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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
December 2006 - March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Dermal application without covering. Oral exposure due to grooming has occurred.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
The dermal applied dose is not covered, grooming and oral exposure has occurred.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: F344/NTac
Details on species / strain selection:
For many years, the NTP used the inbred F344/N rat for its toxicity and carcinogenicity studies. However, this model has exhibited sporadic seizures, idiopathic chylothorax, and consistently high rates of mononuclear cell leukemia and testicular neoplasia. Therefore the F344 rat from the Taconic commercial colony (F344/NTac) was chosen as a more fecund rat model that could be used in both reproductive and carcinogenesis studies for comparative purposes.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female F344/NTac rats were obtained from the commercial colony at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the rats were 5 to 6 weeks old
- Weight at study initiation: 82-84 g (female) 83-86 g (male)
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hardwood bedding, changed weekly, omnischield papaer cage filter changed every 2 weeks, Stainless steel Racks changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 (males) or 13 (females) days

DETAILS OF FOOD AND WATER QUALITY:
Diet Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water Tap water (Washington Suburban Sanitary Commission, Potomac Plant) via automatic watering system (Edstrom Industries, Inc., Waterford, WI)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F (22° ± 1.668° C)
- Humidity (%): 50% ± 15%
- Air changes (per hr): ≥ 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day, 12 hours/night

IN-LIFE DATES:
Date of First Dose: December 12 (males) or December 13 (females), 2006
Necropsy Date: March 13 (males) or March 14 (females), 2007


Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: dorsal surface just posterior to the scapulae to the base of the tail
- % coverage: approximately 10% of body surface area (corresponds to approximately 25 cm2)
- Time intervals for shavings or clipplings: 1 week (shaved)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL/kg body weight (rats)
- Concentration (if solution): 6.25%, 12.5%, 25%, 50% or 100% (neat) OTNE
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Purity: USP-grade 95% ethanol

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not used

CALCULATION OF LOCAL EXPOSURE: average body weight (kg) * exposure concentration (µg/kg bw/day) / surface area (25 cm2)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed three times by the study laboratory using GC/FID by analysis. All 30 of the dose formulations analyzed for rats were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; 28 of 30 were within 10% of the target concentrations.
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Remarks:
100% OTNE
Dose / conc.:
250 mg/kg bw/day
Remarks:
50% OTNE
Dose / conc.:
125 mg/kg bw/day
Remarks:
25% OTNE
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
12.5% OTNE
Dose / conc.:
31.25 mg/kg bw/day
Remarks:
6.25% OTNE
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Probably based on the information of the 28-day oral gavage study.
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily, histopathology at end of study period

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed and clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

