2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Endpoint summary

Description of key information

NOAEL = no determined (OECD 407, GLP, WoE, rel. 1).

NOAEL = 50 mg/kg/day for male and female rats (OECD 407, GLP, WoE, rel. 1).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 10 March To 24 July, 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.

Reason / purpose for cross-reference:

reference to other study

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:

amended December 5, 1986

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 3 October 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed July 28, 2009

Limit test:

no

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 4 4414 Füllinsdorf / Switzerland.
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: Males: 182 g to 197 g (mean 189 g) Females: 135 g to 155 g (mean 145 g).
- Housing: Animals were housed in groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Diet: Pelleted standard Kliba Nafag 3433 (batch no. 76/08) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum
- Water: Community tap-water from Itingen, ad libitum in water bottles
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 ºC
- Humidity: 30-70 %
- Air changes: 10-15 changes/ h
- Photoperiod: 12 h dark/ 12 h light
- Music: at least eight hours music during the light period.

IN-LIFE DATES: from 10 March to 14 April (Satellite A) and 28 April (Satellite B).

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

TREATMENT PREPARATION AND ADMINISTRATION:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared, weekly, at the appropriate concentrations as a solution in PEG 300 (Sigma-Aldrich / Fluka Analytical, Batch Number: 560502-099 / 1368992 43508153). The test item was weighed into a tared glass container on a suitable precision balance and the vehicle, PEG 300, was added to give the appropriate final concentration of the test item in the dose formulation.
The mixtures were prepared first by mixing with a homogenizer (i.e. Ultra Turrax) for approximately 2 minutes, and then mixed using a magnetic stirrer for approximately 30 minutes. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. The dose formulations were protected from light (i.e. wrapping in aluminum foil).
The dose formulations were stored in glass beakers at room temperature (20 ± 5 °C). Based upon the results of dose formulation analyses performed during previous studies (RCC Study Number C00630 and Harlan Laboratories Study C00641), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

VEHICLE
- Justification for use and choice of vehicle: had been prooved as appropriate in previous studies ( days repeated dose toxicity study).
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 560502-099 / 1368992 43508153
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis was performed using gas chromatography coupled to a flame ionisation detector. The test item was used as analytical standard.

The samples (generally 2 g each) were delivered to the analytical laboratory. Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

PEG 300 (Group 1)

Dose / conc.:

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

Remarks:

Group 2 (Low dose)

Dose / conc.:

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

Remarks:

Group 3 (Mid dose)

Dose / conc.:

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

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

5 animals/sex for all dose groups (satellite A: Toxicity testing: termination after 28 treatment days).
5 animals/sex for group 1 and group 4 (satellite B: Recovery testing: termination after an additional 14-day recovery period).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on previous study (Harlan, C00641). In this study, the test substance is tested at 100, 300 and 600 mg/kg bw/day. Based on the results of this study, the NOAEL is < 100 mg/kg bw/day.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: the animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum
- Post-exposure recovery period in satellite groups: 14 days after the end of administration (satellite B).

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration and once weekly (weeks 1 to 3) thereafter.
The animals were observed for general clinical signs once before commencement of administration as well as twice daily on days 1 to 3, once daily on days 4-28 (treatment period), and once daily during days 1 to 14 (recovery period).

BODY WEIGHT: Yes
- Time schedule for examinations: recorded before initiation of treatment (day 0), weekly during treatment, recovery period and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: was recorded once during the acclimatization period and weekly during treatment and recovery period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of treatment (day 29/30) and at the end of recovery period (day 43).
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals: all animals
- Parameters checked in Table 7.5.1/2 were examined.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: at termination of treatment (day 29/30) and at the end of recovery period (day 43).
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals: all animals.
- Parameters checked in Table 7.5.1/2 were examined.


PLASMA/SERUM HORMONES/LIPIDS: No
Thyroid Hormone Analysis Preparation for the analysis of thyroid hormones (T3, T4 and TSH) was described in the study plan. However, organ weight changes were not seen that would otherwise indicate that toxicologically relevant changes were present. Therefore, it was decided to not perform these analyses.

URINALYSIS: Yes
- Time schedule for collection of urine: at termination of the treatment period (day 29/30) and at the end of reversal period (day 43).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes
- Parameters checked in Table 7.5.1/2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the fourth week of treatment.
- Dose groups that were examined: 0, 5, 25 and 50 mg/kg bw/day
- Battery of functions tested: grip strength / locomotor activity
IMMUNOLOGY: No

Other: Vaginal Smear for Estrus Stage:
A vaginal smear was taken during week 4 from each female, and the stage of estrus was evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY:
All animals were weighed and necropsied after 4 weeks and 6 weeks (recovery). Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.
Samples of tissues and organs reported on the table 7.5.1/2 were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated).
Organ weight:
Weight of the following organs were weighed (absolute weight): kidneys, liver, adrenals, testes, epididymides, thymus, spleen, brain, heart, ovaries and Seminal vesicles and prostate gland incl. coagulating glands.
Values of these organ weights as percent of body weight, on the day of necropsy, were also estimated (relative organ weights).

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.

HISTOPATHOLOGY:
All organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.
Slides of all organs and tissues listed in boldface type (see table 7.5.1/2) that were collected at scheduled sacrifice from the animals of all control and high-dose groups were examined by a pathologist.The same applies to all occurring gross lesions and to the control animal, which died spontaneously.

