4H-Pyrazino[2,1-a]isoquinolin-4-one, 2-acetyl-1,2,3,6,7,11b-hexahydro-

Endpoint summary

Description of key information

In a subacute oral toxicity study in compliance with OECD TG 407 in the rat the NOAEL was 500 mg/kg bw/d was (reference 7.5.1 -1).

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:

2005-05-04 to 2005-09-06

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:

1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Directive 96 / 54 / EC, B. 7. "Repeated dose (28 days) toxicity (oral)"

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13,1974

Version / remarks:

1986

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HanRcc:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 135 - 156 grams (mean 146 grams) Females: 118 - 132 grams (mean 124 grams)
- Fasting period before study: no
- Housing: groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: PEG 300 is common and accepted as vehicle
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

nominal recovery date_or_preparation date_of_analyis
10 mg/mL 106 % 04-May-05 03-June-05
30 mg/mL 104 % 04-May-05 03-June-05
100 mg/mL 110 % 04-May-05 03-June-05
10 mg/mL 99.2 % 26-May-05 03-June-05
30 mg/mL 101.6 % 26-May-05 03-June-05
100 mg/mL 98.5 % 26-May-05 03-June-05

Duration of treatment / exposure:

Test duration: 28 days / daily treatment

Frequency of treatment:

Dosing regime: daily on 7 days/week

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals / sex at 0 mg/kg bw/day
5 animals / sex at 50 mg/kg bw/day
5 animals / sex at 150 mg/kg bw/day
10 animals /sex at 500 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

In this subacute toxicity study, the test material was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 150 and 500 mglkg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 500 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

From the animals of the low and middle dose groups, liver and thyroid glands were examined to establish a no-effect level.

Positive control:

no

Observations and examinations performed and frequency:

Observations/Measurements (Frequency)

MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

FOOD CONSUMPTION (WEEKLY)
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the computer.

BODY WEIGHTS (WEEKLY)
Body weights were recorded weekly during acclimatization, treatment and recovery and before each necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
The behavioral parameters of the functional observational battery are similar to those evaluated during weekly observations.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with AMS Fohr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.
Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted in metabolic cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Urine was collected during the 18-hour fasting period into a specimen vial.
Sampling after 4 weks and after 6 weeks (post recovery)

The assays were performed under internal laboratory quality control conditions to assure reliable test results.

HEMATOLOGY
The following parameters have been investigated:
RBC, HB, HCT, MCV, MCH, MCHC, Platelets, Reticulocytes, HFR, MFR, LFR, nucleated erythrocytes, Heinz bodies, MET-HB, WBC, diff. WBC Count, red blood cell morphology, PT, APTT

CLINICAL BIOCHEMISTRY
Glucose, Urea, Creatinine, Uric acid, Bilirubin, Cholesterol, Triglycerides, Phospholipids, GOT, GPT, LDH, CK, ALP, GT, Ca, P, Na, K, Cl, Albumin, Protein, Globulin, A/G Ratio

URINALYSIS
Volume, specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin

Sacrifice and pathology:

NECROPSY
after 4 weeks
after 6 weeks (post recovery)

All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist.

All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

Samples of the following tissues and organs were collected from all animals at necropsy and, unless indicated otherwise, fixed in neutral phosphate buffered 4 % formaldehyde solution:

Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (sternum, femur) Brain (cerebrum, cerebellum, pons) Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution) Esophagus
Eyes with optic nerve (fixed in Davidson's solution) Harderian gland (fixed in Davidson's solution) Heart
Ileum, with Peyer's patches Jejunum with Peyer's patches Kidneys
Larynx
Lacrimal gland (exorbital)
Liver
Lungs (infused with formalin)
Lymph nodes (mesenteric, mandibular) Mammary gland area
Nasal cavity
Ovaries Pancreas Pituitary gland
Prostate gland
Rectum
Salivary glands (mandibular, sublingual)
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord (cervical, midthoracic, lumbar)
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland)
Tongue Trachea
Urinary bladder (infused with formalin)
Uterus
Vagina
Gross lesions

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:

Brain Thymus Spleen Ovaries
Heart Kidneys Testes
Liver Adrenals Epididymides

The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist.
As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (liver and thyroid gland) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate were 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) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
- Fisher's exact-test were applied to the macroscopic findings.