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

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: hemoglobin; erythrocyte, reticulocyte, and platelet counts; erythrocyte distribution of width; mean platelet volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Animals fasted: No
- How many animals:10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- gross observation for evidence of disease, the health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program
HISTOPATHOLOGY: Yes
- Histopathology: adrenal gland, bone (femur) with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, oral cavity and larynx, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin (site of application and control sites), spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
- Sperm Motility and Vaginal Cytology: At the end of the studies, sperm samples were collected from male rats in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per gram testis and per testis, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 16 consecutive days prior to the end of the studies from female rats in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for vaginal cytology evaluations.
- Liver Cytochrome P450 Activity: Liver samples were collected from five male and five female special study rats per dose group on day 23 and from five male and five female core study rats per dose group at the end of the studies. Livers were analyzed for total microsomal protein content and CYP2E1 activity.
Statistics:
- The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance between dosed and vehicle control groups and between the 100% OTNE and untreated control groups.
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epididymal spermatozoal data, (skewed distributions) were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus, were constructed based on a Markov chain model proposed by Girard and Sager (1987). Equality of transition matrices among dose groups and between the control group and each dosed group was tested using chi-square statistics.
- A t-test was used to determine significant differences in organ and body weight data, and Wilcoxon’s (1945) rank sum test was used for hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epidymal spermatozoal data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no biologically significant clinical findings related to OTNE administration
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Suppurative inflammation of the dermis in 1/10 Male rats treated with 100% OTNE, and 1/10 Female rats treated with 100% OTNE.
Other skin effects are reported under histopathological findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight gains of dosed (except 100% OTNE) males and females were similar to those of the respective control groups. The mean body weight gain of 100% OTNE males was significantly less than that of the untreated control group.
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:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in the hematology parameters attributable to the dermal administration of OTNE
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum alkaline phosphatase activities were decreased in various male and female dosed groups at all time points (only average value, incidence not given): days 3 (100% (500 mg/kg bw OTNE) and 23 (50% (250 mg/kg bw) and 100% OTNE) and week 13 (all dose groups). Decreases were consistently observed in 100% OTNE males and females at all time points compared to the untreated control groups.
At the end of the study (week 13), alanine aminotransferase activities were decreased in 25% (125 mg/kg bw) OTNE or greater females and in all dosed groups of males compared to their respective control groups (only average value, incidence not given). The significance of these decreases is not known but may be related to an alteration in liver metabolism. Other changes in clinical chemistry results were not considered toxicologically or biologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the twice-daily observations, the behavior of the animals did not indicate any neurotoxic potential of the substance.
Immunological findings:
not examined
Description (incidence and severity):
Not specifically examined, however the obtained results did not indicate any immunotoxic potential of the substance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Only average value provided, incidence is not given:
Increased absolute (males 100% OTNE , females 50% and 100% OTNE) and relative (50% and 100% OTNE males and females) liver weights
Increased relative kidney weight (males and females 100% OTNE)
The concentrations 25, 50 and 100%, relates to 120, 250 and 500 mg/kg bw.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
Not specifically examined, however the behavior of the animals during the study was normal and did not indicate any neurotoxic potential of the substance. Furthermore, no histopathological abnormalities were observed in the brain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Skin (site of application):
- hyperkeratosis (mild to minimal):
1/10 in the 6.25% (31.25 mg/kg bw/day) group;
3/10 in the 12.5% (62.5 mg/kg bw/day) group;
4/10 in the 25% (125 mg/kg bw/day) group;
5/10 in the 50% (250 mg/kg bw/day) group;
8/10 in the 100% (500 mg/kg bw/day) group;

- hyperplasia (mild to minimal)
2/10 in the 31.25 mg/kg bw/day group;
4/10 in the 62.5 mg/kg bw/day group;
5/10 in the 125 mg/kg bw/day group;
8/10 in the 250 mg/kg bw/day group;
10/10 in the 500 mg/kg bw/day group;

Other treatment-related histopathologic, toxicologically relevant, changes were not observed in the organs of male or female rats
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Male rats did not display any OTNE-related changes in testis and epididymis weights or sperm parameters. Female rats administered OTNE did not display any toxicologically significant effects on cycle length, or estrous cyclicity.

Effect levels

open allclose all
Dose descriptor:
other: NOAEC
Remarks:
Local
Effect level:
6.25 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Dermal effects are confounded by grooming
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Systemic effects are confounded by grooming

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
12.5 other: %
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

The effects reported in this study need to be evaluated in light of the used route of exposure. The study was intended as a sub-chronic dermal study; however, the animals were dermally exposed to OTNE in ethanol without occlusion of the treated site. Oral absorption (voluntarily or involuntarily), is expected as a result of oral grooming by the animals. In the present study, the grooming could have a contributed to an unknown extent to the systemic exposure and toxicity. Furthermore, also the local effects observed as a result of dermal exposure are confounded, a , as grooming by the animals can have influenced the actual dermal dose, dermal exposure time and could have given te opportunity for the animals to scratch, rub or lick the exposed site. As a result of these methodological limitations, the derivation of a dose descriptor for systemic or local effects due to dermal exposure is deemed inappropriate.