The examination was carried out on the tissues reported on the table 7.5.1/2.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, 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:

effects observed, non-treatment-related

Description (incidence and severity):

- DAILY OBSERVATIONS:
No test item-related differences of toxicological relevance were noted at any dose level. Salivation was noted in most test item-treated animals immediately after application. This finding was considered to be related to the taste of the dose formulations and was not considered to be related to systemic toxicity. Male rats of the control group and test item-treated groups had slightly soft feces. The mean severity was similar for all groups. This was first evident on (or around) day 7 in test item-treated males of all dose levels. In females treated with 5, 25 and 50 mg/kg/day, soft feces was first recorded on days 6, 7 and 3 of treatment, respectively. In control males and females, this finding was first noted on day 9 of treatment. This finding was considered to be due to the vehicle, PEG 300, and is a typical effect. Soft feces was no longer evident during the recovery period.
- WEEKLY BEHAVIORAL OBSERVATIONS: No symptoms were evident during the weekly detailed clinical observations (weeks 1-3).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female treated with 50 mg/kg/day died shortly after treatment on day 18. Although the precise cause of death could not be established by pathomorphological evaluation, the macroscopical changes (discolored, inflated lungs) were considered to be evidence of a dosing error. All other animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects upon the mean body weights of the test item-treated males or females after 28 days of treatment. After the recovery period, the mean body weights of the males previously treated with 50 mg/kg/day were elevated on days 1, 8 and 14. The differences to the control males attained statistical significance on days 8 (p<0.05) and 14 (p<0.05). These differences were considered to be of no toxicological relevance as no differences were seen at the end of the treatment period. The mean body weights of the corresponding females were unaffected.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption values after 28 days of treatment were unaffected by the test item. No late effects of differences were ascertained after the recovery period.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males treated with 50 mg/kg/day, differences in the reticulocyte maturity indices were noted when compared with the controls. The low-fluorescence reticulocytes were significantly reduced (p<0.05) and the high-fluorescence reticulocytes were elevated, which is generally indicative of a trend towards a younger cell population and increased turnover. However, as females at this dose level were unaffected, these changes were considered to be incidental. Females and males treated with 5 mg/kg/day or 25 mg/kg/day were unaffected.
After the recovery period, reduced hemoglobin levels, hematocrit and mean corpuscular volume were noted in males. In the absence of such differences after the treatment period, and in the absence of similar changes in the females, these differences were considered to be incidental and of no toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences in the mean clinical biochemistry parameters were noted at any dose level. In males treated with 50 mg/kg/day, lower mean glucose levels attained statistical significance (p<0.05) when compared with the control males, yet remained within the lower limit of the historical control data. In females at 50 mg/kg/day, marginally lower glucose levels were seen but were well within the ranges of typical variation. Total bilirubin was significantly lower (p<0.01) in females treated with 50 mg/kg/day, as were triglyceride levels (p<0.05). Bile acids and phospholipid levels were also lower and the latter value remained within the lower limit of the historical control data (bile acids is a new guideline requirement; historical data are still being collected). Total protein levels were similar in the females treated with 50 mg/kg/day and in the females of the control group, but the mean albumin was significantly reduced (p<0.05), the globulin level was elevated and the resulting albumin/globulin ratio was lower. The latter two values were outside the ranges of the historical control data. Lactate dehydrogenase activity, significantly elevated in males treated with 25 mg/kg/day (p<0.05), was within the range of typical variation and was not related to dose. Hence it was considered to be incidental. Glutamate dehydrogenase activity was significantly reduced in males at all dose levels (p<0.05 or p<0.01). All values – including those of the control males – exceeded the lower limit of the historical control data. These differences were considered to be of no toxicological relevance. After the recovery period, there were no differences that would be indicative of late changes of toxicological relevance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted in the urinalysis parameters after treatment or recovery. All differences noted after the end of the treatment period remained within the ranges of the historical control data. Any changes noted after the end of the recovery period were generally not present after the end of the treatment period and were therefore considered to be unrelated to the test item treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery: No symptoms were evident during the functional observational battery (week 4).
Grip Strength: The mean fore- and hindlimb grip strength values were similar in test item-treated and control rats of both sexes.
Locomotor Activity: The mean locomotor activity of test item-treated males and females compared favorably with those of the respective controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related differences were noted in organ weights at any dose level,. After the treatment period, a significantly lower mean heart-to-body weight ratio (p<0.05) was noted in females treated with 25 mg/kg/day. The mean heart-to-body weight ratio of the females treated with 50 mg/kg/day was also lower than the controls, but the difference did not attain statistical significance. In the absence of any microscopical changes, these findings were considered to be incidental. After the recovery period, significantly lower mean absolute heart weights, lower heart-to-body weight and heart-to-brain weight ratios (all p<0.05) were noted in females previously treated with 50 mg/kg/day. In the absence of any microscopical changes, these findings were considered to be incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no gross 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. In a single female treated with 5 mg/kg/day, unilateral enlargement of the adrenal glands was noted. In a single female treated with 50 mg/kg/day, dilation of the uterine horns and dark red discoloration of the mandibular lymph nodes were noted. These findings were considered to be typical background changes without toxicological relevance.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic findings recorded were considered to be within the range of normal background lesions, which may be seen in rats of this strain, age and study type, and were considered incidental, reflecting the usual individual variability.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Vaginal Smear for Estrus Stage:
Analysis of the vaginal smears taken from the surviving females during the fourth week of treatment showed generally similar distributions of the estrus cycle stages

Details on results:

ANALYSIS OF DOSE FORMULATIONS:
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to exceed 0.99. The GR-86-2406 peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms, no significant peak appeared at the retention time of GR-86-2406 and, therefore, it was confirmed that only PEG 300 was administered in the control part of the experiment. The application formulations investigated during the study were found to comprise GR-86-2406 in the range of 74.3% to 99.6%. Fifteen out of 18 samples met the required content limit of ±20% with reference to the nominal concentration. Because single results did not deviate more than 1.5% (<15%) from the corresponding mean, the test item was considered to be homogeneously distributed in the vehicle. In addition, the test item was found to be stable in application formulations when kept 7 days at room temperature: Recoveries met the variation limit of 10% from the time-zero (homogeneity) mean. The results of dose formulation analysis indicate the accurate use of the test item GR-86-2406 and PEG 300 as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficiently stable under storage conditions.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Critical effects observed:

no

Conclusions:

Under the test condtions, the no-observed-effect-level (NOAEL) of the test substance in male and female rats, following oral administration for 28 days was found to be 50 mg/kg bw/day. No adverse effects were observed up to 50 mg/kg bw/day. The test substance is not classified for damage to organs through prolonged oral dose repeated exposure according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and the GHS.

