Registration Dossier

Administrative data

Description of key information

Three groups of ten male and ten female rats received the substance at doses of 30, 100 or 200 mg/kg/day by oral gavage administration. The 200 mg/kg bw dose was lowered to 125 mg/kg bw after two weeks of treatment due to bodyweight loss in both sexes and mortality in females. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, corn oil, at the same volume-dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

Treatment at 200 mg/kg bw/day revealed reduced body weight development for animals of either sex, which was also seen in the males receiving the test item at 100 mg/kg bw/day and above. The mean overall body weight gain in these animals at the end of the dose administration period was approximately 58% and 33% (200/125 and 100 mg/kg bw/day males, respectively) lower than controls. There were no effects evident in males treated with 30 mg/kg bw/day.

The food intake and food conversion efficiency showed similar trends to body weight development for high dose males: no such effects were detected for males at 30 or 100 mg/kg bw/day. However, for females this remained unaffected by treatment throughout the study in comparison to controls. There were no detrimental effects on behavioural/functional or sensory performance at any dose level.

The microscopic examination revealed adverse kidney changes in animals of either sex at 200/125 mg/kg bw/day and males at 100 mg/kg bw/day.  These changes mainly consisted of damage to the tubules but urothelial hyperplasia was also present. Crystalline deposits were also apparent and these may be due to deposition of the test item or its metabolites during the excretion process. The changes in the kidneys correlate with the macroscopic findings, changes in electrolytes and blood urea and with the weight increase evident for 200/125 and 100 mg/kg bw/day males.

Adverse changes in the liver for all treatment groups were also apparent in a dose-related manner. Whilst bile duct hyperplasia occurred only in animals administered with 100 mg/kg bw/day and above there was an increased cell turnover noted in males and females from all dose groups, which included degeneration/apoptosis and an increase in mitosis. Diffuse inflammatory cell infiltration was seen only in males administered with 100 mg/kg bw/day and above.  Centrilobular hypertrophy was also noted in a few 30 mg/kg bw/day males and females from 100 or 200/125 mg/kg bw/day dose groups.  Vacuolation was seen occasionally in males at 30 or 100 mg/kg bw/day.  The pathology in the liver correlates with the clinical chemistry changes and the increase in liver weight in males.

There was a marginal increase in hematopoiesis seen in the spleen of the high dose males; this correlated with a weight increase and is likely to be linked to the hematology changes.

Therefore, due to the adverse microscopic changes to the kidney and liver a NOAEL for systemic toxicity could not be established for either sex. The lowest dose level 30 mg/kg bw is considered to be a LOAEL.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
repeated dose reproduction study OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2017 to 04 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:CD(SD) rat
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 and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: Males: 11 weeks;Females: 14 weeks
- Weight at study initiation: Males: 274 to 361 g; Females: 199 to 244 g.
- Fasting period before study: no
- Housing: solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (during pairing grid bottomed polypropylene cages)
Pre-pairing up to 3 animals of one sex
Pairing one male and one female
Males after mating up to 3 animals
Gestation one female
Lactation one female + litter
- Enrichment: wooden chew blocks and cardboard fun tunnels (except during pairing and lactation)
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ad libitum
- Water: water ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3ºC:
- Humidity: 50 ± 20%
- Air changes (per hr): at least 15.
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
gavage with stainless steel cannula attached to a disposable plastic syringe (dosing volume adjusted to latest measured body weight)
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing formulation was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were made daily from Days 1 to 8 of dosing, and dosed within four hours of being prepared. Following confirmed stability
and homogeneity results, formulations were prepared weekly and stored refrigerated in the dark.

VEHICLE: corn oil
- Concentration in vehicle: 0, 7.5, 25, 50/31.25 mg/mL
- Amount of vehicle (if gavage): 4 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analyzed on three occasions for concentration of the substance at Envigo Research Limited, Shardlow, UK, Analytical Services and analysed by HPLC/UV.

