Hexahydrocyclopenta[c]pyrrol-2(1H)-amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

4 may 2017 to 16 november 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 10, 30 and 75 mg/kg bw/day. A control group of five males and five females was dosed
with vehicle alone (Distilled water).
Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.
All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydrocyclopenta[c]pyrrol-2(1H)-amine

EC Number:

259-201-9

EC Name:

Hexahydrocyclopenta[c]pyrrol-2(1H)-amine

Cas Number:

54528-00-6

Molecular formula:

C7H14N2

IUPAC Name:

octahydrocyclopenta[c]pyrrol-2-amine

Test material form:

liquid

Remarks:

Colourless

Specific details on test material used for the study:

batch 18128549

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Deviations from these targets were considered not to have affected the purpose or integrity of the study;
see deviations from Study Plan. The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Animals were allocated to treatment groups as follows:
Treatment Group / Dose Level(mg/kg bw/day) / Treatment Volume (mL/kg) / Concentration(mg/mL) / Animal Numbers Male / Female
Control / 0 / 5 / 0 / 5 (1-5) / 5(6-10)
Low / 10 / 5 / 2 / 5 (11-15) / 5 (16-20)
Intermediate / 30 / 5 / 6 / 5 (21-25) / 5 (26-30)
High / 75 / 5 / 15 / 5 (31-35) / 5 (36-40)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.
The test item was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Distilled water.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show
the formulations to be stable for at least eleven days. Formulations were therefore prepared weekly during the treatment period and stored at approximately 4 ºC in the dark.
Samples of each test item formulation were taken and analyzed on two occasions for concentration of 852 AMINAZA PURIFIE at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained
are given in Annex 2. The results indicate that the prepared formulations were within +7% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Examinations

Observations and examinations performed and frequency:

Clinical observations, Bodyweight, Food and Waterconsumption
Functionnal Observations, Behavioral assessment,
Motor activity, Forelimb/Hindlimb Grip Strenth, sensory reactivity.
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28).

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate followed by exsanguination.
Thyroid Hormone Assessment, Organ Weights, Histopathology, pathology, data evaluation, statistical analysis

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse clinical signs detected.
Increased salivation was evident in all males and females treated with 75 mg/kg bw/day on Day 17 only. An observation of this nature is commonly observed following the oral administration of an unpalatable test item formulation and in isolation is considered not to represent an adverse effect of treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 and 30 mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Statistical significance was achieved for males treated with 75 mg/kg bw/day (p<0.05-0.01) throughout the treatment period and for males treated with 30 mg/kg bw/day (p<0.05) during the first week of treatment. Overall body weight gain for 75 and 30 mg/kg bw/day males was reduced by 39% and 17% respectively.
Females treated with 75 mg/kg bw/day showed a reduction in body weight gain during Weeks 1, 2 and 4. Although statistical significance was not achieved during these weeks, overall body weight gain for these females was reduced by 28%.
No such effects were detected in females treated with 30 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 mg/kg bw/day showed a 15% reduction in overall food consumption.
Food conversion efficiency for these males was reduced throughout the treatment period.
Females treated with 75 mg/kg bw/day and males treated with 30 mg/kg bw/day also showed a slight reduction (5%) in overall food consumption and fluctuations in food conversion efficiency were evident in these animals during the treatment period.
No such effects were detected in females treated with 30 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 75 mg/kg bw/day showed an increase in overall water consumption (42% for males and 33% for females).
No such effects were detected in animals of either sex treated with 30 or 10 mg/kg bw/day.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the hematological parameters examined.
Females from all treatment groups showed a statistically significant reduction (p<0.05) in hematocrit and a statistically significant increase (p<0.01) in reticulocyte count. Females treated with 75 and 30 mg/kg bw/day also showed a statistically significant (p<0.05) increase in mean corpuscular hemoglobin concentration. With the exception of one hematocrit value for one female treated with 30 mg/kg bw/day, all individual values were within historical control ranges. In the absence of true dose related responses or any associated histopathological findings, the intergroup differences were considered not to be of toxicological significance.
Males treated with 75 and 30 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in reticulocyte count. All individual values were within historical control range and in the absence of any associated histopathological findings, the intergroup difference was considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
Females treated with 75 mg/kg bw/day showed statistically significant reductions (p<0.05- 0.05) in alanine aminotransferase and alkaline phosphatase and a statistically significant increase in albumin/globulin ratio. All of the individual values were within the historical control ranges and whilst these intergroup differences may indicate minor perturbations in metabolism, in the absence of any histopathological hepatic changes in this sex, these were considered unlikely to be of an adverse toxicological response.
Males treated with 75 mg/kg bw/day showed a statistically significant increase (p<0.05) in glucose. Males treated with 75 and 30 mg/kg bw/day also showed a statistically significant reduction (p<0.01) in triglyceride levels. With the exception of two glucose values, all individual values were within historical control ranges and a true dose related response was not evident for triglycerides, therefore, the intergroup differences were considered not to be of toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 and 30 mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Statistical significance was achieved for males treated with 75 mg/kg bw/day (p<0.05-0.01) throughout the treatment period and for males treated with 30 mg/kg bw/day (p<0.05) during the first week of treatment. Overall body weight gain for 75 and 30 mg/kg bw/day males was reduced by 39% and 17% respectively.
Females treated with 75 mg/kg bw/day showed a reduction in body weight gain during Weeks 1, 2 and 4. Although statistical significance was not achieved during these weeks, overall body weight gain for these females was reduced by 28%.
No such effects were detected in females treated with 30 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected.
One control male had increased renal pelvic space in both kidneys, however, in the absence
of treatment, this was considered to be incidental.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic changes were evident:
Kidneys: Increased hyaline droplets were noted in one male treated with 10 mg/kg bw/day, four males treated with 30 mg/kg bw/day and four males treated with 75 mg/kg bw/day.
There was a minor increase in the appearance of basophilic tubules with the change noted as multifocal in one male treated with 30 mg/kg bw/day and in three males treated with 75 mg/kg bw/day, suggesting an increase in pathological change associated with the hyaline droplet accumulation. Minimal, focal basophilic tubules are considered to be incidental and the appearance of increased hyaline droplets in one male treated with 10 mg/kg bw/day is not considered to be significant within the confines of the study.
After examination of all female kidneys it was considered that all animals were within expected limits and no correlation with the weight increase was apparent at histopathology.