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 5, 25 and 50 mg/kg 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 50 mg/kg/day. 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 the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were evaluated 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. 

 

No test item-related unscheduled deaths occurred and the death of one female treated with 50 mg/kg/day on day was considered to be dosing error.

No test item-related differences of toxicological relevance were noted during daily observations at any dose level. No symptoms were evident during the weekly detailed clinical observations (weeks 1-3). No symptoms were evident during the functional observational battery (week 4). The mean fore- and hindlimb grip strength values were similar in test item-treated and control rats of both sexes. The mean locomotor activity of test item-treated males and females compared favorably with those of the respective controls. Analysis of the vaginal smears taken from the surviving females during the fourth week of treatment showed generally similar distributions of the estrus cycle stages.

Test article did not have any adverse effect on the body weight gain and average daily food consumption by the male and female rats treated up to the dose level of 50 mg/kg bw/day.

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

In hematology parameters, in males treated with 50 mg/kg/day, differences in the reticulocyte maturity indices were noted when compared with the controls. The low-fluorescence reticulocytes were reduced, and the high-fluorescence reticulocytes were elevated, which is generally indicative of a trend towards a younger cell population and increased cell turnover. However, as females at this dose level were unaffected, these changes were considered to be incidental. Females and males treated with 5 mg/kg/day or 25 mg/kg/day were unaffected. After the recovery period, reduced hemoglobin levels, hematocrit and mean corpuscular volume noted in males were not seen after the treatment period. In the absence of similar changes in the females, these differences were considered to be incidental and of no toxicological relevance.

No test item related differences were noted in organ weights at any dose level. No treatment related gross and microscopic pathological alterations in the tissues of male and female rats treated up to the level of 50 mg/kg bw/day.

 

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) of the test substance in Wistar rats, following oral administration for 28 days was found to be 50 mg/kg bw/day. 

Under the test conditions, the test substance is not classified according to the criteria of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.

This study is acceptable and satisfies the requirement for repeated dose toxicity endpoint.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 06 August 2008 To 06 February 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: "Repeated Dose 28-Day Oral Toxicity Study in Rodents", EPA Health Effects Test Guidelines: OPPTS 870.6050.

Version / remarks:

Adopted August 1998.

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:

amended December 5, 1986

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 27, 1995.

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 30, 1996.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed February 18, 2009

Limit test:

no

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 4 4414 Füllinsdorf / Switzerland.
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: Males: 176 g to 195 g (mean 183 g) Females: 137 g to 157 g (mean 147 g).
- Housing: Animals were housed in groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Diet: Pelleted standard Kliba Nafag 3433 (batch no. 44/08) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum
- Water: Community tap-water from Itingen, ad libitum in water bottles
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 ºC
- Humidity: 30-70 %
- Air changes: 10-15 changes/ h
- Photoperiod: 12 h dark/ 12 h light
- Music: at least eight hours music during the light period.

IN-LIFE DATES: from 06 August To 01 October 2008 (Satellite A) and 15 October (Satellite B).

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

TREATMENT PREPARATION AND ADMINISTRATION:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared, weekly, at the appropriate concentrations as a solution in PEG 300 (Fluka AG, Batch Number: 1349048 53807070). The test item was weighed into a tared glass container on a suitable precision balance and the vehicle, PEG 300, was added to give the appropriate final concentration of the test item in the dose formulation.
The mixtures were prepared first by mixing with a homogenizer (i.e. Ultra Turrax) for approximately 2-3 minutes, and then mixed using a magnetic stirrer for approximately 30 minutes. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. The dose formulations were protected from light (i.e. wrapping in aluminum foil).
The dose formulations were stored in glass beakers at room temperature (20 ± 5 °C). Based upon the results of dose formulation analyses performed during previous studies (RCC Study Number C00630), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

VEHICLE
- Justification for use and choice of vehicle: had been prooved as appropriate in previous studies.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 1349048 53807070
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis was performed using gas chromatography coupled to a flame ionisation detector. The test item was used as analytical standard.

The samples (generally 2 g each) were delivered to the analytical laboratory. Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

PEG 300 (Group 1)

Dose / conc.:

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

Remarks:

Group 2 (Low dose)

Dose / conc.:

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

Remarks:

Group 3 (Mid dose)

Dose / conc.:

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

Remarks:

Group 5 (High dose), replaced Group 4 (1000 mg/kg bw/day), which was removed from the study and sacrificed for ethical reasons.

No. of animals per sex per dose:

5 animals/sex for all dose groups (satellite A: Toxicity testing: termination after 28 treatment days).
5 animals/sex for group1 and group 5 (satellite B: Recovery testing: termination after an additional 14-day recovery period).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on previous dose range-finding toxicity study, Harlan Laboratories study C00630, and adapted according to findings noted in the early stages of this study.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: the animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum
- Post-exposure recovery period in satellite groups: 14 days after the end of administration (satellite B).

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration and once weekly (weeks 1 to 3) thereafter.
The animals were observed for general clinical signs once before commencement of administration as well as twice daily on days 1 to 3, once daily on days 4-28 (treatment period), and once daily during days 1 to 14 (recovery period).
The animais were observed in their home cages, outside their home cages in a standard arena and in the band.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded before initiation of treatment (day 0), weekly during treatment, recovery period and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: was recorded once during the acclimatization period and weekly during treatment and recovery period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of treatment (day 29/30) and at the end of recovery period (day 43).
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals: all animals
- Parameters checked in Table 7.5.1/2 were examined.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: at termination of treatment (day 29/30) and at the end of recovery period (day 43).
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals: all animals.
- Parameters checked in Table 7.5.1/2 were examined.


PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: at termination of the treatment period (day 29/30) and at the end of reversal period (day 43).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes
- Parameters checked in Table 7.5.1/2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the fourth week of treatment.
- Dose groups that were examined: 0, 100, 300 and 600 mg/kg bw/day
- Battery of functions tested: grip strength / locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY:
All animals were weighed and necropsied after 4 weeks and 6 weeks (recovery). Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.
Samples of tissues and organs reported on the table 7.5.1/2 were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated).
Organ weight:
Weight of the following organs were weighed (absolute weight): thymus, testes, spleen, ovaries, liver, kidneys, heart, epididymides, brain and adrenal glands.
Values of these organ weights as percent of body weight, on the day of necropsy, were also estimated (relative organ weights).

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.

HISTOPATHOLOGY:
All organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.
Slides of all organs and tissues listed in boldface type (see table 7.5.1/2) that were collected at scheduled sacrifice from the animals of all control and high-dose groups and all gross lesions from all animais were examined by the study pathologist. Organ and tissue samples taken from animais which died spontaneously or which were killed for ethical reasons were evaluated similarly to those organs taken from animais of the high-dose group.

As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (heart, liver, stomach and thymus) from animals of the mid- and law-dose groups were examined.

The examination was carried out on the tissues reported on the table 7.5.1/2.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, 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:

effects observed, treatment-related

Description (incidence and severity):

Clinical symptoms of excessive toxicity were seen in the rats treated with 1000 mg/kg bw/day and they were subsequently removed from the study. A new high dose group, treated with 600 mg/kg bw/day, was added to the study and was treated for 28 days.


- DAILY OBSERVATIONS:
At 600 mg/kg bw/day, clinical signs recorded in females included ataxia (days 2-13 and day 24), slight sedation (days 3-8) and decreased activity (days 25-27).
Hunched posture, ruffled fur and visible weight loss were recorded in several high-dose females beginning on or about treatment day 2 and persisted for much of the treatment period. Bilateral ptosis was noted from day 7 to day 12 of treatment.
Dyspnea, bilateral chromodacryorrhea and/or salivation (reddish in males with staining of the fur on the lower mandible) were recorded sporadically, and soft feces was recorded in all males and all females on day 26 of treatement, and in females ond ays 27-28 of treatment.
No clinical signs of toxicological relevance were evident in the males and females treated with 100 mg/kg bw/day or 300 mg/kg bw/day.
Salivation was noted in animals at all dose levels shortly after administration. This finding was considered to be due to the taste of the test item formulations rather than systemic toxicity.


- WEEKLY BEHAVIORAL OBSERVATIONS:
At 600 mg/kg bw/day, ptosis, hunched posture and ruffled fur were noted in females during week 1. The iridic light reflex was absent in both eyes of one female and the righting reflex was slowed in two females during week 1. Bilateral chromodacryorrhea was noted in one female during week 1 of treatement. Ruffled fur was noted in one female during week 2.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 600 mg/kg bw/day, two females were found dead during the study. Three further females were removed from the study for ethical reasons.
All other animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

AFTER 4 WEEKS OF TREATMENT:
At 600 mg/kg bw/day, the mean absolute body weight and the mean body weight gain of the males were unaffected by the test item. Females treated with this dose had significantly reduced mean body weight (a body weight loss) on day 8 (p<0.01). The mean absolute body weights and the mean body weight gain of these females remained below that of the control females. At termination, the mean absolute body weights of these females remained approximately 5% lower that those of the control females.
There was a loss of body weight from days 1-8 of treatment, and the mean body weight gain remained lower on days 8-15, 15-22 and 22-28 of treatment.
At 300 mg /kg bw/day and 100 mg/kg bw/day, no differences in the mean absolute body weights were noted in males or females during the treatment period.
AFTER 2 WEEKS OF RECOVERY PERIOD:
During recovery, the mean body weight of test item-treated males remained below that of control males, but the mean body weight gain compared favorably. None of the test item-treated high dose females survived until scheduled necropsy after recovery.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

AFTER 4 WEEKS OF TREATMENT:
At 600 mg/kg bw/day, the mean daily food consumption of the males was unaffected. In females, a marked reduction in food consumption was noted during days 1-8 of treatment. During the subsequent measurement intervals -treatment days 8-15 and 15-22), the food consumption compared favorably, but was marginally reduced during treatment days 22-28.
At 300 and 100 mg/kg bw/day, the main daily food consumption was unaffected in males or females.
AFTER 2 WEEKS OF RECOVERY PERIOD:
Males rats previously treated with 600 mg/kg bw/day consumed less feed during week 1 of recovery, but compared favorably during week 2. There were no surviving females.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