Ultra performance liquid chromatograph (UPLC):
Column: Waters Acquity Phenyl C18, 1.7 µm, 2.1 × 50 mm
Column temperature: 45°C
Sample temperature: Ambient
Mobile Phase A: ACN/ 50 mM Ammonium Acetate 10/90 vv
Mobile Phase B: ACN/ 50 mM Ammonium Acetate 90/10 vv
Gradient Time (min) A% B%
0.00 98 2
0.20 98 2
1.60 30 70
1.70 30 70
1.80 98 2
2.40 98 2

Flow rate: 0.25 mL/min
Weak rinse solvent: ACN/water 50/50 v/v, 600 µL
Strong rinse solvent ACN 100%, 200 µL
Detector wavelength: 435 nm
Injection volume: 1.0 µL
Run time: 2.4 minutes
Approximate retention time: 1.3 minutes

Calibration: linear 0.99-4.95 ug/mL (r 0.9999); CV of 6 determinations 0.60% at 1 ug/mL and 0.75% at 5 ug/mL
Recovery at 1 and 200 mg/mL: 100.4% of nominal (CV 0.48%) and 98.8% of nominal (CV 2.46%)
QC samples at 1 and 100 mg/L: 95.6-103.9% of nominal (in duplicate on day 0,1, 8 and 15)
Homogeneity: 1 mg/mL CV 1.47%, 100 mg/L CV 1.74% on t= 0 hours; 1 mg/mL CV 0.27%, 100 mg/L CV 1.38% on day 15
(homogeneity was measured at 0, 1 and 2 h and on day 1 , 8 and 15 all CV at 1 and 100 mg/mL < 2.37%, except after 1 day in 100 mg/ml samples at 21 °C, where the CV was 7.42%)
Stability: at 1 and 2 hours and 1 day at 21 °C:1 mg/mL 99.7-101% of nominal, 200 mg/mL 98-104% of nominal
on day 1, 8 and 15 at 4 °C: 1 mg/mL 99.6-105% of nominal, 200 mg/mL 100-107% of nominal
LOD = 0.00785 µg/mL ; LOQ = 0.0262 µg/mL
Duration of treatment / exposure:
Males Two weeks pre-pairing up to necropsy after ca 6 weeks of treatment
Females Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation (total ca 8 weeks)
Frequency of treatment:
daily
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
At the initial dose of 200 mg/kg bw/day, one female each was terminated on Day 20 and 21 on animal welfare grounds with the remaining Group 4 animals, in particular the females, showing a decline in body weights. It was therefore decided to not dose the high dose animals on Day 21 relative to the start of dosing and the dose level was reduced to 125 mg/kg bw/day from Day 22
onwards.
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 2-week dose range finding study
In that study (animals dosed at 0, 250, 500 or 1000 mg/kg/day) due to rising animal welfare concerns following the start of treatment the dose levels of the high and intermediate dose groups were reduced to 150 or 50 mg/kg bw/day respectively from Study Day 5. A control group of three males and three females received vehicle alone (Corn oil). No animals died during the study. Following the Day 5 reduction in dose levels the majority of animals showed weight gains and normal food consumption. Water consumption was unaffected by treatment.
Macroscopic examination identified a pale and/or mottled appearance to the kidneys of all animals treated at 500/150 and 1000/50 mg/kg bw/day and in males only that received 250 mg/kg bw/day. In addition, the kidneys of all 1000/50 mg/kg bw/day animals were noted to be larger than those of the concurrent controls.
No macroscopic abnormalities were detected in the females treated at 250 mg/kg bw/day.
It was concluded that the doses selected for the Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) with
Comet Assay (Envigo Study Number XC92TB) would be 0 (Control), 30, 100 and 200 mg/kg bw/day.

Due to deteriorating health and clinical signs including associated body weight losses in the main study, two females receiving 200 mg/kg bw/day were terminated humanely during Week 3 of treatment. Microscopic findings revealed one female had notable kidney changes along with liver toxicity and lymphoid depletion.  The deterioration of the other female was likely to be related to the dosing procedure, although the animal also had changes in the liver and kidney. On review of the body weight effects, it was decided to reduce the high dose level to 125 mg/kg bw/day from Day 22 of treatment, where improvement was evident thereafter.  
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily immediately before dosing, soon after dosing, and one hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-dose, post-dose and at the end of the day
F0 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in a standard arena during each week of treatment and for females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum detailed physical examination and arena observations (Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation and Lachrymation)

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males Before dosing on the day that treatment commenced (Day 1) and weekly thereafter (at termination)
F0 females Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4 and 7 of lactation and at termination

FOOD CONSUMPTION : Yes
- Time schedule for examinations:
Weekly, from the day that treatment commenced until animals paired for mating.
For females after mating food consumption:
Days 0-7, 7-14 and 14-20 after mating
Days 1-4, 4-76 and 7-13 of lactation.

