Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Mar 2011 - 30 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

other: Amendment

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted in September 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.30 (Chronic Toxicity Studies)

Version / remarks:

adopted 1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: M.A.F.F N°8147 (Japan, notification 12 Nousan N°8147), adopted in November 2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

EC Number:

620-056-5

Cas Number:

874195-61-6

Molecular formula:

C14H13F3N4O5S

IUPAC Name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Green Hill 2001 Srl, Montichiari, Italy
- Age at study initiation: 8-9 months
- Weight at study initiation: 8.8-11.7 kg (males), 6.9-10.2 kg (females)
- Housing: individually in stainless steel kennels with a floor surface area of 2 m², pair housed overnight when possible (2 dogs from the same sex and dose group)
- Diet: certified canine meal 125C3-P1 (Scientific Animal Food and Engineering, Augy, France),
- Water: tap water, ad libitum
- Acclimation period: at least 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-21
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-03-10 To: 2012-03-30

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): ground diet
- Storage temperature of food: at room temperature

The test item was ground to a fine powder before being incorporated into the diet by dry mixing. Nine diet formulations (F1 to F9) were prepared at each dose level to provide the treated diet required for the study. Test diet formulations at 800 ppm were stored at room temperature and those at 300 and 100 ppm were stored at approximately -18 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in the diet at 600 and 15000 ppm was determined in a previous study under storage and usage conditions similar to those of the current study. Stability of the test substance in the diet at 100 ppm was determined during the chronic study for a period that covers the storage conditions used. Homogeneity was verified for all concentrations on the first preparation of the first formulation and for the lowest and highest concentrations on the first preparation of the fifth formulation to demonstrate adequate formulation procedures. In addition, the homogeneity of the second load of the first formulation (F1) at 100 ppm was checked in error, due to a mistake of labeling or of order of formulation.

Results:
AE 1887196 was stable at concentrations of 600 and 15000 ppm in the dry diet stored for at least 59 days at room temperature. In addition, the stability of the test substance at concentrations of 600 and 15000 ppm in the moistened diet (as distributed to the dog) was demonstrated for a period of 4 hours which corresponded approximately to the time of food preparation and distribution. The test substance was found to be stable at 100 ppm in the dry diet stored frozen for up to 50 days followed by 15 days of storage at room temperature. In addition, the stability of AE 1887196 at concentrations of 100 ppm in the moistened diet as distributed to the dog was demonstrated for a period of 4 hours which corresponded approximately to the time of food preparation and distribution.

Homogeneity reached 88-101% of the nominal concentration and measured concentrations of the formulations ranged between 86 and 99% of nominal concentrations and were therefore within the in-house target ranges (85-115%). According to the available results, homogeneity and concentration of AE 1887196 in diet mixtures were acceptable.

Duration of treatment / exposure:

1 year

Frequency of treatment:

daily (7 days/week)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 300 and 800 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2.8, 8.8 and 21.9 mg/kg bw/day (males)
Basis:
other: mean dose value as calculated from food intake

Doses / concentrations 3

Remarks:

Doses / Concentrations:
3.0, 9.7 and 24.3 mg/kg bw/day (females)
Basis:
other: mean dose value as calculated from food intake

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dose levels were selected based on the results from a previous subacute toxicity study in dogs
- Rationale for animal assignment (if not random): similar body weight distribution among groups for each sex, whilst ensuring full siblings were not placed in the same treatment group (computerized randomization procedure (Pristima System, version 6.1.0 Build 19))

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs: at least once daily; detailed physical examinations: weekly in an open area; behavioral changes, ill-health, moribundity and mortality: twice daily or once daily on weekends and public holidays
- Elevated parameters: general behavior and appearance, skin and fur, teeth and gum, eyes, ears, mucous membranes, gait, posture and response to handling

PHYSICAL EXAMINATION: Yes
- Time schedule: monthly except on Study months 4 and 12 (in error)
- Elevated parameters: fur and skin, eyes, ears, teeth, gum, mucous membranes, rectal temperature, gait, stance, general behavior, chest including heart and respiratory rate, abdomen including palpitation, external genitalia and mammary glands, mental state, posture, gait and motor function, muscle tone, postural reactions, spinal nerve reflexes, sensation and cranial nerve reflexes

