Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to relevant guidelines performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
The study was conducted as part of a sub-chronic, 28-day repeated-dose toxicity study under OECD TG 407 and EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP. The study was modified according to the relevant listed regulatory guidelines in order to incorporate micronucleus assessment following exposure to the test item in a manner compatible with OECD TG 474 (2014) and listed guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2015; signature: September 2015
Type of assay:
mammalian germ cell cytogenetic assay
Species:
rat
Strain:
other: Crl:CD(SD) IGS BR
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 guideline and consistent with the OECD TG 474 guideline.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 to 8 weeks
- Weight at study initiation: males 205 - 311 g and females 139 - 215 g; individuals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Fasting period before study: None
- Housing: Polypropylene body with a stainless steel mesh lid with softwood flake bedding, changed at appropriate intervals; group housed (5 per group) by sex. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. Cage distribution within the holding rack was randomized.
- Diet (e.g. ad libitum): Rodent 2014C, Global Certified Diet, ad libitum
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 5 days.
DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent 2014C, Global Certified Diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2016-03-04 To: 2016-04-15
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Arachis oil BP was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable for at least eleven days when stored refrigerated. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored at approximately 4 ºC in the dark.
- Concentration of test material in vehicle: Samples of the test item formulations were taken on two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ± 4% of the nominal concentration. Arachis oil formulations was assessed and confirmed at nominal concentrations of 3.75 mg/mL and 250 mg/mL, during refrigerated storage.
- Amount of vehicle (if gavage or dermal): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group; positive control was 10 mL/kg. For further information see 'Doses / concentrations'.
- Type and concentration of dispersant aid (if powder): Not applicable.
- Other: Dose-formulations were analysed during the study and were reported as within ± 10% applied limits.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period (treatment free) for the OECD 407 part of the study. The last dose was administered on Day 28. Micronucleus assessment proceeded after in non-recovery groups thereafter.
Frequency of treatment:
Once daily at approximately the same time each day.
Post exposure period:
24-hours following final dosing in relevant treatment groups only
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control - Group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High - Group
No. of animals per sex per dose:
10 per sex per dose (5 male / 5 female)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): The choice of positive control was made in accordance with the OECD TG 474 guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 1 dose; 25 mg/kg bw
- Other: five (5) males - single dose were used for micronucleus test, treated with cyclophosphamide at 25 mg/kg bw/day - dosed on Day 28 only.
Tissues and cell types examined:
Bone marrow was extracted and smear preparations were made and stained Polychromatic (PCE) and nomtochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCEINCE ratio was calculated as an indicator for toxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The test item dose levels used in the main study were 100, 350 and 1000 mg/kg bw/day. The maximum dose level was considered to be more than adequate to investigate the genotoxic endpoint in this study (confirmed as part of investigations in 7-d range finder and 28-day repeated dose toxicity testing).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Males/females were treated daily for 28-days at the specified dose levels. 24-hours following the last treatment (day 28) the treatment groups were terminated and then bone marrow was extracted from the left femur as part of sampling. Smear preparations were subsequently made and stained.

DETAILS OF SLIDE PREPARATION:
24-hours following last treatment, left femurs were extracted, aspirated with foetal bovine serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald / Giemsa. Then allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
Stained smears were coded and examined blind using microscopy (x1000) magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with the appropriate group mean values and standard deviations.

OTHER: Not applicable.
Evaluation criteria:
1. Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group:
- A positive response would be demonstrated when: there is a statistically significant dose response, toxicologically relevant increases in the number of micronucleated polychromatic erythrocytes observed at the 24-hour termination time compared with the vehicle control group
- A negative response would be fulfilled if the positive response criteria were not fulfile (no statistically significant dose responses, no toxicologically relevant increases in micronucleated polychromatic erythrocytes.

