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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Non GLP study. Study was limitedly reported and the severity of these effects cannot be judged. Additionally, no caution was taken to prevent oral intake so the exact dose cannot be determined. The methodology was conducted without collar or occlusion to prevent oral intake

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1980

Materials and methods

Principles of method if other than guideline:
The purpose of the study was to screen for possible neurotoxicity. 15 female rates were dermally topically unoccluded exposed to AHTN dose levels of 1, 10, 100 mg/kg bw/day as a 1% ethanolic solution. Observations included mortality, clinical signs, behavioural and motor function and (limited) haematology, serum chemistry, organ weights, macroscopy and histopathology.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
AHTN (purity approximately 99.9%) was analyzed for purity by means of gas chromatography, infrared spectroscopy and nuclear magnetic resonance.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
13 week study: 15 female rats, body weight 156-232 g
26 weeks study: 20 female rats, body weight 156-232 g
Rat strain Crl:COBS CD (SD) BR

Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
Dermal exposure to AHTN was topically unoccluded (gentle inunction to the anterior dorsal shaven skin).
Area of application not reported.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
For dosing purposes, ethanol dilutions of each material were prepared weekly.
Duration of treatment / exposure:
Test materials were applied by gentle inunction to the anterior dorsal (scapular region) shaved skin of each animal once daily, seven days per week. No attempt was made during the treatment period to preclude ingestion.
Frequency of treatment:
one treatment daily
No. of animals per sex per dose:
see table 1 Test conditions
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
Animals were weighed on the first treatment day and weekly thereafter, doses being adjusted correspondingly. Throughout the test period, observations were made daily for any specific overt toxicological signs, including mortality, general appearance, and condition of the skin at the site of application. Once weekly, each animal was further examined with particular attention being paid to any changes in behavioural and motor function.
Positive control:
Acetyl Ethyl Tetramethyl Tetralin (AETT) (neurotoxic)

Examinations

Observations and examinations performed and frequency:
During the seventh and thirteenth weeks of treatment, for animals on the thirteen week exposure regimen, Five rats from each group were selected at random end fasted overnight (16 hours). The following morning, blood samples were obtained from each rat, under light ether anaesthesia, by means of orbital sinus puncture. All animals on the twenty six week exposure regimen had blood samples drawn one week prior to their scheduled interim (week 13) or terminal (week 26) necropsy. The following parameters were evaluated: hemetocrit, hemoglobin, red blood cell count, mean corpuscular volume, fasting blood glucose, urea nitrogen, glutamic oxaloacetic transaminase, glutemic-pyruvic transaminase and alkaline phosphatase.
Sacrifice and pathology:
Two animals from each group on the 13 week exposure regimen were selected for special neuropathological examination based on consistent display of clinical signs of neurotoxicity. In the absence of such signs, animals were randomly selected. At the interim sacrifice of the 26 week study, two animals each from the ethanol control, end 36, 18 and 9 mg/kg AETT groups, were selected for special neuropathological examination. In addition, at the terminal sacrifice, two animals from each group (except untreated controls) were similarly chosen. All other animals at all sacrifice points were subjected to routine necropsy.
For the routine interim and terminal necropsies, animals were fasted overnight, anesthetized with ether and the exsanguinated. The adrenals, brain, heart, kidneys, liver, lungs, spleen and uterus were removed and weighed. The following tissues were fixed in 10% neutral buffered formalin, paraffin embedded, and sectioned and stained with hematoxylin and eosin for microscopic examinations: brain, spinal cord, sciatic nerve, liver and kidney. Animals selected for special neuropathologic examination were anesthetised with sodium barbital and perfused in-situ through the heart with 4% paraformaldehyde followed by 5% buffered glutaraldehyde solution. Brain, spinal cord and peripheral nerves were then stained with osmium tetroxide, embedded in epoxy resin, sectioned and stained with toluidine blue.

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 examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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: neoplastic:
effects observed, treatment-related
Details on results:
Clinical changes
Two treatment related deaths occurred and both were in the 100 mg/kg AETT treatment group. The first was after 28 doses and the second after 69 doses. Both animals suffered severe weight loss and exhibited the blue tissue discoloration and histopathologic changes in the central nervous system characteristic of AETT toxicity
High dose (100 mg/kg) animals in the 13 week exposure regimen from the AETT, Tonalid and Phantolid groups, as well as the 30 mg/kg AETT group, exhibited a significant depression in body weight gain compared to controls. A similar effect was seen at both interim and terminal sacrifice in the 26 week study for animals treated with 36 mg/kg of AETT, Tonalid, as well as with 18 mg/kg AETT. By 26 weeks, the 9 mg/kg AETT group also had reduced body weight gain.

Behavioural treatment-related changes were noted only among animals given 30 mg/kg AETT or higher. These changes consisted of hyperexcitability, aggressive and irritable behaviour, and impaired motor function, the latter characterized by ataxia, wobbly gait and hind limb weakness.

