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Diss Factsheets

Administrative data

Description of key information

- Key study: Subchronic oral study in rats, OECD TG 408, GLP: male NOAEL = 1 mg/kg bw/day ; female NOAEL = 3 mg/kg bw/day.

- Supporting study: Subacute oral study in rats, OECD TG 407, GLP: male LOAEL = 5 mg/kg/day ; female LOAEL = 20 mg/kg/day.

Both studies (and 2 other supporting studies) showed specific toxicity targeting the liver, with effects observed in males at lower doses compared to females.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Remarks:
(The study was performed to comply with a Regulation outside Europe)
Adequacy of study:
key study
Study period:
From 21 February 2012 to 11 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline 408 without any major deviation
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese METI (Ministry of Economy, Trade and Industry) of 13 July 1974 and subsequent revisions
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy and supplied by Harlan Nederland, Kreuzelweg, 53, Horst NL-5960 AD Horst, The Nederlands.
- Age at study initiation: 27-29 Days old
- Weight at study initiation: 94.1-123.4 g for males and 98.2-109.7 g for females
- Housing: The animals were housed in a limited access rodent facility. The animals were housed 5 of one sex to a cage, in clear polycarbonate cages measuring 59.5x38.5x20 cm with a stainless steel mesh lid and floor (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) laboratory rodent diet, ad libitum
- Water: drinking water supplied via water bottles, ad libitum (except before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis).
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 to 20 per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 21 February 2012 to 01 June 2012 and 12 July 2012 for the main and recovery groups, respectively.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
On a daily basis, a sufficient amount of the test item was diluted 1:100 with purified water, to obtain a concentration of 3.76 mg/mL (stock solution). Then further independent solutions were prepared with the same vehicle from the stock solution to give the required concentrations of 0.03, 0.1, 0.3 and 1 mg/mL. Concentrations were calculated and expressed in terms of dry salt.

VEHICLE
- Concentration in vehicle: 0.03, 0.1, 0.3 and 1 mg/mL
- Amount of vehicle (if gavage): 10 mL/ kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical analysis was carried out at RTC according to a validated method (RTC Study nos. 82340 and 82350) in the range from 0.03 to 100 mg/mL.
- Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable. Samples of the formulations prepared on Week 1 and 13 were analyzed to check the concentration.
- Acceptance criterion: Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (95-105%).

Results: Results of all analyses were within the limits of acceptance.
Duration of treatment / exposure:
13 consecutive weeks
Frequency of treatment:
Once daily, 7 days a week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
0.3 mg/kg bw/day (nominal)
Dose / conc.:
1 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were defined in agreement with the Sponsor based on available information from other studies of shorter duration.
- Post-exposure recovery period in satellite groups: 10 additional animals for each sex were included in the medium-high, high dose and control groups for recovery assessment (6 consecutive weeks)
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- Mortality and morbidity: Twice a day
- Clinical signs: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: the day of group allocation, on the first day of treatment and then once a week until the end of the study and before sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal was recorded once a week and mean daily diet consumption was calculated as g food/animal/day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on control and high-dose animals on one occasion during Week 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from the abdominal vena cava of the surviving male and female animals from each main phase group, at the end of the treatment period, just prior to necropsy. At the end of week 6 of the recovery period, blood was also taken under identical conditions in order to re-evaluate any parameters which showed potential treatment-related changes at measurements performed during the treatment period.
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Yes
- Parameters checked: hematocrit, hemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes and large unstained cells, monocytes, eosinophils, basophils), platelets, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the abdominal vena cava of the surviving male and female animals from each main phase group, at the end of the treatment period, just prior to necropsy. At the end of week 6 of the recovery period, blood was also taken under identical conditions in order to re-evaluate any parameters which showed potential treatment-related changes at measurements performed during the treatment period.
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Yes
- Parameters checked: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, bile acids, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, phosphorus, total bilirubin, total cholesterol, total proteins, albumin, globulin, albumin/globulin ratio, sodium, potassium, calcium, chloride.

URINALYSIS: Yes
- Time schedule: Urinalysis was performed in all animals at the end of the treatment period. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. At the end of week 6 of the recovery period, urine was also taken under identical conditions in order to re-evaluate any parameters which showed potential treatment-related changes at measurements performed during the treatment period.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Appearance, volume, specific gravity, pH, proteins, glucose, ketones, bilirubin, blood, urobilinogen, cytology of sediment (epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the beginning of the treatment period and then once a week until the end of the study, during the detailed clinical examination.
- All the animals were observed in an open arena for a minimum of 3 minutes.
Battery of functions tested: removal from cage, handling reactivity, lachrymation, palpebral closure, salivation, piloerection, earing, spasms, myoclonia, mobility impairment, arousal (animal activity), vocalization, stereotypies, unusual respiratory pattern, bizarre behavior, urination, defecation, tremors, gait (normal, ataxia, hunched, pronation, forelimbs drag, hindlimbs drag.
- Once during weeks 12/13 of treatment and once during week 6 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and assessment of grip strength were also performed.
Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; Animals completing the scheduled test period were killed by exsanguination under isofluorane anaesthesia. All animals, including that found dead, were subjected to necropsy.
- ORGAN WEIGHTS: Body weight of each animal was recorded before sacrifice and the organs specified in the table 7.5.1/2 were weighed. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
- HISTOPATHOLOGY: Yes; For all animals, the tissues specified in the table 7.5.1/2 were preserved in 10 % neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol). An extra liver sample was taken from all animals and fixed in 1% glutaraldehyde and 4% formaldehyde in phosphate buffer for evaluation by electron microscopy. All tissues required for microscopic examination were dehydrated and embedded in paraffin wax, sectioned at a thickness of approximately 5 µm and stained with hematoxylin and eosin (except for testes and epididymides of main group animals which were cut at 2-3 µm thickness and stained with PAS).
Microscopic examination was performed on:
- all tissues listed in the table 7.5.1/2 from all animals in the control and high dose dying during the treatment period or sacrificed at the end of the 13-week treatment period.
- all tissue listed in the table 7.5.1/2 from all animals killed or dying during the treatment period.
- all abnormalities from all main phase groups.
The examination was then extended to include the liver from all animals of groups 2, 3 and 4 killed after 13 weeks of treatment and those of the recovery groups (1, 4 and 5) and the thyroid in the males of the same groups.
Other examinations:
Testes and epididymides of main group animals were cut at 2-3 µm thickness and stained with PAS. The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in all animals in the control and high dose groups killed after 13 weeks of treatment.
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathology findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
Clinical signs:
no effects observed
Description (incidence and severity):
- No daily clinical signs of toxicological significance were observed.
- No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity.
Mortality:
mortality observed, treatment-related
Description (incidence):
- One male animal from the medium-high dose group was found dead on Day 78 of treatment period. No clinical signs or other signs of toxicity were observed in this animal during the study. The cause of the death cannot be clearly established on the basis of the post mortem macroscopic and microscopic findings.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences of toxicological significance were noted in body weight and body weight changes between treated and controls groups
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were seen at the ophthalmic examination.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Dosing phase: See table Table 7.5.1/3. The most relevant finding observed was the decrease of circulating white blood cells in males treated with 10 mg/kg bw/day. Decrement was 26% when compared with controls and included lymphocytes and monocytes. The other minor changes were of minimal magnitude and therefore considered of no toxicological relevance. No changes were recorded for treated females.

- Recovery phase: Changes recorded during the dosing phase showed complete reversibility and leucocytes were even slightly higher than controls.
- Coagulation: Slight decrement of prothrombin time was also recorded in few animals dosed with 10 mg/kg bw/day. Those changes observed during the treatment and recovery periods of the study were of low severity and therefore not considered of toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Dosing phase: Fluctuations of a number of metabolic parameters were recorded in treated animals, mainly those receiving 3 and/or 10 mg/kg bw/day (decrement of triglycerides (-57% and -60%, respectively) and increment of alanine aminotransferase (61% and 53%, respectively), glucose (39% and 13%, respectively), urea (9% and 13%, respectively) and albumin (4% and 14%, respectively) in males treated with 3 and 10 mg/kg bw/day; slight decrease of cholesterol (-17%) and increase of glucose (36%) and albumin (9%) in the females dosed at 10 mg/kg bw/day).

- Recovery phase: At the end of the recovery phase, no reversibility was observed for alanine aminotransferase, triglyceride and urea in males, cholesterol and albumin in females. The other statistically significant changes were considered unrelated to treatment.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- Dosing phase: Increased ketonuria was observed in males treated with 3 and 10 mg/kg bw/day. No other findings were recorded.
- Recovery phase: Increased ketonuria was still present in males treated with 10 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- No differences between treated animals and controls which could be considered of toxicological relevance were noted at evaluations of sensory reaction and motor activity measurements performed at the end of the treatment and recovery periods.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Dosing phase:
LIVER: dose-related, statistically significant increases in the absolute weight of the liver were observed at the end of the treatment period animals dosed at 3 and 10 mg/kg bw/day (+30% and +81%, respectively in males and +15% and +37%, respectively in females). The relative weights of the liver were also significantly increased at statistical analysis in the males dosed at 1, 3 and 10 mg/kg bw/day (+10%, +29% and +81%, respectively) and in the females dosed at 3 and 10 mg/kg bw/day (+11% and +34%, respectively).
KIDNEYS: A slight but statistically significant increase in the relative weight of kidneys was seen in the males dosed at 10 mg/kg bw/day (+8%).

- Recovery phase:
at the end of recovery, the absolute and relative weights of the liver were still slightly increased in the males dosed at 3 and 10 mg/kg bw/day (+16% and +43%, respectively) and in the females dosed at 10 mg/kg bw/day (+27%). No other significant changes were observed at the end of treatment or recovery periods.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Final sacrifice: treatment-related changes were seen on the liver of the males treated with the high dose. This organ was enlarged and/or swollen in some of the animals of this group.
- Recovery sacrifice: No treatment related changes were noted.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FINAL SACRIFICE:
- LIVER: treatment-related, mild to moderate diffused hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was found in the males treated with the medium/low (1 mg/kg bw/day), medium/high (3 mg/kg bw/day) and high dose (10 mg/kg bw/day). In a single male animal treated with the high dose, the hypertrophy was associated with minimal multifocal hepatocytic vacuolation and focal fibrosis and mineralization. The effects were of minimal degree in the medium/low dose group (1 mg/kg bw/day) and no effects were seen in the low dose group (0.3 mg/kg bw/day). In the females, mild hypertrophy was observed in the group treated with the high dose (10 mg/kg bw/day). No effects were seen in the other treated groups.
- THYROIDS: most follicles in the male high ad medium/high dose treated groups (10 and 3 mg/kg bw/day) were lined by columnar epithelium when compared to controls, showing most follicles lined by cuboidal. This treatment-related change was associated with reduction of the follicular colloid contents, compared to the concurrent control group. The changes in the thyroids are suggested to be secondary, due to primary stimulatory effect of the liver P-450 microsomal enzymes, associated with increased hepatic metabolism of thyroid hormone.

