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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study which meets basic scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 39 to 42 days
- Weight at study initiation: 59.2 - 85.4 g (males), 57.0 - 79.3 g (females)
- Fasting period before study: No
- Housing: Individual in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 35 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: an area extending from the animal's mid-back to dorsal interscapular region
- Time intervals for shavings or clipplings: at least 24 hours prior to initial dose, and once weekly thereafter


REMOVAL OF TEST SUBSTANCE
No data


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0, 1.0, 0.5, 0.25, 0.125 and 0 g/kg bw
- Concentration (if solution): 1120, 560, 280, 140, 70 and 0 mg/ml
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
125; 250; 500; 1000; 2000 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
20; 10 special study animals (designated for periodic urinalysis, hematology, and clinical chemistry determinations), 10 base study animals (subject to the collection of clinical observations data, sperm morphology and vaginal cytology evaluations, necropsy with gross examination and tissue collection, and histopathologic examination).
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: no
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION: No


FOOD EFFICIENCY: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 11
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters examined were erythrocyte count (RBC), hemoglobin (HgB), hematocrit (HCT), leukocyte count (WBC), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leukocyte differential count, reticulocyte count, erythrocyte and platelet morphology.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 11
- Animals fasted: No data
- How many animals: All
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
The following tissues were examined: gross lesions and tissue masses (and regional lymph nodes, if possible), blood smear (if required by the pathologist), mandibular and mesentric lymph nodes, salivary gland, sternebrae, femur, or vertebrae, including marrow, thyroids, heart, esophagus, stomach (to include forestomach and glandular stomach), uterus, brain (three sections, including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), thymus, trachea, parathyroids, small intestine (duodenum, jejunum, ileum), cecum, colon and rectum, liver, prostate, testis, epididymis, seminal vesicle, ovaries, lungs and mainstem bronchi, nasal cavity and nasal turbinates (3), preputial or clitoral glands (paired), pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurologie signs were present), eyes (if grossly abnormal), mammary gland (including surface skin), pharynx (if grossly abnormal), skin (lesions in dosed area, unaffected skin in dosed area, and undosed control skin).

Complete histopathologic evaluation of all tissues listed was performed on all base study rats from the 2.0 and 0 g/kg dose groups. On the basis of those findings, the skin at the site of application (both males and females) and the kidney (females only)were selected as the target tissues. These tissues were examined in rats in successively lower dose groups to a no-effect level; in addition, any gross lesions detected at necropsy were examined microscopically.
Other examinations:
Sperm morphology and vaginal cytology were evaluated in the control and the three highest dose groups.

Results and discussion

Results of examinations

Details on results:
BODY WEIGHTS
The 2.0 g/kg male dose group exhibited a pronounced depression in body weight gain; the differential weight gain, relative to control, for this group was -32.8 percent, whereas those for the other male dosed groups ranged from -4.8 to +8.4 percent. The mean weight gains for all female dosed groups were depressed when compared with that of the female control group; the differential weight gains (relative to control group weight gain) for the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg female dose groups were -13.1, -10.6, -6.0, -21.4, and -35.9 percent, respectively.

MORTALITY
All animals in all dose groups survived until scheduled termination.

CLINICAL OBSERVATIONS
The only clinical abnormalities associated with treatment with triethanolamine occurred at the site of dermal dose application. Discoloration of the skin was seen in males from all dose groups (including control); it was present in 6 males in the 0 g/kg dose group, and in all 10 base study animals in each of the dosed groups. Irritation at the site of application was seen in the three highest triethanolamine dosed groups (0.5, 1.0, and 2.0 g/kg), with incidence increasing and time to onset decreasing with increasing dose level. Scaliness was observed only at the 1.0 and 2.0 g/kg levels; this was observed in 1 rat in the 1.0 g/kg group (first seen on Day 13), and in 5 rats in the 2.0 g/kg group (first seen on Day 6). Crustiness at the site of application was recorded for 10 rats in the 2.0 g/kg dose group; two males from this group were also observed to have ulceration at the application site. Other clinical abnormalities occurred sporadically.
All treatment-related abnormalities in females were seen at the site of dermal dose application. Irritation was observed at the 1.0 (7 rats) and 2.0 (10 rats) g/kg dose levels; scaliness was also seen in female rats from these groups (4 animals dosed at the 1.0 g/kg level, and 5 animals dosed at the 2.0 g/kg level). Crustiness was present in 3 female rats dosed at 2.0 g/kg. Other abnormalities were determined not to be compound induced.

