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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
repeated dose toxicity: dermal, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 12, 2003-October 20, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diallyl 2,2'-oxydiethyl dicarbonate
EC Number:
205-528-7
EC Name:
Diallyl 2,2'-oxydiethyl dicarbonate
Cas Number:
142-22-3
Molecular formula:
C12H18O7
IUPAC Name:
diallyl 2,2'-oxydiethyl dicarbonate
Details on test material:
- Name of test material (as cited in study report): diallyl diglycol carbonate (ADC)
- Substance type: pure active substance
- Physical state: liquid
- Purity test date: 2002-11-19
- Stability under test conditions: considered stable for study duration
- Storage condition of test material: at room temperature

Test animals

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
- Age at study initiation: eight weeks (six at arrival plus two-week acclimation period)
- Weight at study initiation: All animals placed on study had body weights within ±20% of the mean body weight for each sex. Forty male rats (weighing 223 to 292 g at randomization) and 80 female rats (weighing 163 to 205 g at randomization)
- Fasting period before study: No, except overnight prior to blood sample collection.
- Housing: Animals were individually housed in stainless steel cages with wire mesh fronts and bottoms, suspended over pans containing absorbent liners.
- Diet (e.g. ad libitum): yes, except during designated fasting periods. Diet (meal Lab Diet® Certified Rodent Diet® #5002, PMI Nutrition International, Inc.)
- Water (e.g. ad libitum): Tap water was available ad libitum to all animals and supplied via an automatic watering system.
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67 to 74°F [monitored and recorded daily]
- Humidity (%): 21 to 69% [monitored and recorded daily]
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day via an automatic timer.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Test areas: an anterior and posterior site on the back. The dose sites were alternated daily between the anterior and posterior areas.
- % coverage: no less than 10% of the total body surface.
- Type of wrap if used: The control and test article were drawn into a plastic 1 mL syringe (except for the 150 mg/kg/day groups, which required a
100 µL glass syringe), administered from the hub of the syringe, and distributed evenly over the appropriate site.
- Time intervals for shavings or clipplings: at weekly intervals throughout the study, all animals had hair clipped from the test areas.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): the sites were washed with warm soapy water, and rinsed with tepid tap water.
- Time after start of exposure: six hours after test article administration.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at dose levels of 150, 454 and 1030 mg/kg/day at respective dose volumes of 0.13, 0.4, and 0.9 mL/kg.
- Constant volume or concentration used: yes. The dose volumes were derived on the basis of the density of the test article, 1.143 g/mL.


CONTROL MATERIAL

- Amount(s) applied (volume or weight with unit): based on the most recent body weightt at dose volume of 0.9 mg/mL
- Concentration (if solution): not applicable
- Lot/batch no. (if required): 03-144-JT-03, 04-146-JT, 04-100-JT
- Purity: Documentation on the strength, purity, composition, stability, physical properties, and other pertinent information on each batch of control article (0.9% Sodium Chloride for Injection, USP) was limited to that information listed on the label of this commercially available control article.


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. Immediately following dosing, the application site was covered with gauze dressing and
secured with nonirritating tape. Six hours after test article administration, the gauze and tape were removed, the sites washed and rinsed. The dose site of each animal was blotted dry prior to returning to the cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A compositional analysis was conducted on test article samples (2 g/sample) collected prior to initiation of dosing (start of study) and after completion of test article administration (end of study). The analysis was performed to confirm the identity of the test article by HPLC TAN and NMR procedures.All compositional analytical work was conducted by Ricerca, LLC, Concord, Ohio. The work performed by Ricerca in conjunction with this study was conducted in compliance with GLP regulations and subjected to review by the Quality Assurance Unit (QAU) of that laboratory.
Duration of treatment / exposure:
The test and control articles were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period), at least 42 days to females in the repeat dose component, and up to 48 days in females in the reproductive component, depending on reproductive performance (14 days premating, up to 14 days of mating, and through Day 20 of gestation).
Frequency of treatment:
once per day at approximately the same time each day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
454 mg/kg bw/day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
1030 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
-ten male and ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in repeat dose component
-ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in reproductive component
Control animals:
other: concurrent with saline
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the Sponsor, on the basis of available data from previous studies.
- Rationale for animal assignment (if not random): randomized
- Rationale for selecting satellite groups: not applicable
Control groups, treated only with vehicle alone, were needed to exclude any effect of vehicle and make possible to interpret the effects of test material
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): randomized
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily (once during test or control article administration, and once following completion of the six-hour exposure and removal of the wrap) for morbidity, mortality, signs of injury, and the availability of food and water.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of treatment and weekly during the study.
Deatiled clinical observations checked in table [No.1] were included

