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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May - 29 Jun 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

ORSZÄGOS GYÖGYSZERESZETI INTEZET, National Institue of Phyrmacy, Budapest, Hungary

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:

rat

Strain:

other: Crl:Wistar rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 302-341 g (males), 195-236 g (females)
- Housing: 5 animals of the same sex per cage in type II and/or III polycarbonate cages; individual caging was employed after the onset of treatment to avoid ingestion of the jackets/bandage/test item between the animals housed in the same cage; deep wood sawdust bedding was used (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Germany).
- Diet: ssniff® SM R/M “Autoclavable Complete diet for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6–24.3
- Humidity (%): 30-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark/hrs light): 12/12

IN-LIFE DATES: From: 2012-05-31 To: 2012-06-29

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: unchanged (the test substance was moistened sufficiently with water (no more than 300 µL))

Details on exposure:

TEST SITE
- Area of exposure: clipped flank (area starting approximately at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal
- % coverage: 10% of body surface area
- Type of wrap if used: test substance was held in contact with the skin with a porous gauze dressing (less than or equal to 8 plies); the test site was further covered with non-irritating tape to retain the gauze dressing
- Time intervals for shavings or clipplings: approximately 24 hours before the test (repeated as required and as practical depending upon hair growth of individual animals and/or the effects observed)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test substance was gently wiped from the skin with lukewarm water
- Time after start of exposure: 6 hours

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Lomir jackets with inserts and collars as required), but no complete immobilization was used

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily (7 days/week)

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

10

Control animals:

other: control animals were similarly treated with gauze patches and restrainers (Lomir jackets with inserts and collars).

Details on study design:

- Route selection rationale: The dermal route was selected to provide information for hazard assessment as skin contact is one of the possible routes of human exposure.
- Dose selection rationale: Based the absence of signs of toxicity following a single dermal application of 2000 mg/kg bw for 24 hours to the shaved flank of male and female Wistar rats, the applied concentrations were selected.
- Animal assignment: according to body weight

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: inspection for morbidity and mortality: twice daily, general clinical observation: at least daily, detailed examinations performed outside the home cage: prior to first exposure and and once weekly thereafter

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily, following patch removal

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during the last week of exposure (Day 26)
- Dose groups that were examined: prior to treatment: all animals, Day 26: control and high-dose groups (as no effects were observed in the high-dose group, the low- and mid-dose groups were not further examined)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy on Day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hgb), haematocrit (Het), mean corpuscular (erythrocyte)
volume (MCV), mean corpuscular (erythrocyte) haemoglobin (MCH), red cell (erythrocyte) volume (RDW), platelet (thrombocyte) count (Plt), mean platelet (thrombocyte) volume (MPV), reticulocyte count (RETIC), white blood cell (leukocyte) count (WBC); coagulation: activated partial thromboplastin time (APTT), prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy on Day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, total bilirubin concentration (T-BIL), urea, cholesterol (Chol), triglycerides (TRIG), creatinine concentration (CREAT), phosphorus concentration (Phos), sodium, potassium, calcium and chloride concentrations, total protein concentration (Tot Prot), albumin (Alb), Alb/glob ratio (A/G), aspartate aminotransferase activity (U/L), alanine aminotransferase activity (U/L), alkaline phosphatase activity (ALKP), gamma glutamyltransferase activity (GGT), bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy on Day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: leukocyte (LEU), nitrite (NIT), pH, protein (Pro), glucose (GLU), urobilinogen (UBG), bilirubin (BIL), ketones (KET), blood/erythrocytes (BLD/ERY), specific gravity (SG), sediment (SED), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of exposure (on Day 26)
- Dose groups that were examined: all dose groups
- Battery of functions tested: functional observation battery / grip strength / motor activity / other: measurements of landing foot splay, sensory reactivity