2. A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically lower than the vehicle control group.

Data is subject to statistical analysis, where appropriate.
Statistics:
Wherea appropriate, data is subject to statistical analysis using appropriate methods as recommended in UKEMS Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No statistically significant PCE/NCE decreases at up to 1000 mg/kg bw/day observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: positive control male (number 65) slide was disregarded due to excessive bone marrow toxicity. The loss of one individual from the PC scoring data was considered to have no impact on the outcome of the study.
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not applicable.
- Induction of micronuclei (for Micronucleus assay):
Vehicle Control, 0 mg/kg bw/day: Group mean (SD) = 12.0 (10.6) PCE with micronuclei per 4000 PCE
Low dose, 100 mg/kg bw/day: Group mean (SD) = 8.5 (8.9) PCE with micronuclei per 4000 PCE
Intermediate dose, 350 mg/kg bw/day: Group mean (SD) = 6.8 (4.5) PCE with micronuclei per 4000 PCE
High dose, 1000 mg/kg bw/day: Group mean (SD) = 8.8 (5.0) PCE with micronuclei per 4000 PCE
Positive Control, 25 mg/kg bw/day: Group mean (SD) = 83.8 ** (71.7) PCE with micronuclei per 4000 PCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01
- Ratio of PCE/NCE (for Micronucleus assay):
Vehicle Control, 0 mg/kg bw/day: Group mean (SD) = 1.29 (0.39) PCE/NCE
Low dose, 100 mg/kg bw/day: Group mean (SD) = 1.22 (0.62) PCE/NCE
Intermediate dose, 350 mg/kg bw/day: Group mean (SD) = 0.90 (0.52) PCE/NCE
High dose, 1000 mg/kg bw/day: Group mean (SD) = 1.01 (0.39) PCE/NCE
Positive Control, 25 mg/kg bw/day: Group mean (SD) = 0.50 ** (0.06) PCE/NCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01
- Appropriateness of dose levels and route: The highest dose was set in accordance with the findings of the 7-day Range Finding study and the OECD TG 407 study. The appropriateness of the dose and route was confirmed during the study (recorded in the full study report). Applicant assessment: The maximum dose 1000 mg/kg bw/day is in accordance with the OECD TG 474 guideline for a period of administration greater than 14-days and three dose levels with a separation factor less than 4.0.
- Statistical evaluation: No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw/day relative to the concurrent vehicle control.
- Other: positive control male (number 65) slide was disregarded due to excessive bone marrow toxicity. The loss of one individual from the PC scoring data was considered to have no impact on the outcome of the study. A statistically significant (P < 0.01) response was observed in PCE with micronuclei per 4000 PCE therefore confirming the sensitivity of the test system under the conditions of the test.

Table 1.0 – Table of results

Treatment Group

Number PCE with micronuclei per 4000 PCE

PCE/NCE Ratio

 

Group mean

SD

Group mean

SD

Vehicle Control, 0 mg/kg bw/day

12.0

10.6

1.29

0.39

Low dose, 100 mg/kg bw/day

8.5

8.9

1.22

0.62

 

Intermediate dose, 350 mg/kg bw/day

6.8

4.5

0.90

0.52

High dose, 1000 mg/kg bw/day

8.8

5.0

1.01

0.39

 

 

 

 

 

Positive Control, 25 mg/kg bw/day

83.8**

71.7

0.50**

0.06

Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01

0.01

Conclusions:
Under the conditions of this study, the test item was considered to be non-genotoxic.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Additional parameters covering an in vivo, micronucleus assessment was included under the following guidelines OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 as part of the study. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) IGS BR rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 100, 350 or 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Furthermore, as part of micronucleus assessment a concurrent positive control group of five males was exposed to 25 mg/kg bw/day cyclophosphamide on day 28 only. Within the micronucleus assessment, there was no indications of genotoxicity. The vehicle control group frequency of micronucleated PCE and PCE/NCE was within the normal range. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item groups when compared with the vehicle control group. There were no statistically significant decreases in the PCE/NCE ratio in any of the test item groups when compared with the vehicle control group. Additionally, the vehicle control groups were within the normal range for the frequency of micronucleated PCEs and for PCE/NCE ratio. The positive control group showed a marked increase in the incidence of mononucleated polychromatic erythrocytes. Confirming sensitivity of the test system to the known mutagenicity of the cyclophosphamide under the conditions of the test. Under the conditions of this study, the test item would not be considered as genotoxic.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003
Deviations:
no
Principles of method if other than guideline:
The micronucleus assessment phase of the study was compatible with the following regulatory guidelines:
1. OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 26 September 2014)
2. Commission Regulation (EC) 440/2008 Method B12 “Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test”, dated May 30, 2008
3. US EPA (TSCA) OPPTS 870.5395 “Mammalian Erythrocyte Micronucleus Test” EPA 712-C-98-226, August 1998
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2016; signature: October 2016
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark
- Other: clear colourless