AFTER 4 WEEKS OF TREATMENT:
Test item-related changes in the hematology parameters were largely restricted to shifted reticulocyte maturity indices (towards younger cells) in rats at 600 mg/kg bw/day. the reduction in lymphocyte count was considered to be a stress-related change.
A number of other (statistically significant) changes were noted. nearly all remained within the ranges of the historical control data. Many differences were unrelated to dose and were therefore considered to be of no toxicological relevance.
At 600 mg/kg bw/day, males showed statistically elevated red blood cell counts (p<0.05) when compared with control males. Females were unaffected. Although the red cell count of females treated with 600 mg/kg bw/day was unaffected, the mean hemoglobin concentration was signficantly reduced (p<0.05). In both sexes, the mean corpuscular hemoglobin was significantly lower (p<0.05 in males, p<0.01 in females). In females, the mean corposcular volume was significantly lower (p<0.05) than the controls.
Changes in the reticulocyte maturity indices were noted in bioth sexes. The low fluorescence reticulocyte counts were significantly reduced in both sexes (p<0.01 in males and p<0.05 in females), the medium fluorescence reticulocytes were elevated in males (p<0.05) and the high fluorescence reticulocytes were significantly elevated in both sexes (both p<0.01). Slight differences in the mean absolute and reticulocyte counts were noted primarily in females, but the differences were neither dose-related nor statistically significant. In males, the red cell and hemoglobin distribution widths were largely unaffected.
The white blood cell count in males was very slightly reduced and significantly reduced in females (p<0.05) when compared with the respective controls. The differences primarily resulted from statistically significant reductions in mean absolute lymphocyte counts (booth p<0.05). Further significant differences in the absolute differential count of males included elevated neutrophill count (p<0.05), elevated monocytes count (p<0.01), and elevated large unstained cell count (p<0.01), whreas the relative differential count showed significantly elevated neutrophils (p<0.01), elevated basophil (p<0.01). Changes in the absolute and relative differential count of females included reduced absolute neutrophil (not significant), increases monocytes (absolute and relative, both p<0.01) and increased large unstained cell counts (absolute, p<0.05 and realtive, p<0.01).
The mean patelet counts of males and females were significantly reduced (p<0.01) when compared with the respective controls. Signficantly reduced methomoglobin concentration, recorded in males and females (both p<0.01), represents a change which is generally not associated with systemic toxicity and therefore considered to be incidental.
The activated partial thromboplastin time was significantly prolonged in males (p<0.05) and foreshortened (p<0.05) in females. These contrary changes were considered to be unrelated to the treatment with the test item.
At 300 mg/kg bw/day, males showed a significantly elevated mean red blood cell count (p<0.05) when compared with control males. Hemoglobin was significantly elevated in males (p<0.05) and in females(p<0.01). Females, however, showed a statistically significant increase in the mean corpuscular hemoglobin concentration (p<0.01) that was not seen at the higher dose and also remained within the upper range of the historical control values. Although the mean red cell and hemoglobin distribution widths in females were significantly higher than those of the controls (p<0.01 and p<0.05, respectively), the differences were not dose-related although the latter exceeded the ranges of the historical control data.
The white blood cell count in males was very slightly reduced and significantly reduced in females (p<0.01) when compared with the respective controls. The diffences largely resulted from statistically significant reductions in mean absolute eosinophils (p<0.05) and lymphocytes (p<0.01). A significant reduction of the mean relative differential count was noted only in monocytes (p<0.01) of females.
The mean (relative) thromboplastin times of males and females were significantly less (p<0.05 and p<0.01, respectively) than those of the controls, whereas the mean activated partial thromboplastion time of the females was significantly prolonged (p<0.05)
At 100 mg/kg bw/day, the mean absolute differential counts of eosinophiles and basophils were significantly elevated (both p<0.05) in females when compared with controls. The mean (relative) thromoplastion time was significantly lower (p<0.01)when compared with the controls.
AFTER 2 WEEKS OF RECOVERY PERIOD:
After two weeks' recovery, males previously treated with 600 mg/kg bw showed several statistically significant differences: the mean corpuscular hemoglobin was reduced (p<0.01), the mean corpuscular hemoglobin concentration was reduced (p<0.05), the low fluorescent reticulocytes were reduced (p<0.05), the high fluorescent reticulocytes were increased (p<0.05) and the mean partial activated thromboplastin time was foreshortened (p<0.05). There were no surviving test item-treated females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

AFTER 4 WEEKS OF TREATMENT:
Changes noted in the clinical biochemistry findings were considered to be indicative of metabolic activation in the liver at 600 mg/kg/day and ofimpaired function in the kidney at 100,
300 and 600 mg/kg/day. The changes in the liver enzyme activity were considered to be
metabolic changes, confirming at least some degree of bioavailability. The elevations in urea were considered to indicate slightly impaired kidney function.

Increased urea levels were noted in females at 100 mg/kg/day (p<0.01l), males at 300 mg/kg/day (not significant) and females at 300 mg/kg/day (p<0.01), and in both sexes at 600 mg/kg/day (p<0.01). All statistically significant differences also exceeded the range of the historical control data.

Creatinine was slightly lower in males and females but the differences to the control values remained within the ranges of the historical control data.

Significantly lower mean total bilirubin levels were seen in males (p<0.01) and females (p<0.05) when compared with the controls, this finding is generally not indicative of a toxic response and both values remained within the ranges of the historical control data.

The mean cholesterol level in males at 600 mg/kg/day was significantly lower (p<0.05) than in controls. This parameter was mildly reduced in females but the difference did not attain statistical significance. The statistically significant reduction in the cholesterol level of females treated with 100 mg/kg/day (p<0.05) was neither dose-related nor did it exceed the historical control data and was considered to be incidental. Triglycerides, in contrast, were significantly higher in males (p<0.05), but females were largely unaffected.

The respective activities of aspartate aminotransferase were higher in males and females. The differences were not only statistically significant, but they exceeded the upper ranges of the historical control data. In the females, the differences were primarily due to two outlying values.

The activity of lactate dehydrogenase was higher in males and females (both p<0.01), but within the ranges of the historical control data. The clinical relevance of this parameter is considered to be somewhat limited in rats; it is generally considered to be a quality control indicator for hemolytic samples, but can also indicate effects upon cellular integrity, particularly when seen together with the significantly elevated creatine kinase levels (p<0.01 in males and females at
600 mg/kg/day) noted in this study.

In the glutamate dehydrogenase activity of males and females treated with 300 mg/kg/day, contrary statistically significant differences to the control values were noted (reduced in males (p<0.05), and increased in females (p<0.05)). Insofar as these differences were also dose­ unrelated, they were considered to be incidental.

Changes in electrolytes were noted in both sexes, although more markedly in females. Males treated with 600 mg/kg/day had significantly elevated sodium levels.

