Quinazolin-4(1H)-one

Endpoint summary

Description of key information

Repeated dose toxicity: via oral route:

One subacute toxicity study was available, conducted with rats of both sexes at dose levels of 0, 10, 30 or 100 mg/kg body weight/day for a period of 28 days. NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) were established to be 30 mg/kg/day and 100 mg/kg/day, respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2011-07-19 to 2011-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch No.: 1176335
Purity: 99.8%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by international guidelines as a recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 163.0 to 190.2 g (176.9 ± 7.2 g); Females: 127.1 to 167.0 g (145.2 ± 9.6 g)
- Fasting period before study:
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C, ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum
- Acclimation: Under test conditions after health examination. Duration of Pre-Random Acclimatization Period: 1 day; Duration of Post-Random Acclimatization Period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle, with at least eight hours music during the light period.

IN-LIFE DATES: From: 19-Jul-2011 To: 23-Aug-2011/06-Sep-2011

Route of administration:

oral: gavage

Details on route of administration:

Administration by gavage is a common and accepted route of exposure for studies of this type.

Vehicle:

polyethylene glycol

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a glass beaker on a tared Mettler balance. Approximately three quarters of the total amount of vehicle was added and a suspension was prepared. Then, the remaining amount of vehicle was added. The mixtures were stirred using a magnetic stirrer and stored in the refrigerator (5 ± 3 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

- VEHICLE
- Concentration in vehicle: Group 1: 0 mg/mL; Group 2: 2.0 mg/mL; Group 3: 6.0 mg/mL; Group 4: 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): STBB2046M9

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day duplicate samples of the control group formulation as well as three samples (top, middle and bottom) of about 2 g of each dose group formulation were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each dose group were taken four hours and 8 days after completion of the dose formulation preparations to confirm stability.
During week 3 (i.e. day 15), duplicate samples of the control group as well as of each dose group were taken prior to dosing for analysis of concentration.
The dose formulations were analyzed using a HPLC-UV method. The test item was used as analytical standard. Since the achieved concentration results were within the target range only one sample was analyzed, the results were accepted and the second sample was discarded.
The following acceptance criteria were met: The achieved content was ±20% of the nominal content. The formulations were considered homogenous as the individual recoveries did not deviate more than ±15% from the corresponding mean and he results obtained from storage stability samples did not deviate more than 10% from time-zero reference (content or mean of homogeneity samples).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

Group 1

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

Group 1: 10 males and 10 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a dose range finding toxicity study in Wistar rats, Harlan Laboratories study D28671, using dose levels of 10, 30 or 100 mg/kg/day. In an acute oral toxicity study, Harlan Laboratories study D28658, which was not completed at the finalization of this study plan, the LD50 estimate using the Up-and-Down-Procedure was 300 mg/kg.

Observations and examinations performed and frequency:

Observation:
- Viability / Mortality: Twice daily
- Clinical Signs: Acclimatization Period: Once daily; Treatment Period: Twice daily on days 1 to 3, once daily thereafter; Recovery Period: Once daily
- Detailed Behavioral Observations: Acclimatization Period: Once weekly; Treatment Period: Once weekly; Recovery Period: Not performed during recovery
- Food Consumption: Acclimatization Period: Weekly; Treatment Period: Weekly; Recovery Period: Weekly
- Body Weights: Acclimatization Period: Once weekly; Treatment Period: Once weekly; Recovery Period: Once weekly
- Ophthalmoscopy: Acclimatization Period: Once in all animals; Treatment Period: Once during week 4 in animals of the control and high dose groups, using a direct ophthalmoscope.
Functional Observational Battery (FOB Screen):
- Time: During week 4 (day 24/25), relevant parameters from a modified Irwin screen test were performed on all rats in place of the usual weekly behavioral observation.
- Parameters tested: Appearance, Motor, Behavior, Respiration, Reflexes, Grip Strength, Locomotor Activity
Clinical Laboratory Investigations:
- Blood and Urine Sampling: After 4 Weeks: 23-Aug-2011 (allocation A and B); After 6 Weeks: 06-Sep-2011 (allocation B)
- Blood collection: Blood samples were drawn from the retro-orbital plexus under light isoflurane anesthesia from every animal. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by the diurnal rhythm. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- Urine collection: collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded.

HISTOPATHOLOGY: Yes, Slides of all organs and tissues which were collected at scheduled sacrifices from all animals of the control and high-dose groups, animal nos. 18, 20 and 41 and all gross lesions from all animals were examined by the study pathologist. A histopathology peer review was performed and the results compared favourably.