RECOVERY SACRIFICE:
A trend for recovery, but still incomplete, was noted in the liver and thyroid effects previously seen in the terminal sacrifice animals.
- LIVER: moderate to mild diffused hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was still present in the high and medium/high dose males, respectively. In females, treatment-related findings were found in the liver of the high dose group.
- THYROIDS: follicles were still lined by columnar epithelium in the high and medium/high dose males. This treatment-related change was associated with reduction of the follicular colloid contents, comparing to the concurrent control group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Spermatogenic staging: Histopathological evaluation was performed only on the testes of control and high dose groups. No treatment-related changes were seen in the testes.
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
3 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

Table 7.5.1/3: Haematology changes in male rats during the dosing phase

 

Control
(Group1)

Group2

Group3

Group4

Group5

RBC (%)

8.611

-1

-2

-6**

-8**

HGB (%)

14.96

-2

-1

-4**

-5**

HCT (%)

46.5

-1

-2

-5**

-5**

MCV (%)

54

-1

1

1

3*

MCH (%)

17.36

-1

2

1

3**

MCHC (%)

32.14

0

1

1

1

PLT (%)

763.1

-2

-2

-2

1

WBC (%)

8.004

-6

-1

-12

-26**

NEU (%)

0.991

18

43*

8

-1

LYM (%)

6.519

-11

-8

-15

-31**

MON (%)

0.233

28

16

9

2

EOS (%)

0.147

8

-1

-10

-22

BAS (%)

0.048

0

-2

-29

-33*

LUC (%)

0.067

-6

-25

-1

-36

RET (%)

151.57

-4

6

-2

-7

* Significantly different from control at level p<0.05
** Significantly different from control at level p<0.01

Control data are expressed in absolute values, other treated groups values are % differences with controls

Histopathological findings in liver and thyroid

   Mt Mt  Mt  Mt  Mt    Ft  Ft  Ft  Ft  Ft    Mrec  Mrec  Mrec   Frec   Frec Frec 

 Doses (mg/kg/d)

  0 0.3   1  3  10    0  0.3  1  3  10    0  3  10    0 3   10
 # examined  10  10  10  9  10    10  10  10  10  10    10  10  10    10  10  10
 LIVER                                      
 inflammatory cell foci  9  10  10  8  6    9  9  10  10  10    10  8  6    10  10  9
 Hepatocytic hypertrophy (total)  0  0  10*  9*  10*    0  0  0  0  10*    0  10*  10*    0  0  10*

grade: minimal

      10                      5          10
grade: mild        9              10      5 10         
grade: moderate          10                            
                                       
 Centrilobular hepatocytic vacuolation  0  0  0  0  1    0  0  0  0  0    0  0  0  

 0

 0

 0

 Fibrosis

 0

 0

 0

 0

 1

 

 0

 0

 0

 0

 0

 

 0

 0

 0

 

 0

 0

 0

 Mineralisation

 0

 0

 0

 0

 1

 

 0

 0

 0

 0

 0

 

 0

 0

 0

 

 0

 0

 0

 Hepatocytic vacuolation

 0

 0

 0

 0

 0

 

 0

 0

 1

 0

 0

 

 0

 0

 0

 

 0

 0

 0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 # examined

 10

 10

 10

 9

 10

 

 10

 -

 -

 -

 10

 

 10

 10

 10

 

 -

 -

 -

 THYROID

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 Follicle lining epith., mostly cuboidal

 10

 10

 10

 0

 0

 

 0

 -

 -

 -

 0

 

 10

 10

 0

 

 -

 -

 -

 Follicle lining epith., mostly columnar

(grade: moderate)

 0

 0

 0

 9*

 10*

 

 0

 -

 -

 -

 0

 

 0

 0

 10*

 

 -

 -

 -

Mt: males, end of treatment period ; Ft: females, end of treatment period

Mrec: males, end of recovery period; Frec: females, end of recovery period

* statistical significance compared to control at the 0.05 level using the Kolmogorov-Smirnov one-tailed test.

Conclusions:
Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of cC604 was considered to be 1 and 3 mg/kg bw/day for males and females, and the Lowest Observed Adverse Effect Level (LOAEL) was considered to be 3 and 10 mg/kg bw/day for males and females, respectively.
Executive summary:

In a GLP-compliant subchronic repeated dose oral toxicity study conducted according to the OECD Guideline 408, cC604 diluted in water was administered daily by gavage to groups of Sprague-Dawley rats (10/sex/dose), for 13 weeks at the dose-levels of 0 (vehicle), 0.3, 1, 3 or 10 mg/kg bw/day. Ten additional animals for each sex were included in the medium-high, high dose and control groups for a 6 consecutive weeks recovery assessment. Examinations during the study included: mortality, daily clinical signs, weekly detailed (open field observations) clinical signs, evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmology, clinical pathology investigations, terminal body weight, hematology, blood chemistry, urinalysis, organ weights, macroscopic observations and histopathological examination which included the evaluation of the staging of spermatogenesis.

One male animal dosed at 3 mg/kg bw/day was found dead on Day 78 of the treatment period. Although treatment-related findings were observed at post-mortem observations (changes in the liver), the cause of this death cannot be clearly established. No test item treatment-related clinical signs were observed during the study. In addition, no adverse effects were observed during the Functional Observation Battery or at ophthalmology examinations. Body weight and food consumption were not affected by treatment with the test item. However, signs of evident treatment-related effects of the test item were observed in males dosed at 3 and 10 mg/kg bw/day and in females dosed at 10 mg/kg bw/day. These effects were demonstrated by changes in clinical pathology parameters and post mortem findings. A main target organ, the liver was identified on the basis of the organ weights data and histopathological findings. A second target organ appeared to be the thyroid, whose changes were considered to be a secondary effect of the liver toxicity. The above effects showed a dose-related trend and appeared to be only partially reversible after the 6-week recovery period.

Changes observed in females dosed at 3 mg/kg bw/day were limited to the very slight increase in liver weights, which was fully reversible. Therefore, this change was not considered to be adverse. In the males, no adverse effects were seen following treatment at a dosage of 1 mg/kg bw/day, changes being limited to a very slight increase in relative liver weight associated to a minimal hepatocytic hypertrophy. No changes were observed in the females doses at 1 mg/kg bw/day and animals of both sexes dosed at 0.3 mg/kg bw/day.

Therefore, the test item cC604, when administered daily to rats for 13 consecutive weeks to both sexes, showed a No Observed Adverse Effect Level (NOAEL) of 3 mg/kg bw/day in the females and 1 mg/kg bw/day in the males. The Lowest Observed Adverse Effect Level (LOAEL) was considered to be 3 and 10 mg/kg bw/day for males and females, respectively.

This study was considered as acceptable as it satisfied the main criteria of the OECD guideline.


 

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2010-07-28 until 2010-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to guideline
Guideline:
other: Japanese METI (Ministry of Economy, Trade and Industry) of 13 July 1974 and subsequent revisions.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Harlan Italy S.r.l., 33049 San Pietro al Natisone (UD), Italy
- Number and sex : 130 rats (65 males and 65 females)
- Age at study initiation: 27-29 days old
- Weight at study initiation: Males: 86.2 to 110.0 grams; Females: 81.4 to 101.4 grams
- Caging
No. of animals/cage : up to 5
Housing : Polycarbonate cages measuring 59x38.5x20 cm, with stainless steel mesh lid and floor.
Cage tray control : Daily inspected and changed as necessary (at least 3 times/week):
- Water : drinking water supplied to each cage via a water bottle, ad libitum
- Diet : 4 RF 21 throughout the study, ad libitum
- Acclimation period: 19-20 days

Temporary identification : by means of a coloured mark on the tail within each cage
Veterinary health check : after arrival

Housing conditions:
Room lighting: : Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: : Approximately 15 to 25 air changes per hour
Temperature range: : 22°C ± 2°C
Relative humidity range: : 55% ± 15%
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
sterile
Details on oral exposure:
The test item was administered orally, by gavage, at a dose volume of 10 ml/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
On a daily basis, a sufficient amount of the test item was diluted 1:10 with purified water. Then further independent solutions were prepared with the same vehicle from the diluted stock to give the required concentrations of 0.5, 2, 6, 10 or 20 mg/ml.
Concentrations were calculated and expressed in terms of dry salt.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in a separate RTC Study in the range from 0.1 to 100 mg/ml.
Duration of treatment / exposure:
Main Groups - All animals were dosed for a minimum of 4 consecutive weeks followed by a recovery period of 4 weeks for 5 males and 5 females from Groups 1 and 4. All animals from the main phase groups were dosed up until the day before necropsy.
Frequency of treatment:
Main Groups - All animals were dosed once a day, 7 days a week. No treatment was given during the recovery period.
Satellite Groups - All animals received a single dose only.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Males & Females
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Males (expressed as dry salt)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Males (expressed as dry salt)
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Males (expressed as dry salt)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Females (expressed as dry salt)
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Females (expressed as dry salt)
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Females (expressed as dry salt)
200 mg/kg/day (dry salt) from D1 to D 19, then decreased due to toxicity
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Females (expressed as dry salt)
200 mg/kg/day (dry salt) from D1 to D 19, then decreased to 100 mg/kg/day (dry salt) from D20 to D28
No. of animals per sex per dose:
Each main group, for toxicological evaluation, comprised 5 male and 5 female rats. Control and high dose groups included 5 additional animals per sex to be sacrificed after 4 weeks of recovery.
Satellite groups, for toxicokinetic evaluation, included 3 or 6 animals/sex/group for control or treatment groups respectively.
Control animals:
yes
Details on study design:
- Dose selection rationale: The starting dose levels were defined on the base of a 7-day preliminary toxicity study in Sprague Dawley rats (male and female) after daily oral administration at dose levels of 200, 400 and 800 mg/kg/day (with a dose volume of 10 ml/kg using water as vehicle) -RTC Study no. 76230EXT (non GLP study)- .
Clinical signs, body weight, haematology, clinical chemistry, gross observation at necropsy with organ weight analysis and histopathology of a selected number of organs were carried out in 4 animals/sex/group. An exploratory toxicokinetics was carried out on the animals treated with the test item using 6 animals/sex/group.
Based on the result of this study the dose level of 200 mg/kg/day may be considered as the NOAEL for the females. The dosages of 100 mg/kg/day and 25 mg/kg/day may be considered as the high dose levels for females and males, respectively.

