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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:
From 02 FEB 2006 to 11 JUN 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (according to OECD 422 and GLP)
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

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:
yes
Remarks:
Minor deviations (e.g. relative humidity ranged from 23 to 83% instead of 30 to 70% during the study) by which study integrity is not affected adversely
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Perhydroazepine
EC Number:
203-875-9
EC Name:
Perhydroazepine
Cas Number:
111-49-9
Molecular formula:
C6H13N
IUPAC Name:
azepane
Details on test material:
- Name of test material (as cited in study report): Hexamethyleneimine (HMI)
- Substance type: clear colourless liquid
- Physical state: fluid
- Analytical purity: 99.6 %
- Lot/batch No.: 06702JC
- Expiration date of the lot/batch: 2007-01-01
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 10 weeks
- Housing: pre-mating: 5 animals/sex/cage, post mating: individually, lactation: offspring was kept with dam
- Diet: standard pelleted laboratory diet (from Altromin code VRF1), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-U
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw, actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations prepared during the treatment period was performed according to the following scheure:

Day of Date Group Analysis (type of sample)
study

Day 12* 17 February 1 acc (M)
2006 2 acc + hom + stab. 0 (TMB), stab. 5, RT (S)
3 acc (M)
4 acc + hom + stab. 0 (TMB), stab. 5, RT (S)
Day 26** 03 March 1 acc (M)
2006 2 acc + hom + stab.0 (TMB), stab. 5, RT (S)
3 acc (M)
4 acc + hom + stab.0 (TMB), stab.5, RT (S)
Day 32 09 March 1 acc (M)
2006 2 acc + hom + stab.0 (TMB), stab. 5, RT (S)
3 acc (M)
4 acc + hom + stab. 0 (TMB), stab. 5, RT (S)
Day 53 30 March 1 acc (M)
2006 2 acc + hom + stab. 0 (TMB), stab. 5, RT (S)
3 acc (M)
4 acc + hom + stab. 0 (TMB), stab. 5, RT (S)
Duplicate samples were analysed
acc=accuracy, hom=homogeneity, stab=stability (hours), T=top, M=middle, B=bottom position of container S=stability sample taken at middle position of container, RT=room temperature
The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 456773).
* These samples were kept in the freezer as the analytical method was not completed at that time. These samples were (without being analysed) discarded on 12 April 2006.
** Because the criterion that mean recoveries of the procedural recovery samples should be between 70% and 110% was not met (they were 125 and 138%), the results for the test samples were not accepted. These results were not reported.

Duration of treatment / exposure:
males: 28 days (2 weeks prior to mating, during mating and up to termination)
females: 38-56 days (2 weeks prior to mating, during mating, during post-coitum, and at least 3 days of lactation)
Frequency of treatment:
Once daily for 7 days per week, approx. the same time each day. Animals were dosed up to the day prior to scheduled necroscopy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 25, 50 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results of a 5-day range finding study (NOTOX Project 457436), the dose levels for this study were selected to be 0, 10, 25 and 50 mg/kg/day. In the dose range finding study, severe toxicity (clinical symptoms, body weight loss, decreased organ weights, macroscopic and microscopic findings in the stomach and small intestine) was noted at 200, 400 and 500 mg/kg body weight. In animals treated at 50 mg/kg body weight/day, yellow urine was noted and microscopic examination revealed minimal hyperplasia of the forestomach in one male and minimal erosion of the forestomach in one female.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: males: during week 4, females: during lactation
- Dose groups that were examined: all
- How many animals: 5 per sex

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: 5 per sex

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: 5 per sex

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: durind week 4, females: during lactation
- Dose groups that were examined: all
- Battery of functions tested: motor activity
- How many animals: 5 per sex
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyse the data:

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Student's t-test was applied for motor activity data.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off . Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
deatails see below
Mortality:
mortality observed, treatment-related
Description (incidence):
deatails see below
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 specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
details see below
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
deatails see below
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
One group 2 female was killed in a moribund state which was due to failed parturitian. This was not considered to be caused by treatment of the test substance. All other rats survived the scheduled duration of the study.
Yellow discolouration of urine was observed for all animals treated at 25 and 50 mg/kg body weight/day from Week 2 of study onwards.
The animal that was killed in extremis showed piloerection and pale appearance an the day of death.
Incidental findings that were noted included alopecia of the forelegs, chromodacryorrhoea, and rales. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

CLINICAL CHEMISTRY:
Females of the 50 mg/kg dose group showed slightly decreased sodium levels. No other differences between control and treated animals occurred.

GROSS PATHOLOGY:
At the high dose group, four females showed stomach abnormalities. These consisted of glandular mucosa thickened, forestomach thickened, and/or glandular mucosa gelatinous. One female of the intermediate dose group showed an isolated dark red focus on the glandular mucosa of the stomach.
The animal that was killed in extremis showed pale discolouration of the liver, the uterus contained thirteen foetuses, the cervix was obstructed by one foetus, and the mandibular lymph nodes were enlarged.
Incidental findings included enlarged mandibular lymph nodes, many gray-white foci on the papillary process of the liver, cervix and uterus dilated and containing fluid, isolated tan focus on the right clitoral gland, watery-clear cyst on the ovaries, and adrenal glands grown together with the kidneys. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental NOAEL for local effects
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental NOAEL for systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no changes for mortality, clinical signs, body weight, food consumption, functional observations, clinical laboratory investigations, organ weights, microscopic examination, reproduction, breeding data and pup development that were considered to be an effect of treatment.
Based on the findings on the stomach observed macroscopically, the parental No Observed Adverse Effect Level (NOAEL) for local effects was established at 25 mg/kg body weight/day. The parental NOAEL for systemic toxicity of 50 mg/kg body weight/day was established for the test substance.
Executive summary:

In a guideline study (OECD 422) the test substance was administered by daily oral gavage to male and female Wistar rats at dose levels of 0, 10, 25 or 50 mg/kg body weight/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum,and at least 3 days of lactation (for 38 to 56 days). There were no changes for mortality, clinical signs, body weight, food consumption, functional observations, clinical laboratory investigations, organ weights, or microscopic examination, that were considered to be an effect of treatment. Based on the findings on the stomach observed macroscopically, the parental No Observed Adverse Effect Level (NOAEL) for local effects was established at 25 mg/kg body weight/day. The parental NOAEL for systemic toxicity of 50 mg/kg body weight/day was established for the submission substance.