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimatisation phase and treatment period, and before final necropsy (terminal body weight)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimatisation period and at the end of treatment
- Dose groups that were examined: all animals from all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to dosing and on Study days 87-88, 178-179 and 360-361
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters checked: red blood cell count, white blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count and % reticulocytes, leukocyte differential count, platelet count, activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to dosing and on Study days 87-88, 178-179, 248-249 and 360-361
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters checked: total bilirubin, glucose, urea, creatinine phosphokinase, total protein, total cholesterol, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities, cholinesterase, creatinine, lactic acid dehydrogenase, Gamma glutamyltransferase, sorbitol dehydrogenase, glutamate dehydrogenase, albumin, globulins, trigylcerides and serum protein electrophoresis, calcium, chloride, inorganic phosphorous, potassium and sodium

URINALYSIS: Yes
- Time schedule for collection of urine: prior to dosing and on Study days 85, 176-177, 354, 358-359 in the morning
- Animals fasted: access to water was restricted
- Parameters checked: appearance, volume, specific gravity, osmolality, refractive index, pH, sediment, protein, glucose, ketones, bilirubin, blood/red blood cells, nitrate, urobilinogen

OTHER:
Bioanalytical examination:
- Time schedule: test animals: at the end of the study just prior to food distribution and 1, 2 and 4 hours after food distribution (2 dogs/sex/group); . control animals: at the end of the study just prior to food distribution (2 dogs)
-Elevated parameter: plasma was prepared for determination of the test item and its major metabolites

Sacrifice and pathology:

SACRIFICE: Study days 365-368 - 103 (animals were diet fasted prior to sacrifice)
GROSS PATHOLOGY: Yes
- Parameters checked: tongue, submandibular (salivary) gland, esophagus, stomach, duodenum, jejunum, ileum, ceacum, colon, rectum, liver, pancreas, gallbladder, trachea, lung, pharynx, larynx, aorta, heart, bone marrow, sternum, lymph node (retropharyngeal and mesenteric), spleen, thymus, kidney, urinary bladder, testis, epididymis, prostate gland, ovary, uterus (with cervix), mammary gland, vagina, oviduct, brain, sciatic nerve, spinal cord (cervical, thoracic and lumbar), eyes, optic nerves, pituitary gland, adrenal gland, parathyroid gland, thyroid gland (weighed with parathyroid), bone, sceletal muscle, skin, all gross lesions, articular surface (femur head)

HISTOPATHOLOGY: Yes
- Parameters checked: tongue, submandibular (salivary) gland, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, liver, pancreas, gallbladder, trachea, lung, aorta, heart, bone marrow, sternum, lymph node (retropharyngeal and mesenteric), spleen, thymus, kidney, urinary bladder, testis, epididymis, prostate gland, ovary, uterus (with cervix), mammary gland, vagina, oviduct, brain, sciatic nerve, spinal cord (cervical, thoracic and lumbar), eyes, optic nerves, pituitary gland, adrenal gland, parathyroid gland, thyroid gland (weighed with parathyroid), bone, sceletal muscle, skin, all gross lesions, articular surface (femur head)

Statistics:

Mean and standard deviations were calculated for each group. All statistical analyses were carried out separately for males and females using Path/Tox System V4.2.2. (Module Enhanced Statistics). For determination of statistically significant differences, the data were analysed with Bartlett Test followed by ANOVA Test and DUNNETT Test (if not significant in BARTLETT Test) or KRUSKAL-WALLIS Test and DUNN Test (if significant in BARTLETT Test). Quantitative urinalysis parameter (pH) were analysed with KRUSKAL-WALLIS TEST followed by DUNN TEST in case of a significant result. If one or more group variance(s) equaled 0, means were compared using non-parametric procedures. Comparison between the high-dose group and the control group was performed with F test combined with Modified t-test (in case of a significant result) or T-test (in case of a non-significant result).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the high dose females, there was a minor decrease of 11% in mean food consumption throughout the study in comparison to the controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Lower mean total cholesterol concentrations in males at 800 ppm; lower mean albumin concentrations, lower total protein concentrations and albumin/globulin ratio at 800 and 300 ppm in both sexes; data not always statistically significant