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) IGS BR
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 to 8 weeks
- Weight at study initiation: males 222 - 356 g and females 154 - 223 g; individuals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Fasting period before study: None
- Housing: Polypropylene body with a stainless steel mesh lid with softwood flake bedding, changed at appropriate intervals; group housed (5 per group) by sex. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. Cage distribution within the holding rack was randomized.
- Diet (e.g. ad libitum): Rodent 2014C, Global Certified Diet, ad libitum
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent 2014C, Global Certified Diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2016-03-03 To: 2016-04-14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Arachis oil BP was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable for at least eleven days when stored refrigerated. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored at approximately 4 ºC in the dark.
- Concentration in vehicle: Samples of the test item formulations were taken on two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ± 4% of the nominal concentration. Arachis oil formulations was assessed and confirmed at nominal concentrations of 3.75 mg/mL and 250 mg/mL, during refrigerated storage. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group; positive control was 10 mL/kg. For further information see 'Doses / concentrations'.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10% applied limits.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed in Arachis oil BP formulations at nominal concentrations of 3.75 mg/mL and 250 mg/mL , during refrigerated storage for at least eleven (11) days.
- Refrigerated formulations were also analysed after refrigeration on receipt, then on day 11: the formulation was removed from storage and equilibrated to ambient temperature. The formulations were mixed according to mixing procedure (as used in the definitive test) and single samples were removed for analysis from the top, middle and bottom of the mixed formulation.
- The analysis consisted of GC FID analysis with internal calibration (within a dedicated formulation analysis report attached to the full study report). Samples of Arachis oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These were then subjected to analysis by GC FID and using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period (treatment free). The last dose was administered on Day 28.
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High - Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Recovery High - Group
No. of animals per sex per dose:
10 per sex per dose (5 male / 5 female); 10 per sex per dose for recovery phase groups
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 7-day sighting study (Report number attached to and cited in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: 14 days.- Dose selection rationale: Dose levels were based on the results of a previously conducted 7-day sighting study (Report number attached to and cited in the full study report). Dose levels were selected following 7-day sighting test as: Group 1: 0 mg/kg/day (Arachis oil BP) Group 2: 100 mg/kg/day, Group 3: 350 mg/kg/day Group 4: 1000 mg/kg/day (regulatory limit dose). In the 7-day range finder (administered consecutively, for 8-days) determined: no significant adverse effects detected at up to 1000 mg/kg/day using dose levels of 250, 500 and 1000 mg/kg/day. The plasma analysis indicated exposure of the bone marrow to the test item, and therefore the suitability of incorporating a micronucleus assessment as part of a 28 day toxicity study. Basis: other: nominal in vehicle (Arachis Oil BP)
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: 14 days.
Positive control:
Positive control males: five (5) males - single dose were used for micronucleus test, treated with cyclophosphamide at 25 mg/kg bw/day - dosed on Day 28 only. No concurrent positive control was used for other effects.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All individuals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment free period, animals were observed daily. All observations were recorded. Additional functional observations were made as ‘special evaluations’. This was prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination and, in the case of recovery group animals, on Days 36 and 43 prior to termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

FOOD EFFICIENCY: Yes.
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily. Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically. A possible intergroup difference was detected during Week 3, therefore water consumption was continued to be measured and recorded for each cage group until the termination.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed. Additional histopathology was conducted on eyes after termination.
- Dose groups that were examined: All test animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all non-recovery test and control group individuals. End of recovery period (day 42) for all recovery group individuals.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic). Additionally: Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all non-recovery test and control group individuals. End of recovery period (day 42) for all recovery group individuals.
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Urea, Aspartate aminotransferase (ASAT), Glucose, Alanine aminotransferase (ALAT), Total protein (Tot.Prot.), Alkaline phosphatase (AP), Albumin, Creatinine (Creat), Albumin/Globulin (A/G) ratio (by calculation), Total cholesterol (Chol), Sodium (Na+), Total bilirubin (Bili), Potassium (K+), Triglycerides (Tri), Chloride (Cl-), Bile acids, Calcium (Ca++), Gamma glutamyltranspeptidase, Inorganic phosphorus (P)