The following statistical methods were used for statistical analysis of clinical laboratory data:
- Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett.
- Alternatively, if the variances are considered to be heterogenous (p50.05), a non-parametric Kruskal-Wallis test was used. -
- Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted during daily observations at any dose level.
No test item-related clinical signs were noted during the weekly detailed observations (weeks 1-3) at any dose level.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted in the mean body weights and mean body weight gain of males and females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test item-treated rats was unaffected by the treatment with the test item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Although all values remained within their respective ranges of the historical control data, the observed reticulocytosis and "left-shifted" reticulocyte maturity indices were considered to be a test item-related effect. These changes either partly or completely reverted during the recovery period and therefore considered not adverse.

After the end of the treatment period, elevated mean absolute and relative reticulocyte counts were noted in males treated with 150 mg/kg/day and 500 mg/kg/day. After the end of the recovery period, the mean absolute and relative reticulocyte counts remained slightly elevated in males previously treated with 500 mg/kg/day. Females were unaffected after treatment and after recovery.

In males treated with 150 mg/kg/day and 500 mg/kg/day, the reticulocyte maturity indices were shifted towards high fluorescence reticulocytes. In males at 150 mg/kg/day, a very marginal increase in medium fluorescence reticulocytes was noted. In males at 500 mg/kg/day, there were reductions of low fluorescence and medium fluorescence reticulocytes, accompanied by increased high fluorescence reticulocytes. Females treated with 500 mg/kg/day had only slightly elevated high fluorescence reticulocytes when compared with the controls. In recovery males previously treated with 500 mglkglday, the reticulocyte maturity indices were shifted only slightly towards high fluorescence reticulocytes, and this was considered to be indicative of partial reversibility. Still, reduced low fluorescence reticulocytes, increased medium fluorescence reticulocytes and increased high fluorescence reticulocytes persisted in these males.

All other changes were considered to be unrelated to the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In females treated with 150 and 500 mg/kg/day, elevated cholesterol levels were seen. In both sexes treated with 500 mg/kg/day, elevated triglycerides and phospholipids were noted when compared with the respective controls. Although the affected parameters remained within the historical control ranges for males, all three parameters (cholesterol, triglycerides and phospholipids) exceeded the historical control data in females, and were considered to be indicative of a test item-induced change in lipid metabolism.
All other changes were considered to be unrelated to the test item.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related differences in the urinalysis parameters were noted after the treatment or recovery periods.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted during the functional observational battery (week 4) at any dose level. Test item-related reductions of mean locomotor activity were noted in males at 150 mg/kg/day and in both sexes at 500 mg/kg/day.
No test item-related reductions in mean fore- and hind limb grip strength were noted at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After the end of the treatment period, elevated mean absolute and relative liver weights were noted in male rats treated with 150 mg/kg/day, and in both sexes treated with 500 mg/kg/day. These changes were considered to be test item related adaptive changes, and were largely, but not completely, reversible after the end of the recovery period.
All other organ weights and ratios were considered to be incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

A small number of typical background macroscopical findings were noted in test item-treated rats of both sexes. None were considered to be of toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Minimal to mild hepatocellular hypertrophy, mainly centrilobular, at doses of 150 and 500 mg/kg/day. Following a 14-day recovery period, this hepatic change showed advanced but incomplete regression.
- Follicular cell hypertrophy in thyroid gland was associated with hepatocellular hypertrophy and the consequently enhanced metabolic liver activity.
These findings were not considered to be of adverse character.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: findings were reversible after 2 weeks recovery period

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this study, 50 mg/kg/day of the test material were established as the no-observed-effect-level (NOEL).
Based on the type of findings observed and their general tendency towards reversibility, the no-observed-adverse-effect-level (NOAEL) of the test material was considered to be 500 mg/kg/day.

Executive summary:

The test material was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of SPF-bred Wistar rats kept under conventional conditions at the test facility and in accordance with OECD TG 407. A similarly constituted group received the vehicle, PEG-300, and served to generate contemporary control data. 5 rats per sex of the control group and of group 4 were scheduled for a 2 week treatment-free follow-up period.
Daily dose levels and number of rats used:

Group		Doses	Number of animals
		(mg/kg)	Main	Recovery
1	Control	0	5M/5F	5M/5F
2	Test Group	50	5M/5F
3	Test Group	150	5M/5F
4	Test Group	500	5M/5F	5M/5F

The total number of rats was 60 (30M, 30F).
Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, liver and thyroid glands were examined to establish a no-effect level.