- Rationale for animal assignment: The rats were allocated to the 8 groups (4 main groups plus 4 satellite groups) by computerised stratified randomisation to give approximately equal initial group mean body weights. The computerised system used in this study was the Xybion Path/Tox System, Version 4.2.2.
Observations and examinations performed and frequency:
IN VIVO OBSERVATIONS
Clinical signs and neurotoxicity assessment
All clinical signs were recorded for individual animals.
Main Groups - Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. The observations performed before the testing period are not reported in this report.
Once during week 4 of treatment and once during week 4 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.
Satellite Groups - Animals of the satellite groups were observed for general clinical signs once before commencement of treatment, 2 hours after dosing, then daily thereafter up to sacrifice.

Motor activity assessment (Main groups)
The motor activity (MA) of all animals was measured once during week 4 of treatment and once during week 4 of recovery by an automated activity recording. Measurements were performed using a computer generated random order.

Body weight
Each animal was weighed on the day of allocation to treatment groups and on the day that treatment commenced. In addition, animals of the main groups were weighed at weekly intervals and just prior to necropsy.

Food consumption (Main groups)
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

CLINICAL PATHOLORY INVESTIGATIONS (Main groups)
At the end of week 4 of treatment, just prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava of all surviving male and all surviving female animals from each group, under conditions of food and water deprivation.
At the same time interval, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 ml/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. At the end of week 4 of the recovery period, blood and urine samples were also taken from all surviving animals under identical conditions in order to re-evaluate all parameters at measurements performed during the treatment period.
Blood samples were collected and analysed in the same order.
The blood samples collected were divided into tubes as follows:
EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements performed on blood and urine samples are listed below:
-Haematology: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Heinz bodies, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time
-Clinical chemistry: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium
Chloride
- Urinalysis: Appearance, Volume, Specific gravity, pH, Protein, Total reducing substances, Glucose, Ketones, Bilirubin, Urobilinogen, Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components.

-Hormone determination:
At the end of week 4 of treatment, prior to necropsy, additional blood samples (as much as possible, taking into account the amount required for clinical pathology investigations – see above) were taken from the abdominal vena cava of all surviving male and female animals under isofluorane anaesthesia and from 5 male and 5 female spare untreated rats from the same batch.
Blood samples were collected into tubes without anticoagulant, centrifuged and serum samples were frozen at approximately -80°C until analysis for T3 , T4 and TSH evaluation.
At the end of week 4 of the recovery period, blood samples were also taken from all surviving animals under identical conditions in order to re-evaluate the three hormones, since treatment-related changes were noted at measurements in samples withdrawn at the end of the treatment period.

TOXICOKINETICS
Samples collection
Satellite groups
Blood samples were withdrawn from the animals at 8 time points from the day of dosing, while urine samples were collected at 6 intervals from the day of dosing.
Main groups: On Day 27, blood samples were withdrawn from the first 3 main phase animals per sex, approximately 24 hours from the last dosing. Urine and faeces were not collected for these animals. Food and water were provided during this period.
Blood collection - Approximately 0.8 ml blood samples were collected from the tail vein of the animals at each sampling time as indicated above (3 animals/sex/time point; up to 4 bleedings/animal). Samples were transferred into tubes containing heparin anticoagulant, centrifuged and the plasma frozen in two aliquots at –20°C pending analysis.
One aliquot was analysed at the end of the in vivo phase and the other was retained at RTC until successful analyses of plasma.
The second aliquots will be eliminated after the finalisation of the report.
Urine and faeces collection - Animals were individually caged in a metabolic cage for the indicated time period. Urine and faeces were collected daily and preserved at 4°C. After the end of collection for each time point, a cage wash was performed with approximately 5 ml of water (instead of 3 ml as indicated in the protocol). Each urine sample collected during the interval and the correspective volume of water used for the cage wash were pooled for each single animal, mixed and the total volume was measured. Faecal weight was determined.
Urine and faeces samples were frozen (without dividing them into aliquots as indicated in the protocol) and stored at -20°C, pending analysis. Remaining urine samples will be used for further analysis in a non GLP study (RTC Enq. No. 84764EXT). On the contrary, remaining faeces samples will be eliminated after finalisation of the report.

Chemical analysis
Analysis of the samples was carried out by the Analytical Chemistry Department of RTC. The plasma samples were assayed to determine cC6O4 according to a validated method (under RTC Study no. 82330).

Assessment of toxicokinetic data
Plasma samples
For satellite groups, the toxicokinetic analysis was conducted according to a standard non-compartmental analysis. The following toxicokinetic parameters were obtained or calculated, when appropriate, from the mean plasma values: Cmax, tmax, t ½, AUC (0-tlast), AUC
For Day 27 samples, the following toxicokinetic parameters were obtained or calculated, when appropriate, from the mean plasma values: C24h
All the toxicokinetic parameters were estimated or calculated by the Kinetica™, version 4.4.1, PK/PD Analysis (Thermo Electron Corporation Informatics, Philadelphia - USA) software.
Means and/or medians, standard deviations and coefficients of variation were obtained using a Microsoft Excel worksheet.

Assessment of urinary excretion
The following parameters were calculated: Urinary excretion (Ae) of the test item in each urine sample for the time interval, Urinary excretion in the time interval expressed as percentage of the administered dose (Ae, %), the renal clearance of the unchanged free acid, the plasma terminal half-life.
Further parameters of excretion data were evaluated.

Assessment of faecal excretion
The following parameters were calculated : Faecal excretion of the test item, Faecal excretion in the time interval expressed as percentage of the administered dose.
Sacrifice and pathology:
Mortality (All groups)
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Severely debilitated animals were observed carefully. Animals judged to be in extremis were killed.

TERMINAL STUDIES
Euthanasia
Main Groups - Animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. All animals, including those found dead, were subjected to necropsy and supervised by a pathologist, as detailed below.
Satellite Groups - Animals were killed after the last bleeding/withdrawal procedure by carbon dioxide inhalation. No post mortem examinations were performed on any animal.
Necropsy (Main groups): The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
Organ weights (Main groups): From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. The liver was also weighed from animals dying prior to terminal kill.
Tissues fixed and preserved (Main groups): Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin (except eyes which were fixed in Davidson's fluid; and testes and epididymides which were fixed in Bouin's solution and all preserved in 70% ethyl alcohol). Extra liver samples were taken from all main group animals for further detailed evalutations.
Histopathological examination (Main groups)
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In the first instance the examination was restricted as detailed below:
a) Tissues from all animals in the control and high dose groups killed after 4 weeks of treatment.
b) Tissues from all animals dying during the treatment period.
c) All abnormalities in all main phase groups.
The examination was then extended to include, from all other animals killed after 4 weeks of treatment and 4 weeks of recovery, the following tissues.
- In both sexes: liver, spleen, thymus, bone marrow (from sternum) and kidneys
- Males: seminal vesicles and prostate
- Females: uterus, ovaries, cervical and mesenteric lymph nodes and ears
Other examinations:
Based on the findings in the preliminary non-GLP compliant study (RTC Study no. 76230EXT), additional investigations were carried out in the main group for a better unravelling of liver toxicity features.
- Peroxisome proliferation marker (Main groups – Delegated phase): a liver extract was prepared from one of the two frozen samples. Extracts were assayed for each of the following endpoints: A) protein content B) cyanide-insensitive palmitoyl-CoA oxidation.
- Hepatic microsomal cytochrome P-450 enzyme content: Liver microsomal fractions were prepared from a second aliquot of frozen liver and assayed for microsomal protein content (Lowry et al (spectrophotometer) method) and total cytochrome P450 content (Omura and Sato (spectrophotometer) method).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the males, some clinical signs such as, hunched posture, pallor, damaged whiskers, thin aspect and prolapse of the penis were recorded in a few animals of Group 4 (high dose) from Day 26. Recovery was completed in the recovery male animals by Day 38.
No treatment-related signs were recorded in control males or males from the low- and mid-dose groups.
Other signs occasionally recorded in the males of the control group (hairloss) or treatment groups (abrasion) are not considered toxicologically significant.

In the females, several clinical signs were recorded in most of Group 4 animals (high dose group), starting from Day 6 and with increasing occurrence and incidence during the treatment period.
The most commonly recorded clinical signs were hunched posture, pallor, thin aspect, staining of the perigenital region, hairloss in various body regions.
Occasionally decreased activity and presence of scabs were also recorded. After the decrease of dose level from 200 to 100 mg/kg (Day 20), a partial recovery from these signs occurred. A more effective recovery started after the end of treatment. Recovery from most signs occurred by Day 35 even though some minor signs (hairloss and whiskers missing) were recorded up to Day 56.
No treatment-related signs were recorded in control females or females from the low- and mid-dose groups with the exception of damaged ear recorded on Day 29 in animals treated with the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the main groups, mortality occurred in three female animals of Group 4 (high dose group).
Animal nos. 82430043 and 82430057 died on Day 13, while animal no. 82430053 on Day 19.
Animal no. 82430057 showed a staining of the head and of perigenital region from Day 8. On Day 12, the animal was additionally emaciated and showed hunched posture, decreased activity and pallor.
Animal no. 82430043 was emaciated and showed hunched posture, a marked staining of the perigenital region and a moderate hairloss of the head on Day 12. Animal no. 82430053 showed from Day 13 damaged whiskers (later missing) and a slight hairloss of the muzzle from Day 13. On Day 15, the animal showed rales and from this day on the animal was emaciated, showing additional signs such as: pallor, hunched posture, decreased activity, moderate staining of the muzzle, moderate piloerection, slight hairloss of the muzzle.
At post mortem examination, the liver of these animals was weighed and the weight was found increased, when compared to the mean liver weight of female control animals at the final sacrifice.
Macroscopically, the range of lesions seen in these animals included the following: the liver was swollen, enlarged and pale (a statistically significant increase was recorded both for absolute and relative liver weight in all groups with a dose-related trend.The increase in relative liver weight was approximately 98 to 130% in males and 70 to 136% in females), the kidneys, lungs, stomach, caecum, ears, thymus, adrenals, pituitary and brain had dark and/or red colour; the contents in the jejunum were green/yellow dark; the uterus, spleen, thymus and mesenteric lymph nodes were reduced in size.

Microscopically, the range of lesions seen in these animals are reported under "details on results".

On the basis of these results, the cause of death in these animals is considered to be related to the test compound.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Dosing Phase:
Statistically significant reductions of body weight were recorded from the second or third week of treatment respectively in females and males of the high dose group. A trend for reduction was also evident in the intermediate treatment groups of both sexes at the end of the treatment period.

