ethynyl cyclopropane

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)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Method B.7. Sub-acute toxicity (oral) .

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-430-1

EC Name:

-

Cas Number:

6746-94-7

Molecular formula:

C5 H6

IUPAC Name:

ethynylcyclopropane

Details on test material:

- Name of test material (as cited in study report): EthynyI cyclopropane (SD 957)
- Physical state: Colourless liquid
- Analytical purity: 97-100 %
- Lot/batch No.: 3590-129-D4
- Expiration date of the lot/batch: January 1999
- Storage condition of test material: -20°C, under nitrogen and in the dark
- Intended use: Pharmaceutical intermediate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, England
- Age at study initiation: four to six weeks
- Weight at study initiation: group means about 100g (females) and 104 g (males)
- Fasting period before study: none
- Housing: housed by sex in groups of 5
- Diet: Rat and Mouse No. 1 maintenance diet (Special Diet Services) ad libitum
- Water: drinking water ad libitum
- Acclimation period: for a minimum period of 1 week prior to the start of the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

The batch(s) of diet used for the study was analysed for certain nutrients, possible contaminants and microorganisms.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution in com oil. Control animals received corn oil alone. Formulations were prepared in the Pharmacy Department at Huntingdon Research Centre. The test substance was removed from the cold store (ca -20°C) and allowed to warm to ambient temperature. A solution of the test substance, in corn oil, was prepared for the High dose group by initially shaking and stirring the two liquids together. Formulations were then prepared for the Intermediate and Low dose groups by serial dilution. The formulations were stirred using a magnetic stirrer until such time the operator considered that adequate mixing had taken place, prior to removal of an aliquot for subsequent dilution.
Fresh batches of formulation were made for each week of the study . Each batch was subdivided into bottles, transported to Building Y11 and stored in a refrigerator. One bottle was used for each group on each day of dosing. Unused formulation was discarded each day following dosing. The formulations
were stored in a refrigerator at 4°C.

Formulation sampling and analysis
Prior to the commencement of the study, the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the stability of the formulation was satisfactory under the conditions of the study.

Administration of test and control formulations
During dosing, the formulations were held in a fume cabinet which was held in a walk-in ventilated cabinet separate from those cabinets used to house the animals and maintained at a pressure negative to that of the main room. The walk-in ventilated cabinet drew its air supply from the main room and the air extracted from the cabinet was scrubbed by passage through activated charcoal filters before venting to atmosphere. The test formulation containers were held in the fume cabinet within the ventilated cabinet and kept closed when not in use to prevent loss of test substance through evaporation. Filling of dosing syringes and associated procedures took place in the fume cabinet within the ventilated cabinet with the doors shut to ensure containment of any vapour.
Dosing of rats was staggered for logistical reasons. Ten rats (5 male and 5 female) were dosed from 9 April to 6 May 1997 (Subgroup A) and ten rats (5 male and 5 female) were dosed from 10 April to 7 May 1997 (Subgroup B) . The treatment period was reported as Days 1 to 28 of the study and any data
from the two subgroups were combined in order to calculate overall group means.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 150, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose level

Control animals:

yes

Details on study design:

- Dose selection rationale: The high dosage level was selected upon available toxicity data, in particular a preliminary seven-day oral toxicity study
(Huntingdon Life Sciences report number DTL 15/971456). Generally, other levels were selected on the basis of the key dosages relative to EEC labelling requirements.

Groups of twenty rats (ten males and ten females) were dosed as follows :
Group Dosage of SD957 Dose concentration Dose volume Measured dose concentrations
(mg/kg bw/day) (mg/ml) (ml/kg) (mg/ml)
Day 1 Day 3
1 (Control) 0 0.0 10 0.0 0.0
2 (Low dose) 15 1.5 10 1.18 1.32
3 (Inter dose) 150 15.0 10 12.5 13.9
4 (High dose) 1000 100 10 87.8 95.7

All dose groups were dosed once a day, for 28 consecutive days.
Twice during each week of the treatment period (Days I and 4), after the animals had been weighed, the volume of vehicle and test formulation to be administered to individual animals was calculated according to the most recent individual bodyweight data. Volumes of test formulation or vehicle to be
administered were rounded to the nearest 0.1 ml.
Prior to dosing of each group, the test formulations were mixed by inversion. Each cage of animals was transferred from the holding cage to a closed cage for transportation to the ventilated dosing cabinet. The doors of the dosing cabinet remained closed during the administration of the test formulation. Dosing was performed using a suitably graduated glass syringe and stainless steel catheter. The rats were then returned to the appropriate holding cage.
Control animals received a similar volume of the vehicle and were dosed in the main room. Each cage of animals was dosed separately and animals were dosed at approximately the same time each day, where possible.