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher mean absolute and relative liver weights were considered to be toxicologically relevant only in males at 800 ppm and in females at 800 and 300 ppm, since these changes were associated with histopathological findings.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological findings were observed in the liver and thyroid gland.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities during the study.
At 800, 300 and 100 ppm, no treatment-related clinical signs were noted in either sex. The clinical signs recorded in both sexes were those commonly recorded in untreated dogs of this strain and age or distributed between different groups with no evidence of a dose-related effect and/or with sporadic occurrence and were thus considered not to be related to AE 1887196 administration. Some females from different groups, including the control, were recorded with genital discharge, which correlated with animals being in estrus at the time of clinical examination.
No changes at physical examination were noted in either sex. Rectal temperature recordings in all animals revealed no treatment-related changes.

BODY WEIGHT AND WEIGHT GAIN
At 800, 300 and 100 ppm, mean body weight parameters were unaffected by the treatment in both sexes throughout the study. The few changes observed had no dose-relationship and were thus considered to be incidental.

FOOD CONSUMPTION AND COMPOUND INTAKE
At 800 ppm in females, mean food consumption was marginally reduced during the first week of treatment (-20%, not statistically significant) and at several other occasions during the study (maximum of -26%, not statistically significant), when compared to the controls. Overall in the high dose females, there was a minor decrease of 11% in mean food consumption throughout the study in comparison to the controls.
At 800 ppm in males and at 300 and 100 ppm in both sexes, mean food consumption was unaffected by treatment throughout the study. The few increases in weekly mean food consumption noted in the high and mid dose male groups compared to the controls were considered to be incidental and within the range of expected variations for Beagle dogs of this age kept under laboratory conditions.

OPHTHALMOSCOPIC EXAMINATION
No changes at the ophthalmological examination were noted at any dietary level in either sex.

HAEMATOLOGY
No treatment-related changes were noted at any dietary level in either sex throughout the study.
The few differences noted, even if statistically significant, were not considered to be treatment-related in view of their incidental or sporadic occurrence and/or their low magnitude.

CLINICAL CHEMISTRY
Higher mean alkaline phosphatase activities were observed at all dietary levels in both sexes throughout the study compared to the controls, though no clear dose-effect relationship was seen between 800 and 300 ppm in females. The differences were lower in males than in females.
At 100 ppm in both sexes, the increase in mean alkaline phosphatase activities was not considered to be an adverse effect, because it was the only clinical pathology alteration potentially indicative of liver toxicity and had no associated histopathological changes.
Slightly lower mean total cholesterol concentrations were noted at 800 ppm in males only: -29%, -40%, -34% and -30% at Month 3, 6, 9 and 12, respectively, compared to the controls (p ≤ 0.05).
Lower mean albumin concentrations were observed at 800 and 300 ppm in males (between -9 and -13%) and in females (between -10 and -21%), compared to the controls. As a consequence, lower total protein concentrations (between -5 and -10% in males and -3 and -16% in females) and lower albumin/globulin ratios (between -5 and -13% in males and -10 and -18% in females) were noted in these groups. As the data were non-homogeneous, the differences were not always statistically significant. Lower mean total bilirubin concentrations were noted at 800 and 300 ppm in males and at any dietary level in females, compared to the controls. However, lower total bilirubin concentrations are not considered to be adverse effects of the test item as they do not represent any functional impairment to the test organism. Other changes, even if statistically significant, were not considered to be treatment-related in view of their sporadic occurrence.

URINALYSIS
No relevant change was noted in the parameters assayed. The few differences noted were not considered to be treatment-related in view of their incidental or sporadic occurrence and/or their low magnitude.

TERMINAL BODY WEIGHT AND ORGAN WEIGHTS
There were no treatment-related changes in mean terminal body weight. The lower mean terminal body weight observed in high dose females (-6% compared to the controls, not statistically significant), was in line with the lower mean body weight seen at start of treatment for this group (-8% compared to the controls, not statistically significant). At 800 and 300 ppm in males and at all doses in females, a tendency towards higher mean absolute and relative liver weights was observed, when compared to the controls (not statistically significant).
These changes were considered to be toxicologically relevant only in males at 800 ppm and in females at 800 and 300 ppm, since they were associated with histopathological findings. The few other organ weight changes were considered to be incidental and not treatment-related.

