Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

EC Number:

682-238-0

Cas Number:

1190931-27-1

Molecular formula:

C6H4F9NO6

IUPAC Name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): cC604 (alternative name: C604 cyclic)
- Physical state: colourless liquid
- Analytical purity (in terms of dry salt): 37.6% (water solution)
- Composition of test material, percentage of components: Anti/(anti+sin) = 52.6% w; cC904NH4 = 0.4% w; Structural H = 20 ppm (as H); H by total organic contaminations < 10 ppm (as H); other fluorinated organic compound = 400 ppm (as F); H by total organic contaminations < 10 ppm (as H)
- Lot/batch No.: SPI-011-NH4-CA
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: In RTC Study nos. 82340 and 82350, 24 hour stability was verified in the range from 0.03 to 20 mg/mL.
- Storage condition of test material: ambient condition

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

0.3 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

3 mg/kg bw/day (nominal)

Doses / concentrations 5

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

3

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

4

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

Target system / organ toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.