Positive control:

not included

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGNS
All signs of ill health, together with any behavioural change or response to treatment, were recorded. Individual animal records were maintained on the basis of daily observations made at approximately 1 and 5 hours post-dose; any observation made at any time during the study. An entry was made on the individual sheets at 1 and 5 hours post-dose even if abnormalities were not seen.

MORTALITY AND MORBIDITY
All cages were checked early in each working day and again in the late afternoon for dead or moribund animals

BODYWEIGHT
The weight of each rat was recorded before dosing on Days I and 4 of each week and prior to termination on Day 29.

FOOD CONSUMPTION
The quantity of food consumed by each cage of rats was recorded twice weekly from weighday to weighday. The quantity of food consumed was calculated from the amount of food given to and left by each cage of rats for the specified time period. Mean food intake (g/rat/day) was calculated for each dosage group from the amount of food consumed by each cage group during the specified time period and the number of rats in each cage. This consumption value was calculated using a computer program and adjustments made if unscheduled deaths had occurred. Food consumption was not adjusted for spillage.

LABORATORY INVESTIGATIONS
Sample collection
Blood samples were collected from all animals, where practicable, during Week 4 (prior to dosing on Day 27). Rat number 64F (Intermediate dose) died under anaesthesia prior to removal of the blood sample. Food was withheld from the animals overnight, prior to blood sampling; free access to water was
available to all rats overnight. Samples of venous blood were obtained from the orbital sinus using sterile glass pipettes while the rats were held under light ether anaesthesia. The blood samples collected were divided into tubes containing the following anticoagulants:
EDTA for haematological investigations
Citrate for clotting tests
Heparin for blood chemistry investigations

The haematological and blood biochemical investigations performed are listed below, together with an abbreviated title, methods and the units of measurement.

Haematology
Units
The following estimations were performed using a Bayer-Technicon HlE haematology analyser, using standard Bayer-Technicon HIE methodology:
Packed cell volume (PCV)
Haemoglobin (Hb)
Red cell count (RBC)
Mean corpuscular haemoglobin concentration (MCHC) by calculation, Hb (g/dl) x 100 / PCV (%) in (g/dl)
Mean corpuscular volume (MCV) - by calculation, PCV (%) x 10 / RBC (x 10e12/l) in (fi)
Mean corpuscular haemoglobin (MCH) - by calculation, Hb (g/dl) x 10 / RBC (x10e12/l) g/dI x 1Oe12/l in(pg)
Total white cell count (WBC total) x 10e9/l
Differential WBC count (Diff) x 10e9/l
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)

Cell morphology : The most common morphological changes (anisocytosis, micro/macrocytosis, variation in colour, hypo/hyperchromasia, left shift, atypical/blast cells) were recorded as follows:
No abnormality detected
+ Slight
++ Moderate
+++ Marked

In the case of atypical blast cells, or other abnormalities, confirmation or a written description from a blood film was made.
Platelet count (Plt) x 10,71
Thrombotest (TT) - method of Owren (1959)

The following estimations were performed using an ACL 3000 Plus analyser using the appropriate IL reagents:
Activated partial thromboplastin time (APTT)
- method of Proctor and Rapaport (1961)

Biochemistry
The following estimations were performed with an Hitachi 737 clinical chemistry analyser, using standard Hitachi 737 methodology:
Glucose - hexokinase mediated assay mg/dl
Total protein g/dl
Albumin(Alb) g/dl
Globulin (Glob) - by subtraction
total protein (g/dl) minus albumin (g/dl) g/dl
Albumin/globulin ratio (A/G)
Calculated from Total protein and Albumin concentrations
Urea nitrogen (Urea Nitr) mg/dl
Creatinine mg/dl
Alkaline phosphatase (AP) - reaction temperature 30°C mU/ml
Glutamic-pyruvic transaminase (GPT) also known as 'Alanine aminotransferase - reaction temperature 30*C mU/ml
Glutamic-oxaloacetic transaminase (GOT) also known as 'Aspartate aminotransferase - reaction temperature 30°C mU/ml
Total bilirubin mg/dI
Sodium (Na) mEq/l
Potassium (K) mEq/1
Calcium (Ca) mEq/l
Inorganic phosphorus (P) mEq/1
Chloride (CI) mEq/I
Cholesterol (Chol) mg/dl

