(6R,7R)-7-[((2Z)-2-[({2-[(1,1-dimethylethyl)oxy]-1,1-dimethyl-2-oxoethyl}oxy)imino]-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetyl)amino]-8-oxo-3-(1-pyridiniumylmethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-8-2017-15-12-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

elevated humidity for 5 day of study, no adverse impact on the result

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

as above

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-{[(6R,7R)-7-[(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetamido]-2-carboxylato-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}pyridin-1-ium

EC Number:

617-560-2

Cas Number:

84377-83-3

Molecular formula:

C45H44O7S2.5/2(C3H7NO)

IUPAC Name:

1-{[(6R,7R)-7-[(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetamido]-2-carboxylato-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}pyridin-1-ium

additive 1

Reference substance name:

N,N-dimethylformamide

EC Number:

200-679-5

EC Name:

N,N-dimethylformamide

Cas Number:

68-12-2

Molecular formula:

C3H7NO

IUPAC Name:

N,N-dimethylformamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification : Substance CQ
Physical State/Appearance : White Powder
Purity : 99.112%
Batch Number : G315830
Label : CQP BX G315830 DOM 26 APR 2017 Shelf Life
Expires: 26 OCT 2017
Date Received : 04 May 2017
Storage Conditions : Store cold at approximately 4ºC in darkness,
used/formulated in light and ambient temperature
Expiry Date : 26 October 2017

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were
examined for signs of ill-health or injury. The animals were acclimatized for nine days
during which time their health status was assessed. A total of forty animals (twenty males
and twenty females) were accepted into the study. At the start of treatment the males
weighed 192 to 218g, the females weighed 146 to 177g, and were approximately seven weeks
old.
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.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for up to 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 4 mL/kg of Arachis oil BP.
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.

Vehicle:

arachis oil

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 Arachis oil BP. The stability and homogeneity of the test item formulations were
determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results
showed the formulations to be stable for at least 12 days. Formulations were therefore
prepared weekly during the treatment period and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken on three occasions and analyzed for
concentration of Substance CQ at Envigo Research Limited, Shardlow, UK, Analytical
Services. The results indicate that the prepared formulations were within ± 7% of the
nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 (five)

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were randomly allocated to treatment groups using a 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.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.


DETAILED CLINICAL OBSERVATIONS: Yes - as above

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 then weekly intervals until terminal kill



FOOD CONSUMPTION: Yes
Food consumption was recorded for each cage group at weekly intervals throughout the
study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION : Yes
Water intake was observed daily, for each cage group, by visual inspection of the water
bottles for any overt changes except during Week 3 where water intake was measured
gravimetrically.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all surviving animals from each test and control group at the end of the study
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11
mol/L).

CLINICAL CHEMISTRY: Yes
-- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals:
all surviving animals from each test and control group at the end of the study
The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea, Inorganic phosphorus (P)
Glucose, Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.), Alanine aminotransferase (ALAT)
Albumin, Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat)
Sodium (Na+), Triglycerides (Trigs/Tri)
Potassium (K+), Total cholesterol (Chol)
Chloride (Cl-), Total bilirubin (Bili)
Calcium (Ca++), Bile acids

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
Prior to the start of treatment and on Days 6, 13, 20 and 27 all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different
stimuli. Observations were carried out from approximately two hours after dosing on each
occasion.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: behavior assessment

IMMUNOLOGY: No


OTHER:

Sacrifice and pathology:

Necropsy
On completion of the dosing period all surviving animals were killed by intravenous
overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum
from each animal was stored frozen at approximately -80 °C. No treatment-related effects on
the pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
The following organs, removed from animals that were killed at the end of the dosing period
were dissected free from fat and weighed before fixation:
Adrenals, Liver
Brain, Ovaries
Epididymides, Spleen
Hear,t Testes
Kidneys, Thymus
Pituitary (following partial fixation), Thyroid/Parathyroid (following partial fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids), Uterus with Cervix

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered
10% formalin, except where stated:
Adrenals, Ovaries
Aorta (thoracic), Pancreas
Bone & bone marrow (femur including stifle joint), Pituitary
Bone & bone marrow (sternum), Prostate
Brain (including cerebrum, cerebellum and pons), Rectum
Salivary glands (submaxillary)
Caecum, Sciatic nerve
Colon, Seminal vesicles (with coagulating glands and fluids)
Duodenum
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions, Spleen
Heart, Stomach
Ileum, Testes ♦
Jejunum, Thymus
Kidneys, Thyroid/Parathyroid
Liver, Trachea
Lungs (with bronchi)#, Urinary bladder
Lymph nodes (mandibular and mesenteric), Uterus & Cervix
Mammary gland , Vagina
Muscle (skeletal)

* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid

All tissues were dispatched to the histology processing Test Site (Propath UK Ltd., Willow
Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal
Investigator: N Fower). The tissues shown in bold from all control and 1000 mg/kg bw/day
dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5
µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any
macroscopically observed lesions were also processed. In addition, sections of testes from all
Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain
and examined.
Since there were indications of treatment-related changes, examination was subsequently
extended to include similarly prepared sections of the liver (both sexes), thyroids (both
sexes), kidneys (males only) and mesenteric lymph nodes (females only) from animals in the
low and intermediate groups.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values
where appropriate. All data were summarized in tabular form.
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.
Data were analyzed using the decision tree from the Provantis
TM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method.
The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with
appropriate covariates. Any transformed data were analyzed to find the lowest treatment
level that showed a significant effect using the Williams Test for parametric data or the
Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity
of means,
the
data
were
analyzed
by
a stepwise
Dunnett’s (parametric)
or
Steel