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily for the first 20 days and weekly for the remainder of the study for erythema and edema.
The evaluations were made according to a skin reaction scale based on the Draize1 scale for scoring skin irritation. Evaluations were conducted prior to test or control article administration that day. The daily observations occurring between weekly intervals are not reported, but are maintained in the study data.
Dermal irritation checked in table [No.1] were included

BODY WEIGHT AND BODY WEIGHT GAIN: Yes
- Time schedule for examinations: the day after arrival, at randomization, at initiation of test or control article administration, weekly during the study, and at termination.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, but in g food/animal/day
Individual food consumption for males was measured and recorded weekly during the study, except during the two-week mating period. Individual food consumption for repeat dose females was measured and recorded weekly during the study.

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes, carbon dioxide/oxygen
- Animals fasted: Yes, overnight prior to sample collection, but had free access to drinking water
- How many animals: all males and repeat dose females
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:at termination from all males and repeat dose females
- Animals fasted: Yes, overnight prior to sample collection, but had free access to drinking water
- How many animals: from all males and repeat dose females
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of test or control article administration, at the start of treatment, and weekly during the study.
- Dose groups that were examined: each repeat dose male and female
- Battery of functions tested in table [No.1] were included.

BEHAVIORAL OBSERVATION-REPEAT DOSE COMPOMENT:

- Time schedule for examinations: prior to the first exposure and during the last week of study.
- Dose groups that were examined: All males and repeat dose females
- Battery of functions tested in table [No.1] were included.

OTHER:
Fo BREEDING PROCEDURES- REPRODUCTIVE COMPONENT (see in section 7.8 Toxicity to reproduction)
Fo PARTURITION AND Fi LITTER OBSERVATIONS - REPRODUCTIVE COMPONENT (see in section 7.8 Toxicity to reproduction)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (table 2), all adult animals dying spontaneously or euthanized at scheduled necropsies were externally examined and any abnormalities were identified and correlated with antemortem findings. The carcasses were discarded without further evaluation.
Organs and tissues from all adult animals were removed, trimmed of any adherent tissues, weighed (where required) and placed in neutral buffered formalin, except for the testes and epididymides which were placed in Bouin's fixative, and the eyes which were placed in Davidson's fixative. The lungs were infused via the trachea with formalin. The urinary bladder was inflated with formalin at necropsy and examined internally after fixation.

HISTOPATHOLOGY: Yes, were performed for all males and repeat dose females in the control and high dose groups.
Microscopic examination of formalin-fixed hematoxylin and eosin-stained paraffin sections of the tissues were performed for all males and repeat dose females in the control and high dose groups. For processing of the testes, hematoxylin and period acid Schiff (PAS) staining and paraffin embedding was performed.

NEUROPATHOLOGICAL EVALUATIONS
Nervous system tissue (cervical, thoracic, and lumbar spinal cord) from female animals from the mid and high repeat dose groups (454 and 1030 mg/kg bw/day, respectively) as well as the control were sent to Experimental Pathology Laboratories, Inc. (EPL*), in Henidon, Virgina, for further processing and/or special neuropathology staining which allowed for better examination of possible nervous system effects. The sciatic nerve, when available, was processed to glycomethacrylate blocks and then sectioned and stained with Toluidine Blue or Bielschowsky for histopathologic evaluation.
Other examinations:
ORGAN WEIGHTS
Body and protocol-specified organ weights were recorded at necropsy and appropriate organ weight ratios were calculated (absolute and relative to body and brain weight) for all adult animals. Paired organs were weighed together. Organs were not weighed for animals dying spontaneously.
Statistics:
Group Pair-wise Comparisons
For each specified endpoint and for all collection intervals, Levene's test 5 was used to assess homogeneity of group variances. If Levene's test was not significant (p>0.01), Dunnett's test6 was used to compare each treatment group with the control group. If Levene's test was significant (p<0.01), comparisons with the control group were made using Welch's t-test7 with a Bonferroni correction. Results of all pair-wise comparisons were reported at the 0.05 and 0.01 significance levels. All endpoints were analyzed using two-tailed tests. Besides these tests, Log Transformation, Fisher's Exact Test, Covariate Analysis and Arcsin-Square-Root Transformation were used