Significantly elevated potassium levels were seen in females at 300 mg/kg/day (p<0.05) and at
600 mg/kg/day (p<0.01), whereas reduced calcium levels were seen only in females (p<0.01). Significantly increased phosphorus levels were noted in females at 100 mg/kg/day (p<0.05), males at 300 (p<0.05), females at 300 mg/kg/day (p<0.01), males at 600 mg/kg/day (p<0.01) and females at 600 mg/kg/day (p<0.01) when compared with the controls.

Reduced protein levels were noted in males and females treated at 600 mg/kg/day. Both attained statistical significance (p<0.01), but both had slightly high control values and the differences remained within the ranges of the historical control data. In females at 600 mg/kg/day. Albumin was significantly lower (p<0.01), as was globulin (p<0.05). The albumin/globulin ratio was, however, nearly identical to that of the control females.

AFTER 2 WEEKS OF RECOVERY PERIOD:

Glucose levels were reduced (p<0.01) in male previously treated with 600 mg/kg/day. Although triglycerides were significantly higher (p<0.05) in males previously treated with 600 mg/kg/day, this was a trend already seen after the treatment period that remained within the ranges of the historical control values on both occasions. The activities of alanine aminotransferase and alkaline phosphatase were significantly elevated (both p<0.05) in males, but these differences remained within the range of the historical control data.

There were no surviving test item-treated females.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

AFTER 4 WEEKS OF TREATMENT:
Elevated urine output noted in both sexes at 600 mg/kg/day was considered to be possibly related to the impaired ability of the kidney to concentrate urine, whereas a dose-dependent increase in ketone concentration were noted in all males at all dose levels.

Increased urinary output was noted in males and females treated with 600 mg/kg/day. In males, the difference attained statistical significance (p<0.05) when compared with controls.

Elevated levels of ketones were detected in the urine of males at all dose levels, attaining statistical significance at 300 mg/kg/day (p<0.05) and at 600 mg/kg/day (p<0.01). These differences exceeded the upper range of the historical control data at all dose levels. In females, one female treated with 300 mg/kg/day was considered to be an outlier that resulted in statistical significance (p<0.01). Females treated with 600 mg/kg/day were unaffected.

AFTER 2 WEEKS OF RECOVERY PERIOD:

There were no differences of toxicological relevance noted in males. There were no surviving test item-treated females.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery: at 600 mg/kg/day, dyspnea was evident in one male and one female during week 4 of treatment, whereas salivation was seen in several males. All other animais were unaffected
Grip Strength: the mean fore- and hindlimb grip strength values of the test item-treated males compared favorably with those of the controls. In females treated with 600 mg/kg/day, the mean forelimb grip strength was significantly lower (p<0.01) than that of the control females. The hindlimb grip strength of these females was similar to those of the controls. The difference noted in the forelimb grip strength was considered to be incidental
Locomotor Activity: no toxicologically relevant changes in locomotor activity were noted in males at any dose level.
In females treated with 600 mg/kg/day, the mean locomotor activity was elevated during 40- 50
minutes and 50 - 60 minutes, whereby only the difference recorded during 50 - 60 minutes
attained statistical significance (p<0.01) when compared with the controls. The overall locomotor activity (0- 60 minutes) of the females treated with 600 mg/kg/day was generally similar to that of the controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

AFTER 4 WEEKS OF TREATMENT:
In animals of allocation A, the mean relative heart-to-body weight ratio was significantly increased in males at 600 mg/k/day (p<0.05). The liver weights of test item-treated rats were elevated, although a clear dose-response relationship was not seen.
In females treated with 300 mg/kg/day, the mean absolute liver weight was statistically elevated (p<0.05) when compared with control females. The mean liver-to-body weight ratio of test item-treated males were slightly elevated, with rats treated with 300 mg/kg/day attaining statistical significance (p<0.05). The mean liver-to-body weight ratios of the females were elevated at 300 mg/kg/day (p<0.01) and 600 mg/kg/day (p<0.05). Mean absolute or relative weights of the thymus were significantly decreased in females at 600 mg/kg/day (p<0.01).

AFTER 2 WEEKS OF RECOVEY PERIOD:
Following the recovery period, the mean absolute and relative heart weights were significantly increased in males at 600 mg/kg/day (p<0.01 or p<0.05) and correlated with minimal to marked ventricular dilation. Only the reductions in the mean absolute thymus weight attained statistical significance (p<0.05). The relative thymus weights were also lower when compared with the control males. Mean kidney-to-body weight ratio was significantly increased (p<0.05) when compared with the controls. This finding was not associated with microscopie changes and therefore considered to be incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In allocation A, one of five males at 600 mg/kg/day bad accentuated lobular pattern in the liver. In four of five females at 100 mg/kg/day and five of five females at 300 mg/kg/day, the liver was clay-colored. These changes were associated with increased periportal hepatocellular vacuolation. The thymus was reduced in size in two of five females at 600 mg/kg/day, due to thymic atrophy.

In the premature decedent females at 600 mg/kg/day, 2/5 animais had black brown focus/foci in the forestomach, correlating with ulceration or squamous cell hyperplasia and hyperkeratosis.

The other macroscopic findings recorded were considered to be within the range of normal background lesions which may be seen in rats of this strain, age and study type, and were considered incidental, reflecting the usual individual variability. Such findings were esophagus with food-like contents; reddish or black foci in the stomach; reddish, firm nodule(s) in the epididymides; yellowish or dark red focus/foci in the liver, left lateralliver lobe reduced in size; reddish discoloration of the ovaries; Peyer's patches not visible; general dark red discoloration; reddish discoloration of jejunum or ileum; reddish discoloration of the uterus; dark red discoloration of the thymus or the mesenteric lymph node; the mandibular lymph node reduced in size; and black focus/foci in the Harderian glands.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Heart with Auricles:
In test item-treated animais, there was myocardial vacuolation and myocardial necrosis. Vacuoles were mostly about the size of cell nuclei and had round shape. In fat stain carried out on a typical specimen, they did not stain positively, indicating that they were not caused by fatty change. Occasionally there were very small vacuoles separating the myofibrils a giving the myocardium pale appearance. Focal/multifocal myocardial necrosis was characterized by eosinophilia and fragmentation of myocardium fibers, and presence of reactive cells, mainly lymphocytes and occasional granulocytes. These changes were observed in the ventricular walls, apex, interventricular septum and papillary muscles.