(non-parametric)
test
to
determine
significant
difference
from
the
control
group.
Where
the

data
were
unsuitable
for these analyses,
pair-wise
tests
was performed
using
the
Student
t-test

(parametric)
or the
Mann-Whitney
U
test
(non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased salivation was evident in all males and females treated with 1000 mg/kg bw/day
between Days 11 and 26 and in one female treated with 300 mg/kg bw/day on Day 22. Noisyrespiration was also evident in one female treated with 1000 mg/kg bw/day on Day 18 only.
The female treated with 1000 mg/kg bw/day that was killed in extremis on Day 21 showed
ptosis, pilo-erection, decreased respiratory rate, lethargy, hypothermia, hunched posture,
pallor of the extremities and ataxia on Day 21. The female that was found dead on Day 18
only previously showed increased salivation.
No such effects were evident in males treated with 300 mg/kg bw/day or in animals of either
sex treated with 30 mg/kg bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female treated with 1000 mg/kg bw/day was found dead on Day 18. This female had a
dark liver and pale lungs and a thin non-glandular region of the stomach at necropsy. At
histopathology, there was marked centrilobular necrosis and hemorrhage in the liver (cause of
death). Lymphocytolysis and plasmacytosis were present in the mesenteric lymph node and
decreased cellularity in the bone marrow. A further female from this treatment group was
killed in extremis on Day 21. This female had a pale brain and pituitary, a dark liver with a
mass attached to all lobes and an enlarged spleen at necropsy. At histopathology, there was
marked centrilobular necrosis, hemorrhage in the liver (cause of death) and a lobar torsion
which accounted for the mass. Lymphocytolysis was present in lymph nodes and the thymus
and increased hematopoiesis in the spleen.
There were no further unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect on body weight development for treated animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption or food conversion efficiency for treated
animals.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption or food conversion efficiency for treated
animals.

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

no effects observed

Description (incidence and severity):

There was no adverse effect on water consumption for treated animals.
Hematology

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the hematological parameters
examined.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 and 300 mg/kg bw/day showed an increase in
cholesterol. Males treated with 1000 and 300 mg/kg bw/day also showed a reduction in
triglycerides whilst females treated with 1000 mg/kg bw/day also showed an increase in total
protein. No toxicologically significant effects were evident in animals of either sex treated
with 30 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

here were no toxicologically significant changes in functional performance. There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 and 300 mg/kg bw/day showed an increase in liver
weight both absolute and relative to terminal body weight. Males treated with 1000 mg/kg
bw/day also showed an increase in kidney and thyroid weights both absolute and relative to
terminal body weight. No toxicologically significant effects were detected in animals of
either sex treated with 30 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three males treated with 1000 mg/kg bw/day had mottled kidneys. No macroscopic
abnormalities were detected in surviving females treated with 1000 mg/kg bw/day or in
animals of either sex treated with 300 or 30 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys: Mild hyaline droplets were present in the cortical tubules of four males treated with 1000 mg/kg bw/day. No such effects were evident in treated females or in males treated with 300 or 30 mg/kg bw/day.
Liver: Minimal to marked centrilobular necrosis was present in four males and two females
treated with 1000 mg/kg bw/day, one hemorrhagic, along with one female treated with 300
mg/kg bw/day. Centrilobular vacuolar degeneration was present in two males treated with
1000 mg/kg bw/day and in one female treated with 300 mg/kg bw/day. Increased mitosis was
seen in three males and one female treated with 300 mg/kg bw/day. Moderate centrilobular
hepatocyte hypertrophy was present in four males and one female treated with 1000 mg/kg
bw/day. Minimal or mild centrilobular hypertrophy was present in three males and three
females treated with 300 mg/kg bw/day.
Mesenteric Lymph Node: Mild lymphocytolysis was present in two females treated with
1000 mg/kg bw/day. No such effects were evident in treated males or in females treated with
300 or 30 mg/kg bw/day.
Thyroid Glands: Minimal follicular cell hypertrophy was present in one male treated with
1000 mg/kg bw/day. No such effects were evident in treated females or in males treated with
300 or 30 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

ca. 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of Substance CQ for up to twenty-eight consecutive days, to
Wistar rats of both sexes at dose levels of 30, 300 or 1000 mg/kg bw/day resulted in the early
death of two females treated with 1000 mg/kg bw/day and adverse microscopic liver changes
in animals of either sex treated 1000 and 300 mg/kg bw/day. Secondary to the marked liver
necrosis, microscopic mesenteric lymph node changes were also evident in females treated
with 1000 mg/kg bw/day. Non-adverse microscopic kidney and thyroid changes were also
evident in males treated with 1000 mg/kg bw/day. The No Observed Effect Level (NOEL)
was therefore considered to be 30 mg/kg bw/day for both sexes.