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Basic toxicokinetics

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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
5th May 1987 to 28th August 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Housing: individually in screen bottom stainless steel cages (metabolism cages for the kinetic phase)
- Diet: Purina Rodent Laboratory Chow® #5002 ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 ºF
- Humidity: 50 ± 20 %
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: 5th May 1987 To: 28th August 1987
Route of administration:
other: oral: feed (toxicity phase); oral: gavage (kinetic phase)
Vehicle:
other: corn oil (toxicity study); polyethylene glycol (kinetic study)
Details on oral exposure:
DIET PREPARATION (TOXICOLOGY PHASE)
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Rodent Laboratory Chow® #5002
- Storage temperature of food: refrigerated

PREPARATION OF DOSING SOLUTIONS (KINETIC PHASE):
Radiolabelled test material was dissolved in a minimum amount of hexane and transferred to a tared serum vial. The hexane was evaporated with nitrogen and 18 mL polyethylene glycol 300 was added and the test material dissolved. The amount of radioactivity administered to each animal was calculated from the weight of dose solution (g) and the dose solution radioactivity concentration (dpm/g).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
10 g of feed was weighed into a 250 mL Erlenmeyer flask, 50 mL chloroform added and the flask shaken for 1 hour. Chloroform extract was vacuum filtered through a Whatman #2 11cm filter along with two 25 mL chloroform washing aliquots. The chloroform extracts were transferred to 250 mL boiling flasks and evaporated to dryness using a rotary flash evaporator. The dry flask were extracted with 10 mL of hexane and place on a florisil column. The flask were then extracted with two additional 25 mL rinses and the column rinsed with an additional 50 mL hexane. The test material was eluted from the column with 100 mL of 5 % acetone in hexane and collected in a 250 mL boiling flask. The 5 % acetone:hexane was evaporated to dryness using a rotary flask evaporator and the dry flask extracted with chloroform rinses and transfered quantitatively to a 10 mL volumetric flask and diluted to volume.

A 2 mL aliquot was injected into a GC-FID under the following conditions:
Column: 3 %, SP 2100 6' x 4 mm ID
Temperature:
Injector - 300 ºC
Column - 235 ºC 8 min, 32 ºC/min, 280 ºC 8 min
Detector - 300 ºC
Carrier: N2 at 60 mL/min
Duration of treatment / exposure:
13 weeks (toxicity study)

Once (kinetic study)
Frequency of treatment:
Daily (toxicity study)
Dose / conc.:
100 mg/kg diet
Dose / conc.:
1 000 mg/kg diet
Dose / conc.:
2 000 mg/kg diet
No. of animals per sex per dose:
Twelve (toxicity study)
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: study initiation, weekly thereafter and at terminal sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes
- Time schedule: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before study initiation and at 13 weeks

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13
- Anesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: 10 per sex per group
- Parameters checked: differential white blood cell count (relative and absolute), nucleated red blood cell count, corrected white blood cell count, segmented neutrophil count, band neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count, red blood cell count, haematocrit, hemoglobin, white blood cell count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13
- Anesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: 10 per sex per group
- Parameters checked: albumin, calcium, chloride, creatinine, globulin (and albumin/globulin ratio), glucose, phosphorus, potassium, aspartate aminotransferase/serum glutamic-oxaloacetic transamine, alanine aminotransferase/serum glutamic-pyruvic transaminase, sodium, total bilirubin, total protein, urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Parameters checked: external surfaces, all orifices, cranial cavity, carcass, external surfaces of brain and spinal cord, nasal cavity and paranasal sinuses and thoracic, abdominal and pelvic cavities and their viscera. For each sacrificed animal from the toxicity study, the kidneys, liver and testes with epididymides (males only) were weighed.

HISTOPATHOLOGY: Yes
- Parameters checked: adrenals, aorta, bone/bone marrow (proximal femur including articular surface of head), brain, cecum, colon, duodenum, oesophagus, heart, ileum, jejunum, kidneys, lesions, liver, lungs, mesenteric lymph node, ovaries, pancreas, pituitary, rectum, salivary glands, sciatic nerve, spleen, stomach, testes with epididymides, thymus, thyroid (parathyroid), trachea, urinary bladder and uterine horn. all tissues were examined microscopically from control and high dose group animals and all animals that died or were sacrificed in a moribund condition. Lungs, liver and kidneys were examined microscopically from all low and mid dose animals.