FOOD EFFICIENCY: Yes, calculated retrospectively for males throughout the study period (with the exception of the mating
phase) and for females during the pre-pairing phase.

WATER CONSUMPTION: Visual observation

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: not indicated
- Animals fasted: no
- How many animals: 5/sex/group
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT) and Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: not indicated
- Animals fasted: no
- How many animals: 5/sex/group
- Parameters checked: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
-- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: grip strength, sensory activity (includes: Grasp response, Touch escape, Vocalization,Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex and Finger approach ), motor activity(beam crossing over 30 min period)

IMMUNOLOGY: Yes
- Time schedule for examinations: at termination
- Anaesthetic used for blood collection: not indicated
- Animals fasted: no
- How many animals: all adult males
- Dose groups that were examined: all
- Parameters checked: serum samples of the adult males for thyroxine (T4) levels (additional taken samples from all adult females were not further examined)

ESTOUS CYCLE: Yes
Vaginal smears: daily during pre-pairing period and on day of necropsy
Sacrifice and pathology:
ORGAN WEIGHTS: Yes (see tables)

GROSS PATHOLOGY: Yes on all animals

HISTOPATHOLOGY: Yes (see tables)
Premature deaths All F0 animals from all groups.
Scheduled kill 5 F0 animals/sex in Groups 1 and 4:
All F0 animals. Abnormalities only.

Based on findings in liver, kidney and spleen additional histopatlogy was performed on the
kidneys of 5, 6, 5, 11 males and 5, 5, 5, 5 females at 0, 30, 100 and 125/200 mg/kg bw
liver of 5, 10, 9, 11 males and 5, 5, 5, 5 females at 0, 30, 100 and 125/200 mg/kg bw
spleen of 5, 5, 5, 6 males and 5, 0, 0, 5 females at 0, 30, 100 and 125/200 mg/kg bw

Other examinations:
F0 Females:
Each ovary:Number of corpora lutea
Each uterine horn: Number of uterine implantation sites were recorded.

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted
Statistics:
Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).

The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA. Any transformed data were analyzed 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 nonhomogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel test.
Where the data were unsuitable for these analyses, the Student -test (parametric) or the Mann-Whitney U test (non-parametric).

The distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows: p<0.01 **, p<0.05 *, p>0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
males/females: transient post dose increased salivation at 100 and 200/125 mg/kg bw associated with noisy respiration (increased in incidence ith dose)
males/females at 200/125 mg/kg bw sporadic instances of pilo-erection, hunched posture and staining around the mouth.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the final days of the third week of treatment it was considered necessary to humanely sacrifice two females at 200 mg/kg bw due to deteriorating animal health. One female had notable kidney changes along with liver toxicity and lymphoid depletion. The death of another female is likely to have been related to dosing procedure although it also had changes in the liver and kidney. Furthermore, due to concerns for the remaining animals from this test group the dose level was reduced from Study Day 22 to 125 mg/kg bw/day. Following the reduction in dose level there were no further unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males: body weight gain decreased at 100 and 200/125 mg/kg bw (33 and 56%)
females:body weight gain sign increased during gestation (27%) at 200/125 mg/kg bw
(see attached table)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
in the first two weeks decreased in males at 200/125 mg/kg bw (after lowering of the dose improvement)
Food efficiency:
no effects observed
Description (incidence and severity):
in the first two weeks decreased in males at 200/125 mg/kg bw (after lowering of the dose improvement)
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
visual inspection did not show intergroup differences
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
males:
200/125 mg/kg bw: sign decreased Hb and MCHC (marginal), sign increase of WBC (mainly neutrophils and lymphocytes), sign increase of reticulocytes (within historical controls) and sign increase in number of platelets (without and effect on clotting time)
100 mg kg bw: sign decreased Hb and MCHC (marginal), sign increase of WBC (mainly neutrophils and lymphocytes), sign increase of reticulocytes (within historical controls)
30 mg/kg bw: sign increase of reticulocytes (within historical controls)

females: at 30, 100 and 200/125 mg/kg bw: increas of reticulocytes (not clearly related to dose)

