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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-04-23 to 2005-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted GLP guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005
Reference Type:
publication
Title:
Oral toxicity study of 2-Pentenenitrile in rats with reproductive toxicity screening test.
Author:
Lewis J.M., Maslanka J.C., Malley L.A., Everds N.E., Mann P.C. and Kennedy G.L.
Year:
2006
Bibliographic source:
Drug and Chemical Toxicology, 29:345-361.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): H-26232
- Physical state: very pale yellow liquid
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 65 days old at the start of treatment (May 5, 2004)
- Weight at study initiation: Males: 277.9–385.1 grams; Females: 203.7-244.8 grams
- Housing:
1/ Pretest and Premating: All rats were housed individually in stainless steel, wire-mesh cages suspended above cageboards.
2/ Cohabitation Period: All rats were housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboard. At the end of the
cohabitation period, females without evidence of copulation were housed individually in polycarbonate pans.
3/ Gestation Period (Females with evidence of copulation):
• Days 0-19: Dams were housed individually in stainless steel, wire-mesh cages suspended above cageboards.
• Day 20-Delivery: Females were housed individually in polycarbonate pans with bedding.
4/ Lactation Period Dams were housed with their litters in polycarbonate pans with bedding.
- Diet: All rats were fed pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002 ad libitum, except when fasted
- Water: Tap water from United Water Delaware was provided ad libitum.
- Acclimation period: rats were quarantined for 12 days of the 13-day pretest period (from April 22, 2004 (reception date) to May 3, 2004)


ENVIRONMENTAL CONDITIONS
- Temperature: 18-26ºC (targeted at 22º-24ºC)
- Humidity: 30%-70% (targeted at 40%-60%)
- Photoperiod: artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions of the test substance in deionized water were prepared daily and used after preparation.

VEHICLE
- Concentration in vehicle: 0, 0.1, 0.3 or 1.0 mg/mL.
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions at concentrations of 0.1, 0.3, and 1.0 mg/mL of H-26232 were collected for concentration verification and stability analysis on May 5, 2004. Dosing solutions at the same concentrations of H-26232 were collected for concentration verification and stability analysis on May 7, 2004. Dosing solutions at same concentrations of H-26232 were collected for concentration verification analysis on June 1, 2004 and June 29, 2004. In
addition, 0 mg/mL (control) samples were submitted for analysis with the set of samples.
Concentrations of H-26232 in separate dosing solutions were measured by high performance liquid chromatography (HPLC).
Duration of treatment / exposure:
Adult males: 44 days.
Pregnant female: until day 3 postpartum (days 55-68) except during the process of delivery.
Nonpregnant females: 58 and 62 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 3 or 10 mg/kg bw/day (P, m/f)
Basis:
other: gavage
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected for this study were based on results from a repeated dose oral toxicity study in rats in which male and female rats were dosed with the test substance by oral gavage at once daily dosages of 0, 10, 30, or 100 mg/kg/day for 28 days (See Rep. dose tox. oral V1 2001 MACK).
- Rationale for animal assignment: randomization
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during quarantine and predosing and twice daily thereafter.

GENERAL CLINICAL OBSERVATIONS: Yes
- Time schedule: daily at approximately the same time of day (± 2 hours) for acute/systemic toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and weekly thereafter at approximately the same time of day (+ 2 hours).
Observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior.

BODY WEIGHT: Yes
- Time schedule for examinations:
1. Premating Period
All P1 rats were weighed once a week and at terminal sacrifice.
2. Gestation and Lactation Periods
P1 females were weighed on days 0, 7, 14, and 21 of gestation and on days 0 and 4 of lactation.
Females without evidence of copulation, those that copulated and did not deliver a litter, and males were weighed on a weekly schedule.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
Each feeder was weighed at the beginning and end of the weekly food consumption interval and the final weight of the feeder and the amount of spillage from the feeder during the interval was subtracted from the initial feeder weight. From the food consumption and body weight data, the mean daily food efficiency was calculated.
1. Premating and Cohabitation Periods
Individual food consumption was determined weekly throughout the period, ending on test day 29. Food consumption was not measured during cohabitation for males and females or after cohabitation for males.
2. Gestation and Lactation Periods
Individual food consumption of pregnant P1 females was recorded on gestation days 0, 7, 14, and 21, and for lactating females on lactation days 0 and 4. Food consumption was not measured for females without evidence of copulation, females that did not deliver a litter, or for males.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: test day 24
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes, fasted after 3 p.m. (at least 15 hours)
- How many animals: 5 animals/sex/group
- Parameters checked in table 7.5.1/1 were examined.

COAGULATION: Yes
- Time schedule for collection of blood: from males on test day 45, and from females on test days 56-69
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- How many animals: 5 animals/sex/group
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: test day 24
- Animals fasted: Yes, fasted after 3 p.m. (at least 15 hours)
- How many animals: 5 animals/sex/group
- Parameters checked in table 7.5.1/1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Pretest (male rats on day –2 and female rats on day –1) and once after initiation of test substance administration (male rats on day 43, near the end of the dosing period, and female rats on day 21, near the end of the premating period).
- Dose groups that were examined: all animals
- Battery of functions tested: Functional Observational Battery and Motor Activity
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals, test day 45
- Maternal animals: All surviving animals, day 4 of lactation

GROSS NECROPSY
The tissues indicated in Table 7.5.1/2 were observed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 7.5.1/2 were prepared for microscopic examination and weighed, respectively.
Other examinations:
No
Statistics:
See table 7.5.1/3 for details on statistical methods used in this study.
For each parameter analyzed with a trend test, the test will be applied to the data sequentially. If a significant dose-response is detected, data from the top dose group will be excluded and the test repeated until no significant trend is detected.(14) For litter parameters, the proportion of affected
fetuses per litter or the litter mean will be used as the experimental unit for statistical evaluation.(17) The level of significance selected is p < 0.05.
Additional statistical tests will be used, and other parameters analyzed, if deemed necessary.