- Recovery Phase
The group mean body weight difference was statistically significant during the whole recovery period. However, the statistical significance is likely to be due to the high difference in the starting body weight at the beginning of the recovery period. A partial recovery was evident in both sexes at the end of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A reduction of food consumption was recorded in Group 4 males during the 4th week of treatment. Recovery occurred at the beginning of the recovery period. On the contrary, food consumption was unaffected by treatment in females.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Dosing Phase
Thrombocytosis, leucopenia and reticulopenia were recorded in males treated with 20 and/or 60 mg/kg/day. In particular, platelets were increased in males dosed with 60 mg/kg/day only (27% above controls).
White blood cells were decreased in a number of males treated with 20 and 60 mg/kg/day, being 25% to 39% lower than controls.
The same animals showed decrement of reticulocytes, which were 29% to 87% lower than controls.
Concerning females, platelets were increased in 2 animals receiving high dose (200 and then 100 mg/kg/day) with a maximum increment of 27% when compared to the control.
Leucocytes and reticulocytes were comparable with controls, with the exception of eosinophils which were decreased in almost all treated females (maximum decrement of 60% when compared to the controls). Prothrombin time was decreased in treated females, without dose-relation (approximately 20%).

- Recovery Phase
In males, thrombocytosis and leucopenia showed a partial reversibility, being approximately 19% above and 18% below controls respectively. In the same group, reticulocytopenia completely recovered.
In addition, a slight reduction of erythrocytes and haemoglobin was observed in treated males. However, changes were insufficient in magnitude (approximately 8%) to represent an appreciable anaemia.
In females, platelets were slightly higher than controls (21% increment) in one animal only. Eosinopenia completely recovered.
With reference to coagulation parameters, prothrombin time showed a slight decrement (12%) in males, while in females a partial recovery was observed, being the value 17% below controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Dosing Phase
In general, liver and kidneys markers showed increment in some animals treated with medium and/or high dosages. Changes were moderate to severe and involved 1 to 3 animals/group.
Transaminases enzymes and bile acids were increased in some animals dosed with the medium and/or high dosages. Changes increased in magnitude and/or incidence with the dose.
Moreover, animals receiving the highest dose showed increment of alkaline phosphatase (females only), bilirubin (males only), cholesterol, triglycerides, urea, phosphorus and potassium and decrement of creatinine, protein and globulin. In addition, animals treated with the medium dosage showed increment of cholesterol, triglycerides (females only), urea and potassium and decrement of globulin.

- Recovery Phase
Compared with controls and data of the dosing phase, an almost complete recovery was observed for aspartate aminotransferase, alanine aminotransferase (females only), alkaline phosphatase, bile acids, bilirubin, urea, creatinine, globulin and potassium.
A partial reversibility was recorded for alanine aminotransferase in males.
In females triglycerides, glucose and phosphorus did not show recovery.
In addition, animals from both sexes showed a slight increment in protein, albumin, and cholesterol; a decrement of triglycerides in males and an increment of calcium in females.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- Dosing Phase: Urinary pH was slightly decreased in animals treated with the highest dosage.

- Recovery Phase: pH was still lower than controls in males only. In addition, ketonuria was increased in the same animals.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- Weekly detailed clinical examinations
During the treatment period, a slight decrease in rearing activity was recorded in treatment groups when compared to controls. No significant alterations were recorded at the examinations performed at the end of the recovery period.
Variations recorded in the other measurements may be considered within the acceptable range.

- Sensory reactivity to stimuli
Variations recorded in the measurements may be considered within the acceptable range. No significative variations were recorded in treated animals when compared to controls.

- Motor activity
At the end of treatment, a decrease in motor activity was recorded in animals of Group 4 when compared to controls. This variation was statistically significant only in Group 4 females.
No significant alteration in motor activity was recorded in Group 4 at the examinations performed at the end of recovery period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Consistent and partially dose-related increase in liver weights were recorded at all dose levels tested with a maximum increase of 190% and 276% at absolute and relative level, respectively.
In the high dose group, consistent organ weight reductions were also recorded in thymus, spleen, heart (males only), prostate and ovaries.
A slight increase of the relative kidney weight was recorded in the high dose animals and in the mid-dose males, even though not consistently with the absolute weight.
At the end of recovery, increase of the liver weight still persisted both at relative and absolute level.
Consistent with the observations performed at the end of the dosing phase, slight increase of relative kidney weight in both sexes, decrease of spleen and ovaries weights in females, decrease of heart absolute weight in males were still observed in Group 4.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Final sacrifice
Treatment-related changes were seen in the liver, thymus, spleen, seminal vesicle, prostate, uterus and ovaries.
Liver: Treatment-related changes were seen in all treatment groups, with no apparent dose relation. The range of changes consisted of swollen, enlarged and pale or dark aspect.
Thymus, spleen, seminal vesicle, prostate, uterus and ovaries: These organs were described as smaller in size and were limited to the high dose group.
The ears in few animals from all female dosed groups were described as red in colour.

- Recovery sacrifice
Treatment-related changes were seen in the liver, consisting of swollen, enlarged and pale or dark aspect.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Final sacrifice
Treatment-related findings were found in the high dosed animals of both sexes, involving the liver, spleen, thymus and kidneys. In addition, treatment-related changes were seen in the high dosed males in the bone marrow (sternum), seminal vesicles and prostate and in the high dosed females, in the uterus, ovaries, cervical and mesenteric lymph nodes and ears.
In the intermediate and lower groups, treatment-related changes were mostly seen in the liver, with singular cases of treatment effects seen in the thymus and kidneys of male and/or female rats from Group 3. Frequent treatment-related effects were seen in the ear of female rats of Group 3.
The changes in the liver are considered to be a primary toxic effect of the test compound, while all the rest of the changes seen in the other mentioned organs are considered to reflect a non-specific high dose stress effect, which was associated with a significant reduced body weight gain.
The following are details of the treatment-related lesions seen in the high dosed animals.
Specific notes are added in those organs in which a treatment-related effect was observed in the intermediate and low dosed groups.
Liver: Treatment-related changes seen in the high dose included diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia. This was occasionally associated with the presence of minimal to mild multifocal hepatocytic necrosis, minimal to mild multifocal accumulation of brownish pigment-laden macrophages and mild multifocal bile duct hyperplasia. In the intermediate groups (i.e., Groups 2 and 3),
diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted. This was occasionally associated with the presence of minimal to moderate multifocal hepatocytic necrosis (not seen in the female animals from Group 2), minimal to mild hepatocytic degeneration (vacuolation), mild fibrosis (seen only in a male rat from Group 3) and minimal multifocal accumulation of brownish pigment-laden
macrophages (seen in a female from Group 3).
The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of smooth endoplasmic reticulum (See Maronpot et al, 2010).
Kidneys: Treatment-related changes seen in the high dose consisted of mild to moderate cortical tubular dilation. In a single male rat from Group 3, minimal cortical tubular dilation was noted. In a single female rat from Group 3, minimal pelvic cavity dilation, associated with minimal pelvic inflammation and pelvic epithelium hyperplasia, were noted.
Thymus: Treatment-related changes seen in the high dose included diffused minimal to severe cortical atrophy, resulting from loss of the lymphocytes. This change was usually associated with multifocal lymphocytic necrosis. In a single female rat from Group 3, minimal cortical atrophy was noted.
Spleen: Treatment-related changes seen in the high dose included multifocal mild to moderate atrophy of the lymphoid tissue (i.e., involving the periarteriolar lymphoid sheath).
Bone marrow (sternum): Treatment-related changes seen in the high dose included diffused mild reduced cellularity (i.e., atrophy) of the hematopoietic tissue.
Prostate and seminal vesicle: Treatment-related changes seen in the high dose included diffused mild reduced secretion, associated with mild atrophy of the lining glandular epithelium.
Uterus and ovaries: Treatment-related changes seen in the high dose included diffused moderate atrophy of the myometrium and follicles (i.e., atresia), respectively in the uterus and ovaries.
Ears: Treatment-related changes seen in the females of Groups 2, 3 and 4 (low, intermediate and high dose groups), included mild to moderate necrosis of the epidermis and underlying subcutis. This change was usually seen at the tip of the ear.
All other observed changes had comparable incidence in the control and treated groups, and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.

- Recovery sacrifice
Treatment-related findings were found in the liver, including diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia. This was frequently associated with the presence of minimal multifocal hepatocytic necrosis. In two male rats, these changes were also associated with minimal multifocal accumulation of brownish pigment-laden macrophages and hepatocytic degeneration.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
TOXICOKINETICS
The 28 days oral toxicity study results have showed a high variability between different doses; a high variability was seen also comparing animals of the same sex at the same administered dose. They were not considered significant for understanding the toxicokinetic behavior of the substance.

CYTOCHROME P450 ACTIVITY
- Final sacrifice
In male rats, at dose levels of 5, 20 and 60 mg/kg/day, the mean activities (per g protein) of Cytochrome p450 was increased with statistical significance by 263%, 245% and 258%, respectively, with statistical significance only in mid- and high-dose group. Statistically significant increase of the mean activities (per g liver) was also observed (345%, 380% and 338%, respectively at low-, mid- and high dose).
In female rats, at dose levels of 20, 60 and 200/100 mg/kg/day, the mean activities (per g protein) of Cytochrome p450 was increased by 102%, 181% and 176%, respectively. Statistically significant increase of the mean activities (per g liver) was also increased (296%, 448% and 395%, respectively at low-, mid- and high dose).

- Recovery sacrifice
Following a 4-week recovery period, statistically significant increases were observed in male rats at 60 mg/kg/day relative to mg of protein and g of liver (both p<0.01, respectively increased of 339% and 527%).
In females, statistically significant increases were observed at 200/100 mg/kg/day relative to mg of protein and g of liver (both p<0.01, respectively increased of 191% and 343%).


THYROID HORMONES
- Dosing Phase
Thyroxine (T4) showed a dose-related decrement in many treated animals, with the exception of females receiving 20 mg/kg/day. Changes were approximately 30 to 50% and were not associated with relevant decrement of triiodothyronine (T3).
Concerning thyroid stimulating hormone (TSH), most of the values were below the limit of quantification. One male treated with 20 mg/kg/day and one female treated with the high dose (200 and then 100 mg/kg/day) showed high levels of TSH. These values were associated with low levels of T4, even though other animals, which showed low T4, did not display high TSH.

- Recovery Phase
In males, T4 showed partial reversibility, being 28% below controls, while T3 was increased in the same animals (34%). One treated male had high level of TSH, while the other males showed values below the limit of quantification. On the contrary, females showed an increment of T4 (24% above controls) and no relevant changes of T3. TSH was below the limit of quantification in each female.
Details on results:
CLINICAL SIGNS IN ANIMALS FOUND DEAD
In the main groups, mortality occurred in three female animals of Group 4 (high dose group).
Animal nos. 82430043 and 82430057 died on Day 13, while animal no. 82430053 on Day 19.