In animals of allocation A, minimal to moderate myocardial necrosis affected four of five males at 300 mg/kg/day and two of five males at 600 mg/kg/day, as well as two of five females at
100 mg/kg/day and two of five females at 600 mg/kg/day. Minimal to marked myocardial
vacuolation occurred in three of five males at 300 and four of five males at 600 mg/kg/day, as well as four of five females at 100, five of five females at 300, and four of five females at
600 mg/kg/day. Among the premature decedents (females assigned to recovery group), minimal to slight myocardial necrosis occurred in two of five females at 600 mg/kg/day and minimal to slight myocardial vacuolation in four of five females at 600 mg/kg/day.

Following the recovery period (which only the males survived), three of five males at
600 mg/kg/day had minimal to marked ventricular dilation and one of these animais had moderate epicarditis and myocardial necrosis.

Liver:
In animals of allocation A, the mean grade of minimal to moderate periportal hepatocellular vacuolation was increased in males at 300 and 600 mg/kg/day and in females at 100, 300 and
600 mg/kg/day. These vacuoles were very small, giving the hepatocytes foamy appearance. Such
changes were not observed in premature decedent females at 600 mg/kg/day. Following the recovery period, one of five males at 600 mg/kg/day had minimal hemorrhagic necrosis and slight periportal fibrosis. This animal also had increased alveolar histiocytosis in the lung and necrosis and epicarditis in the heart.

Stomach:
In the premature decedent females at 600 mg/kg/day, there were changes in the forestomach. One of five females had moderate ulceration and three of five females had moderate squamous cell hyperplasia with hyperkeratosis.

Thymus:
In animals of allocation A, slight to moderate atrophy was seen in two of five females at
600 mg/kg/day. In the premature decedent females at 600 mg/kg/day, all five animals had marked to severe atrophy of the thymus.

Testes/Epididymides:
Following the recovery period, minimal to moderate cellular debris was found in the epididymides of three of five males at 600 mg/kg/day. Two of these animais had minimal to moderate tubular atrophy of the testes.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

ANALYSIS OF DOSE FORMULATIONS:

The identity of the test item was confirmed by its retention time which was similar to that measured in the working standards.

The application formulations investigated during the study contained the test item in the range of
91.9% to 110.8% and fulfilled the required content limit of ±20% with reference to the nominal concentration. Because single results found did not deviate more than 8.9% (<15%) from the corresponding mean, the test item was considered to be homogeneously distributed in the dosing solutions.

Although the values of three stability samples were out of the required limit, the application formulations were considered to be stable for at least 7 days when kept in the refrigerator (5 ± 3 °C).

In conclusion, the results obtained within this part confirm the correct preparation and storage of application formulations during the conduct of this study.

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable

Remarks:

Based on the results of this study, it was not possible to set a NOAEL for the test substance. The NOAEL was below the lowest dose level test in this study.

Key result

Critical effects observed:

no

Conclusions:

Under the test condtions, it was not possible to set a no-observed-effect-level (NOEL) or a no-observed-adverse-effect-level (NOAEL). The NOAEL for the test substance was below the lowest dose level test in this study.

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only. However, the animals of the high dose group developed symptoms of unexpected severity and were subsequently removed from the study. An additional dose level, treated with 600 mg/kg bw/day, was added and treated as the high dose group. 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 600 mg/kg/day. 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 the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were evaluated 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, heart, liver stomach and thymus were examined to establish a no-effect level.

 

At all tested doses, no effects on the fore-and hindlimb grip strength values of the test item-treated rats were noted, and no toxicologically relevant changes in locomotor activity were noted at any dose level.

 

Test item-related findings included the premature deaths of five females treated with 600 mg/kg bw/day, daily clinical signs in females (ataxia, sedation, decreased activity, hunched posture, ruffled fur and visible weight loss) and males (dyspnea, bilateral chromodacryorrhea and/or salivation) at 600 mg/kg bw/day, weekly clinical signs (ptosis, hunched posture and ruffled fur in females during week 1 at 600 mg/kg bw/day, slowed righting reflex in two females during week 1 at 600 mg/kg bw/day), a marked reduction in food consumption of females during week 1 at 600 mg/kg bw/day, reduced mean body weights (a body weight loss) in females during week 1 at 600 mg/kg bw/day, changes in hematology parameters (shifted to high-fluorescent reticulocytes) at 600 mg/kg bw/day and a stress-related reduction in lymphocytes, changes in clinical biochemistry parameters (indicative of metabolic activation in the liver at 600 mg/kg bw/day and of impaired function in the kidney at 100, 300 and 600 mg/kg bw/day), changes in urinalysis parameters (elevated urine output noted in both sexes and ketones in all males) at 600 mg/kg bw/day, increased mean absolute and relative heart weights in males at 600 mg/kg bw/day, elevated absolute and/or relative heart weights in males at 600 mg/kg bw/day, elevated absolute and/or relative liver weights at all dose levels, decreased mean absolute or relative weights of the tymus in females at 600 mg/kg bw/day, ulceration or squamous cell hyperplasia in the forestomach and thymic atrophy in premature decedent rats at 600 mg/kg bw/day, and microscopical findings in the hearts of the females at 100 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day, and increased periportal hepatocellular vacuolation in the livers of females at 100 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day).