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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
All treated animals in the repeat dose component survived to scheduled euthanasia.
No effect of treatment with ADC was evident from the general and detailed clinical examinations of the repeat dose animals. The few findings seen among the treated animals occurred with low incidence or with similar frequency as controls. These findings (i.e. red material around the eyes or nose, sparse amounts of hair on the fore- and hindlimbs/paws) are seen commonly in this laboratory with this strain and age of animal and the occurrences in this study were considered unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN
No effect of treatment with ADC was evident from body weight data for males and females in the repeat dose component. Mean body weights for the treated animals were comparable to controls throughout the seven-week treatment study.
No effect of treatment with ADC was evident from body weight gain for males and females in the repeat dose component. Mean week-to-week body weight gain for the treated animals was comparable to controls. In the females there was a mean weight loss during the last week of study. This was seen in all groups (control and treated) and was attributed to half of the females in each group being fasted for blood collection (clinical pathology) overnight prior to euthanasia and necropsy. The remaining females in each group were necropsied the following day and were not fasted overnight.
FOOD CONSUMPTION
No effect of treatment with ADC was evident from food consumption for the males and females in the repeat dose component. Mean food consumption for the males during the two weeks prior to pairing and two weeks following completion of the pairing (mating) period was comparable to controls. Likewise, food consumption for the treated females in the repeat dose component over the entire six-week treatment period was comparable to controls.

HAEMATOLOGY
There were no adverse test article-related effects on hematology parameters evaluated. The red cell indices (MCHC, MCV, and MCH) were statistically decreased in males at 454 mg/kg/day. The differences in MCHC, MCV, and MCH were minimal, however, and not considered to be treatment related.

CLINICAL CHEMISTRY
No adverse effects were seen in clinical chemistry analytes. Few analytes in the treated groups exhibited statistically significant differences from controls, but none were considered physiologically or toxicologically relevant.

NEUROBEHAVIOUR
No effect of treatment with ADC was evident from the neurobehavioral evaluations of the repeat dose animals.

BEHAIVIOUR
Auditory Response, Corneal Reflex, Pupil Response, and Grip Strength

No effect of treatment with ADC was evident from auditory response, corneal reflex or pupil response. With the exception of one control female which did not pass the corneal reflex evaluation, all other animals (control and treated groups) passed these behavioral tests at terminal evaluation. Similarly, no effect of treatment was evident in either males or females from forelimb grip strength evaluations. Forelimb grip strength in the 150 mg/kg/day males was statistically lower than controls; however, in the absence of a similar response in males at the higher dose levels, this was considered spurious and unrelated to treatment. In both sexes, forelimb grip strength at termination was greater than at the pretest evaluation.

No effect of treatment with ADC to males was evident from hindlimb grip strength. These values in the treated males at terminal examination were comparable to controls. Grip strength in the control and treated males at terminal examination was notably lower than at pretest and differences ranged from about 43% lower in controls to 23% lower in the 1030 mg/kg/day group. During this pretest interval, hindlimb grip strength in the 150 and 1030 mg/kg/day males was statistically lower than controls and comparable to controls in the 454 mg/kg/day group.

Mean hindlimb grip strength at terminal examination among females treated at 454 and 1030 mg/kg/day was statistically significantly lower than the control group; both the mid-and high-dose groups displayed a very similar decrease. This finding appears to be spurious and not toxicologically significant because: 1) the effect was not dose responsive (grip strength was similar for the two dose groups despite the two-fold increase in dose between the mid- and high-dose groups), 2) mean hindlimb grip strength was significantly lower at termination than prior to the start of the study for all groups (treatment and control), 3) the effect was noted only in females and not in males, 4) there were no microscopic changes noted in H&E stained sections of the spinal cord or sciatic nerve or hindlimb muscle, and 5) further processing and/or special neuropathology staining of the spinal cord and sciatic nerve also did not reveal any microscopic changes that were believed to be treatment related.

Motor activity
Motor activity evaluated as horizontal activity, vertical activity, total distance, and stereotypy over a 20-minute trial was comparable to controls in the treated females and in the 150 mg/kg/day males at the terminal evaluation. Motor activity was higher than control in the 454 and 1030 mg/kg/day males during the 5-10 minute testing interval, and in the 454 mg/kg/day males over the entire 0-20 minutes of testing and in most instances these differences were statistically significant. For all other intervals (0-5, 10-15, and 15-20 minutes) motor activity for these treated males did not differ statistically from controls and was considered comparable between the groups. The increased activity in these treated males during this one five minute interval over the entire 20-minute period was not considered toxicologically meaningful since there was no clear dose response in any of the activity parameters. Mean values for each activity (horizontal, vertical, total distance, and stereotypy) were similar between the 454 and 1030 mg/kg/day groups. Likewise, cumulative evaluations over the entire 20-minute testing period were also not dose responsive, and only differed statistically from control at the 454 mg/kg/day dose level. During the pretest evaluation, motor activity for the treated males and females (all groups) was comparable to controls.
Emotionality
No effect of treatment with ADC was evident from the emotionality evaluations conducted during the first five minutes of the motor activity testing. The parameters recorded during these evaluations (intervals of urination and counts of defecation, grooming, backing and rearing) for the treated groups were comparable to controls.