Applicant's summary and conclusion

Conclusions:
As a result of methodological limitations of this dermal repeated dose toxicity study (confounding of exposure route due to oral grooming), the derivation of a dose descriptor for systemic or local effects is deemed inappropriate by the assessor. Nevertheless, the syetemic (hepatic) effects at the high doses (>=500 mg/kg bw) support the findings in the two oral repeated dose toxicity studies.
Executive summary:

Introduction: In the rat study, OTNE was tested in a sub-chronic dermal toxicity study according to OECD Guideline 411. The study is rated Klimisch 2, because the substance was administered through dermal application without coverage resulting in oral exposure (due to grooming), which is not in line with the required coverage according to the guideline and a real systemic dermal NOAEL cannot be derived.

Method: Male and female F344/NTac rats were administered OTNE dermally for 3 months. Groups of 10 male and 10 female rats received no treatment (untreated control) or dermal application of OTNE in 95% aqueous ethanol at concentrations of 0% (vehicle control), 6.25%, 12.5%, 25%, 50%, or 100% (neat) 5 days per week for 3 months (31.25, 62.5, 123, 250 and 500 mg/kg bw). Animals in the 100% OTNE groups were compared to untreated controls, and the remaining dose groups were compared to the vehicle controls. Formulations were administered at a volume of 0.5 mL/kg body weight, on approx. 10% of body surface area (25 cm2).

Results:

Local dermal effects: For the local irritant effects the dose in concentration will be used because concentration is a better indicator for irritancy than the dose. In male and female rats administered 12.5 to 100% OTNE, the incidences of minimal to mild hyperplasia and hyperkeratosis (except in 12.5% OTNE males) at the site of application were significantly greater than those in the vehicle control groups. The dermal exposure and effects are confounded for the following reasons: 1) grooming by the animals decreased the actual dermal dose; 2) dermal exposure time is decreased and; 3) the scratch, rubbing and licking of the exposed site may have enhanced the dermal penetration. In view of these confounders a dose descriptor for local effects via the dermal exposure route is deemed inappropriate.

Systemic effects

Clinical signs and body weight: All rats survived to the end of the study. There were no biologically significant clinical or behavioural effects. Final mean body weights and body weight gains of dosed male and female rats were similar to those of the respective control groups with the exception of the 500 mg/kg bw/day OTNE male rats, which had a statistically significant but minor decrease in mean body weight gain (-6%) compared to the untreated control.

Clinical chemistry: Alanine aminotransferase and Alkaline phosphatase are significantly decreased becoming dose depending from 125 mg/kg bw onwards (starting with a decrease of 25%) in all dosed groups of males and in 125 to 500 mg/kg bw/day OTNE females. In general, the toxicological relevance of a decrease of these enzymes is generally doubtful but it is consistently seen.

Organ effects:

Liver: Relative liver weights at 250 and 500 mg/kg bw/day OTNE were significantly increased (> 10-31%) compared to the control. At 250 mg/kg bw this increase is considered adaptive at 500 mg/kg bw these may be considered adverse. 

Kidney: The relative kidney weights of the 500 mg/kg bw/day OTNE male and female rats were significantly increased by 10 and 8%, respectively. These increased are not considered adverse.

Other effects: There were no neurotoxic and immunological effects seen. There were no biologically significant neoplastic findings found.

Conclusion

A local long-term repeated dose NOAELcould not be established because the concentration to which the animals are exposed doubtful due to oral grooming

A systemic dermal repeated dose NOAELcannot be established because beside dermal exposure the oral exposure route is a major route e.g. the relative liver weights in the oral and dermal studies is similar increased (ca 50% at 500 mg/kg bw) in the oral 90-day IFF study as in this NTP study. Only a combined systemic dermal/oral NOAEL can be established. This can be set at 250 mg/kg bw considering the relative liver weight increase of < 30% as an adaptive effect in absence of other liver related findings of toxicological relevance.