered test substance-related. The observation of dark eye in one rat treated at 100 mg/kg was short in duration, was not observed in any other rats, and therefore is not considered to be test substance-related.
No dermal irritation was observed during the study.
No deaths occurred.

HAEMATOLOGY
There were no adverse changes in hematologic parameters in males. The following statistically significant changes in mean hematologic parameters were considered to be non-adverse or not related to exposure to the test substance:
Mean red cell parameters (red cell count, hemoglobin, and hematocrit) were similarly decreased in males dosed with 10, 100, or 1000 mg/kg/day (statistically significant for red cell count at 10 mg/kg/day, and for hemoglobin and hematocrit at 100 and 1000 mg/kg/day). There was no dose-relationship to the changes, despite the 100-fold range of doses. There were no correlative changes in other red cell parameters (reticulocytes, indices, or morphology) that would be expected with processes that alter red cell mass. In addition, red cell mass parameters for the three dosed groups were similar to that of the control groups on a recent dermal study. It is likely that the apparent decrements in red cell mass were due to higher than usual red cell mass in the control group, rather than a direct effect of the compound. These changes were considered to be unrelated to treatment.
Eosinophils were minimally increased in males dosed with 1000 mg/kg/day (147% of control group mean). Due to the consistency of change, this increase was likely to be due to treatment. There were no other alterations in leukocyte counts, and no correlative histologic changes. In addition, eosinophils counts of all individual rats dosed with 1000 mg/kg/day were within the range of the control group counts. Although this change was possibly related to treatment, it was considered to be non-adverse.
Clinical Chemistry
Aspartate and alanine aminotransferase activities were minimally (aspartate aminotransferase) to mildly (alanine aminotransferase) increased in some males dosed with 100 or 1000 mg/kg/day. In these groups, mean activities were statistically significant, and were 133-144% of control for aspartate aminotransferase, and 200-222% of control for alanine aminotransferase. In individual affected rats, generally activities of both enzymes were elevated. Sorbitol dehydrogenase was also increased in affected rats in both groups, although mean activities were not statistically different from the control mean. The presence of increased transaminases and sorbitol dehydrogenase indicates hepatocellular injury. Histologically, there were no alterations in the liver. Due to the magnitude of the change, this effect was considered to be potentially adverse.

CLINICAL CHEMISTRY
There were no other adverse changes in clinical chemistry parameters. The following statistically significant changes in mean clinical chemistry parameters were considered to be non-adverse or not related to exposure to the test substance:
Alkaline phosphatase was minimally increased in males dosed with 1000 mg/kg/day (mean was 136% of control group mean). This change was possibly related to treatment. The magnitude of change was very small. There were no alterations in correlative clinical or anatomic pathology parameters. Increased alkaline phosphatase activity did not occur in the same rats that had increased transaminase and sorbitol dehydrogenase activities. Although this change was possibly related to treatment, it was considered to be non-adverse.
Bilirubin was statistically and minimally increased in males dosed with 1000 mg/kg/day (133% of control group mean). Mean and individual bilirubin concentrations for this group (mean: 0.12 mg/dL, individual values: <0.10 to 0.16 mg/dL) were well within the historical age-matched control range (control mean range: 0.09-0.29 mg/dL; range of individual animals: 0.07-0.33 mg/dL). Based on individual rat data compared to the control and other treated groups, this change was unlikely to be related to treatment. The magnitude of change was very small, and non-adverse.
Cholesterol was minimally increased in males dosed with 10, 100, or 1000 mg/kg/day. Although this change may have been related to treatment, the magnitude of change was very small and was not expected to result in adverse events. This change was considered to be non-adverse.
Triglycerides were minimally increased in males dosed with 10, 100, or 1000 mg/kg/day (147%, 150%, and 187% of control group means; statistically significant only at 1000 mg/kg/day). Increased triglycerides were likely to be treatment-related. The magnitude of change was very small and was not expected to result in adverse events. This change was considered to be non-adverse.
The following statistically significant change in mean clinical chemistry parameters was considered to be non-adverse because it was unrelated to dose: Minimally decreased albumin in males dosed with 100 mg/kg/day.

URINALYSIS
There were no adverse changes in urinalysis parameters. The following statistically significant changes in mean urinalysis parameters were considered to be non-adverse:
Urine osmolality was mildly decreased (62% of control group means) in males dosed with 1000 mg/kg/day. This was associated with an appropriate tendency (not statistically significant) towards minimally higher urine volumes in this group. There were no alterations in other renal parameters on this study. The presence of decreased osmolality with increased urine volume, in the absence of other changes in renal parameters, has no toxicologic significance. This change was considered to be non-adverse.
Urine protein concentration was decreased in males dosed with 1000 mg/kg/day. Decreases in urine protein concentration were secondary to increased urine volume. Decreases in urine protein have no toxicologic significance. This change was considered to be non-adverse.

TOTAL BLOOD FLUORINE ANALYSIS
There was no effect of treatment on plasma fluoride concentration. Urine fluoride (total fluoride excreted overnight) was increased in males dosed with 1000 mg/kg/day. There was some fluorine present in the day 21 blood from rats dosed with the test substance. This indicated exposure to a fluoride-containing compound and is considered to be treatment related but not adverse. Two rats treated at 10 mg/kg/day had fluorine levels below the limit of quantification (LOQ). The remaining 3 rats had levels ranging from 0.52 to 5.96 ppm. The rats treated at 100 mg/kg had levels of fluorine ranging from 0.54 to 0.78 ppm. Two rats treated at 1000 mg/kg/day had fluorine levels below the LOQ. The remaining 3 rats had levels ranging from 1.22 to 1.38 ppm. All values for the control rats were below the LOQ.

Effect levels

Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Based on presence of mildly increased liver enzymes at 1000 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Dermal administration of the test substance to male rats 6 hours/day for 28 days resulted in increased aspartate and alanine aminotransferase activities along with increased sorbitol dehydrogenase activities at 1000 mg/kg/day. The no-observed-effect level (NOEL) was 100 mg/kg/day based on the presence of mildly increased liver enzymes at 1000 mg/kg/day.
Executive summary:

There were no effects on body weight, food consumption, food efficiency, or mortality at any dosage. No clinical signs attributable to systemic toxicity occurred. No dermal irritation was observed during the study.

Some fluorine was present in the day 21 blood from rats dosed with the test substance, indicating exposure to a fluoride-containing compound. This finding was considered to be treatment-related but not adverse.

Organ weights were not different in a dose response fashion in either incidence or severity. Gross observations and microscopic findings occur spontaneously in rats of this strain and age and were not present in a dose response fashion in either incidence or severity.

There were no adverse changes in hematology parameters. Aspartate and alanine aminotransferase activities were increased along with increased sorbitol dehydrogenase activities in rats treated at 1000 mg/kg/day. These increases were considered to be potentially adverse. Urine fluoride was increased in rats treated at 1000 mg/kg/day, indicating exposure to and metabolism of a fluoride-containing compound. This urine fluoride increase was considered to be treatment-related but not adverse.

Based on the presence of mildly increased liver enzymes at 1000 mg/kg/day, the no-observed effect level (NOEL) for the dermal exposure of the test substance under the conditions of this study was 100 mg/kg/day for male rats.