Sacrifice and pathology:

TERMINAL STUDIES
Sacrifice
On completion of the 28-day treatment period, all surviving rats were killed.
The rats were killed by carbon dioxide asphyxiation. The terminal bodyweight of each animal was recorded prior to necropsy.
Macroscopic pathology and organ weight analysis
A detailed post mortem macroscopic examination of all rats was performed. The following organs were dissected from each rat and weighed:
adrenals
liver
seminal vesicles
brain
lungs
spleen
epididymides
ovaries / testes
kidneys
prostate
thymus
heart

Testes and epididymides were identified as left and right and weighed separately.

Tissue preservation
Samples, or the whole, of the following organs/tissues, were preserved . Eyes were preserved in Davidson's fixative. Testes and epididymides were fixed in Bouin's solution and then transferred to 70% industrial methylated spirits. All other tissues were preserved in neutral buffered 10% formalin. The lungs were distended with fixative prior to immersion. In addition, samples of any macroscopically abnormal tissues, together with samples of adjacent tissue where considered appropriate, were preserved.

Organ preservation list:
adrenals*
alimentary tract
oesophagus*
Stomach (glandular and non-glandular)*
duodenum*
jejunum*
ileum*
caecum*
colon*
rectum *
animal identification marks
aorta (arch and abdominal)
brain (3 levels)*
epididymides*
eyes
femur with joint (for bone and marrow in situ)
gross abnormalities *
head
heart*
kidneys*
larynx (2 levels)*
liver *
lungs (all lobes and mainstern bronchi)*
lymph nodes (cervical, mesenteric and tracheobronchial)
mammary gland
ovaries*
pancreas
pharynx*
pituitary
prostate*
salivary gland*
sciatic nerve*
seminal vesicles*
skeletal muscle (thigh)*
skin
spinal column
spinal cord (cervical, thoracic and lumbar)
spleen*
sternum (bone and marrow)*
testes 2 *
thymus*
thyroids (with parathyroids)*
tongue*
trachea (including bifurcation)*
urinary bladder*
uterus (corpus and cervix)*
vagina

Microscopic pathology
Tissues which were examined using a light microscope are identified by an asterisk (*) in the list above. These tissues were embedded in paraffin wax and sections, approximately 4 µm thick, were cut, processed routinely and stained with haernatoxylin and eosin. For testes and epididymides, a transverse section of each testis and a U1 longitudinal section of each epididymis was cut as near as possible to 2 µm in thickness, and was stained with PAS I - haematoxylin.

The microscopic examinations consisted of the following:
Groups 1 and 4 - all identified tissues
Groups 2 and 3 - all identified tissues from decedent animals
- other, macroscopically abnormal tissues and tissue(s) with treatment-related findings in the High dose group (Group 4) .

Statistics:

All statistical analyses were carried out separately for males and females. Food consumption data were analysed using cumulative totals. Bodyweight data were analysed using weight gain. The following sequence of statistical tests was used for bodyweight gain, food consumption, haematology, biochemistry and organ weight data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of animals with values different from the mode was analysed by appropriate methods.

Otherwise:
Bartlett's test (Bartlett 1937) was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to determine if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found), a oneway analysis of variance was carried out . If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks (Kruskal and Wallis 1952/3) was used. Analysis of variance was followed by Student's 't' test and Williams' test (Williams 1971/2) for a dose-related response although only Williams' test was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test (Shirley 1977).
Additionally, for organ weight data. analysis of covariance was carried out adjusting for the final bodyweight where the regression coefficient describing the linear relationship between organ weight and the covariate was significantly different from zero at the 10% level. Where there was no such relationship, analysis of variance was performed on the unadjusted values.
Significance testing was performed at the 5% and 1% levels and the results are indicated in the tables using an asterisk notation as follows :
Williams' test
* p<0.05
** P<0.01

Results and discussion

Results of examinations

Details on results:

CLINICAL OBSERVATIONS
Mortality
There were no deaths associated with the administration of SD957.
one rat (Intermediate dose) died under ether anaesthesia prior to the scheduled removal of blood for laboratory investigations in Week 4 .