OTHER:
Examination of vaginal smears prior to necropsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.
Organs checked: brain, epididymides, heart, kidney, liver, spleen, prostate incl. seminal vesicles with coagulating glands, testes, thymus, uterus incl. cervix, adrenals, ovaries, pituitary and thyroids with parathyroids
HISTOPATHOLOGY: Yes (full histopathology was performed in the control and high-dose group and in all organs with macroscopic abnormalities.
Organs checked: gross findings, larynx, skeletal muscle (quadriceps), treated skin area, liver (all dose groups), skin and subcutis (inguinal), adrenals, lungs with bronchi, small intestine, lymph nodes, spinal cord, aorta, mammary gland, spleen, brain, nose/nasal cavity, sternum with marrow, epididymides, ovaries with oviduct, stomach, eyes with the optic nerves, pharynx, testes, external lachrymal glands, pancreas, thymus, oesophagus, pituitary, thyroid with parathyroids, femur with marrow, prostate, tongue, harderian glands, salivary glands, trachea, heart, sciatic nerve, urinary bladder, kidneys, seminal vesicles with coagulating glands, uterus, large intestine, vagina

Statistics:

Mean and standard deviations were calculated and statistical analysis was performed using SPSS PC+4.0. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

all dose groups: slight skin lesions (scab and scar) were observed due to friction with the restraint (non-adverse)

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day: transient statistically increased body weight in females during Week 3 (non-adverse)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

males: all dose groups: lower PTT values; 500 mg/kg bw/day: lower WBC counts and higher platelet counts, 1000 mg/kg bw/day: higher platelet counts (non-adverse); females: 250 mg/kg bw/day: lymphocyte count statistically different (non-adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increased cholesterol in males and females: all dose groups: increased creatinine (non-adverse)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

males, all dose group: slight differences in urine specific gravity (non-adverse)

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

grip strength test: increased values for males from all dose groups and high-dose females (non-adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increased relative liver weight in males and females, increased weight of thyroid glands in females (non-adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

all dose groups: incidental gross abnormalities (non-adverse)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: minimal centrilobular hepatocellular hypertrophy and minimal/mild diffuse hepatocellular vacuolation in males and females

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality or clinical signs of toxicity were observed during the study period. No signs of skin irritation were noted. Occasionally slight skin lesions (scab and scar) were observed with similar low incidence across the groups in males (2/10, 1/10, 1/10 and 0/10) and females (3/10, 0/10, 1/10 and 0/10) in the control, low, mid and high dose groups, respectively. These observations were ascribed to minor effects of friction with the restraint and not related to the test item.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related effect on body weight and weight gain was noted. Due to the transient occurence, the statistically significant higher body weight gain in mid-dose females only during Week 3 (p < 0.05) is not considered as toxicologically significant or related to treatment (Table 1).

FOOD CONSUMPTION
Food consumption was comparable among the groups.

OPHTHALMOSCOPIC EXAMINATION
Opthalmology did not reveal differences between control and test groups.

HAEMATOLOGY
Variations, attaining statistical significance were noted in males in lower WBC counts in the mid-dose group (p < 0.05) and higher platelet counts in the mid- and high-dose groups (p < 0.05 and p < 0.01, respectively). Lower PTT values were noted in all treated males, due to relatively high variations in the control individual values. Furthermore, statistical difference (p < 0.05) was noted for relative lymphocyte count in low-dose females (Table 2). As all parameters fall within the normal physiological and historical control ranges for the age, strain and sex of rats, these effects are not considered as adverse.

CLINICAL CHEMISTRY
High-dose males and females revealed a slight but statistically significant increase in cholesterol concentration around 23-24% , respectively. In mid-dose females, 2/10 animals reached cholesterol values above the control range. As both values still remained within the physiological range, the effect in the mid-dose group is not considered as adverse. Creatinine levels were slightly increased in all dose groups. In the high-dose group, no more than 17-19% difference was noted without any effect on urea concentration in males. As the observed changes in creatinine were of small magnitude, the mean and individual values remained within normal ranges and no consequent effect on urea concentration was observed, the effect on creatinine is not considered as adverse (Table 3). Other minor variations noted in chloride concentration, total protein or urea achieved statistical significance occasionally (Table 2). Due to the absence of consistent patterns or other correlates, these changes were regarded as incidental or individual findings, and are therefore not considered as treatment-related or of toxicological significance.