Liver: Centrilobular hepatocyte hypertrophy was present in three males treated with 75 mg/kg bw/day. No such effects were evident in females treated with 75 mg/kg bw/day or in animals of either sex treated with 30 or 10 mg/kg bw/day.
No other changes were noted which could be attributed to the administration of the test item.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOEL

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of 852 AMINAZA PURIFIE to animals of either sex at dose levels of 10, 30 or 75 mg/kg bw/day resulted in a reduction in body weight gain and food consumption in animals of either sex treated with 30 (males only) and 75 mg/kg bw/day, microscopic kidney changes in males treated with 75 and 30 mg/kg bw/day and microscopic liver changes in males treated with 75 mg/kg bw/day.
The microscopic liver changes identified in males treated with 75 mg/kg bw/day was considered to represent an adaptive response to mixed function oxidase induction and was considered not to represent an adverse toxicological finding. Although the kidney finding of multifocal tubular basophilia in male kidneys treated with 75 mg/kg bw/day could be considered an adverse effect, this finding was considered to be associated with alpha 2uglobulin
and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans.
The effect on body weight gain in animals of either sex treated with 75 mg/kg bw/day and in males treated with 30 mg/kg bw/day was considered to represent an adverse effect of treatment. Therefore, the “No Observed Effect Level” (NOEL) was considered to be 30 mg/kg bw/day for females and 10 mg/kg bw/day for males.

Executive summary:

The oral (gavage) administration of 852 AMINAZA PURIFIE to animals of either sex at dose levels of 10, 30 or 75 mg/kg bw/day resulted in a reduction in body weight gain and food consumption in animals of either sex treated with 30 (males only) and 75 mg/kg bw/day, microscopic kidney changes in males treated with 75 and 30 mg/kg bw/day and microscopic liver changes in males treated with 75 mg/kg bw/day.

The microscopic liver changes identified in males treated with 75 mg/kg bw/day was considered to represent an adaptive response to mixed function oxidase induction and was considered not to represent an adverse toxicological finding. Although the kidney finding of multifocal tubular basophilia in male kidneys treated with 75 mg/kg bw/day could be considered an adverse effect, this finding was considered to be associated with alpha 2uglobulin

and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans.

The effect on body weight gain in animals of either sex treated with 75 mg/kg bw/day and in males treated with 30 mg/kg bw/day was considered to represent an adverse effect of treatment. Therefore, the “No Observed Effect Level” (NOEL) was considered to be 30 mg/kg bw/day for females and 10 mg/kg bw/day for males.