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld during time of urine collection; overnight)
- Parameters checked: urine volume, urine appearance, urine density, pH, ketones, bilirubin, urobilnogen, blood pigments, protein, sodium, potassium, chloride, creatinine, glucose. Microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Casts, Spermatozoa, Other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles, Uterus with Cervix (with coagulating glands and fluids)

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 10% formalin: Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and Rectum pons), Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides (Preserved in modified Davidson’s fluid), Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (fixed in Davidson’s fluid), Gross lesions, Spleen, Heart, Stomach, Ileum ,Testes (Preserved in modified Davidson’s fluid), Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver,Trachea, Lungs (with bronchi) - inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina, Muscle (skeletal).
Microscopic analysis was conducted thereof. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Other examinations:
- Thyroid Hormone Assessment: blood samples were taken at exsanguination and the plasma was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

- Micronucleus assessment (non-recovery groups only):
- Following termination (i.e. 24 hours following dosing), from the left femur: fetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip applied using mounting medium. Separate positive control slides were prepared. Stained bone marrow smears were coded and examined blind using light microscopy.
- The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
- An additional group of five males were given a single oral dose of cyclophosphamide (25 mg/kg), to serve as a positive control.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Micronucleus results, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Where appropriate, data transformations were performed using the most suitable method. Data were analysed using the decision tree from proprietary tables and statistics modules incorporating, homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day dose level: Increased salivation was reported from Day 2 (females) and Day 5 males) and these animals also showed an increase in overall water consumption. Increased salivation was also evident in animals of either sex treated with 350 mg/kg bw/day from Day 9. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are generally considered not to be of toxicological importance.

No such effects were detected in either sex treated with 100 mg/kg bw/day dose level. There were no treatment-related changes in the behavioral parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.

During the treatment-free period, no clinical observations were detected for any of the recovery group. Therefore there was evidence of recovery.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a reduction in body weight gain during Weeks 1, 2 and 4 of treatment and during the final week of the treatment free period. No adverse effects were detected in body weight development in treated females or in males treated with 350 or 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. A slight reduction in body weight gain remained evident in these males during Weeks 2 and 4 although statistical significance was not achieved. Overall body weight gain for these males was reduced by 24%. No adverse effects were evident in food consumption for these individuals and some recovery was evident following the ceasation of treatment therefore in isolation the reduced body weight gain was considered not to reflect an overall adverse effect of treatment.

No toxicologically significant effects were detected in treated females at 1000 mg/kg bw/day or in males/females treated with 350 or 100 mg/kg bw/day.

Females treated with 350 mg/kg bw/day showed a reduction in body weight gain during the final week of treatment. This was considered to be the result of two females showing an actual body weight loss. In the absence of a similar effect in 1000 mg/kg bw/day females the intergroup difference was considered to reflect normal biological variation.

There was no effects observed on females during the recovery period.

Recovery males that were previously given 1000 mg/kg bw/day showed a statistically significant (p<0.05) reduction in body weight gain during the second week of the treatment free period which resulted in slightly lower overall gains during the treatment free period. However, body weight gain in these males during the first week of the treatment free period was actually greater than controls therefore the intergroup difference was considered not to be of toxicological importance.

Therefore, there was limited effects seen on bodyweight post-exposure and evidence of recovery at all dose levels in males and females.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a slight reduction in food consumption and food conversion efficiency between Days 1 and 3. Recovery was evident thereafter. No treatment-related effects on overall food consumption were detected in females treated with 1000 mg/kg bw/day or in either sex treated with 500 or 250 mg/kg bw/day. Applicant assessment considers this observation of limited or no toxicological relevance.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males and females treated with 1000 mg/kg bw/day showed an increase in overall water consumption (24% for males and 35% for females) during the final two weeks of the treatment period. Following the cessation of treatment, water consumption for both males and females was comparable to controls. Therefore there was evidence of recovery.

Males and females treated with 350 mg/kg bw/day also showed a slight increase in overall water consumption (18% for males and 16% for females).

No such effects were detected in either sex treated with 100 mg/kg bw/day.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined at the end of the treatment or treatment free periods.

Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in activated partial thromboplastin time. Similar differences were not evident following two weeks of recovery, indicating full reversibility of these changes.