URINALYSIS
Slight differences were observed in urine specific gravity in males from all dose groups, which attained statistical significance in low- and high-dose males (p < 0.05 and p < 0.01, respectively). As no dose-dependency was present, and due to the lack of any evident renal pathology, these observations were regarded as incidental findings.
No abnormalities were observed in the remaining evaluated parameters.

NEUROBEHAVIOUR
Increased values were noted in grip strength tests for hind limb in all treated male groups and in females of the high-dose group compared to the controls. The differences reached statistical significance in mid-dose males and high-dose females (p < 0.05), but were considered to be without toxicological significance. Increased vocalization was observed on occasion throughout all the dose groups at the modified Irwin test (functional observation battery). However, as there were no treatment-related differences to the control, and the incidence was similar in all groups and no dose-, or gender-related responses were noted, these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations. Thus, no toxicologically significant changes in behaviour, general physical condition, or neurobehaviour (including the reactions to different type of stimuli, grip strength or motor activity) were noted on Day 26.

ORGAN WEIGHTS
Test item related organ weight differences were found in liver (males and females) and thyroid (females) weights in the high-dose group. As the increased liver weights in low- and mid-dose animals were of low magnitude (3-10%) without any morphological change or adverse effect in clinical pathology, they were considered as adaptive (Table 1). Thyroid weights were dose-dependently increased in females of the mid-and high-dose group. The increase in the mid-dose group was of low magnitude, with almost all individual weights lying in the control range. Compared to control, the thyroid weights in the high-dose females were significantly elevated by approximately 22% (absolute value) and 19-21% (relative values). 4 of 10 individual values were above the control, but within the physiological range. As the changes were not associated with any morphological change suggesting increased TSH activity or utilization of thyroglobulins, the observations on the thyroid weights were not considered adverse. Absolute kidney weights were slightly increased in all treatment groups of both sexes by approximately 4-9%. Differences were statistically significant in high-dose males. However, as the differences remained minor and were within the physiological range, they were not considered as adverse. No biologically significant differences were observed among the groups in the weights of the remaining evaluated organs compared with the control, although statistically significant variations without trend were noted (i.e thymus). Therefore, in the absence of a dose- or gender-related response, or consistent correlation with histopathological results, these variations were not considered to reflect an adverse effect.

GROSS PATHOLOGY
Multifocal/focal dark red discoloration of the thymus was observed in 1/10 control female and 2/10 high-dose males which was regarded as terminal procedure related. Furthermore, multifocal/focal dark red/brown discolouration of glandular mucosa of the stomach (1/10 mid-dose female), enlarged adrenal (1/10 mid-dose female), unilateral enlargement of the testes (1/10 mid-dose male) were observed. Due to the low incidence and missing dose-response, these findings are regarded as incidental. Unilateral pelvic dilatation of the kidney (2/10 high-dose males) or dilated uterine horns (1/10 control and 2/10 high-dose females) were observed in single animals. As these variations represent common observations in Wistar rats, they are not interpreted as incidental and not adverse due to the incidence. In addition, at external examination, small scars, or scabs on the dorsal neck, thoracic region and on forelimbs were observed with low incidence in control and mid-dose males which were regarded as procedure related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were observed in the liver of high-dose males and females visible as minimal/mild, periportal/diffuse hepatocellular vacuolation which was found in 3/10 males and females. Minimal centrilobular hepatocellular hypertrophy was observed in 3/10 males and in 2/10 females. As the changes were correlated with increased organ weights, they are considered as treatment-related. No similar observations were made in animals of the lower dose groups. Examination of the skin revealed signs of minimal, multifocal hyperkeratosis/parakeratosis which was observed in 7/10 and 3/10 high-dose males and females, respectively, in 1/10 mid-dose males and in 3/10 control females (skin in the low- and mid-dose animals were not examined, except necropsy findings). In addition, focal purulent inflammation and mixed cellular infiltrate in the skin were observed in 3/10 control females. As these abnormalities occured also in the control group, the changes were considered to be procedure- and not test item-related. Changes observed in various organs including the prostate (only in the control), kidney (1/10 control and 1/10 high-dose male and 1/10 control female), testis (1/10 control and 1/10 mid-dose male, but not in the high-dose group) or thymus (2/10 control and 2/10 high-dose males and 1/10 control female) were regarded as incidental because of the randomly distribution across control and treated groups. Microscopic findings in the uterus (like luminal dilatation and glandular proliferation) seen in 1/10 control and 2/10 high-dose females were related to typical physiological changes observed during oestrus cycle in female rats and are therefore not toxicologically significant and/or associated with test item administration.