(see attached table)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
males: at 200/125 mg/kg bw: sign increase ASAT/ALAT , ALP and bile acids (all within historical controls; sign increase Cholesterol (49%)
males at 100 mg/kg bw: sign increase ASAT/ALAT , ALP and bile acids (all within historical controls; sign increase Cholesterol (54%)
females: at 200/125 mg/kg bw: sign increase ALP and bile acids (all within historical controls; sign increase Cholesterol (22%)
females: at 100 mg/kg bw: sign increase ALP and bile acids (all within historical controls; sign increase Cholesterol (26%)
females: at 30 mg/kg bw: sign increase ALP within historical controls

(see attached table)
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity observations and grip strength: no treatment related effects
Motor activity: males statistically significant increase in overall activity at all dose levels (see attached table)
Immunological findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males (see table)
The microscopic examination of thymus, thyroid, adrenal and pituitary glands and the reproductive organs was also unremarkable.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males: 200/125 mg/kg bw: sign increased liver, kidney, seminal vesicles and spleen weight (absolute and relative to body weight)
males:100 mg/kg bw: sign increased liver, kidney, seminal vesicles and spleen weight (absolute and relative to body weight)
males 30 mg/kg bw: sign increased liver weight (absolute and relative to body weight)
females: 100 and 200/125 mg/kg bw sign increased adrenal weight

All values are within historical control values
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
males: 200/125 mg/kg bw: yellow discoloration and/or yellow contents of the GI-tract, yellow discoloration of the testes, enlarged spleen (3/12); enlarged, mottled/pale kidneys, pale mottled liver (10/12)
Males at 100 mg/kg bw: yellow discoloration and/or yellow contents of the GI-tract, pale kidneys, pale mottled liver (4/12)
Males at 30 mg/kg bw: pale mottled liver (6/12)
Females: at 30, 100 and 200/125 mg/kg bw: pale mottled liver (2/12, 3/12 and 3/10 respectively)

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys
Tubular nephropathy was present in eleven males (mild to marked) and three females (minimal or mild) at 200/125 mg/kg bw/day. This finding was also present (mild or moderate) in five 100 mg/kg bw/day males. This term was used to describe when 2 or more of the following occurred: tubular basophilia; tubular dilation; tubular atrophy/degeneration, inflammatory cell infiltration acute to chronic; occurring in both the cortex and medulla. The glomeruli were largely unaffected.
Crystalline deposits in the tubules were present in the same animals. Multifocal basophilic tubules were present one 200/125 mg/kg bw/day female.
Urothelial hyperplasia was present in six 200/125 mg/kg bw/day males and in one 100 mg/kg bw/day male.
Pyelonephritis (moderate) was present in one 200/125 mg/kg bw/day male. The kidneys examined for animals of either sex at 30 mg/kg bw/day and 100 mg/kg bw/day females were considered to be within normal limits.
Liver
Bile duct hyperplasia (minimal to moderate) was present in nine 100 mg/kg bw/day and eleven 200/125 mg/kg bw/day males and in three 100 mg/kg bw/day and six 200/125 mg/kg bw/day females.
Increased cell turnover or multifocal degeneration on its own (Group 4 females) was present in four 30 mg/kg bw/day, eight 100 mg/kg bw/day and eleven 200/125 mg/kg bw/day males and in two 30 mg/kg bw/day, three 100 mg/kg bw/day and three 200/125 mg/kg bw/day females. Increased cell turnover was recorded when there was degeneration, necrosis, apoptosis, mitosis, variation in nuclear and/or cell size, increased bi or multi-nucleated cells.
Diffuse inflammatory cell infiltration was present in three 100 mg/kg bw/day and ten 200/125 mg/kg bw/day males. Centrilobular hypertrophy was present in three 30 mg/kg bw/day males, one 100 mg/kg bw/day and two 200/125 mg/kg bw/day females. Vacuolation, midzonal/centrilobular in distribution, was present in two 30 mg/kg bw/day and two 100 mg/kg bw/day males.
Spleen
Hematopoiesis was present in one control, one 100 mg/kg bw/day and four 200/125 mg/kg bw/day males. While this increase was marginal, it correlated with a weight increase at 200/125 mg/kg bw/day for spleens.

