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REACH

Pentanedioic acid, compd. with (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (1:1)

EC number: 469-910-7 | CAS number: 847842-48-2

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Two studies conducted on an analogue material to assess repeated dose toxicity in rats provided the following information:

Natvig & Brodie (1998) - Subacute (28 day): Doses equivalent to 35, 135 and 530 mg 1592U89/kg/day were selected for this study based on a previous 90 day (twice daily) oral dose range finding toxicity study in rats (Myer 1998). Decreases in serum total protein and albumin, increased cholesterol, and liver hepatocellular hypertrophy were effects noted in that study.

Decreases in albumin and total protein were observed at 135 and 530 mg/kg/day doses and increased cholesterol values were seen in the 530 mg/kg/day dose. The no observable effect level (NOEL) was 35 mg/kg/day.

All findings showed evidence of reversibility following a 14 day recovery period.

Slauter (1997) - Subchronic (90 day): The test article related effects noted in the serum biochemistry parameters in the 135 and 530 mg/kg/day dosage groups were correlated with liver weight changes, the presence of hepatocellular hypertrophy, and Kupffer's cell pigment accumulation in the 530 mg/kg/day rats. Liver weight changes were correlated microscopically in the 530 mg/kg/day rats with hepatocellular hypertrophy and pigment accumulation. There was also trace hepatocellular hypertrophy observed in the 135 mg/kg/day male dosage group. Test article related hypertrophy of the thyroid follicular epithelium and germ cell loss in the testis were also present in the 530 mg/kg/day male dosage group.

Based on the results, the no observable effect level (NOEL) is considered to be 35 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

18 December 1996 and 18 March 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

: Please see "Principles of method.." for further details.

Deviations:

yes

Remarks:

: Please see "Principles of method...." below for details.

Principles of method if other than guideline:

Study Guideline:
This was a nonclinical laboratory study and was conducted in the spirit of the United States Food and Drug Administration (FDA) Good Laboratory Practice Standards, 21 CFR Part 58, effective 5 October 1987. However, this study had no Quality Assurance inspections conducted and did not appear on the Quality Assurance Master Schedule. The protocol for this study met the United States Food and Drug Administration requirements for a study of this type. All procedures were conducted according to the study protocol.

Deviations:
This study was consucted in conformance with the protocol and Standing Operating Procedures and in compliance with the Good Laboratory Practice Regulations.
On 7 January 1997, when the rats were moved into clean cages as scheduled, the technicians did not review the schedule and offered the rats clean feeders prior to the day they were to receive measured food consumption. Therefore, this resulted in the Week 3 food consumption being lost.
Toxicokinetic rats received an opthalmoscopic examination at study termination. The protocol specified that only main study rats were to receive an opthalmoscpic examination at study termination. This data has not been included in this report but will be maintained in the study records.
In the opinion of the Study Director the minor deviations that occurred did not affect the quality or integrity of the study.

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

other: Han Wistar (Glx:Han:WlfBR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal acquisition and maintenance
Seventy male and 70 female Han Wister (Glx:Han:WIfBR) rats were received on 5 December 1996 from Taconic Farms, Inc., Gennantown, New York, and acclimated for a 14 day period. The rats were housed 3-5 to a cage. The rats were approximately 4 weeks of age at arrival and 6 weeks at study initiation. The bodyweight range 8 days prior to study initiation was 128-196 grams for males and 99-157 grams for females.

Each rat was identified by cage, group, sex, and individually with an animal number by tail tattoo. Animals numbers were verified after initial tagging, at each cage change, at blood sample collection, and prior to euthanasia. The individual animal number plus the MPI Research project study number comprised a unique identification for each rat All unassigned rats were euthanized and discarded.

The rats were housed 3-5 per cage in clear, polycarbonate "shoe-box" type cages. The rats were maintained in an environmentally-controlled room with a 12 hour light/dark cycle, a temperature range of 68 to 74°F (20 to 23°C), and a humidity range of46 to 58%.

The basal diet was certified Opti-Diet #5K15 Rodent Pellets, PMI Feeds, Inc. Food and water were available ad libitum except during designated fasting periods. Each lot number used was identified and recorded. Certification analysis of each diet lot was performed by the manufacturer. The water supply was analyzed on a quarterly basis for the presence of heavy metals, pesticides, and specified contaminants. Neither the Sponsor nor the Study Director is aware ofany potential contaminants likely to be present in the diet or water which would interfere with the results of this study.