Other examinations:

- Organ Weights: weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, ophthalmoscopic findings, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Clinical signs:

no effects observed

Description (incidence and severity):

- Clinical Signs (Daily): No clinical signs were detected in any animal throughout the scheduled treatment or recovery periods.
- Detailed Clinical Observations (Weekly): No abnormalities were detected

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled treatment or recovery periods.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During treatment with the test item, absolute body weight and body weight gain was slightly reduced in males treated with 100 mg/kg/day and body weight gain was slightly reduced females treated with 100 mg/kg/day, however, this effect was reversible after two weeks of recovery.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related effect on food consumption was noted.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were noted. Findings such as slight corneal opacity, persistent pupillary membrane and persistent hyaloid vessels are within the normal range of background findings recorded for animals of this strain and age.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hemoglobin levels (HB) were slightly, but statistically significantly increased in males treated with 100 mg/kg/day and females treated with 30 or 100 mg/kg/day. However, these changes are within the range of historical control data and were not observed after two weeks of recovery. All other changes attaining statistical significance are considered incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related findings were observed. All changes attaining statistical significance are considered incidental, as they are opposite in direction in males and females, or as there is no dose-relationship observed. No statistically significant differences were observed after the recovery period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related findings were observed. All changes attaining statistical significance are considered incidental, as there is no dose-relationship observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormalities were detected in any animal throughout the scheduled treatment or recovery periods.
Grip Strength: No differences were detected between test item-treated groups and controls
Locomotor Activity: No differences were detected between test item-treated males and controls. Females treated with 100 mg/kg/day showed a statistically significantly increased locomotor activity during the first 10 minute interval and slightly, but not statistically significantly increased activity at the remaining measuring intervals, leading to a statistically significantly increased total locomotor activity. Taking into account the highly variable data and the absence of an effect in males, this finding is considered to be of no toxicological relevance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes in organ weights were noted after 4 weeks of treatment: increased organ/body weight ratio of testes in males treated with 100 mg/kg/day and increased organ/body weight ratios of liver, kidneys and uterus in females treated with 100 mg/kg/day. However, these changes are all within the range of historical control data, did not show statistically significant weight differences after two weeks of recovery.
All statistically significant differences between animals treated with 100 mg/kg/day and controls observed after two weeks of recovery are considered incidental, as these organs were not affected after four weeks of treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings that are considered test item-related were detected.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A slight increase in incidence and severity of retinal degeneration was found in recovery animals treated with 100 mg/kg/day. There were no microscopic lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

organ weights and organ / body weight ratios

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

haematopoietic

Conclusions:

Considering the absence of any test item-related histopathological findings and the full reversibility of all other changes observed after treatment with the test item, a NOEL (No
Observed Effect Level) of 30 mg/kg/day and a NOAEL (No Observed Adverse Effect Level) of 100 mg/kg/day could be established.

Executive summary:

The subacute toxicity study of test item was performed according to OECD407. The test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 10, 30 and 100 mg/kg body weight/day for a period of 28 days, followed by a 14-day recovery period. A control group was treated with the vehicle only, polyethylene glycol 300 (PEG 300).

Treatment with the test item at these dose levels was well tolerated, and no test item-related effects on mortality, clinical signs, FOB, grip strength, locomotor activity, food consumption, ophthalmoscopic findings and macroscopic findings were observed. A slight decrease of body weight gain was noted in both sexes treated with 100 mg/kg/day, but the effect was reversible during the recovery period.

Hemoglobin levels (HB) were slightly but statistically significantly increased in males treated with 100 mg/kg/day and females treated with 30 and 100 mg/kg/day. However, these changes are within the range of historical control data. All other changes observed in hematology, clinical biochemistry and urinalysis parameters are considered to be not test item-related.

Statistically significantly increased organ/body weight ratio of testes in males treated with 100 mg/kg/day and statistically significantly increased organ/body weight ratios of liver, kidneys and uterus in females treated with 100 mg/kg/day were observed. As these changes in organ/body weight ratios are small in magnitude and all range within the historical control data, these findings are considered to be of minor toxicological concern. Moreover, these changes were not observed any more after two weeks of recovery. Microscopically, no test item-related histopathological findings were observed.

Considering the absence of any test item-related histopathological findings and the full reversibility of all other changes observed after treatment with the test item, a NOEL (No Observed Effect Level) of 30 mg/kg/day and a NOAEL (No Observed Adverse Effect Level) of 100 mg/kg/day could be established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

System:

haematopoietic

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

NOAEL: 100 mg/kg bw, no test item-related histopathological findings and all other changes (body weight, hemoglobin levels (HB)) observed after treatment full reversible.

According to Regulation (EC) No 1272/2008, table 3.9.1 and 3.9.3, this substance should not be classified for specific target organ toxicity-repeated exposure.