Microscopically, the range of lesions seen in these animals included the following:
Liver: Treatment-related changes included diffused moderate hepatocytic hypertrophy, which consisted of cytoplasmic eosinophilia. This was occasionally associated with presence of multifocal hepatocytic necrosis. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum (see Maronpot et al, 2010).
Kidneys: Treatment-related changes consisted of mild papillary necrosis.
Thymus: Treatment-related changes included diffused moderate to severe cortical atrophy, resulting from loss of the lymphocytes. This change was usually associated with multifocal lymphocytic necrosis.
Spleen: Treatment-related changes included multifocal moderate atrophy of the lymphoid tissue (i.e., involving the periarteriolar lymphoid sheath).
Cervical and mesenteric lymph nodes: Treatment-related changes included multifocal mild to moderate atrophy of the lymphoid tissue (i.e., involving the lymphoid follicles).
Bone marrow (sternum): Treatment-related changes included diffused mild reduced cellularity (i.e., atrophy) of the hematopoietic tissue.
Uterus and ovaries: Treatment-related changes included diffused mild to moderate atrophy of the myometrium and follicles (i.e., atresia), respectively in the uterus and ovaries.
Adrenals: Diffused moderate reduction of the usually present cortical (Zona Fasciculata) cytoplasmic vacuolation was noted. This change was associated with increased cytoplasmic acidophilia of these cells, suggesting that these cells secreted the normally present corticoid hormone contents.

On the basis of these results, the cause of death in these animals is considered to be related to the test compound.
Key result
Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
other: dry salt
Sex:
male
Basis for effect level:
body weight and weight gain
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

 

Haematology results in male and females rats treated with cC6O4 ammonium salt for 28 days, with a 4-week recovery period - main findings

 

End of treatment

 

End of recovery period

 

Males

  

Females

  

Males

Females

Dose (mg/kg/day)

Control

5

20

60

  

Control

20

60

200/100

  

Control

60

Control

200/100

Number of animals

5

5

5

4

 

5

5

5

4

 

5

5

5

3

Mean + SD

                            

MCHC, g/dl

33.46

33.62

33.84

34.05

  

34.53

34.26

34.06

32.90**

  

33.42

33.14

34.32

33.70

0.53

0.28

0.38

0.53

0.21

0.23

0.27

0.36

0.29

0.50

0.35

0.35

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RET, %

1.976

1.770

1.448**

0.265**

  

1.900

2.178

1.864

2.153

  

1.984

2.830*

1.896

2.190

0.356

0.154

0.158

0.192

0.652

0.257

0.461

0.860

0.224

0.515

0.374

0.835
                             

RET, 10E9 /l

154.24

134.18

109.54**

19.53**

  

138.08

148.98

128.80

148.83

  

163.28

212.08

149.12

159.83

25.95

10.12

12.92

12.97

39.59

16.72

22.24

60.77

22.61

41.93

21.64

55.46
                             

WBC, 10E3/µl

10.122

11.57

7.616

6.188*

  

5.505

6.026

6.358

5.338

  

11.020

9.084

7.322

5.697

1.714

1.506

2.099

1.572

0.916

1.574

0.768

0.997

1.965

1.518

2.178

1.178
                             

LYM, 10E3 /µl

7.87

8.816

6.224

4.965*

  

4.385

5.062

5.43

4.28

  

8.890

6.792*

5.506

4.657

1.600

1.471

1.817

1.077

0.831

1.398

0.855

1.298

1.446

0.936

1.251

0.996
                             

PLT, 10E3 /µl

978.4

981.8

906.6

1240.3**

  

1141.5

1086.8

994.4

1280.3

  

802.2

951.8

931.2

1018.3

15.1

75.1

77.2

103.6

86.4

62.3

134.4

183.8

34.8

140.1

36.0

96.6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EOS, 10E3 /µl

0.158

0.184

0.092*

0.040**

  

0.163

0.088**

0.092**

0.065**

  

0.164

0.126

0.132

0.133

0.034

0.110

0.022

0.018

0.026

0.019

0.019

0.031

0.059

0.097

0.051

0.102
                             

EOS, %

1.60

1.64

1.26

0.68

  

3.03

1.50**

1.44**

1.18**

  

1.48

1.30

1.78

2.17

0.45

1.11

0.22

0.17

0.77

0.36

0.27

0.43

0.33

0.92

0.34

1.17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PT, sec

20.92

20.10

19.42*

22.04

  

22.80

18.48**

19.32**

17.65**

  

21.56

19.04**

22.24

18.37**

0.43

0.56

0.77

2.97

0.98

0.93

1.18

0.87

0.61

0.64

0.76

1.85

n: group size

SD: standard deviation

*  mean value of group is significantly different from control at p < 0.05

** mean value of group is significantly different from control at p < 0.01

 

 

Main biochemistry data

 

 

End of treatment

 

End of recovery period

 

Males

 

Females

 

Males

Females

Dose (mg/kg/day)

Control

5

20

60

 

Control

20

60

200/100

 

Control

60

Control

200/100

Number of animals

5

5

5

5

 

5

5

5

4

 

5

5

5

3

Mean (+SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

AP, U/l

269.06

(20.29)

308.04

(12.49)

422.90**

(30.80)

289.86

(49.34)

 

177.24

(36.19)

155.52

(14.61)

280.64*

(37.49)

345.40**

(87.44)

 

221.18

(16.48)

241.56

(24.65)

171.26

(44.20)

161.63

(26.11)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALAT, U/l

42.68

(12.42)

49.76

(6.32)

114.98*

(49.92)

149.94

(99.19)

 

32.98

(8.89)

34.86

(10.32)

83.80

(58.28)

141.33*

(41.66)

 

49.58

(4.13)

121.52*

(54.40)

31.82

(5.34)

31.40

(4.5)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ASAT, U/l

90.20

(16.67)

85.32

(4.97)

109.80

(17.19)

174.48

(72.47)

 

86.94

(7.36)

72.10

(9.86)

133.02

(73.93)

263.80*

(86.51)

 

86.00

(7.19)

90.10

(17.78)

77.78

(8.80)

57.53*

(6.50)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Total bilirubin,mg/dl

0.182

(0.053)

0.128

(0.013)

0.100*

(0.025)

0.490

(0.253)

 

0.188

(0.020)

0.080**

(0.026)

0.058**

(0.019)

0.205

(0.124)

 

0.206

(0.166)

0.082

(0.008)

0.232

(0.174)

0.090

(0.010)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cholesterol,mg/dl

69.50

(8.88)

59.00

(5.57)

87.92

(6.41)

81.40

(21.61)

 

68.52

(11.25)

76.92

(11.99)

86.92

(13.64)

102.33**

(19.75)

 

65.80

(4.13)

122.48**

(7.73)

72.46

(13.87)

139.27**

(18.84)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Triglycerides,mg/dl

27.06

(4.64)

20.34

(6.87)

20.92

(2.69)

38.44

(12.23)

 

28.30

(3.50)

36.18

(5.43)

60.68**

(12.56)

51.95**

(6.10)

 

56.82

(7.05)

31.66**

(3.78)

30.08

(7.47)

57.03**

(3.58)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Bile acids,µmol/l

6.54

(4.65)

1.40

(1.27)

7.80

(4.66)

173.34**

(61.34)

 

7.72

(5.12)

6.30

(2.81)

15.32

(6.80)

34.25**

(6.59)

 

11.84

(12.91)

9.92

(1.90)

10.34

(6.03)

15.67

(2.30)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A/G ratio

1.77

(0.16)

1.72

(0.20)

2.39*

(0.32)

2.38

(0.62)

 

1.99

(0.10)

2.55*

(0.16)

2.91**

(0.20)

2.73**

(0.62)

 

1.63

(0.21)

1.82

(0.24)

1.68

(0.38)

2.24

(0.26)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Creatinine, mg/dl

0.310

(0.037)

0.246*

(0.026)

0.448

(0.482)

0.272

(0.180)

 

0.388

(0.029)

0.334

(0.036)

0.320*

(0.026)

0.275**

(0.051)

 

0.350

(0.047)

0.304

(0.025)

0.384

(0.057)

0.320

(0.026)

n: group size

SD: standard deviation

*  mean value of group is significantly different from control at p < 0.05

** mean value of group is significantly different from control at p < 0.01

 

 

Thyroid hormones levels in male and female rats treated with cC6O4 ammonium salt for 28 days, with a 4-week recovery period.

 

 

End of treatment

 

End of recovery period

Sex

 

 

 

Males

 

 

 

Females

 

 

 

Males

 

Females

 

Dose (mg/kg/day)

 

Control

5

20

 60   Control 20 60 200/100   Control 60 Control 200/100
                             
T3 (ng/ml)                            
  Mean 0.644 0.65 0.572 0.584 0.616 0.604 0.548 0.543   0.558 0.746** 0.39 0.8
(+SD) (0.101) (0.091) (0.053) (0.077) (0.102) (0.104) (0.063) (0.054) (0.034) (0.048) (0.092) (0.078)
  n 5 5 5 5 5 5 5 4   5 5 5 3
T4 (µg/dl)                            
  Mean 3.65 2.608** 2.086** 1.914** 3.22 3.142 2.038** 1.560**   3.316 2.396* 2.634 3.257*
(+SD) (0.674) (0.434) (0.295) (0.411) (0.647) (0.403) (0.488) (0.477) (0.528) (0.355) (0.276) (0.388)
  n 5 5 5 5 5 5 5 4   5 5 5 3
TSH (ng/ml)                            
  Mean 1.750† 1.280† 6.600† -† 2.81 2.6 3.975 10.047   -† 19.530† -† -†
(+SD) (1.311) - (6.59) - (2.907) (1.058) (0.908) (14.076) - - - -
  n 3/5 1/5 2/5 0/4§ 4/5 4/5 4/5 3/4§   0/5 1/5 0/5 0/3

n  group size

SD standard deviation

*  mean value of group is significantly different from control at p < 0.05

** mean value of group is significantly different from control at p < 0.01

†  the values reported are based on the number of animals indicated that had quantifiable levels. The levels were below the limit of quantification (xx) in the other animals of the group.

