Bis(cyclohexyl)ammonium(2S)-2-{[(benzyloxy)carbonyl]amino}-3,3-dimethylbutanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-250-9

EC Name:

-

Cas Number:

62965-37-1

Molecular formula:

C26H42N2O4

IUPAC Name:

(2S)-2-{[(benzyloxy)carbonyl]amino}-3,3-dimethylbutanoic acid; N-cyclohexylcyclohexanamine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch number 25557

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 43-49 days (at the start of treatment)
- Weight at study initiation: males 253-295 g, females 168-211 g (at the start of treatment)
- Fasting period before study: overnight
- Housing: 5 of one sex/cage
- Diet: standard rodent diet
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22°C
- Humidity (%): 23-61%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11.01.2007 To: 08.02.2007

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v methylcellulose in water

Details on oral exposure:

1.0%w/v Methylcellulose in water for formulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed at
concentrations of 1 and 100 mg/mL to assess the homogeneity and stability of the test substance in the vehicle. Samples of each formulation
prepared for administration in Week 1 of treatment were analysed for achieved concentration of the test substance.
Apparatus and instrumentation
High performance liquid chromatograph (HPLC): High performance liquid chromatograph (HPLC) fitted with a UV-Vis detector.
Chromatography data handling: Waters Empower
Balances fitted with printers: Capability of weighing to accuracies of 0.1 mg, 0.01 mg and 0.001 mg.
General laboratory apparatus and glassware.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 150 and 300 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Mortality:

mortality observed, treatment-related

Description (incidence):

See details on results below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
During the entire study period there was no unscheduled mortality noted at any dose level.
Clinical signs of toxicological importance comprised partially closed eyelids, hunched
posture and piloerection, which were observed in both sexes receiving 300 mg/kg/day and
underactive behaviour which was observed in females receiving 300 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
Lower overall bodyweight gains were evident for males at 300 mg/kg/day,
whilst females at 150 or 300 mg/kg/day showed higher weight gains.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Lower food intake was evident in males at 300 mg/kg/day,
whilst females dosed with 300 mg/kg/day had a higher than control food intake.

FOOD EFFICIENCY
No data

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

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
Lower than control group mean haematocrit, haemoglobin and red blood cell counts were
recorded for females receiving 150 mg/kg/day and both sexes receiving 300 mg/kg/day.
These female groups also showed lower than control MCH and MCV values. In addition
females receiving 15 mg/kg/day had a tendency to exhibit lower than control haematocrit
with males at 150 mg/kg/day also showing lower haemoglobin levels.

CLINICAL CHEMISTRY
for all treated male groups. Mean creatinine values for females receiving 300 mg/kg/day
were also lower than controls. Lower than control group mean cholesterol values were
recorded for both sexes receiving 300 mg/kg/day. In addition lower than control total protein
(due to both a lowering in albumin and globulin) was recorded for females receiving 300
mg/kg/day, with lower globulin also being apparent in males at this dose level.

URINALYSIS
No data

NEUROBEHAVIOUR
Hindlimb grip strength values for males and females receiving 300 mg/kg/day were low
when compared with controls. Additionally, when compared with controls, high beam scores
(rearing activity) and low beam scores (cage floor activity) for males and females receiving
300 mg/kg/day were low throughout most of the 1-hour recording period, with the differences
being somewhat more marked in females.
Most or all of the individual total scores for these treated animals were lower than those of
the concurrent control animals.
High beam scores and, to a lesser extent, low beam scores for males receiving 150 mg/kg/day
were also low when compared with controls, with some evidence of a dose-relationship
between these animals and the males receiving 300 mg/kg/day. Motor activity scores for
females receiving 150 mg/kg/day and both sexes at 15 mg/kg/day were, however, unaffected.

ORGAN WEIGHTS
Heavier than control group bodyweight-adjusted mean kidney weights were recorded for both
sexes receiving 150 or 300 mg/kg/day. Males dosed with 300 mg/kg/day also showed
elevated bodyweight-adjusted mean liver weight compared with controls. Heavier than
control mean adrenal weight was recorded for both sexes receiving 300 mg/kg/day.

GROSS PATHOLOGY
Enlarged adrenals were seen in 3/5 females dosed at 300 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment with 300 mg/kg/day was associated with cortical hypertrophy and hyperplasia in
the adrenals of female rats, an increased degree of aggregations of foamy alveolar
macrophages in the lungs of male and female rats and extramedullary haemopoiesis in spleen
of female rats. Treatment with 150 mg/kg/day was associated with cortical hypertrophy in
the adrenals and extramedullary haemopoiesis in the spleen of female rats. Treatment with
15 mg/kg/day was associated with cortical hypertrophy in the adrenals of female rats. As this
alteration was only slight and seen in 5/5 females each dosed at 15 and 150 mg/kg/day but only
in 2/5 dosed at 300 mg/kg/day, the finding could not be attributed to the treatment with the test
item. Therefore it was not considered for the overall assessment concerning the NOAEL.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data

HISTORICAL CONTROL DATA (if applicable)
No data

OTHER FINDINGS
None

Effect levels

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Gross pathology table

Dose Level (mg/kg/day)/Sex	0/M	15/M	150/M	300/M	0/F	15/F	150/F	300/F
Adrenals:
Enlarged	0	0	0	0	0	0	0	3
Total examined	5	5	5	5	5	5	5	5

 

Histopathology table

Dose Level (mg/kg/day)/Sex	0/M	15/M	150/M	300/M	0/F	15/F	150/F	300/F
Adrenals:
Cortical hyperplasia
Minimal	0	0	0	0	0	0	0	5
Cortical hypertrophie
Minimal	0	0	0	0	0	0	0	0
Slight	0	0	0	0	0	5	5	2
Moderate	0	0	0	0	0	0	0	3
Total affected	0	0	0	0	0	5	5	5
Total examined	5	5	5	5	5	5	5	5
Lungs:
Aggregations of Foamy alveolar macrophages
Minimal	1	0	1	3	0	0	0	2
Slight	0	0	0	1	0	0	0	1
Total affected	1	0	1	4	0	0	0	3
Total examined	5	5	5	5	5	5	5	5

Applicant's summary and conclusion

Conclusions:

As toxicologically important findings were evident for both sexes dosed at 300 mg/kg/day and 150 mg/kg/day the No Observed Adverse Effect
Level (NOAEL) on this study for VRT-126017 dcha is 15 mg/kg/day.
Due to the disturbances in red blood cell parameters at all treatment levels a No Observed Effect Level (NOEL) was not established on this study.