ORGAN WEIGHTS
No test article-related organ weight changes were noted in rats of either sex that received ADC in the repeat dose phase of the study.
Absolute mean prostate weights and relative prostate weights to body and brain weights were statistically significantly increased in male rats that received 454 mg/kg/day and 1030 mg/kg/day ADC compared to controls. However, there were no corresponding microscopic changes in prostates of males in these groups. The prostate weight increase was not considered to be toxicologically significant. Absolute mean heart weight and relative heart weight to body and brain weights were statistically significantly increased in female rats that received 1030 mg/kg/day ADC compared to controls, but males were not affected and there were no corresponding microscopic changes in hearts. The heart weight increase was not considered to be toxicologically significant.

GROSS PATHOLOGY
No test article-related macroscopic changes were observed in rats of either sex.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test article-related microscopic changes were noted in rats of either sex that received repeated doses of ADC. Microscopic changes observed were typical of those commonly found in animals of the same strain and age and were considered to be incidental.

OTHER FINDINGS
NEOROPATHOLOGICAL EVALUATIONS
The purpose of these additional neuropathology evaluations was to determine if the decrease in hindlimb grip strength in mid-and high dose females was associated with any neuropathological findings. Cervical, thoracic and lumbar spinal cord tissues were submitted for histopathological evaluation
In the neuropathological evaluations of selected nervous tissues conducted by EPLS (Report No: 139-019), minimal axonal degeneration characterized by either the presence of digestion chambers and /or swollen axons (spheroids) was observed in one level of spinal cord from one high-dose (1030 mg/kg/day) female and from one mid-dose (454 mg/kg/day) female. The sciatic nerve of one high-dose female and two mid-dose females also had minimal axonal degeneration. Similar minimal changes were not observed in the control animals. These minimal lesions were considered well within the range of what could occur as an incidental background finding. Thus, it was concluded that since there was no dose response, the changes seen were probably incidental background findings unrelated to test article treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 030 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: There were no toxicologically significant treatment related effects in all parameters tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.
Executive summary:

This study was conducted in accordance with OECD Guideline 422, which is comprised of two components, a repeat dose toxicity study with neurobehavioral evaluations and a reproduction/developmental toxicity screening study (see section 7.8 Toxicity to reproduction for summary).

The purpose of the repeat dose toxicity component was to provide information on possible target organ effects and the potential for neurobehavorial effects arising from repeated exposures. Each repeat dose group contained ten male and ten female Sprague-Dawley rats [Crl: CD® (SD)IGS BR]. The ten male animals of each repeat dose group were also utilized for the reproductive/developmental component of the study. Animals were administered dermally once daily via occlusion for 6 hours at dose levels 150, 454, and 1030 mg/kg/day for at least 42 consecutive days. The control animals received 0.9% Sodium Chloride, USP, at a volume of 0.9 mL/kg for the same duration as the treated animals. Observations of the animals included clinical signs, neurobehavioral observations (repeat dose component only), dermal evaluation, body weights, and food consumption. Evaluations for motor activity, emotionality, and other behavioral observations, including auditory response, grip strength, pupil reflex, and corneal reflex, were conducted for all repeat dose animals, pretest and prior to scheduled terminal euthanasia (after seven weeks of treatment). Blood collections for clinical pathology evaluations were conducted at study termination. Complete necropsies were performed on all animals (repeat dose and reproductive components) and organs and tissues were collected, weighed, and preserved. Organs and tissues from control and high-dose animals in the repeat dose component were examined microscopically. Nervous system tissues (cervical, thoracic, and lumbar spinal cord and sciatic nerve) from female animals from the mid and high dose groups (454 and 1030 mg/kg bw/day) as well as the control were evaluated for neuropathology.

No effect of treatment was evident from mortality, clinical evaluations, neurobehavioral evaluations, dermal evaluations, body weights, food consumption, hematological evaluations, serum clinical chemistry, organ weights, macroscopic, or microscopic evaluations. Decreased hindlimb grip strength as well as axonal dgenerations in spinal cord and sciatic nerve were noted at termination for female animals in the 454 and 1030 mg/kg/day dose groups. These findings appear to be spurious and not toxicologically significant.

Thus, in this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.