Results and discussion

Results of examinations

Clinical signs:
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:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
microscopic findings in the nose of female rats
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In P1 females, a statistically significantly lower weight gain occurred during the third week of premating (62% lower than control on test days 15-22)
at 10 mg/kg/day. Body weight was also significantly lower than control (9%) on day 0 of lactation. These findings were considered test substance
related but not adverse since they were transient, and overall weight gain was not significantly affected during premating, gestation, or lactation in
this group.
In P1 females, a statistically significantly lower food efficiency occurred during the third week of premating (59% lower than control group on test
days 15-22) at 10 mg/kg/day and was consistent with the reduced weight gain during this weekly interval.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Test substance-related microscopic findings were present in the nose of female rats (See Table 7.5.1/4)
The degeneration consisted of decreased cellularity, disorganization and thinning of the olfactory mucosa of Level II of the nose in 2/10 female rats
given 10 mg/kg/day. These changes were minimal, focal, and located in the dorsal aspect of the nose. These changes were similar to those seen in
the previous repeated-dose oral toxicity study at 10 mg/kg bw/day (Mackenzie, 2001), although other histopathologic changes present in the previous study (submucosal fibrosis and degeneration of subjacent olfactory nerve fibers) were not present in the current study.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: Based on degeneration of the olfactory mucosa in females at 10 mg/kg bw/day observed in 2/10 females.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 7.5.1/4: Test substance-related effects on the incidence of microscopic findings in female rats

 

Female rats

 

Dose (mg/kg bw/day

0

1

3

10

Nose

 

Number examined:

10

10

10

10

 

Degeneration, olfactory mucosa, Level II

0

0

0

2

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in P1 rats was 3 mg/kg/day, based on
degeneration of the olfactory mucosa in P1 females at 10 mg/kg/day.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (Lewis, 2005) was conducted with cis-2-pentenenitrile according to the OECD test guideline No. 422 and in compliance with GLP.

Crl:CD®(SD)IGS BR rats (10/sex/dose level) were dosed once daily by gavage at dose levels of 0, 1, 3, or 10 mg/kg/day (dose volume of 10 mL/kg in deionized water) for approximately 28 days prior to cohabitation and during the cohabitation period. Male rats and female rats showing no evidence of copulation or delivery of a litter were dosed after the end of the copulation period until the day before sacrifice. Females showing evidence of copulation were dosed throughout gestation and up to and including day 3 postpartum (except during the process of delivery).

General clinical observations were recorded once daily during dosing; detailed clinical observations were recorded once during pretest and weekly thereafter. Body weights and food consumption were recorded weekly for P1 males and females (premating), on days 0, 7, 14, and 21 of gestation, and on days 0 and 4 of lactation; food consumption was not measured during cohabitation or thereafter for males or females with no evidence of copulation. A neurobehavioral evaluation consisting of a functional observational battery and motor activity was conducted in all P1 rats (10/sex/group). Prior to study start and on approximately test day 43 for males and test day 21 for females, clinical pathology parameters (hematology, clinical chemistry, coagulation) were measured in P1 rats (first surviving 5/sex/group). F1 litter examinations (pup viability and clinical observations) were performed at birth and on lactation days 1 and 4. Individual pup weights were recorded at birth and on lactation day 4.

After litter production, all P1 rats were euthanized and given a gross pathological examination.

The testes and epididymides from all male rats were weighed. The liver, kidneys, adrenals, thymus, spleen, brain, and heart from all rats were weighed. Relative organ weights were calculated. Small and large intestines, bladder, lungs, trachea, heart, spleen, thymus, lymph nodes, bone marrow, thyroid, adrenals, brain, spinal cord, sciatic nerve, and femur were saved from all P1 animals. Gross observations, potential target organs (liver, kidneys, nose, spleen, testes, and eyes), and reproductive organs were also saved from all P1 animals. Uterine implantation sites and ovarian corpora lutea were counted in P1 female rats. A histological examination of saved tissues was conducted for the control and 10 mg/kg/day groups. Examination of tissues from the remaining groups was limited to relevant gross lesions and those tissues that demonstrated treatment-related histological effects in the 10 mg/kg/day group. Results related to reprotoxic effects were summarise section 7.8.1.

 

The general parameter results of this study were:

There were no effects considered to be related to treatment on the following parameters at any dose level:

- Mortality and clinical signs of toxicity in P1 males and females

- Body weight and body weight gain in P1 males

- Food consumption in P1 males and females

- Functional observational battery, motor activity, or grip strength in P1 males and females

- Hematology and coagulation parameters in P1 males and females

- Organ weights and gross pathology in P1 males and females

- Microscopic pathology in P1 males

 

Effects considered by the authors to be related to treatment were limited to the 10 mg/kg/day group and comprised:

Adverse effects:

- Degeneration of the olfactory mucosa in Level II of the nose in 2/10 P1 females

Non-adverse effects:

- Decreased body weight, body weight gain, and food efficiency in P1 females

- Increased albumin in P1 males and females

 

Conclusion:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in P1 rats can be identified as 3 mg/kg/day, based on degeneration of the olfactory mucosa in P1 females at 10 mg/kg/day observed in 2/10 females.

This value is supported by Mackenzie, 2001study in which same effects were observed with more serious histopathologic changes such as submucosal fibrosis and degeneration of subjacent olfactory nerve fibers at 10mg/kg bw/day in male and female rats after 28 day exposure

The NOAEL neurobehavioral toxicity in P1 rats was 10 mg/kg/day, the highest dose level tested.