§  one sample had no sufficient volume for analysis

 

Mean liver weight, and incidence/severity of microscopic findings in the liver of male and female rats treated with cC6O4 for 28 days, with a 4-week recovery period

  End of treatment period   End of recovery period
Males   Females Males   Females
Dose (mg/kg/day) 0 5 20 60 0 20 60 200/100 0 60 0 200/100
Number of rats examined 5 5 5 5   5 5 5 4   5 5   5 3
Mean body weight, g 338.56 339.52 323.38 228.00**   223.18 222.98 199.76* 172.43**   371.34 343.80*   239.74 227.30*
  (+SD) (13.42) (12.61) (18.58) (26.72) (16.54) (5.62) (16.55) (14.04) (14.67) (17.41) (5.36) (6.99)
Mean Liver weight
  Absolute, g 9.45 14.711** 20.210** 19.506** 5.777 11.501** 13.693** 16.781** 9.276 21.025** 5.959 12.264**
  (+SD) (0.670) (1.163) (1.728) (2.055) (0.425) (0.64) (1.36) (1.072) (0.439) (1.299) (0.431) (0.255)
  Relative, % bw 2.789 4.326** 6.247** 8.576* 2.591 5.162** 6.882** 9.750** 2.498 6.114** 2.484 5.398**
  (+SD) (0.094)  (0.264) (0.332) (0.47) (0.12 (0.358) (0.805) (0.454) (0.057) (0.185) (0.14) (0.189)
Microscopic findings
Liver
Hepatocytic hypertrophy (total) 0 5 5 5 0 5 5 4 0 5 0 3
    Grade = mild - - - - - 1 - - - - - -
    Grade = moderate - 5 5 5 - 4 5 4 - 5 - 3
Hepatocytic necrosis (total) 0 1 2 3 0 0 1 2 0 3 0 3
     Grade = slight - 1 - 2 - - - - - 3 - 3
     Grade = mild - - 1 1 - - - 2 - - - -
     Grade = moderate - - 1 - - - 1 - - - - -

SD: standard deviation

% bw: organ weight expressed as % relative to body weight ratio

*   Analysis of variance, Dunnett’s test, p < 0.05

** Analysis of variance, Dunnett’s test, p < 0.01

 

Protein concentration and cytochrome P450 activity in the liver of male and female rats treated with cC6O4 for 28 days, with a 4-week recovery period

  End of treatment period         End of recovery period  
    Protein concentration Cytochrome P450 activity     Protein concentration   Cytochrome P450 activity
    mg/ml nmol/g liver nmol/g protein     mg/ml   nmol/g liver nmol/g protein
Doses (mg/kg/day) n Mean SD Mean SD Mean SD   n Mean SD   Mean SD Mean SD
Males   †   †   †       ††     ††   ††  
   Control 5 7.195 0.456 8.9 2.28 0.247 0.059   5 6.622 1.09   18.02 4.49 0.539 0.079
   5 5 8.854** 0.492 39.67** 5.29 0.898** 0.128                  
   20 5 9.985* 2.176 42.75** 14.96 0.853** 0.249                  
   60 5 8.818 1.479 39.01* 20.32 0.885* 0.427   5 9.571++ 0.686   112.97++ 4.99 2.370++ 0.2
                                 
Females   ††   ††   ††       ††     ††   ††  
   Control 5 5.896 2.427 6.96 1.27 0.258 0.025   5 4.925 1.201   8.97 0.32 0.33 0.069
   20 5 10.404+ 3.602 27.58+ 10.61 0.522 0.129                  
   60 5 10.944+ 2.932 38.13++ 6.05 0.726++ 0.167                  
   200/100 4 9.476 0.748 34.48++ 14.02 0.714++ 0.239   3 8.334++ 1.324   39.74++ 2.82 0.962++ 0.08

 n: group size

 SD: standard deviation

 † Data inhomogeneous by Bartlett’s test, a modified t-test of significance was used.

 *    Significance level of comparison with control, using modified t-test, p<0.05

 **  Significance level of comparison with control, using modified t-test, p<0.01

 †† Data homogeneous by Bartlett’s test, a Dunnett’s test of significance was used.

 +    Significance level of comparison with control, using modified t-test, p<0.05

 ++  Significance level of comparison with control, using modified t-test, p<0.01

 

 

Protein concentration and cyanide-insensitive Palmitoyl CoA oxidase activity in the hepatic supernatant of male and female rats

treated with cC6O4 ammonium salt for 28 days, with a 4-week recovery period

  End of treatment period   End of recovery period
    Protein concentration   Palmitoyl CoA oxidase activity     Protein concentration   Palmitoyl CoA oxidase activity
    mg/g liver   nmoles/min/mg protein nmoles/min/g liver     mg/g liver   nmoles/min/mg protein nmoles/min/g liver
Dose (mg/kg/day) n Mean SD   Mean SD Mean SD   n Mean SD   Mean SD Mean SD
Males         ††   ††                    
   Control 5 151 7   5.05 0.35 761 61   5 167 6   4.52 0.51 754 71
5 5 158 3   7.99++ 0.58 1265++ 106                  
60 5 133++ 2   29.31++ 1.44 3879++ 161   5 159* 3   16.46** 6.61 2621** 1080
                                   
Females                                  
   Control 5 131 4   2.9 0.42 380 47   5 146 7   3.7 0.24 541 35
20 5 149 14   15.59++ 2.78 2333++ 528                  
   200/100 4 139 11   31.30++ 3.44 4379++ 774   3 140 7   19.33*** 2.73 2695*** 374
                                   

n: group size

SD: standard deviation

†† For males: Data log transformed prior to analysis to stabilize the variance, observed mean presented

++Significance level of comparison with control, using William’s test, p<0.01

*   Significance level of comparison with control, using a t-test, p<0.05

** Significance level of comparison with control, using a t-test, p<0.01

*** Significance level of comparison with control, using a t-test, p<0.001

Conclusions:
Toxicity in females was lower than in males at the same dose level tested. This was probably linked to a faster elimination of the test item (or at least of the free acid which is mainly excreted by the renal route).
The liver was identified as main target organ. Liver damages consisted of treatment related dose-dependent degenerative microscopic findings, associated with high increase of organ size and weight, threshold-like increase of liver enzymatic activity (Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase). The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of smooth endoplasmic reticulum.
At the low doses, 5 mg/kg/day in males and 20 mg/kg/day in females, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, occasionally associated with minimal to mild hepatocytic degeneration still noted.
Secondary findings in a number of organs were recorded in males treated at 60 mg/kg/day and in females treated at 200/100 mg/kg/day, which reflect a non-specific high dose stress effect associated with a significantly reduced body weight loss.
Partial, recovery of liver findings was found in the high dose animals after a 4 week recovery period (including the assayed enzymatic activities, macroscopic and microscopic findings). Additionally, some secondary findings in blood parameters and organ weights were also still observed in both sexes at the end of the study. This demonstrates that a 4-week recovery period is not sufficient to allow complete recovery from the findings observed after treatment in the experimental conditions used.

Based on the above results, it is concluded that the NOAEL cannot be defined for both sexes in this study. The LOAEL was 5 mg/kg/day in males.
Executive summary:

The toxicity of cC6O4 was investigated, when given daily by oral administration to the Sprague Dawley rat for a 4-week period followed by a recovery period of 4 weeks from the last administration.

Males were dosed at 5, 20 and 60 mg/kg/day. Females were dosed at 20, 60 and, in the high dose group, 200 mg/kg/day from Day 1 to Day 19 and 100 mg/kg/day from Day 20 to Day 28.

The findings are described for each sex and dose level tested:

Males 60 mg/kg (high dose)

Some clinical signs such as, hunched posture, pallor, damaged whiskers, thin aspect and prolapse of the penis were recorded in a few animals from Day 26. Recovery was completed in the recovery animals by Day 38.

A slight decrease in rearing activity was recorded at the end of treatment but recovered within the end of recovery period.

Statistically significant reductions of body weight were recorded from the third week of treatment associated to a consistent reduction in food intake. A partial recovery was evident after the end of recovery.

At the haematology level, thrombocytosis (+27%), leucopenia (-39%) and reticulopenia (-87%) were recorded. Reticulocytopenia completely recovered, while thrombocytosis and leucopenia only showed a partial reversibility. Prothrombin time showed a slight decrement only at the end of recovery period.

Increment of transaminase enzymes, bile acids, bilirubin, cholesterol, triglycerides, urea, phosphorus and potassium and decrement of creatinine, protein and globulin were observed.

An almost complete recovery was observed for alkaline phosphatase, bile acids, bilirubin, urea, creatinine, globulin and potassium. A partial reversibility was recorded for alanine aminotransferase in males. In addition, animals showed a slight increment in protein and albumin; an additional increment in cholesterol and a decrement of triglycerides.

T4 showed a decrement in many animals. No relevant concomitant decrement of T3, or alteration in TSH was observed. At the end of recovery, T4 showed partial reversibility, while T3 was increased in the same animals. One treated male had high level of TSH, while the other males showed values below the limit of quantification.

Urinary pH was slightly decreased at the end of treatment and still at the end of recovery period.

At post mortem examination performed at the end of the dosing phase, swollen, enlarged and pale or dark aspect of the liver was observed. Liver showed the main organ weight alteration, with a marked increase recorded both for absolute and relative liver weight (2.1 and 3.1 fold, respectively).

Microscopically, the liver showed treatment-related changes such as diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia. This was occasionally associated with the presence of minimal to mild multifocal hepatocytic necrosis, minimal to mild multifocal accumulation of brownish pigment-laden macrophages and mild multifocal bile duct hyperplasia. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of smooth endoplasmic reticulum (See Maronpot et al, 2010).

With reference to hepatic enzyme activity, the mean activities (per g protein) of Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase activity was increased by 3.6 and 6 fold, respectively.

Small size of thymus, spleen, seminal vesicles and prostate was additionally recorded at final sacrifice. These were associated with consistent organ weight reductions recorded in thymus, spleen, heart and prostate. A slight increase of the relative kidney weight was recorded, even though not consistently with the absolute weight.

The following microscopic alterations were found in the other organs: in the kidneys, mild to moderate cortical tubular dilation in the thymus: diffused minimal to severe cortical atrophy, resulting from loss of the lymphocytes, usually associated with multifocal lymphocytic necrosis.

In the spleen: multifocal mild to moderate atrophy of the lymphoid tissue (i.e., involving the periarteriolar lymphoid sheath); in the bone marrow (sternum): diffused mild reduced cellularity (i.e., atrophy) of the hematopoietic tissue; in the prostate and seminal vesicle diffused mild reduced secretion, associated with mild atrophy of the lining glandular epithelium.

At post mortem examination performed at the end of recovery period, treatment-related changes consisted of swollen, enlarged and pale or dark aspect of the liver which still showed increase of the weight both at relative and absolute level (2.3 and 2.4 fold, respectively).

Microscopically, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia was noted, frequently associated with the presence of minimal multifocal hepatocytic necrosis and, in two male rats, with minimal multifocal accumulation of brownish pigment-laden macrophages and hepatocytic degeneration.

The mean activities (per g protein) of Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase activity was still increased by 4.4 and 3.5 fold, respectively.