All animals survived until scheduled necropsy. No test item-related clinical signs were noted during daily observations at any dose level. No test item-related clinical signs were noted during the weekly detailed observations (weeks 1-3) at any dose level. No test item-related clinical signs were noted during the functional observational battery (week 4) at any dose level. No test item-related reductions in mean fore- and hind limb grip strength were noted at any dose level. Test item-related reductions of mean locomotor activity were noted in males at 150 mg/kg/day and in both sexes at 500 mg/kg/day. In males treated with 150 mg/kg/day, reductions were noted from 0-10 minutes, 10-20 minutes and 30-40 minutes, resulting in an overall reduction from 0-60 minutes. In females treated with 150 mg/kg/day, reduced locomotor activity was seen only during the 50-60 minute measurement interval. At 500 mg/kg/day, male rats had reduced locomotor activity during 0-10 minutes, 10-20 minutes, 20-30 minutes, 30-40 minutes and the overall locomotor activity (0-60 minutes) was also reduced. The mean locomotor activity of female rats was reduced from 20-30 minutes and 30-40 minutes, resulting in an overall reduction (0-60 minutes) in mean locomotor activity. The mean daily food consumption of the test item-treated rats was unaffected by the treatment with the test item. No test item-related effects of toxicological relevance were noted in the mean body weights and mean body weight gain of males and females. Although all values remained within their respective ranges of the historical control data, the observed reticulocytosis and "left-shifted" reticulocyte maturity indices were considered to be a test item-related effect. These changes either partly or completely reverted during the recovery period and therefore considered not adverse. After the end of the treatment period, elevated mean absolute and relative reticulocyte counts were noted in males treated with 150 mg/kg/day and 500 mg/kg/day. After the end of the recovery period, the mean absolute and relative reticulocyte counts remained slightly elevated in males previously treated with 500 mg/kg/day. Females were unaffected after treatment and after recovery. In males treated with 150 mg/kg/day and 500 mg/kg/day, the reticulocyte maturity indices were shifted towards high fluorescence reticulocytes. In males at 150 mglkglday, a very marginal increase in medium fluorescence reticulocytes was noted. In males at 500 mg/kg/day, there were reductions of low fluorescence and medium fluorescence reticulocytes, accompanied by increased high fluorescence reticulocytes. Females treated with 500 mg/kg/day had only slightly elevated high fluorescence reticulocytes when compared with the controls. In recovery males previously treated with 500 mglkglday, the reticulocyte maturity indices were shifted only slightly towards high fluorescence reticulocytes, and this was considered to be indicative of partial reversibility. Still, reduced low fluorescence reticulocytes, increased medium fluorescence reticulocytes and increased high fluorescence reticulocytes persisted in these males. AU other changes were considered to be unrelated to the test item. In females treated with 150 and 500 mg/kg/day, elevated cholesterol levels were seen. In both sexes treated with 500 mg/kg/day, elevated triglycerides and phospholipids were noted when compared with the respective controls. Although the affected parameters remained within the historical control ranges for males, all three parameters (cholesterol, triglycerides and phospholipids) exceeded the historical control data in females, and were considered to be indicative of a test item-induced change in lipid metabolism. All other changes were considered to be unrelated to the test item. No test item-related differences in the urinalysis parameters were noted after the treatment or recovery periods. After the end of the treatment period, elevated mean absolute and relative liver weights were noted in male rats treated with 150 mg/kg/day, and in both sexes treated with 500 mg/kg/day. These changes were considered to be test item related adaptive changes, and were largely, but not completely, reversible after the end of the recovery period. All other organ weights and ratios were considered to be incidental. A small number of typical background macroscopical findings were noted in test item-treated rats of both sexes. None were considered to be of toxicological relevance. Some microscopical changes were noted: - minimal to mild hepatocellular hypertrophy, mainly centrilobular, at doses of 150 and 500 mg/kg/day. Following a 14-day recovery period, this hepatic change showed advanced but incomplete regression. - Follicular cell hypertrophy in thyroid gland was associated with hepatocellular hypertrophy and the consequently enhanced metabolic liver activity. These findings were not considered to be of adverse character.