OTHER FINDINGS
Oestrus evaluation: All animals showed normal distribution of oestrus phases.

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on adverse effects observed on the liver (minimal/mild, periportal/diffuse hepatocellular vacuolation and minimal centrilobular hepatocellular hypertrophy correlated with increased liver weight and increased cholesterol in both sexes)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Body weight [g]

Sex	Dose group (mg/kg bw/day)	Treatment day
		0	7	14	21	27
Male	Control	319.3 ± 11.5	357.8 ± 17.2	383.6 ± 20.5	414.3 ± 23.4	423.1 ± 24.8
	250	320.1 ± 10.0	361.9 ± 14.0	389.8 ± 19.3	422.3 ± 23.1	433.2 ± 23.6
	500	318.0 ± 8.4	355.7 ± 9.6	381.1 ± 15.1	411.1 ± 14.3	419.5 ± 17.1
	1000	319.9 ± 12.8	361.0 ± 15.5	389.7 ± 19.2	420.0 ± 28.0	427.6 ± 29.7
Female	Control	214.5 ± 11.9	228.3 ± 17.7	236.8 ± 20.5	243.7 ± 17.5	246.1 ± 20.7
	250	215.0 ± 10.8	232.0 ± 14.2	244.9 ± 23.9	253.4 ± 24.1	259.0 ± 26.4
	500	216.2 ± 10.7	228.1 ± 13.6	238.4 ± 14.9	255.5 ± 20.7	254.8 ± 28.3
	1000	210.2 ± 10.8	227.0 ± 13.1	241.5 ± 11.6	253.2 ± 11.7	252.2 ± 10.9

Table 2: Haematology and Clinical Chemistry: mean values of altered parameters on Day 28.

Sex	Dose group (mg/kg bw/day)	Haematology				Clinical Chemistry			Urinalysis
		WBC [K/µL]	PLT [K/µL]	PTT [seconds]	LYMPH [%]	Chloride [mmol/L]	Tot. Prot. [g/L]	Urea [mmol/L]	Specific Gravity
Male	Control	4.533 ± 1.333	1062.5 ± 94.2	22.01 ± 1.19	66.130 ± 6.815	103.51 ± 1.20	54.78 ± 1.82	5.749 ± 0.868	1.0091 ± 0.0007
	250	5.271 ± 2.260	1035.8 ± 188.4	21.18* ± 0.47	67.140 ± 3.940	101.71** ± 0.94 (DN)	54.96 ± 1.71	5.323 ± 0.474	1.0107* ± 0.0021
	500	3.370* ± 0.796 (U)	1157.1* ± 65.6 (U)	20.97* ± 0.93 (DN)	65.150 ± 7.770	102.89 ± 1.20	56.23 ± 1.47	6.040 ± 0.599	1.0109 ± 0.0028
	1000	4.982 ± 1.786	1217.9** ± 112.8 (U)	21.26 ± 0.69	71.380 ± 4.818	103.29 ± 1.95	56.72* ± 2.21 (DN)	5.522 ±	1.0125** ± 0.0030
Female	Control	1.818 ± 1.003	1144.4 ± 209.4	23.03 ± 0.78	65.200 ± 10.071	103.47 ± 1.41	58.01 ± 2.28	6.748 ± 0.834	1.0143 ± 0.0075
	250	2.517 ± 1.131	1119.4 ± 138.3	23.08 ± 0.95	74.250* ± 6.945 (DN)	102.83 ± 1.72	56.84 ± 1.29	5.957 ± 0.985	1.0115 ± 0.0037
	500	2.181 ± 0.845	1153.6 ± 171.4	22.99 ± 1.30	72.770 ± 9.983	102.51 ± 1.36	59.25 ± 3.40	6.124 ± 1.116	1.0146 ± 0.0045
	1000	1.614 ± 0.685	1072.0 ± 102.0	22.20 ± 0.77	68.940 ± 6.428	102.09 ± 1.68	60.33 ± 2.77	5.240** ± 0.482 (DN)	1.0146 ± 0.0056