Consistently with the observation performed at the end of the dosing phase, slight increase of relative kidney weight and decrease of heart absolute weight were still observed, even though no microscopic alterations were noted.

 

Males 20 mg/kg (mid dose)

No treatment related clinical signs were observed, with the exception of slight increase in rearing activity which recovered by the end of the study.

A trend for reduction of body weight (not statistically significant) was recorded while food consumption was unaffected by treatment.

At the haematology level, leucopenia (-25%) and reticulopenia (-29%) were recorded.

Increment of transaminases enzymes, bile acids, cholesterol, urea and potassium and decrement of globulin were observed.

T4 showed a decrement in many animals. One animal showed high levels of TSH (associated with low levels of T4). No relevant concomitant decrement of T3 was observed.

No alterations were recorded in urinalysis.

At post mortem examination, swollen, enlarged and pale aspect of the liver was observed with a high increase of liver weight recorded both at absolute and relative liver weight (2.1 and 2.2 fold, respectively).

At microscopic level, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted in the liver. This was occasionally associated with the presence of minimal to moderate multifocal hepatocytic necrosis, minimal to mild hepatocytic degeneration (vacuolation) and mild fibrosis (seen only in one animal).

With reference to hepatic enzyme activity, the mean activities (per g protein) of Cytochrome P450 was increased by 3.5 fold, when compared to controls.

A slight increase of the relative kidney weight was also recorded even though not consistently with the absolute weight. Microscopically, minimal cortical tubular dilation was noted only in a single male rat.

Males 5 mg/kg (low dose)

No treatment related clinical signs were observed during the in life phase, with the exception of slight increase in rearing activity which recovered by the end of the study.

A trend for reduction of body weight (not statistically significant) was recorded while food consumption was unaffected by treatment.

No significant alteration of haematology, clinical chemistry, urinalysis parameters or thyroid hormones were recorded.

At post mortem examination, swollen, enlarged and pale or dark aspect of the liver was observed with a high increase of liver weight recorded both at absolute and relative liver weight (both 1.6 fold).

At microscopic level, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted in the liver. This was occasionally associated with the presence of minimal to mild hepatocytic degeneration (vacuolation).

With reference to hepatic enzyme activity, the mean activities (per g protein) of Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase activity was increased by 3.6 and 1.5 fold, respectively, when compared to controls.

 

Females 200/100 mg/kg (high dose)

Mortality occurred in three females. In these animals death occurred on Day 13 (two animals) and Day 19. Severe clinical signs were observed before death (i.e. emaciated, hunched posture, decreased activity and pallor). The macroscopic and microscopic findings

noted at the post mortem examination indicated that the cause of death in these animals was related to the test compound.

In the surviving females, several clinical signs were recorded starting from Day 6 and with increasing occurrence and incidence during the treatment period (i.e. hunched posture, pallor, thin aspect, staining of the perigenital region, hairloss in various body region).After

the decrease of dose level from 200 to 100 mg/kg (Day 20) a partial recovery from these signs occurred, while a more effective recovery started after the end of treatment and was completed by Day 56.

A slight decrease in rearing activity and a slight statistically significant decrease in motor activity were recorded. These findings recovered within the end of recovery period.

Statistically significant reductions of body weight were recorded from the third week of treatment even though food consumption was unaffected. A partial recovery was evident after the end of recovery.

At the haematology level, platelets were increased in 2 animals (maximum increment of 27%) at the end of treatment and in one animal only at the end of recovery. Eosinopenia was additionally observed which recovered at the end of the study. Prothrombin time was decreased in treated females (approximately 20%). A partial recovery was observed.

Increment of transaminase enzymes, bile acids, alkaline phosphatase, cholesterol, triglycerides, urea, phosphorus and potassium and decrement of creatinine and globulin were noted.

A complete or almost complete recovery was observed for aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bile acids, bilirubin, urea, creatinine, globulin and potassium. Triglycerides, glucose and phosphorus did not show recovery. In addition, a slight increment in protein and albumin; an additional increment in cholesterol and increment of calcium were observed.

T4 showed a dose-related decrement in many animals. No relevant concomitant decrement of T3 was observed. One animal showed high levels of TSH (associated with low levels of T4).

At the end of recovery, females showed an increment of T4. No relevant changes of T3 were recorded. TSH was below the limit of quantification in all females.

At post mortem examination performed at the end of the dosing phase, swollen, enlarged and pale or dark aspect of the liver was observed. Liver showed the main organ weight alteration, with a marked increase recorded both for absolute and relative liver weight (2.9 and 3.8 fold, respectively).

Microscopically, the liver showed treatment-related changes such as diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia. This was occasionally associated with the presence of minimal to mild multifocal hepatocytic necrosis, minimal to mild multifocal accumulation of brownish pigment-laden macrophages and mild multifocal bile duct hyperplasia. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of smooth endoplasmic reticulum (See Maronpot et al, 2010).

With reference to hepatic enzyme activity, the mean activities (per g protein) of Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase activity was increased by 2.8 and 5 fold, respectively.

Small size of thymus, spleen, uterus and ovaries was additionally recorded at final sacrifice.

This was associated with consistent organ weight reductions recorded in spleen, heart and ovaries. A slight increase of the relative kidney weight was recorded, even though not consistently with the absolute weight. The ears in few animals were described as red in colour.

The following microscopic alterations were found in the other organs: in the kidneys, mild to moderate cortical tubular dilation; in the thymus: diffused minimal to severe cortical atrophy, resulting from loss of the lymphocytes, usually associated with multifocal lymphocytic necrosis; in the spleen: multifocal mild to moderate atrophy of the lymphoid tissue (i.e., involving the periarteriolar lymphoid sheath); in the bone marrow (sternum): diffused mild reduced cellularity (i.e., atrophy) of the hematopoietic tissue; in the uterus and ovaries, atrophy of the myometrium and follicles (i.e., atresia), respectively; in the ears mild to moderate necrosis of the epidermis and underlying subcutis usually seen at the tip of the ear.

At post mortem examination performed at the end of recovery period, treatment-related changes consisted of swollen, enlarged and pale or dark aspect of the liver which still showed increase of the weight both at relative and absolute level (2.1 and 2.2 fold,

respectively).

Microscopically, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia was noted, frequently associated with the presence of minimal multifocal hepatocytic necrosis.

At the end of recovery, the mean activities (per g protein) of Cytochrome p450 and cyanideinsensitive palmitoyl CoA oxidase activity was still increased by 2.9 and 11 fold, respectively. Consistently with the observation performed at the end of the dosing phase, slight increase of relative kidney weight, decrease of spleen and ovaries weights in females were still observed, even though no microscopic alterations were noted.

 

Females 60 mg/kg (mid dose)

No treatment related clinical signs were observed during the in life phase, with the exception of slight increase in rearing activity which recovered by the end of the study. A trend for reduction of body weight (not statistically significant) was recorded while food

consumption was unaffected by treatment.

At the haematology level, eosinopenia was observed and prothrombin time was decreased.

At the clinical pathology level, increment of transaminases enzymes, bile acids, cholesterol, triglycerides, urea and potassium and decrement of globulin and creatinine were observed.

T4 showed a decrement in many animals. No relevant concomitant decrement of T3, or alteration in TSH was observed.

At post mortem examination, swollen, enlarged and pale or dark aspect of the liver was observed with a high increase of liver weight recorded both for absolute and relative liver weight (2.4 and 2.7 fold, respectively).

At microscopic level, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted in the liver. This was occasionally associated with the presence of minimal to moderate multifocal hepatocytic necrosis, minimal to mild

hepatocytic degeneration (vacuolation) and minimal multifocal accumulation of brownish pigment-laden macrophages (seen in a female only).

With reference to hepatic enzyme activity, the mean activities (per g protein) of Cytochrome p450 was increased by 2.8 fold, when compared to controls. Occasionally (single animals), minimal pelvic cavity dilation, associated with minimal pelvic inflammation and pelvic epithelium hyperplasia, was noted in the kidney and minimal cortical atrophy was recorded in the thymus. Additionally, the ears in few animals were described as red in colour. At microscopic level, mild to moderate necrosis of the epidermis and underlying subcutis was usually seen at the tip of the ear.

 

Females 20 mg/kg (low dose)

No treatment related clinical signs were observed during the in life phase, with the exception of slight increase in rearing activity which recovered occurred by the end of the study. A trend for reduction of body weight (not statistically significant) was recorded while food consumption was unaffected by treatment.

At the haematology level, eosinopenia was observed and prothrombin time was decreased.

Clinical pathology, urinalysis parameters and thyroid hormones did not show any significant alterations.

At post mortem examination, swollen, enlarged and pale or dark aspect of the liver was observed with a high increase of liver weight recorded both for absolute and relative liver weight (both 2.0 fold). Additionally, the ears in few animals were described as red in colour.

At microscopic level, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted in the liver.

With reference to hepatic enzyme activity, the mean activities (per g protein) of Cytochrome P450 and cyanide-insensitive palmitoyl CoA oxidase activity was increased by 2.0 and 5 fold, respectively, when compared to controls.

Additionally, the ears in few animals were described as red in colour. At microscopic level, mild to moderate necrosis of the epidermis and underlying subcutis was usually seen at the tip of the ear.

In summary, the administration of cC6O4 for a 4-week period generates the following main changes:

- Toxicity in females was lower than in males at the same dose level tested. This was probably linked to a faster elimination of the test item (or at least of the free acid which is mainly excreted by the renal route).

- The liver was identified as main target organ. Liver damages consisted of treatment related dose-dependent degenerative microscopic findings associated with high increase of organ size and weight, threshold-like increase of liver enzymatic activity (Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase). The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of smooth endoplasmic reticulum. At the low doses, 5 mg/kg/day in males and 20 mg/kg in females, diffused moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, occasionally associated

with minimal to mild hepatocytic degeneration (vacuolation) was still noted.

- Secondary findings in a number of organs were recorded in males treated at 60 mg/kg/day and in females treated at 200/100 mg/kg/day, which reflect a non-specific high dose stress effect associated with a significantly reduced body weight loss.

- Partial, recovery of liver findings was found in the high dose animals after a 4 week recovery period (including the assayed enzymatic activities, macroscopic and microscopic findings). Additionally, some secondary findings in blood parameters and organ weights were also still observed in both sexes at the end of the study. This demonstrates that a 4 week recovery period is not sufficient to allow complete recovery from the findings observed after treatment in the experimental conditions used.