NECROPSY
Gross observations
Gross lesions were common on the skin of high dose rats. The compound-related lesion seen at the application site was dermal crust. Yellow skin was seen in the lumbar region of treated and control animals. This lesion had no corresponding microscopic lesion and is attributed to application of the vehicle (acetone). Other gross lesions were considered incidental, spontaneous lesions.

Final body weight
The mean final body weights for both male and females dosed at 2.0 g/kg body weight were significantly decreased (p <0.01), compared with those of their respective control groups.

Organ weights
The weights of the brain, right kidney, spleen, thymus, liver, lung, heart, right and left testes, and right and left epididymides were recorded at the scheduled necropsy of base study animals.
The mean brain to body weight values for the 2.0 g/kg male and female dose groups were significantly increased relative to control. In the absence of statistical changes in the absolute brain weight for these groups, this increase in relative weight appears to be the result of depressed final body weights in rats dosed at 2.0 g/kg.
Statistically significant increases in mean right kidney absolute weights and weights relative to brain weight occurred in both males and females dosed at 1.0 and 2.0 g/kg. The right kidney to body weight ratios of the 0.25, 0.5, 1.0, and 2.0 g/kg male dose groups and the 1.0 and 2.0 g/kg female dose groups were also significantly increased over that of control.
The mean spleen absolute weight and spleen weight relative to brain weight of the 2.0 g/kg female dose group were significantly decreased, relative to control values. There was a significant increase in the mean spleen to body weight ratio in male rats dosed at 1.0 and 2.0 g/kg. Thymus absolute weight and weight relative to brain weight were increased in males dosed at 2.0 g/kg body weight.
Mean liver to body weight ratios for the 0.5 and 1.0 g/kg male dose groups were significantly elevated over that of control. No statistically significant changes were observed in mean liver weight values for the female dose groups.
The mean lung absolute weight and lung to brain weight ratio of the 2.0 g/kg male dose group were significantly decreased, relative to control. The mean lung to brain weight of the 0.5 g/kg male dose group was also significantly decreased. The only statistically significant change in mean lung weight values for female rats was increased lung to brain weight ratio at the 0.125 g/kg level.
No statistically significant changes were seen in mean heart weight data for male and female rats.
For both testes, there was a significant increase in weight relative to body weight in the 2.0 g/kg male dose group; this was a result of decreased body weight at necropsy.
No statistical changes were observed for mean left epididymis weight values. The 2.0 g/kg male dose group exhibited decreased right epididymis absolute weight, and increased weight relative to body weight. Right epididymis to body and to brain weight ratios were increased in males dosed at 0.25 g/kg.

CLINICAL PATHOLOGY
Hematology
Hematology was performed on blood obtained from special study rats on study Day 80. Male rats dosed at 2.0 g/kg exhibited a significantly increased leukocyte count and decreased mean corpuscular volume. Significantly decreased MCV was also exhibited by the 2.0 g/kg female dose group; the hematocrit of this group was depressed as well.
Male rats in the 2.0 g/kg dose group exhibited significant increases in both relative and absolute number of segmented neutrophils and eosinophils, and a significant reduction in the relative number of lymphocytes. Female rats treated at the same dosage level (2.0 g/kg) also exhibited a significant increase in relative and absolute numbers of segmented neutrophils, as well as a decrease in relative lymphocyte count. These hematological changes in high dose rats of both sexes can be attributed to an inflammatory response resulting from dermal irritation. The mean values for all other dosed groups of both sexes were statistically similar to control.