A number of tissues were examined for possible examination if indicated by signs of toxicity or target organ involvement.
Other examinations:
After at least 13 weeks on test diets, two animals per sex per dose were dosed by gavage with approximately 12.5 µCi 14C-test material in 1 mL PEG-300/animal. The animals were housed in individual metabolism cages for 96 hours after dosing. Blood samples were collected at 1, 2, 4, 8, 24, 48, 72 and 96 hours post-dose. Excreta were collected at 0-6, 6-12, 12-24, 24-48, 48-72 and 72-96 hours after dosing. A cage rinse was collected at the 24 hour collection interval and a cage wash was collected at the end of the 96 hour urine and faeces collection.

After 96 hours, terminal body weights were recorded. The following tissues were collected: lungs, liver, kidneys, spleen, stomach, stomach contents, intestines, intestine contents, fat (perigonadal) and muscle.
Statistics:
Levenes test for variance homogeneity followed by standard one-way analysis of variance. If ANOVA is significant, Dunnetts t-test or the Games and Howell modified Tukey-Kramer test are used for pairwise comparisons. When no transformation established variance homogeneity at p<0.001, the data are also examined by non-parametric techniques. These include the Kruskal-Wallis H-test ANOVA and, if this test is significant, the Nemenyi-Kruskal-Wallis test for multiple group comparisons are evaluated at the 5 % two-tailed probability level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All of the 2000 ppm animals had a rough hair coat with many appearing thin and having a hunched posture, alopecia and rhinorrhea.
Mortality:
no mortality observed
Description (incidence):
There was no mortality throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights for 1000 ppm males and females at week 13 were 30 and 31 % lower, respectively, than the controls (statistically significant). At 2000 ppm, corresponding body weights for males and females were reduced by 69 and 52 %, respectively. Male body weight gains were significantly reduced in the 100 ppm group during week 10, in the 1000 ppm group during weeks 1-6, 8, 10 and 11 and in the 2000 ppm group during weeks 1-11. Male cumulative body weight gains were significantly reduced throughout the study in the 1000 and 2000 ppm groups. Body weight gains in females were significantly reduced during weeks 1 and 5 at 1000 ppm and during weeks 1-4 in the 2000 ppm group (see Table 1).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly reduced in 1000 and 2000 ppm males throughout the entire study, with the exception of week 12 in the 1000 ppm group. Food consumption in 1000 ppm females was reduced during weeks 1, 3, 4, 6 and 10; consumption was reduced throughout the entire study in the 2000 ppm females (see Table 2).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males in the 100 and 2000 ppm groups had significantly higher red blood cell count, while 2000 ppm males had significantly higher haemoglobin. The higher cell count in the 100 ppm males is considered to reflect normal biological variation and is not attributed to exposure to the test material. Mean corpuscular volume and corpuscular haemoglobin was significantly reduced in the 1000 and 2000 ppm males and in the 2000 ppm females. Females administered 1000 ppm had a significantly higher platelet count, although this is considered normal biological variation rather than test material related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose was significantly reduced in males and females receiving 1000 and 2000 ppm. Urea nitrogen was significantly increased, while creatinine was significantly reduced in the 2000 ppm males and females, although these differences were small. Total protein, albumin and globulin were significantly lower in 2000 ppm males and 1000 and 2000 ppm females. The albumin to globulin ratio was significantly higher in males and females administered 2000 ppm. Calcium was lower in males in females in the 2000 ppm group, although the difference was only significant in the females. Inorganic phosphorous and potassium were significantly higher in 2000 ppm males and females, while chloride was higher in the 2000 ppm females.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals administered 1000 and 2000 ppm had significantly lower terminal body weights and tended to have corresponding lower absolute organ weights. Statistically, however, only the 2000 ppm rats had significantly reduced kidney weights, while significant lower liver and testis weights were only seen in this group's males. Organ to body weight percentages for all organs were significantly higher in animals from the 1000 and 2000 ppm groups. There were no treatment-related macro- or microscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant differences for several clinical pathology variables between controls and treated animals, most of which can be attributed to decreased food consumption and body weight.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Equivalent to 7.1 mg/kg/ay (male); 8.3 mg/kg/day (female)
Critical effects observed:
no