No other changes were noted which could be related to the administration of the test item and all are considered to be incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
no treatment related effect (oestrus cycle 4-5 days pre-treatment, at termination all females were in diestrous phase)
Details on results:
Mortality Two 200 mg/kg bw/day females were sacrificed on humane grounds during the third week of treatment due to deteriorating health that was associated with exposure to the test item. One female had notable kidney changes along with liver toxicity and lymphoid depletion. The death of another female is likely to have been related to dosing procedure although it also had changes in the liver and kidney. Furthermore, due to concerns for the remaining animals from this test group the dose level was reduced from Study Day 22 to 125 mg/kg bw/day. Following the reduction in dose level there were no further unscheduled deaths.
Clinical Observations Intermittent episodes of post dose increased salivation were evident in animals of either sex treated at 100 mg/kg bw/day and at a higher incidence in each sex of the high dose group (200/125 mg/kg bw/day). Associated with this condition were isolated instances of noisy respiration identified in high dose animals of either sex and both sexes treated at 100 mg/kg bw/day during the final days of treatment period. Additional findings observed in high dose males and females included sporadic instances of pilo-erection, hunched posture and staining around the mouth.
Behavioral Assessment There were no treatment-related changes in the behavioral parameters measured.
Functional Performance Tests There were no treatment-related changes detected in the functional performance measurements.
Sensory Reactivity Assessments Sensory reactivity assessments did not indicate any adverse effects of treatment.
Body Weight At 200/125 mg/kg bw/day males showed marked reductions in body weight gains during the first three weeks of treatment. Improvement was noted once the dose level was reduced. Also males treated with 100 mg/kg bw/day showed lower body weight gains throughout the treatment period compared to controls. Therefore these reductions influenced the lower overall body weight development in relation to the controls. No such effects were detected for males at 30 mg/kg bw/day. All female treatment groups showed lower body weight gains during the first week of treatment. During gestation females treated with 200/125 mg/kg bw showed an increased body weight gain. There were no apparent effects detected on body weights during the lactation phases of the study.
Food Consumption Males treated with 200/125 mg/kg bw/day showed slight reductions in food consumption during the first two weeks of treatment, thereafter food intake was generally similar to controls. No such effects were evident in males treated with 30 or 100 mg/kg bw/day. There were no adverse effects evident in treated females during maturation, gestation or lactation.
Water Consumption Daily visual assessment of water intake did not reveal any overt intergroup differences in comparison to controls.
Hematology Males at 100 or 200/125 mg/kg bw/day showed a statistically significantly lower hemoglobin and mean corpuscular hemoglobin concentration compared to controls, these findings support the histopathological changes of the spleen. These males also showed statistically higher total leukocyte count, which was attributed to elevated neurophil fractions plus lymphocyte fraction for high dose males only. No such effects were detected for the females. All male treatment groups and high dose females showed a statistically significant increase in reticulocytes. However, all individual values were within the background control data.
Blood Chemistry All treated male dose groups showed an increase sodium concentration and urea in high dose males only. These findings can be associated with the changes in the kidneys. At 100 and 200/125 mg/kg bw/day animals in both sexes showed elevated levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, cholesterol and bile acid when compared to controls.
Necropsy A number of animals of either sex treated with 100 and 200/125 mg/kg bw/day and males at 30 mg/kg bw were observed to have green/yellow coloured contents in the caecum and stomach. At 200/125 mg/kg bw/day eight males had enlarged kidneys, with five males showing mottled appearance. Males across dose groups showed pale areas of the kidneys, to a lesser extent at the lower dose groups. Across all treatment groups there were liver findings including mottled appearance, pale discolouration and pale areas, these findings were at a lesser extent at the lower dose levels. At 200/125 mg/kg bw/day three males had enlarged spleens.
Thyroid Hormone Analysis An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.
Organ Weights All male treatment groups showed a statistically significant increase in liver weights both absolute and relative to terminal body weight, with a dose related response evident to the latter. The increased liver weights correlate with the histopathological changes identified in all treatment groups. At 100 or 200/125 mg/kg bw/day males also showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight, in a dose related manner. The weight increases noted in these males correlate with the histopathological changes in the kidneys. Also at 200/125 mg/kg bw/day males showed a statistically significant increase in spleen weights both absolute and relative to terminal body weight. This can be associated with the increase in haematopoiesis observed in these animals.
Histopathology There were adverse liver changes identified in both males and females from all dose groups in a dose related manner. There were also adverse kidney changes identified in both males and females at 200/125 mg/kg bw/day and males at 100 mg/kg bw/day. There was a marginal increase in haematopoiesis in the spleen of males at 200/125 mg/kg bw/day.

Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The oral administration of the substance to rats by gavage, at dose levels of 30, 100 and 200/125 mg/kg bw/day, resulted in all dose groups showed adverse histopathological changes in the liver and adverse changes in the kidneys for males and females at 200/125 mg/kg bw/day and 100 mg/kg bw/day males. Therefore, the `No Observed Adverse Effect Level' (NOAEL) for systemic toxicity could not be established for either sex.
Executive summary:

Three groups of ten male and ten female rats received the substance at doses of 30, 100 or 200 mg/kg/day by oral gavage administration. The 200 mg/kg bw dose was lowered to 125 mg/kg bw after two weeks of treatment due to bodyweight loss in both sexes and mortality in females. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, corn oil, at the same volume-dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

Treatment at 200 mg/kg bw/day revealed reduced body weight development for animals of either sex, which was also seen

in the males receiving the test item at 100 mg/kg bw/day and above. The mean overall body weight gain in these animals at the end of the dose administration period was approximately 58% and 33% (200/125 and 100 mg/kg bw/day males, respectively) lower than controls. There were no effects evident in males treated with 30 mg/kg bw/day.

The food intake and food conversion efficiency showed similar trends to body weight development for high dose males: no such effects were detected for males at 30 or 100 mg/kg bw/day. However, for females this remained unaffected by treatment throughout the study in comparison to controls. There were no detrimental effects on behavioural/functional or sensory performance at any dose level.

The microscopic examination revealed adverse kidney changes in animals of either sex at 200/125 mg/kg bw/day and males at 100 mg/kg bw/day.  These changes mainly consisted of damage to the tubules but urothelial hyperplasia was also present. Crystalline deposits were also apparent and these may be due to deposition of the test item or its metabolites during the excretion process. The changes in the kidneys correlate with the macroscopic findings, changes in electrolytes and blood urea and with the weight increase evident for 200/125 and 100 mg/kg bw/day males.

Adverse changes in the liver for all treatment groups were also apparent in a dose-related manner. Whilst bile duct hyperplasia occurred only in animals administered with 100 mg/kg bw/day and above there was an increased cell turnover noted in males and females from all dose groups, which included degeneration/apoptosis and an increase in mitosis. Diffuse inflammatory cell infiltration was seen only in males administered with 100 mg/kg bw/day and above.  Centrilobular hypertrophy was also noted in a few 30 mg/kg bw/day males and females from 100 or 200/125 mg/kg bw/day dose groups.  Vacuolation was seen occasionally in males at 30 or 100 mg/kg bw/day.  The pathology in the liver correlates with the clinical chemistry changes and the increase in liver weight in males.

There was a marginal increase in hematopoiesis seen in the spleen of the high dose males; this correlated with a weight increase and is likely to be linked to the hematology changes.

Therefore, due to the adverse microscopic changes to the kidney and liver a NOAEL for systemic toxicity could not be established for either sex. The lowest dose level 30 mg/kg bw is considered to be a LOAEL.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
hepatobiliary
Organ:
liver

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

In the repeated dose-reproduction toxicity study effects on liver were seen in male rats that were exposed during ca 6 weeks. According to Regulation (EC) No 1272/2008 (CLP) an effect level of 10 mg/kg bw in a 90-day study should be multiplied with three when only an 28-day study is available. The effect level in the repeated dose reproduction study on the substance is 30 mg/kg bw, which would lead to classification as STOT 1 based on the rule as described in CLP (the effective dose is directly proportional to the exposure concentration and the duration of exposure ( 3.9.2.9.5.)). However, as in the current study the exposure duration is more than 28 days, a lower multiplication factor seems appropriate here. This will lead to a classification as STOT 2.

Justification for classification or non-classification

Based on the available information where effects on the liver were reported at 30 mg/kg bw in a subacute study where animals were exposed for ca 42 days, the substance needs to be classified as STOT 2 (H373) according to Regulation (EC) No 1272/2008.