Hematology and serum chemistry
In the 13 week regimen, a significant depression in hemoglobin, hematocrit and red blood cell count was observed. For animals given 100 mg/kg of tonalid at 36 mg/kg also produced a depressed hemoglobin level and red blood cell count.
Serum chemistry changes considered to be clinically significant were observed at the 100 mg/kg dose level (13 week regimen). Both produced elevated serum alkaline phosphatase levels.

Pathology
On gross examination, liver discoloration and liver lobular patterns was observed in animals treated for 13 weeks with 100 mg/kg. Similar observations were made in the 36 mg/kg treatment group after 26 weeks of treatment.
At all AETT dose levels down to 9 mg/kg, the characteristic blue discoloration of brain, spinal cord, peripheral nerves, subcutaneous tissues and interne organs was observed. No discoloration was observed at the lower dose levels even after 26 weeks of treatment. The only significant organ weight changes after 13 weeks of treatment were relative and absolute liver weight increases in the 100 mg/kg group. After 26 weeks, animals treated with Tonalid at 36 and 18 mg/kg also demonstrated elevated liver weights.

Microscopically, animals treated for 13 weeks with 100 mg/kg exhibited liver changes consisting of minimal to moderate degrees of hepatocytomegaly and minimal to moderate deposition of an iron positive pigment. A similar, but less pronounced effect, was observed in animals treated for 26 weeks at 36 mg/kg.
No microscopic changes were observed in the nervous tissues of animals treated at any of the dose levels employed. Animals treated with AETT at levels of 130 or 10 mg/kg for 13 weeks, and those treated for 26 weeks et levels of 36, I8 or 9 mg/kg, exhibited the previously reported changes in nervous system tissues. These consisted of vacuolation of brain, spinal cord and peripheral nerves as well as neuronal demyelination and granule deposition.

Effect levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Clinical and pathological neurotoxicity was present in the positive control, but no evidence could be found in the AHTN in both the 13 and 26 weeks study at any dose level. The NOEL of 18 mg/kg/day is unreliable, since it was not possible to accurately determine the dose of exposure.
Executive summary:

In two subchronic dermal toxicity studies to screen for neurotoxicity, rats were topically and unoccluded exposed to dose levels of 1, 10 and 100 mg AHTN /kg bw/day during 13 weeks and 0, 9, 18 and 36 mg AHTN /kg bw/day during 26 weeks in a 1% (w/v) ethanolic solution. Untreated controls, a vehicle control and a positives control, Acetyl Ethyl Tetramethyl Tetralin (AETT), were included. Observations included mortality, clinical signs, behavioural and motor function and (limited) haematology, serum chemistry, organ weights, macroscopy and histopathology.

A significant depression in body weight gain was seen in the 100 mg/kg/day, 13-week group and “a similar effect” was reported at interim and terminal sacrifice in the 26-week study at 36 mg/kg bw/day. A significant depression in haemoglobin, haematocrit and red blood cell count was reported for animals given 100 mg/kg bw/day in the 13-week study and a depressed haemoglobin level and red blood cell count at 36 mg/kg bw/day in the 26-week study. Elevated serum alkaline phosphatase was also seen at 100 mg/kg bw/day. Liver discoloration and prominent liver lobular patterns were observed in animals treated with 100 mg/kg bw /day and “similar effects” were reported at 36 mg/kg bw/day after 26 weeks. Relative and absolute liver weight increases were seen at 100 mg/kg bw/day. After 26 weeks, animals treated with AHTN at 36 and 18 mg/kg bw/day also demonstrated elevated liver weights but it is not stated whether these were relative, absolute or even statistically significant. In the 100 mg/kg/day, 13-week group moderate degrees of hepatocytomegaly and minimal to moderate deposition of an iron positive pigment were observed and “a similar, but less pronounced effect” was reported for the 36 mg/kg bw/day group. For none of these effects is any quantitative data given; thus, it is impossible to determine the severity of the effects or any dose response. However, given the consistency of effects at 100 mg/kg bw/day at 13 weeks and 36 mg/kg bw/day at 26 weeks, along with what appears to be related to the dose, these treatments must be considered to cause adverse effects. The problem is that the studies were limitedly reported and the severity of these effects cannot be judged. Additionally, no caution was taken to prevent oral intake so the exact dose cannot be determined. The most serious omission is data on the increased liver weight at 18 mg/kg for 26 weeks. If this is a mild adaptive effect not associated with any histopathology as it appears, then it could be argued that 18 mg/kg is the NOAEL. There is still the problem, however, of not being able to accurately determine the dose of exposure and the route of intake (dermal or oral). Clear evidence of neurotoxicity, both clinically and pathologically, was seen with the positive control but no such evidence for AHTN was seen in either study at any dose level.

Source: EU Risk Assessment Report AHTN, May 2008, ECB