Table 1: Mean body weights

   Mean body weights (g)
 Week  Sex  0 mg/kg  100 mg/kg  1000 mg/kg  2000 mg/kg
 0  M  119.9  116.9  120.9  118.6
   F  114.5  114.6  113.8  113.0
 1  M  177.1  171.7  147.2  113.0
   F  152.3  148.7  133.4  107.0
 2  M  228.7  223.3  185.5  120.2
   F  176.5  170.6  155.0  112.1
 3  M  280.5  276.6  226.2  137.6
   F  199.7  195.8  174.3  127.1
 4  M  325.0  316.1  254.1  150.9
   F  216.1  208.9  186.0  136.8
 5  M  361.6  353.8  283.2  170.1
   F  232.1  227.1  196.5  150.3
 6  M  390.9  379.8  299.6  182.7
   F  242.6  234.7  202.6  157.4
 7  M  413.9  403.8  319.7  195.8
   F  255.8  247.3  210.1  166.0
 8  M  437.5  423.9  337.4  204.3
   F  263.5  254.1  218.1  173.1
 9  M  451.4  437.0  347.6  210.1
   F  267.8  259.7  219.6  178.9
 10  M  469.1  450.3  357.9  216.3
   F  276.0  264.0  223.6  183.3
 11  M  483.5  466.9  366.8  224.3
   F  282.5  270.4  228.1  188.3
 12  M  485.9  471.9  371.4  227.9
   F  283.3  273.0  228.8  190.7
 13  M  498.4  479.8  385.8  236.0
   F  289.4  278.7  235.2  197.3

Table 2: Food consumption

   Food consumption (g/week)
 Week  Sex  0 mg/kg  100 mg/kg  1000 mg/kg  2000 mg/kg
 1  M  143.3  140.0  117.7  71.8
   F  126.7  119.1  108.6  78.5
 2  M  161.3  154.9  120.3  99.2
   F  125.0  120.2  117.5  75.8
 3  M  177.7  175.4  135.4  77.3
   F  135.2  133.1  109.9  69.2
 4  M  181.5  173.5  133.3  76.3
   F  130.6  129.6  106.6  72.9
 5  M  184.6  179.5  148.0  102.2
   F  134.2  134.2  117.8  79.8
 6  M  186.4  180.0  145.4  95.7
   F  139.4  140.6  108.6  75.9
 7  M  184.5  177.8  138.3  86.4
   F  140.9  133.3  116.7  79.5
 8  M  180.6  178.4  152.2  87.3
   F  138.4  132.6  116.4  89.4
 9  M  178.2  176.6  150.8  98.5
   F  143.8  133.5  120.0  82.7
 10  M  178.6  171.8  145.3  82.1
   F  137.4  130.1  106.6  84.9
 11  M  181.5  180.5  139.0  85.6
   F  144.1  129.7  119.1  87.6
 12  M  169.5  172.3  145.7  104.8
   F  128.4  144.7  111.2  82.5
 13  M  182.6  175.1  157.5  85.4
   F  134.4  136.3  111.2  94.9
Conclusions:
Under the conditions of the test, the No Observed Adverse Effect Level was 100 mg/kg diet which is considered to be equivalent to 7.1 mg/kg/day in males and 8.3 mg/kg/day in females.
Executive summary:

Rats were fed diets containing 0, 100, 1000 or 2000 ppm test material for at least 13 weeks. Body weights, food consumption and overt signs of toxicity were recorded during the study; blood was collected before necropsy for clinical pathology evaluation. Gross changes in the tissues and in the weights of selected organs were recorded at necropsy. Selected tissues were examined microscopically from all animals in the control and high dose groups. In addition, lungs, liver and kidneys were examined for rats treated at 100 and 1000 ppm.

There were no deaths during the 13 week feeding period or during the kinetic phase. All of the animals in the 2000 ppm dose group had a rough hair coat throughout the study; many of these animals were classified as being thin and having a hunched posture, alopecia and rhinorrhea.

Body weights for 1000 and 2000 ppm males and females were significantly lower than those of controls. Body weights for 1000 ppm males and females at week 13 were 30 and 31 % lower, respectively, than those of the controls. For 2000 ppm males and females, body weights were 69 and 52 % lower, respectively, than those of the controls. Male and female cumulative body weight gains in the 1000 and 2000 ppm groups were significantly lower throughout the study. At the end of the study, the 2000 ppm males and females had gained only 31 and 48 %, respectively, of the weight gained by the controls.

Food consumption for males in the 1000 ppm group (except week 12) and males and females in the 2000 ppm group were significantly lower than those of controls for the entire study.

There were statistically significant differences for several clinical pathology variables between control and treated animals. Nearly all of the differences represented treatment-related effects. The effects occurred at 1000 or 2000 ppm or both and, in general, males and females were affected similarly. Most of the effects were considered to be associated with decreased food consumption and body weight.

Animals treated at 1000 or 2000 ppm tended to have lower terminal body weights. Correspondingly these animals tended to have lower absolute organ weights and higher organ-to-body weight percentages. There were no treatment-related macroscopic or microscopic findings.