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose

Details on oral exposure:

Vehicle preparation
The appropriate amount of vehicle was weighed and dissolved in hot, deionized water using a magnetic stir bar and stir plate. The solution was allowed to cool to room temperature. A portion of the solution was transferred into a volumetric flask, and deionized water was added. The flask was shaken thoroughly, and the contents were dispensed into a suitable container. The remaining solution was transferred to the flask, and deionized water was added for rinsing. The flask was shaken thoroughly, and the rinse contents were dispensed into the container. An additional volume of deionized water was measured using the flask and added to the container to yield the prepared vehicle. The contents of the container were mixed using a motor-driven propeller. The prepared vehicle was stored refrigerated when not in use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity
Analysis was carried out using HPLC. Prior to initiation of test article administration, test batches of the low (35mg/kg/day) and high (530mg/kg/day) test article suspensions were analyzed for homogeneity.
Samples ofeach test article suspension at each dose level were collected at Weeks 1, 4, 6, 9, and 13 for analysis oftest article concentration using methods supplied by the Sponsor.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Twice daily, approximately 6 hours apart. The total dose was split equally between the 2 daily doses.

Remarks:

Doses / Concentrations:
35 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
135 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
530 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Test article preparation
The test article was used as received from the Sponsor. No adjustments were made for purity. However, all doses were expressed as 1592U89 (free base) and a correction factor of 1.17 was used to convert 1592U89 (hemisulfate salt) to the free base. Appropriate amounts of the test article were weighed and suspended in the vehicle using a Thomas tissue homogenizer with Teflon pestle. The suspension was transferred into a graduated cylinder. Additional vehicle was added to the cylinder. The cylinder was shaken thoroughly, and the contents were dispensed into a beaker. An additional amount of vehicle was measured into the cylinder; the cylinder was shaken thoroughly, and the contents were dispensed into the beaker to yield the desired concentrations of prepared test material. The contents of the beaker were stirred using a magnetic stir bar and stir plate and, using a syringe, were dispensed into amber glass containers, while stirring. Fresh solutions were prepared weekly during the study, divided into daily aliquots for dosing, and stored at room temperature until used. Dose preparations were stirred throughout the dosing procedure.

Animal maintenance
During the acclimation period, all rats were observed daily for any clinical signs of disease and given a detailed clinical examination and an ophthalmoscopic examination prior to study initiation. The ocular examination consisted of an indirect ophthalmoscopic examination of the cornea, conjunctiva, sclera, iris, fundus, and lens. This examination was conducted by a board certified veterinary ophthalmologist. No ocular lesions were found. All rats with any evidence of disease or other physical abnormalities were euthanized using carbon dioxide inhalation and discarded. Rats considered suitable for study were randomly selected and divided into groups.

Selection for study
Prior to randontization into study groups, the ras were weighed, sexed, and examined for evidence of disease and other physical abnormalities. Rats selected on the basis of best physical condition were randomized utilizing the Xybion block randomization procedure in which they were stratified by bodyweights. Homogeneity of group variance by bodyweight (20% of the mean weight for each group and sex) was used as the criterion for acceptance, then the randomization was accepted and permanent animal numbers were assigned.
Fifty-six males (weighing from 169-219 grams at randomization) and 56 females (weighing from 124-159 grams at randomization) were assigned to the control or treated groups.

Administration
The doses were adjusted weekly to provide constant dose levels of 35, 135, and 530 mg/kg/day at a constant volume of 20mL/kg/day (10mL/kg/dose). The control rats received the vehicle at a dose volume that was comparable to that received by the test rats. Individual doses were based on the most recent bodyweights.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All rats were observed at least twice daily throughout the study for morbidity, mortality, and signs of injury. Any findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed clinical examination of each main study rat was performed once during each study week and included clinical, pharmacological, and toxicological findings as well as the occurrence, size, location, and description of palpable masses. Rats designated for toxicokinetics were examined.

BODY WEIGHT: Yes
Individual bodyweights were measured and recorded for all rats prior to study initiation, then weekly during the study, and prior to necropsy. Postmortem bodyweights taken at terminal necropsy were used for Week 13 bodyweights and food consumption analysis and food efficiency calculation for the main study rats. There were no bodyweight data for Week 13 for the toxicokinetic rats.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
Food consumption was measured and recorded for all main study rats prior to study initiation, then weekly during the study. As these rats were group housed, food consumption was calculated on a per·cage basis. Individual food consumption values were calculated by dividing the per cage food consumption by the number of rats which occupy the cage. Food efficiency was calculated weekly from the food consumption and bodyweight values. Food consumption was not measured for rats designated for toxicokinetics.