In conclusion, oral administration of the test material to Wistar rats at doses of 50, 150 and 500 mg/kg/day, for 28 days resulted in no deaths, no clinical signs during daily or weekly observations (weeks 1-3), no clinical signs during functional observational battery (week 4), no effects on fore- or hindlimb grip strength, no effects on mean daily food consumption or mean body weights. Test item-related non-adverse findings were generally restricted to reductions of mean locomotor activity in males at 150 mg/kg/day and in both sexes at 500 mg/kg/day, reticulocytosis and "left-shifted" reticulocyte maturity indices in males at 150 and 500 mg/kg/day (partly or completely reverted during the recovery period), very marginally "left-shifted" reticulocyte maturity indices in females at 500 mg/kg/day, elevated cholesterol levels in females treated with 150 and 500 mg/kg/day and elevated triglycerides and phospholipids in both sexes treated with 500 mg/kg/day (considered to be a test item-induced change in lipid metabolism), elevated mean absolute and relative liver weights in male rats treated with 150 mg/kg/day, and in both sexes treated with 500 mg/kg/day (adaptive changes that were largely, but not completely, reversible after the end of the recovery period), and microscopical changes which included minimal to mild, mainly centrilobular hepatocellular hypertrophy at doses of 150 and 500 mg/kg/day (with partial regression after recovery) and follicular cell hypertrophy in the thyroid gland was associated with hepatocellular hypertrophy and the consequently enhanced metabolic liver activity. Based on the results of this study, 50 mg/kg/day of the test material were established as the no-observed-effect-level (NOEL). Based on the type of findings observed and their general tendency towards reversal, the no-observed-adverse-effect-level (NOAEL). of the test material was considered to be 500 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

28 -d repeated dose toxicity

The test material was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of SPF-bred Wistar rats kept under conventional conditions at the test facility and in accordance with OECD TG 407. A similarly constituted group received the vehicle, PEG-300, and served to generate contemporary control data. 5 rats per sex of the control group and of group 4 were scheduled for a 2 week treatment-free follow-up period.
Daily dose levels and number of rats used:

Group		Doses	Number of animals
		(mg/kg)	Main	Recovery
1	Control	0	5M/5F	5M/5F
2	Test Group	50	5M/5F
3	Test Group	150	5M/5F
4	Test Group	500	5M/5F	5M/5F

The total number of rats was 60 (30M, 30F). Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, liver and thyroid glands were examined to establish a no-effect level.