In the absence of a similar effect in males or recovery groups, a true dose related response or any associated changes in prothrombin time the intergroup difference was considered to be of no toxicological relevance. Furthermore, all individual values appeared normal
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined at the end of the treatment or treatment free periods.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in alkaline phosphatase.

Females treated with 1000 mg/kg bw/day showed statistically significant reductions in albumin/globulin ratio, aspartate aminotransferase and alanine aminotransferase and statistically significant increases in bilirubin and bile acids. The effects on aspartate aminotransferase, alanine aminotransferase and bile acids also extended to females treated with 350 mg/kg bw/day. Whilst these intergroup differences may indicate minor perturbations in metabolism, in the absence of any histopathological hepatic changes these were considered unlikely to be of an adverse toxicological response.

Females treated with 100 mg/kg bw/day showed a statistically significant increase in triglyceride levels. In the absence of a similar effect seen in females treated with 350 or 1000 mg/kg bw/day or in treated males this observation was considered not to be of toxicological significance.

Males treated with 1000 mg/kg bw/day showed a statistically significant (p<0.05) reduction in phosphorus. At the end of the treatment free period, males that were previously given 1000 mg/kg bw/day showed a statistically significant increase in phosphorus. In the absence of a consistent effect in treated males, a similar effect in females or an associated effect on calcium concentration, the intergroup differences were considered not to be of toxicological importance.

At the end of the treatment free period, females that were previously given 1000 mg/kg bw/day showed statistically significant increases in glucose (p<0.05) and potassium concentration (p<0.05). In the absence of a similar effect observed in animals at the end of the treatment period or any histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the urinalytical parameters.

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the behavioral parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured at the end of the treatment or treatment free periods.

Males and females treated with 1000 mg/kg bw/day and males treated with 350 and 100 mg/kg bw/day showed increases in absolute and relative liver weight. Recovery females that were previously given 1000 mg/kg bw/day also showed an increase in absolute and relative liver weight; however, all individual values were within the control range at the end of the treatment period. Although there were a few minor blood chemical changes which may have indicated minor perturbations in metabolism, in the absence of any histopathological hepatic changes, the intergroup differences were considered unlikely to be of any toxicological importance.

Males and females treated with 1000 mg/kg bw/day and males treated with 350 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in liver weight both absolute and relative to terminal body weight.

At the end of the treatment free period, females that were previously given 1000 mg/kg bw/day also showed a statistically significant increase (p<0.01) in absolute and relative liver weight however male liver weights were comparable to controls.

Although there were a few minor blood chemical changes evident at 1000 mg/kg bw/day which may indicate minor perturbations in metabolism, in the absence of any histopathological hepatic changes, the intergroup differences in liver weights were considered unlikely to be of any toxicological relevance. Females treated with 100 mg/kg bw/day showed a statistically significant increase in absolute and relative spleen weight and males treated with 350 mg/kg bw/day also showed a statistically significant increase in absolute and relative thyroid weight. In the absence of a similar effect seen in animals at 1000 mg/kg bw/day the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Adrenal Glands: hypertrophy of the zona glomerulosa was evident in two females treated with 1000 mg/kg bw/day. No such effects were detected in treated males or in females treated with 350 or 100 mg/kg bw/day and this change was not seen in recovery females that were previously given 1000 mg/kg bw/day following fourteen days without treatment. Therefore there was evidence of recovery.

Minimal cortical vacuolation was evident in the adrenals of two males treated with 1000 mg/kg bw/day. After examination of the adrenal glands from all males, cortical vacuolation was also evident in one control male in the recovery group. This finding was therefore considered not to be related to the administration of the test item.

Thyroids: minimal follicular cell hypertrophy was evident in three females treated with 1000 mg/kg bw/day, two females treated with 350 mg/kg bw/day, one female treated with 100 mg/kg bw/day and in one control female following the fourteen day treatment free period. Due to its presence in one control female, variability across groups and absence in recovery 1000 mg/kg bw/day males/females, this change was considered not to be significant within the study.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
1. Micronucleus Test:
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item groups when compared with the vehicle control group.
- There were no statistically significant decreases in the PCE/NCE ratio in any of the test item groups when compared with the vehicle control group.
- The vehicle control groups were within the normal range for the frequency of micronucleated PCEs and for PCE/NCE ratio.
- The positive control group showed a marked increase in the incidence of mononucleated polychromatic erythrocytes. Confirming sensitivity of the test system to the known mutagenicity of the cyclophosphamide under the conditions of the test.
Note: one PC male slide, was disregarded due to excessive bone marrow toxicity. This was considered to have no impact on the results.