Based on the above results, it can be concluded that the NOAEL cannot be defined for both sexes in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
GLP-compliant study with Klimisch 1.
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

Two repeated dose toxicity studies by the oral route are available (OECD Guidelines 407 and 408):

- In a GLP 13-week repeated dose oral toxicity study conducted according to the OECD Guideline 408 (Key study, reliability 1), cC604 ammonium salt diluted in water was administered by gavage to groups of Sprague-Dawley rats (10/sex/dose) at the dose-levels of 0.3, 1, 3 or 10 mg/kg bw/day. Ten additional animals for each sex were included in the medium-high, high dose and control groups for a 6 consecutive weeks recovery assessment.

One male animal dosed at 3 mg/kg bw/day was found dead on Day 78 of the treatment period, but a clear cause of this death could not be established. During the study, there were no test item treatment-related clinical signs, no adverse effects during the Functional Observation Battery or at ophthalmology examinations. Body weight and food consumption were not affected by treatment with the test item. However, signs of evident treatment-related effects of the test item were observed in males dosed at 3 and 10 mg/kg bw/day and in females dosed at 10 mg/kg bw/day. The liver was identified as the main target organ, on the basis of the organ weights data (increased liver weights), clinical biochemistry and histopathological findings (hepatocytic hypertrophy with a dose-dependent increase in severity grades in male liver, while limited to the high dose female)). A second target organ appeared to be the thyroid, with morphological changes that were considered to be a secondary effect of the liver toxicity. The above effects showed a dose-related trend and appeared to be only partially reversible after the 6-week recovery period.

Changes observed in females dosed at 3 mg/kg bw/day were limited to the very slight increase in liver weights, which was fully reversible. Therefore, this change was not considered to be adverse. In the males, no adverse effects were seen following treatment at a dosage of 1 mg/kg bw/day, changes being limited to a very slight increase in relative liver weight associated to a minimal hepatocytic hypertrophy. No changes were observed in the females doses at 1 mg/kg bw/day and animals of both sexes dosed at 0.3 mg/kg bw/day.

In this study, the Lowest Observed Adverse Effect Level (LOAEL) was considered to be 3 and 10 mg/kg bw/day for males and females, respectively.

The test item cC604 ammonium salt, when administered daily to rats for 13 consecutive weeks to both sexes, showed a No Observed Adverse Effect Level (NOAEL) of 3 mg/kg bw/day in the females and 1 mg/kg bw/day in the males.

The study is appropriate for the purpose of classification and labelling and to derive DNELs for the risk assessment, considering the effects observed at high doses in rats are related to mechanisms known to be species-specific and with rats showing a greater sensitivity.

 

Supporting studies provided additional information on liver effects at higher doses.

In a GLP 4- week repeated dose toxicity study conducted according to OECD Guideline 407 (Supporting study, reliability 1), the toxicity of cC6O4 was investigated, when given daily by oral administration to the Sprague Dawley rat for a 4-week period followed by a recovery period of 4 weeks from the last administration. Males were dosed at 5, 20 and 60 mg/kg/day. Females were dosed at 20, 60 and, in the high dose group, 200 mg/kg/day from Day 1 to Day 19 and 100 mg/kg bw/day from Day 20 to Day 28. The following changes were observed:

- Toxicity in females was lower than in males at the same dose level tested.

- The liver was identified as main target organ. Liver damages consisted of treatment related dose-dependent degenerative microscopic findings associated with large increase of organ size and weight, and increased liver enzymatic activity (Cytochrome p450 and cyanide-insensitive palmitoyl CoA oxidase). The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of smooth endoplasmic reticulum. At the low doses, 5 mg/kg/day in males and 20 mg/kg in females, diffuse moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, occasionally associated with minimal to mild hepatocytic degeneration (vacuolation) was still noted. Bile duct hyperplasia was found in only one male of the high dose group.

- Secondary findings in a number of organs were recorded in males treated at 60 mg/kg/day and in females treated at 200/100 mg/kg/day, which are consistent with a non-specific high dose systemic stress effect associated with a significantly reduced body weight and body weight gain (Everds et al., 2013). Reduction of T4 levels are considered as secondary findings likely related to the increase in the enzymes of phase II involved in T4 catabolism (Zabka et al., 2011).

- Partial recovery of liver findings was found in the high dose animals after a 4 week recovery period (including the assayed enzymatic activities, macroscopic and microscopic findings). Additionally, some secondary findings in blood parameters and organ weights were also still observed in both sexes at the end of the study. This demonstrates that a 4 week recovery period is not sufficient to allow complete recovery from the findings observed after treatment in the experimental conditions used. Based on the above results, a NOAEL could not be established in this study.

 

In addition, the Reproduction/Developmental toxicity screening test (RTC 2012, OECD 421, rel.1, GLP) showed systemic effects that were consistent with the standard repeated dose toxicity studies. Groups of 10 males received cC6O4 ammonium salt by gavage at 5, 20 and 60 mg/kg bw/day and groups of 10 females received the test item at 5, 20 and 80 mg/kg bw/day. A similarly constituted group of animals received the vehicle alone (purified water) and acted as a control. One satellite group of 5 male and 5 female animals was dosed at 0.5 mg/kg bw/day for liver toxicity evaluation. Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of approximately 6 consecutive weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post-partum.

In this study, as in the 28 and 90-day toxicity studies, the treatment-related findings on the liver were seen at lower doses in males compared to females. Macroscopic and microscopic examinations revealed swollen liver, hepatocytic hypertrophy, single cell necrosis in males at 20 and 60 mg/kg/day and only at 80 mg/kg bw/day in females.

There was no treatment-related change at the histopathological examination of the liver performed in satellite group animals, dosed at 0.5 mg/kg/day for 28 consecutive days.

 

Overall, based on the results of the 28- (OECD 407 and OECD 421) and 90-day (OECD 408) studies in rat, the liver was identified as main target organ:

Besides some inter-study differences, in all the studies the incidence and/or severity of liver effects were usually lower in females compared to males treated at the same dose level.

At the higher doses (60 mg/kg/day both sexes and above in females) administered in the 28-day study diffuse moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was occasionally associated with minimal to mild hepatocytic multifocal necrosis. Moderate hypertrophy and minimal necrosis persisted in animals of the recovery group. Signs of severe hepatic toxicity also included alteration in the clinical biochemistry (including increase in serum levels of liver enzymes, cholesterol, bilirubin, bile acids in males and females).

There was no sign of hepatocytic necrosis in the 90-day study at up to 10 mg/kg/day, and the effects in that study were limited to hepatocytic hypertrophy, of minimal to moderate grade, consistent with the macroscopic swollen or enlarged aspect of the liver, and effects on clinical biochemistry were more limited, even at the highest dose.

The lesions in the rat liver are considered to be primary effects of the test compound. The rest of the changes seen in the other organs (kidneys, thymus, spleen, bone marrow, prostate, seminal vesicles, uterus, ovaries and ears) at doses starting from 20 mg/kg/day are considered related to non-specific high dose systemic stress effects, and they were concomitant with significantly reduced body weight and body weight gain.

Hepatic hypertrophy was demonstrated macroscopically by enlarged and swollen liver aspect, marked increase in liver weight, and microscopically by increase in size of hepatocytes, both in the 28-day and the 90-day studies. In the 28-day study liver enlargement and hepatocellular hypertrophy were associated with enhanced hepatic metabolic activity, as evidenced by marked increases in hepatic toxicity markers (ALAT, ASAT, alkaline phosphatase) in plasma, as well as increased activity of cytochrome P-450 and cyanide-insensitive palmitoyl CoA oxidase, and a higher total protein content in liver extracts. Most parameters increased in a dose-dependent manner, although changes were not always statistically significant. A partial reversibility was observed in the recovery groups.

Differences in liver lesions between males and females might be related to the large difference in plasmatic concentration of cC6O4 ammonium salt measured in the various toxicokinetic studies for the same dose administered: males had a higher plasmatic peak concentration (Cmax), and a larger AUC than females, indicating a greater systemic exposure to the substance, and of the liver. However, in the available studies with a single administration, most of the substance appeared excreted in urine within 24 to 48 hours, with only minor levels remaining in liver and kidneys in males, and no detection in those organs in females. In two reliable studies, there was no major difference observed between sexes in the plasma peak time, plasma elimination half-life or urinary half-life. In contrast, while females had a higher urinary clearance rate (2- to 4-fold compared to males).

 

Liver damages consisted of treatment related dose-dependent degenerative microscopic findings associated with significant increases of organ size and weight. The morphological aspect of the hepatocytic hypertrophy was described as consistent with proliferation of smooth endoplasmic reticulum (cytoplasmic eosinophilia), and the related increased P450 activity. In addition, the increased activity of cyanide-insensitive palmitoyl CoA oxidase is a marker of peroxisome proliferation and provides indirect evidence of PPARalpha involvement and likely activation (Maronpot et al., 2010). The rats are the most susceptible to peroxisome proliferation, followed by hamsters, monkeys, and dogs in that order (Doull et al, 1999, Rodricks et al, 1987). Female rats and hamsters are less susceptible than males. The rodents show a more pronounced response to peroxisome proliferators than to primates, including humans, due to differences in PPARalpha receptor gene expression, as well as its transcriptional response (Kane et al., 2006). Male rats seem to be the most sensitive). Therefore, liver effects consistent with increased peroxisome proliferation activity and PPARalpha activity as observed at high doses in male rats 28-day study can be considered as species-specific (Klaunig et al., 2003, Corton et al., 2014, 2018).

 

The reduction in circulating thyroid hormone is also a secondary effect of the increased activity of microsomal enzymes leading to increased catabolism and clearance (Hood et al., 1999, Ennulat et al., 2010). There was no effect on thyroid weight or morphology in the 28-day study. While in the 90-day study, males treated at the highest doses showed a follicle lining epithelium with a more columnar cell morphology and reduced colloid content, compared to control animals, which may indicate early signs of increased follicular activity. These changes were no longer present at the end of the recovery period for the dose 3 mg/kg/day, but still observed at 10 mg/kg/day. As there are also significant biological differences between rats and humans with regard to the regulation of thyroid hormones (longer half-life and bound reserve in human), the decrease in T4, without any significant associated modifications of T3 or TSH levels, was not considered a direct effect of the substance relevant for the risk assessment (Ennulat et al., 2010).

Justification for classification or non-classification

Based on the results of the 28 and 90 days studies in rats, the liver is considered as the target organ. Hepatotoxicity was associated with increase of organ size and weight, increase of liver enzymatic activity (Cytochrome p450 and cyanide-insensitive palmitoyl-CoA oxidase).The Lowest Observed Adverse Effect Level (LOAEL) was considered to be 3 and 10 mg/kg bw/day for males and females, respectively when administered daily to rats for 13 consecutive weeks to both sexes. However, effects involving peroxisome proliferation activity are recognised to be specific to rats and are not relevant for Humans. Therefore, based on expert judgment a classification as STOT RE2 (H373) is proposed according to EU criteria.