*: p < 0.05, **: p < 0.01

DN = Duncan's Multiple Range Test; U = Mann- Whitney U-Test

Table 3: Mean values of cholesterol and creatinine levels on Day 28. 

		Dose group (mg/kg bw/day)
		Control	250	500	1000
Males	Cholesterol (mmol/L)	1.64	1.56	1.75	2.04** (DN)
	Cholesterol (% difference)		-5	7	24
	Creatinine (µmol/L)	53.3	57.7	60.1	63.2* (DN)
	Creatinine (% difference)		8	13	19
Females	Cholesterol (mmol/L)	1.84	1.80	2.13	2.26** (U)
	Cholesterol (% difference)		-2	16	23
	Creatinine (µmol/L)	48.7	53.0	53.5	56.9
	Creatinine (% difference)		9	10	17

*: p < 0.05, **: p < 0.01

DN = Duncan's Multiple Range Test; U = Mann- Whitney U-Test

 

Table 4: Mean weights of liver of males and females on Day 28. 

		Dose group (mg/kg bw/day)
		Control	250	500	1000
Males	Liver weight (absolute [g])	10.8	12.0*	11.4	12.2** (DN)
	Difference [%]		11	6	13
	Body weight relative [%]	2.7	2.9**	2.9*	3.0** (DN)
	Difference [%]		7	7	11
	Brain relative [%]	526	578*	556	598** (DN)
	Difference [%]		10	6	14
Females	Liver weight (absolute [g])	6.64	7.32	7.66*	7.87** (DN)
	Difference [%]		10	15	19
	Body weight relative [%]	2.9	3.0	3.2**	3.3** (DN)
	Difference [%]		3	10	14
	Brain relative [%]	364	384	414*	429**
	Difference [%]		5	14	18

* = p < 0.05, ** = p < 0.01

DN = Duncan's Multiple Range Test

 

Table 5: Mean weights of kidneys and thyroids of males and females on Day 28. 

		Dose group (mg/kg bw/day)
		Control	250	500	1000
Males	Kidney weight (absolute [g])	2.66	2.83	2.76	2.87* (DN)
	Difference [%]		6	4	8
	Thyroid weight (absolute [g])	21.2	21.6	19.6	20.8
	Difference [%]		2	-8	-2
Females	Kidney weight (absolute [g])	1.64	1.78	1.76	1.73
	Difference [%]		9	7	6
	Thyroid weight (absolute [g])	16.7	17.3	18.9	20.3** (DN)
	Difference [%]		4	13	22
	Body weight relative [%]	7.19	7.17	7.91	8.55** (DN)
	Difference [%]		0	10	19
	Brain relative [%]	0.92	0.91	1.02	1.11** (DN)
	Difference [%]		-1	11	21

* = p < 0.05, ** = p < 0.01

DN = Duncan's Multiple Range Test