All animals survived until scheduled necropsy. No test item-related clinical signs were noted during daily observations at any dose level. No test item-related clinical signs were noted during the weekly detailed observations (weeks 1-3) at any dose level. No test item-related clinical signs were noted during the functional observational battery (week 4) at any dose level. No test item-related reductions in mean fore- and hind limb grip strength were noted at any dose level. Test item-related reductions of mean locomotor activity were noted in males at 150 mg/kg/day and in both sexes at 500 mg/kg/day. In males treated with 150 mg/kg/day, reductions were noted from 0-10 minutes, 10-20 minutes and 30-40 minutes, resulting in an overall reduction from 0-60 minutes. In females treated with 150 mg/kg/day, reduced locomotor activity was seen only during the 50-60 minute measurement interval. At 500 mg/kg/day, male rats had reduced locomotor activity during 0-10 minutes, 10-20 minutes, 20-30 minutes, 30-40 minutes and the overall locomotor activity (0-60 minutes) was also reduced. The mean locomotor activity of female rats was reduced from 20-30 minutes and 30-40 minutes, resulting in an overall reduction (0-60 minutes) in mean locomotor activity. The mean daily food consumption of the test item-treated rats was unaffected by the treatment with the test item. No test item-related effects of toxicological relevance were noted in the mean body weights and mean body weight gain of males and females. Although all values remained within their respective ranges of the historical control data, the observed reticulocytosis and "left-shifted" reticulocyte maturity indices were considered to be a test item-related effect. These changes either partly or completely reverted during the recovery period and therefore considered not adverse. After the end of the treatment period, elevated mean absolute and relative reticulocyte counts were noted in males treated with 150 mg/kg/day and 500 mg/kg/day. After the end of the recovery period, the mean absolute and relative reticulocyte counts remained slightly elevated in males previously treated with 500 mg/kg/day. Females were unaffected after treatment and after recovery. In males treated with 150 mg/kg/day and 500 mg/kg/day, the reticulocyte maturity indices were shifted towards high fluorescence reticulocytes. In males at 150 mglkglday, a very marginal increase in medium fluorescence reticulocytes was noted. In males at 500 mg/kg/day, there were reductions of low fluorescence and medium fluorescence reticulocytes, accompanied by increased high fluorescence reticulocytes. Females treated with 500 mg/kg/day had only slightly elevated high fluorescence reticulocytes when compared with the controls. In recovery males previously treated with 500 mglkglday, the reticulocyte maturity indices were shifted only slightly towards high fluorescence reticulocytes, and this was considered to be indicative of partial reversibility. Still, reduced low fluorescence reticulocytes, increased medium fluorescence reticulocytes and increased high fluorescence reticulocytes persisted in these males. AU other changes were considered to be unrelated to the test item. In females treated with 150 and 500 mg/kg/day, elevated cholesterol levels were seen. In both sexes treated with 500 mg/kg/day, elevated triglycerides and phospholipids were noted when compared with the respective controls. Although the affected parameters remained within the historical control ranges for males, all three parameters (cholesterol, triglycerides and phospholipids) exceeded the historical control data in females, and were considered to be indicative of a test item-induced change in lipid metabolism. All other changes were considered to be unrelated to the test item. No test item-related differences in the urinalysis parameters were noted after the treatment or recovery periods. After the end of the treatment period, elevated mean absolute and relative liver weights were noted in male rats treated with 150 mg/kg/day, and in both sexes treated with 500 mg/kg/day. These changes were considered to be test item related adaptive changes, and were largely, but not completely, reversible after the end of the recovery period. All other organ weights and ratios were considered to be incidental. A small number of typical background macroscopical findings were noted in test item-treated rats of both sexes. None were considered to be of toxicological relevance. Some microscopical changes were noted: - minimal to mild hepatocellular hypertrophy, mainly centrilobular, at doses of 150 and 500 mg/kg/day. Following a 14-day recovery period, this hepatic change showed advanced but incomplete regression. - Follicular cell hypertrophy in thyroid gland was associated with hepatocellular hypertrophy and the consequently enhanced metabolic liver activity. These findings were not considered to be of adverse character.

In conclusion, oral administration of the test material to Wistar rats at doses of 50, 150 and 500 mg/kg/day, for 28 days resulted in no deaths, no clinical signs during daily or weekly observations (weeks 1-3), no clinical signs during functional observational battery (week 4), no effects on fore- or hindlimb grip strength, no effects on mean daily food consumption or mean body weights. Test item-related non-adverse findings were generally restricted to reductions of mean locomotor activity in males at 150 mg/kg/day and in both sexes at 500 mg/kg/day, reticulocytosis and "left-shifted" reticulocyte maturity indices in males at 150 and 500 mg/kg/day (partly or completely reverted during the recovery period), very marginally "left-shifted" reticulocyte maturity indices in females at 500 mg/kg/day, elevated cholesterol levels in females treated with 150 and 500 mg/kg/day and elevated triglycerides and phospholipids in both sexes treated with 500 mg/kg/day (considered to be a test item-induced change in lipid metabolism), elevated mean absolute and relative liver weights in male rats treated with 150 mg/kg/day, and in both sexes treated with 500 mg/kg/day (adaptive changes that were largely, but not completely, reversible after the end of the recovery period), and microscopical changes which included minimal to mild, mainly centrilobular hepatocellular hypertrophy at doses of 150 and 500 mg/kg/day (with partial regression after recovery) and follicular cell hypertrophy in the thyroid gland was associated with hepatocellular hypertrophy and the consequently enhanced metabolic liver activity. Based on the results of this study, 50 mg/kg/day of the test material were established as the no-observed-effect-level (NOEL). Based on the type of findings observed and their general tendency towards reversal, the no-observed-adverse-effect-level (NOAEL). of the test material was considered to be 500 mg/kg/day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item does not require classification for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.