GROSS PATHOLOGY
There were no treatment-related macroscopic changes in treated animals, when compared to the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related histopathological findings were observed in the liver and thyroid gland.
In the liver, hepatocellular hypertrophy (with eosinophilic cytoplasmic change) was noted at 800 ppm in both sexes and at 300 ppm in females. Concomitantly, a slight decreased incidence and/or severity of hepatocellular glycogen accumulation was noted at 800 and 300 ppm in both sexes.
In the thyroid gland, minimal follicular cell hypertrophy was noted at 800 ppm in one female only.

BIOANALYTICAL EXAMINATION
AE 1887196 individual plasma concentrations on Month 12 prior to food distribution, and 1, 2 and 4 h after food distribution were below the method validation limit of 0.1 mg/L in all dose groups.
For the metabolite BCS-AA10030, individual plasma concentrations at the same time points were below the validation limit of 0.5 mg/L in all dose groups. For the metabolite BCS-BQ87904, individual plasma concentrations were below the validation limit of 0.5 mg/L prior to food distribution in all dose groups and after food distribution in the low and mid dose groups. In the high dose groups, concentrations after food distribution ranged from the validation limit of 0.5 mg/L up to a maximum of 1.08 mg/L and were similar at the 3 times points (1, 2 and 4 h) .
For the metabolite BCS-BP19252, individual plasma concentrations were similar between the two sexes and at the 4 times points (prior to food distribution and 1, 2 and 4 h after food distribution) and ranged from the validation limit of 0.5 mg/L up to 1.36 mg/L in the low dose groups, from 1.07 mg/L up to 4.95 mg/L in the mid dose groups and from 1.69 mg/L up to 11.0 mg/L in the high dose groups.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on liver effects (reduced absolute and relative liver weights; hepatocellular hypertrophy)

2

Dose descriptor:

NOAEL

Effect level:

2.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: based on liver effects (reduced absolute and relative liver weights; hepatocellular hypertrophy); corresponds to a nominal dose of 100 ppm as calculated for males in regard to group mean food consumption and group mean body weight

3

Dose descriptor:

NOAEL

Effect level:

3.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: based on liver effects (reduced absolute and relative liver weights; hepatocellular hypertrophy); corresponds to a nominal dose of 100 ppm as calculated for females in regard to group mean food consumption and group mean body weight

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 Table 1: Body weight and weight gain (kg)

		Dose level (ppm)
		Control	100	300	800
Males	Initial BW (Day 1) (%C)	10.1 ± 0.4	0.2 ± 0.6 (101%)	10.5 ± 0.4 (104%)	10.4 ± 1.3 (103%)
	BWG Week 1 (Days 1-8)	0.1 ± 0.2	-0.1 ± 0.1	-0.2 ± 0.3	-0.2 ± 0.4
	BWG Weeks 1-13 (Days 1-92)	1.4 ± 0.4	0.7 ± 0.5	0.4 ± 1.0	1.1 ± 0.5
	BWG Weeks 13-26 (Days 92-183)	0.6 ± 0.5	0.3 ± 0.5	0.3 ± 0.5	0.7 ± 0.4
	BWG Weeks 26-52 (Days 183-364)	0.6 ± 1.3	-0.3 ± 0.2	0.3 ± 0.7	0.9 ± 0.6
	BWG Weeks 1-52 (Days 1-364)	2.6 ± 2.0	0.7 ± 1.0	1.0 ± 1.9	2.7 ± 1.1
	Final BW (Day 364) (%C)	12.7 ± 2.2	10.9 ± 1.3 (86%)	11.5 ± 2.1 (91%)	13.1 ± 1.7 (103%)
Females	Initial BW (Day 1) (%C)	8.3 ± 1.4	8.3 ± 1.3 (99%)	7.9 ± 1.0 (95%)	7.7 ± 0.8 (92%)
	BWG Week 1 (Days 1-8)	0.0 ± 0.1	-0.2 ± 0.2	0.1 ± 0.1	-0.1 ± 0.1
	BWG Weeks 1-13 (Days 1-92)	0.6 ± 0.6	0.8 ± 0.3	1.2 ± 0.4	0.9 ± 0.4
	BWG Weeks 13-26 (Days 92-183)	0.2 ± 0.2	0.7 ± 0.5	0.6 ± 0.6	0.3 ± 0.4
	BWG Weeks 26-52 (Days 183-364)	1.0 ± 0.4	0.3 ± 0.1	0.6 ± 1.0	0.6 ± 0.4
	BWG Weeks 1-52 (Days 1-364)	1.8 ± 1.1	1.8 ± 0.9	2.4 ± 1.7	1.8 ± 0.4
	Final BW (Day 364) (%C)	10.1 ± 2.3	10.1 ± 0.9 (100%)	10.3 ± 2.5 (102%)	9.5 ± 1.0 (94%)