The NOAEL was determined to be 100 ppm.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Objective of study:
excretion
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A single oral dose of 1500 mg/kg bw 14C labelled test material was administered to six male rats. Urine samples were taken at the conclusion of 0-24, 24-48 and 48-72 sampling intervals. The quantity of parent compound-equivalents was measured by LSC and expressed as percentage of administered dose. Metabolites were isolated and purified through the use of TLC and HPLC and identified by high resolution NMR spectroscopy and MS.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Propargite
EC Number:
219-006-1
EC Name:
Propargite
Cas Number:
2312-35-8
Molecular formula:
C19H26O4S
IUPAC Name:
propargite
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Omite
- Radiochemical purity (if radiolabelling): 97.5 %
- Specific activity (if radiolabelling): 11.6 mCi/mmole
- Locations of the label (if radiolabelling): phenyl ring
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilington, MA, USA
- Weight at study initiation: 142-163 g
- Fasting period before study: 18 hours
- Housing: Nalgene metabolism cages
- Individual metabolism cages: yes
- Diet: Charles River Rat, Mouse and Hamster formula ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1400 mg unlabelled test material was mixed with 12.6 mg labelled test material in 1 mL hexane. After mixing, the hexane was evaporated and the residue dissolved in 3 mL corn oil and mixed. Each animals was administered approximately 0.7 mL of dosing solution.
Duration and frequency of treatment / exposure:
Once
Doses / concentrations
Dose / conc.:
1.5 other: g/kg
Control animals:
yes, concurrent vehicle
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: 24, 48 and 72 hours after dosing.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on excretion:
Of the applied dose, 12 % was excreted in the urine.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
HPLC radiochromatograms generated for the 0-24, 24-48 and 48-72 hour urine samples all qualitatively resemble each other in that there are five major zones of radioactivity. Moreover, the HPLC profiles are qualitatively the same as profiles obtained in an earlier oral dose study and 13 week dietary study (Banijamali and Tortora, 1998a) in spite of different dose levels and exposure regimes. In addition, the parent compound was not detected in the urine.

The molecular weights of metabolites 1 to 5 were determined to be 294, 382, 296, 296 and 280. In order of increasing polarity the following urinary metabolites were identified:

Metabolite 5: 1-[4-{1.1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane-diol (HOMe-TBOC-diol)
Metabolite 4: 1-[4-{1.1-dimethyl-2-hydroxyethyl)phenoxy]-2,x,x'-cyclohexane-triol (HOMe-TBOC-triol)
Metabolite 3: 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane-triol (HOMe-TBPC-triol)
Metabolite 2: 1-[4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethyl sodium sulphate (HOMe-TBPC-diol sulphate)
Metabolite 1: 1-{4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethyl acetic acid (Carboxy-TBPC diol)

Applicant's summary and conclusion

Conclusions:
Interpretation of results: bioaccumulation potential cannot be judged based on study results
Urinary HPLC radiochromatograms taken over time and mass spectral analysis indicate that the test material is rapidly and completely metabolised in male rats to five major metabolites of greater polarity than the parent compound. The data indicate a metabolic pathway that involves hydrolysis and hydroxylation of the parent compound to 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane-diol (HOMe-TBPC-diol). This metabolite is then subject to either sulphate conjugation to give 1-[4-(2,x-dihydroxycyclohexoxy)phenyl-2,2-dimethylethyl sodium sulphate (HOMe-TBPC-diol sulphate) or further oxidation to give 1-[4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethyl acetic acid (Carboxy-TBPC-diol) or hydroxylation to give 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane-triol (HOMe-TBPC-triol) and 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x,x'-cyclohexane-triol (HOMe-TBPC-triol). Moreover, the same degradation pathway is apparent regardless of dose level or route of administration.
Executive summary:

Male rats were treated with an oral dose (1.5 g/kg) of 14C-test material. Urine and faeces were collected for 72 hours after treatment. During this time, 12 % of the applied dose was excreted in the urine. Five major metabolites were detected by HPLC and identified by high resolution FT-NMR and MS. Spectral analysis indicated that test material was rapidly degraded by rats to more polar products and metabolism of cyclohexyl ring is strongly favoured.

In order of increasing polarity, the following metabolites were isolated in rat urine and identified as:

1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2, x-cyclohexane-diol

1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x,x'-cyclohexane-triol

1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane-triol

1-[4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethylethyl sodium sulfate

1-[4-(2,x-dihydroxycyclohexy)phenyl]-2,2-dimethyl acetic acid

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