After the recovery period, epicarditis, myocardial necrosis and ventricular dilation in the heart, and hemorrhagic necrosis and periportal fibrosis in the liver were noted in males treated with 600 mg/kg bw/day. Cellular debris in the epididymis was evident in three of five animals and tubular atrophy in the testes of two of five rats treated with 600 mg/kg bw/day, possibly representing a delayed effect.

 

Based on the results of this study, it was not possible to set a no-observed-effect-level (NOEL) or a no-adverse-effect-level (NOAEL). The no-observed-adverse-effect-level (NOAEL) for the test substance was below the lowest dose level test in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The studies are GLP-compliant and of high quality (Klimisch score = 1).

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

Repeated dose toxicity - oral:

Two key studies are available and considered as WoE.

- In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP (Harlan, February 2009), the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only. However, the animals of the high dose group developed symptoms of unexpected severity and were subsequently removed from the study. An additional dose level, treated with 600 mg/kg bw/day, was added and treated as the high dose group. 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 600 mg/kg/day. 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 the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were evaluated 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, heart, liver stomach and thymus were examined to establish a no-effect level.

 

At all tested doses, no effects on the fore-and hindlimb grip strength values of the test item-treated rats were noted, and no toxicologically relevant changes in locomotor activity were noted at any dose level.

 

Test item-related findings included the premature deaths of five females treated with 600 mg/kg bw/day, daily clinical signs in females (ataxia, sedation, decreased activity, hunched posture, ruffled fur and visible weight loss) and males (dyspnea, bilateral chromodacryorrhea and/or salivation) at 600 mg/kg bw/day, weekly clinical signs (ptosis, hunched posture and ruffled fur in females during week 1 at 600 mg/kg bw/day, slowed righting reflex in two females during week 1 at 600 mg/kg bw/day), a marked reduction in food consumption of females during week 1 at 600 mg/kg bw/day, reduced mean body weights (a body weight loss) in females during week 1 at 600 mg/kg bw/day, changes in hematology parameters (shifted to high-fluorescent reticulocytes) at 600 mg/kg bw/day and a stress-related reduction in lymphocytes, changes in clinical biochemistry parameters (indicative of metabolic activation in the liver at 600 mg/kg bw/day and of impaired function in the kidney at 100, 300 and 600 mg/kg bw/day), changes in urinalysis parameters (elevated urine output noted in both sexes and ketones in all males) at 600 mg/kg bw/day, increased mean absolute and relative heart weights in males at 600 mg/kg bw/day, elevated absolute and/or relative heart weights in males at 600 mg/kg bw/day, elevated absolute and/or relative liver weights at all dose levels, decreased mean absolute or relative weights of the tymus in females at 600 mg/kg bw/day, ulceration or squamous cell hyperplasia in the forestomach and thymic atrophy in premature decedent rats at 600 mg/kg bw/day, and microscopical findings in the hearts of the females at 100 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day, and increased periportal hepatocellular vacuolation in the livers of females at 100 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day).

After the recovery period, epicarditis, myocardial necrosis and ventricular dilation in the heart, and hemorrhagic necrosis and periportal fibrosis in the liver were noted in males treated with 600 mg/kg bw/day. Cellular debris in the epididymis was evident in three of five animals and tubular atrophy in the testes of two of five rats treated with 600 mg/kg bw/day, possibly representing a delayed effect.

Based on the results of this study, it was not possible to set a no-observed-effect-level (NOEL). The no-observed-adverse-effect-level (NOAEL) for the test substance was <100 mg/kg bw/day in this study. This study is considered as a range-finding for a repeated dose toxicity study (Harlan, July 2009).

 

- In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP (Harlan, July 2009), the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 5, 25 and 50 mg/kg 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 50 mg/kg/day. 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 the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were evaluated 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. 

 

No test item-related unscheduled deaths occurred and the death of one female treated with 50 mg/kg/day on day was considered to be dosing error.

No test item-related differences of toxicological relevance were noted during daily observations at any dose level. No symptoms were evident during the weekly detailed clinical observations (weeks 1-3). No symptoms were evident during the functional observational battery (week 4). The mean fore- and hindlimb grip strength values were similar in test item-treated and control rats of both sexes. The mean locomotor activity of test item-treated males and females compared favorably with those of the respective controls. Analysis of the vaginal smears taken from the surviving females during the fourth week of treatment showed generally similar distributions of the estrus cycle stages.

Test article did not have any adverse effect on the body weight gain and average daily food consumption by the male and female rats treated up to the dose level of 50 mg/kg bw/day.

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

In hematology parameters, in males treated with 50 mg/kg/day, differences in the reticulocyte maturity indices were noted when compared with the controls. The low-fluorescence reticulocytes were reduced, and the high-fluorescence reticulocytes were elevated, which is generally indicative of a trend towards a younger cell population and increased cell turnover. However, as females at this dose level were unaffected, these changes were considered to be incidental. Females and males treated with 5 mg/kg/day or 25 mg/kg/day were unaffected. After the recovery period, reduced hemoglobin levels, hematocrit and mean corpuscular volume noted in males were not seen after the treatment period. In the absence of similar changes in the females, these differences were considered to be incidental and of no toxicological relevance.

No test item related differences were noted in organ weights at any dose level. No treatment related gross and microscopic pathological alterations in the tissues of male and female rats treated up to the level of 50 mg/kg bw/day.

 

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) of the test substance in Wistar rats, following oral administration for 28 days was found to be 50 mg/kg bw/day. 

 

Justification for classification or non-classification

Harmonised classification:

The test material has no harmonised classification for repeated dose toxicity according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification:

Based on the available information, adverse effects on the heart of the rats were observed after oral repeated doses of the test material at 100, 300 and 600 mg/kg bw/day (Harlan February 2009). Therefore a self-classification of STOT RE 2 is proposed for the specific target organ toxicity hazard according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No information is available regarding the dermal and inhalation routes.