BW: body weight; BWG: body weight gain; %C: % versus control, calculated on raw data

 

Table 2: Mean food consumption (g/day)

		Dose level (ppm)
		Control	100	300	800
Males	Week 1 (days 1-8) (%C)	767	739 (96%)	779 (102%)	750 (98%)
	Week 1-52 (days 1-364) (%C)	767	744 (97%)	793 (103%)	791 (103%)
Females	Week 1 (days 1-8) (%C)	673	636 (94%)	614 (91%)	539 (80%)
	Week 1-52 (days 1-364) (%C)	719	680 (95%)	737 (102%)	643 (89%)

%C: % versus control

 

Table 3: Compound intake: The mean dosage intake of AE1887196 per group for each sex was as follows:

		Mean achieved dietary intake of AE 1887196 [mg/kg bw/d]
Dosage level of AE1887196		100	300	800
Males	Week 1 to 13	3.0	9.3	24.4
	Week 1-52	2.8	8.8	21.9
Females	Week 1-13	3.2	10.4	25.3
	Week 1-52	3.0	9.7	24.3

Table 4: Organ weights

INCIDENCE AND SEVERITY OF MICROSCOPIC CHANGES IN THE LIVER
Sex	Males				Females
Dosage level [ppm]	Control	100	300	800	Control	100	300	800
Absolute liver weight (g)	383.78 ± 77.213	336.02 ±45.458 (-12%)	411.02 ±53.465 (+7%)	429.85 ±74.562 (+12%)	304.92 ±62.100	329.29 ±53.712 (+8%)	330.30 ±68.990 (+8%)	363.19 ±44.998 (+19%)
Liver to body weight ratio (%)	3.19 ±1.171	3.18 ±0.872 (0%)	3.73 ±0.706 (+17%)	3.30 ±0.474 (+3%)	3.09 ±0.385	3.27 ±0.401 (+6%)	3.29 ±0.312 (+6%)	3.92 ±0.598 (+27%)
Liver to brain weight ratio (%)	473.58 ±91.697	443.84 ±72.354 (-6%)	534.07 ±48.239 (+13%)	561.60 ±96.386 (+19%)	427.87 ±97.954	455.67 ±67.570 (+6%)	423.53 ±85.174 (-1%)	505.23 ±44.866 (+18%)

 

Table 6: Microscopic changes in the liver

INCIDENCE AND SEVERITY OF MICROSCOPIC CHANGES IN THE LIVER
Sex	Males				Females
Dosage level [ppm]	Control	100	300	800	Control	100	300	800
Number of examined animals	4	4	4	4	4	4	4	4
Hepatocellular hypertrophy: centrilobular to panlobular
Minimal	0	0	0	3	0	0	3	1
Slight	0	0	0	1	0	0	0	3
Total	0	0	0	4	0	0	3	4
Hepatocellular glycogen accumulation: Mainly centrilobular
Minimal	0	3	1	2	0	0	2	2
Slight	1	0	0	0	2	4	2	0
Moderate	2	1	0	0	1	0	0	0
Marked	0	0	0	0	1	0	0	0
Total	3	4	1	2	4	4	4	2

 

Table 7: Microscopic changes in the thyroid galnd

INCIDENCE AND SEVERITY OF MICROSCOPIC CHANGES IN THE THYROID
Sex	Males				Females
Dosage level [ppm]	Control	100	300	800	Control	100	300	800
Number of examined animals	4	4	4	4	4	4	4	4
Follicular cell hypertrophy
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1