2. Thyroid Hormone Assessment: blood samples were taken at exsanguination and the plasma was stored frozen. No treatment-related effects on the pituitary-thyroid axis were identified and therefore further analysis was not conducted.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 1000 mg/kg body weight per day in males and females; findings were either of limited toxicological significance, adaptive changes or have no relevance for human health.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Additional parameters covering an in vivo, micronucleus assessment was included under the following guidelines OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 as part of the study. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) IGS BR rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 100, 350 or 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Furthermore, as part of micronucleus assessment a concurrent positive control group of five males was exposed to 25 mg/kg bw/day cyclophosphamide on day 28 only. Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination, a bone marrow sample was taken from all non-recovery animals for micronucleus assessment. Histopathological examination of selected tissues was performed. There was no unscheduled mortality. Increased salivation post dosing was evident in either sex treated with 1000 mg/kg bw/day from Day 2 (females) and Day 5 (males) onwards and in animals of either sex treated with 350 mg/kg bw/day from Day 9 onwards. This was not seen in recovery groups. The effects observed were not considered to be of toxicological importance. There were no treatment-related changes in the behavioral parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance. Males treated with 1000 mg/kg bw/day showed a reduction in body weight gain however, based on observations in recovery groups was not considered to be of toxicological importance. Therefore, there was limited effects seen on bodyweight post-exposure and evidence of recovery at all dose levels in males and females. There were no significant treatment related effects on food consumption or food conversion efficiency. Males and females treated with 1000 or 350 mg/kg bw/day showed an increase in overall water consumption during the final two weeks of the treatment period. There was evidence of recovery. No toxicologically significant effects were detected in the hematological parameters examined at the end of the treatment or treatment free periods. No toxicologically significant effects were detected in the blood chemical parameters examined at the end of the treatment or treatment free periods. There were no treatment-related effects detected in the urinalytical parameters. There were no treatment-related macroscopic abnormalities detected. There were no toxicologically significant effects detected in the organ weights measured at the end of the treatment or treatment free periods. Animals of either sex treated with 1000 mg/kg bw/day and males treated with 350 and 100 mg/kg bw/day showed increases in absolute and relative liver weight. Recovery females that were previously given 1000 mg/kg bw/day also showed an increase in absolute and relative liver weight, although all individual values were within the control range at the end of the treatment period. Although there were a few minor blood chemical changes which may have indicated minor perturbations in metabolism, in the absence of any histopathological hepatic changes, the intergroup differences were considered not to be of any toxicological importance. In the histopathology assessment: in the adrenal glands: hypertrophy of the zona glomerulosa was evident in two females treated with 1000 mg/kg bw/day only. This change was not seen in recovery females that were previously given 1000 mg/kg bw/day following fourteen days without treatment. Therefore, there was evidence of recovery and this was considered an adaptive change to test item administration. In the thyroids: minimal follicular cell hypertrophy was evident in three females treated with 1000 mg/kg bw/day, two females treated with 350 mg/kg bw/day, one female treated with 100 mg/kg bw/day and in one control female following the fourteen day treatment free period. Due to its presence in one control female, variability across groups and absence in recovery 1000 mg/kg bw/day males/females, this was considered not to be of toxicological significance. Within the micronucleus assessment, there was no indications of genotoxicity. The oral (gavage) administration of the test item to males/females at dose levels of 100, 350 or 1000 mg/kg bw/day resulted in a reduction in body weight gain in males treated with 1000 mg/kg bw/day and microscopic adrenal changes in females treated with 1000 mg/kg bw/day. No adverse effects were evident at 350 or 100 mg/kg bw/day. The reduced body weight gain seen in isolation in males treated with 1000 mg/kg bw/day was considered not to represent an overall adverse effect of treatment. The microscopic adrenal changes in females treated with 1000 mg/kg bw/day were considered to represent adaptive changes and were considered not to be toxicologically significant. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg/day for males/females since findings are either adaptive or have no relevance to human health.