OPHTHALMOSCOPIC EXAMINATION: Yes
All rats received an indirect ophthalmoscopic examination prior to study initiation and during the final week of study. The examination was conducted by a board certified veterinary ophthalmologist.

HAEMATOLOGY: Yes
Clinical laboratory studies were conducted on all surviving main study rats at study termination. Blood samples were taken by the vena cava while the rats were under sodium pentobarbital anesthesia. The rats had free access to food and water prior to blood collection. The order of bleeding and analysis was by alternating 1 rat from each dose group, then repeating, to reduce handling and time biases.

CLINICAL CHEMISTRY: Yes: Plasma Concentration Analysis
Blood samples were collected from 2 rats/sex/group from the rats that were designated for toxicokinetic analysis for determination of the plasma concentration of the test article. Blood was collected from the tail vasculature on Day 3 and Day 90 at 0 (predose), 0.5, 1, 2, 4, and 6 hours following the first daily dose. Each sample was approximately 0.5-1.0mL. The rats were not fasted prior to blood collection. Following collection of the final time interval on Day 90, all of the surviving toxicokinetic rats were humanely euthanized and discarded without necropsy.

URINALYSIS: Yes
On a day separate from the blood collection, the rats were placed individually in stainless steel metabolism cages for urine collection over a period of approximately 16 hours. The rats had access to water but food was removed during the urine collection period.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Anatomic Pathology
Macroscopic
All main study rats received a complete postmortem examination under the direct supervision of a veterinary pathologist. All main study survivors were euthanized by intraperitoneal injection of sodium pentobarbital, followed by exsanguination. All rats designated for toxicokinetic evaluation were euthanized following final blood collection and were discarded without necropsy.
The main study rats were examined carefully for exteroal abnormalities, including palpable masses. The skin was reflected from a ventral midline incision, and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities, and the organs removed and examined.
Representative samples of protocol-designated organs and tissues wete collected and placed in 10% neutral buffered formalin, except the eyes, testes, and epididymides, which were placed in Bouin's fixative. The lungs were infused via the trachea with formalin. The urinary bladder was inflated with formalin at necropsy and was examined internally after fixation.

Organweighls
Protocol-designated organs and tissues collected from all main study rats surviving at the terminal sacrifice were trimmed free of fat and connective tissue and weighed. Absolute organweights were recorded along with postmortem bodyweights, and appropriate weight ratios (relative to bodyweights) were calculated. Paired organs were weighed together. The pituitary and thyroid/parathyroids were weighed after fixation. All other organs and tissues were weighed prior to fixation.

Microscopic
Representative samples of protocol-designated organs and tissues from all main study rats were processed for the preparation and microscopic examination of hematoxylin-eosin stained paraffin sections. A full complement of organs and tissues was prepared and microscopically examined for all main study rats which were found dead and from all rats in the Control and 530mg/kg/day groups which were euthanized at study termination. The liver (male and female), testis, and thyroid (males) were determined to be target organs and were examined in all groups. In addition~ sections of all gross lesions and tissue masses were processed and examined microscopically. The slides were examined by a veterinary pathologist. A 4-step grading system of trace, mild, moderate, and severe was used to define gradable lesions for comparison between dose groups.
Please see Table 1 in "Any other information on materials and methods for details of organs and tissues collected, weighted and microscopically examined.

Statistics:

The table below defines the set(s) of comparisons used in the statistical analyses described below.

Table of Statistical Comparisons
Control Group Treatment Groups
1 2, 3 and 4

Group Pair-wise Comparisons.
Data for each sex were analyzed separately. For each endpoint and time period, Levene's test was used to assess homogeneity of group variances. If the test was not significant (p>0.01), Dunnett's test was used to compare each group receiving test article with the Control group. If Levene's test was significant (p<0.01), comparisons with the Control group were made using Welch's test with a Bonferroni correction. Results of all pair-wise comparisons were reported at the 0.05 and 0.01 significance levels.

Log Transformation.
Historical data indicated that leukocyte counts (total and differential) were not normally distributed; therefore, a log transformation was performed on these data. The transformed data was then analyzed as described in the Group Pair-wise Comparisons section.

Clinical signs:

no effects observed

Description (incidence and severity):