Clinical signs
There were no clinical signs attributable to the administration of SD957.
Isolated incidents of clear salivation were noted at the Intermediate dose (1 male and 1 female) at 1 hour post-dose on Days 6 and 16. Other signs observed included hair loss on the dorsum in a proportion of rats in all groups and malformed toes in a single high dose male rat. These signs were considered incidental in nature and of no toxicological significance.

Bodyweight
The overall mean bodyweight gain for test groups was similar or superior to that of the concurrent controls.

Food consumption
The mean cumulative food consumption for all treated groups was similar to that of controls.

Haematology
There were no treatment-related findings. Any differences achieving statistical separation from control values were inconsistent between sexes, were considered not to be of toxicological importance and did not correlate with any histopathological findings.

Biochemistry
Lower alkaline phosphatase (AP) activities were noted for High dose rats of both sexes, in comparison with concurrent controls, a degree of statistical significance was attained for male rats only. This finding may be related to administration of SD957 but the biological significance is unclear and values
in all groups were at the upper range of historical background values for rats of the strain and age at the investigator's laboratories. Other differences achieving statistical separation from control values were inconsistent between the sexes and were considered not to be of toxicological importance.

TERMINAL INVESTIGATIONS
Organ weights
Differences from control values were generally marginal in degree. Noteworthy findings are summarized as follows:
The mean liver weights of High dose rats (both sexes) were marginally higher than those of the concurrent controls, a degree of statistical significance was attained for High dose female rats. Mean testes weights of High and Intermediate dose rats were higher than control values, attaining a degree of statistical significance for the combined testes and left testis weights. However, control testis weights were at the lower range of our background data records for rats of the strain and age used. Higher, dosage-related mean kidney weights were also noted in male test groups in comparison with controls . A degree of statistical significance was attained for all male test groups.

Other differences from control, some of which attained statistical significance were inconsistent between the sexes and were considered not to be of toxicological importance.

Macroscopic pathology
There were no treatment-related findings noted at necropsy .

Microscopic pathology
Treatment-related findings
Kidneys - Eosinophilic inclusions in the cortical tubular epithelium were seen in males from all groups receiving SD957. The incidence and severity showed a dosage relationship. These findings may account for the increased kidney weights seen in treated males at termination.

Males
Dosage level (mg/kg/day) 0 15 150 1000
Eosinophilic inclusions in cortical Total 0 3 9** 9**
tubular epithelium Minimal 0 3 8** 1
Slight 0 0 0 6**
Moderate 0 0 1 1
Total number of kidneys examined 10 0 0 10
*p < 0.05 **p < 0.01 with Fisher's Exact Test

Other findings
No histopathological findings were seen which would account for the higher liver weights seen in High dose male and female rats or for the higher testes weights seen in High dose male rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

In this study, rats were treated with a solution of SD957 in corn oil, at nominal dosages of 15, 150 and 1000 mg/kg1day, for a period of 28 consecutive days . The actual dosages in the study were approximately 13, 139 and 577 mg/kg/day in the Low, Intermediate and High dosage levels, respectively.

There were no deaths attributable to the administration of SD957. There were no treatment-related clinical signs, and no effect on the rates of bodyweight gain and food consumption, or food consumption parameters. Low alkaline phosphate levels, apparently dose-related in both sexes were within the background control ranges for rats of the age and strain used. Control values were at the high end of the background range for this parameter, and it is considered that the differences in alkaline phosphatase are not of toxicological significance. Control liver and testes weights were towards the lower percentile of background range for the age and strain of rats used in this study. The resulting slight differences that achieved statistical significance were considered not of toxicological significance. In addition there were no treatment-related histopathological changes present in these organs that would account for the minimal weight differences observed. Kidney weight increases recorded for male rats in the SD957 treated groups (approximately 108 -113% of control values) may be associated with an increased incidence of minimal to moderate eosinophilic inclusions in the cortical tubular epithelium. The finding of prominent and/or increased incidence of eosinophilic inclusions in the proximal tubular epithelium of male rat kidney is known to be associated with accumulation of cc2-microglobulin, and has been previously observed in rats treated with hydrocarbon compounds (Alden CL (1996); A Review of Unique Male Rat Hydrocarbon Nephropathology. Toxicol. Pathol. 14, 109 -111). The protein cc2-microglobulin is not found in humans, and this histological finding, peculiar to the male rat is considered of no biological or toxicological significance to man.

Conclusion: The no observed adverse effect level in this study was 1000 mg/kg/day.