Clinical chemistry
The 2.0 and 0.25 g/kg male dose groups exhibited elevated SGOT levels; the mean SGPT level of the 2.0 g/kg male dose group was also significantly higher than control. No other statistically significant changes were seen when mean data from the male dosed groups were compared with that of control. The 2.0 g/kg female dose group exhibited elevated serum urea nitrogen, albumin, SGOT, and SGPT. The only other significant change in the female group mean data was a decrease in serum sorbitol dehydrogenase in rats dosed at 0.5 and 1.0 g/kg.

Urinalyses
Urine, collected from special study group rats on Days 3, 16, 44, and 86, was analyzed.
At study Day 3, mean values for all male dosed groups were statistically similar to those of the male control group. At study Day 16, the only values statistically different from control were urine protein at the 1.0 g/kg level, and the number of leukocytes at the 0.25 g/kg level. By study Day 44, there was a significant reduction in urine protein levels in males dosed at 0.5, 1.0, and 2.0 g/kg; in addition, the specific gravity of urine collected from the 2.0 g/kg male dose group was significantly increased over control. At the final urinalysis (Day 86), the specific gravity of urine from rats in the 2.0 g/kg dose group was again significantly elevated, and the urine protein levels for the 0.5, 1.0, and 2.0 g/kg male dose groups were significantly lower than control. The only other parameter statistically different from control was increased urine volume in males dosed at 0.5 g/kg.
For female rats, there was decreased urine protein concentration at the 0.25 g/kg level and above at the Day 3 urinalysis; the number of crystals found in the urine of rats in the 2.0 g/kg dose group was also significantly greater than control. Increased number of crystals was also present, in females dosed at 1.0 and 2.0 g/kg, on Day 16. Beginning on Day 16, and continuing through Days 44 and 86, the specific gravity of urine from female rats from these two highest dose groups was significantly greater than that of control females. The only other statistically significant changes observed at Day 44 were increased urine protein concentration in females dosed at 0.5 g/kg, and increased number of crystals in females dosed at 2.0 g/kg. At Day 86, in addition to the increased urine specific gravity in the two highest dose groups, the 2.0 g/kg females exhibited an increase in urine glucose concentration.

HISTOPATHOLOGY
The compound-related skin lesions were chronic-active inflammation and acanthosis, and were seen in 2.0, 1.0, 0.5, and 0.25 g/kg male rats and in 2.0, 1.0, and 0.5 g/kg female rats. The chronic-active inflammation contained epidermal and dermal components. The epidermal component was characterized by acanthotic, hyperkeratotic, focally parakeratotic epidermis that occasionally contained rete pegs; these changes were graded separately as acanthosis. Severely affected epidermis contained focal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. The dermal component in severely affected rats was characterized bydermal fibrosis, neocapillarization, minimally distorted adnexal organs, and variably severe mixed inflammatory infiltrates consisting of histiocytes, lymphocytes, neutrophils and eosinophils. Although only the most severe grades of chronic-active inflammation contained all of the previously listed epidermal and dermal features, the lesion was regarded as a continuum, and, therefore, the morphology "chronic-active inflammation" was used in all dose groups. Less severe lesions of chronic-active inflammation had reduced acanthosis, and lacked one or more of the following components: epidermal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. Additionally, less severe lesions contained fewer of the dermal features of the most severe lesion. In minimal inflammatory lesions the epidermal component was predominant. Where only the term acanthosis was used, the least severe lesion contained 2-3 layers of epidermal cells above the basilar layer. Occasionally, there was minimal to mild dermal fibrosis associated with the acanthosis. In males and females there was a dose dependent reduction in severity and incidence of skin lesions from high to no-effect dose.
Non-compound related microscopic lesions were seen in the kidney, liver, lung, prostate, ovary, preputial gland, clitoral gland, eye, Zymbal's gland, nose/nasal cavity; and mediastinal lymph node.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
250 mg/kg bw/day
Sex:
female
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: increased relative kidney weight
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
500 mg/kg bw/day
Sex:
female
Basis for effect level:
other: increased absolute and relative kidney weight

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The compound-induced skin lesions in males and females were chronic-active inflammation and acanthosis. The incidence, severity, and

morphology of the lesions were similar between sexes. In males, however, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding.

Applicant's summary and conclusion