6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic acid

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:

13 June - 12 July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 12 May 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

EC Number:

617-769-9

Cas Number:

858956-08-8

Molecular formula:

C8H8ClN3O2

IUPAC Name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd:Sprague Dawley®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Indianapolis, IN, USA
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 243-267 g (males), 175-199 g (females)
- Housing: individually in suspended stainless steel cages (conform with the Guide for the Care and Use of Laboratory Animals, DHEW (NIH).
- Diet: Purina Certified Rodent Meal #5002, ad libitum
- Water: filtered tap water, ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 57 - 67
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

not specified

Vehicle:

unchanged (no vehicle)

Remarks:

moistened with distilled water

Details on exposure:

TEST SITE
- Area of exposure: control: 2 x 3 inch (males and females), 150 mg/kg bw/day: 1 x 2 inch (males) and 1 x 1 inch (females), 400 mg/kg bw/day: 2 x 2 inch (males) and 1 x 2 inch (females), 1000 mg/kg bw/day: 2 x 3 inch (males and females). (For the range of dermal doses in g, please refer to table 14 in the"Any other information on results incl. tables" section.)
- % coverage: approximately 10
- Type of wrap if used: non-irritating tape (3 inch 3M Micropore™)
- Time intervals for shavings: as needed throughout the study

REMOVAL OF TEST SUBSTANCE
- Washing: wiped with water using clean towels to remove any residual test substance and then gently patted dry with a clean pad
- Time after start of exposure: following the 6 h exposure period

TEST MATERIAL
- Amounts applied: test substance (150, 400 or 1000 mg/kg bw/day) moistened with water
- Constant volume or concentration used: no
- For solids, paste formed: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes/no

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 h per day / 7 days per week for 28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The highest dose (1000 mg/kg bw/day) is the regulatory limit dose. The high level was expected to potentially exhibit some evidence of toxicity and the NOAEL was expected at the low or intermediate exposure level in both male and female rats.
- Fasting period before blood sampling for clinical biochemistry: yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), once daily (general inspection)
- - Cage side observations checked in table No.1 (in the "Any other information and methods incl. tables" section) were examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily before test substance application and weekly thereafter

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: after patch removal for signs of erythema and edema on Day 3, each Friday and on the day of the final application
- Scoring system: "Primary Skin Irritation Scoring System" developed by Draize et al.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first patch application (days 1 and 2 for males and females, respectively) and weekly thereafter (intervals were 7 days ± 1 day)

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and Day 24
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (males) or Day 30 (females), prior to sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes (except for blood samples used for analysis of coagulation parameters)
- How many animals: all animals
- Parameters listed in table No.2 (in the "Any other information and methods incl. tables" section) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (males) or
Day 30 (females), prior to sacrifice
- Animals fasted: Yes
- How many animals: all animals
- Parameters listed in table No.2 (in the "Any other information and methods incl. tables" section) were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Please refer to table 3 in the "Any other information and methods incl. tables" section.)

HISTOPATHOLOGY: Yes (Please refer to table 3 in the "Any other information and methods incl. tables" section.)

Statistics:

Group means and standard deviations were calculated for body weight, daily body weight gain, daily food consumption, daily food efficiency, organ weight, and organ-to-body/brain weight ratio data. Data within groups were evaluated for homogeneity of variances and normality by Bartlett’s test. Where Bartlett’s test indicated homogeneous variances, treated and control groups were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s t-test for multiple comparisons. Where variances were considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT Biostatistics, Graph Pad
Software, San Diego, CA). Differences among groups were judged to be significant at a probability value of p 0.05. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical signs in any test group that were considered to be of toxicological significance.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

400 and 1000 mg/kg bw/day: very slight erythema graded with a score of 1 were noted for the dose sites in single animals (400 mg/kg bw/day: 1/10 males on Day 28; 1000 mg/kg bw/day: 2/10 males on Day 3 and 28, respectively).

(Please refer to table 4 in the "Any other information on results incl. tables" section.)

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Mean body weight and daily body weight gain and overall (Days 2-29) body weight gain were statistically significantly increased in females during Week 4 and/or overall. Since during the same period, control group values for these parameters were notably lower than historical control values for rats of the same age, these findings were not considered treatment-related or adverse and considered not to be of toxicological importance.

(Please refer to table 5 and 6 in the "Any other information on results incl. tables" section.)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

150 and 400 mg/kg bw/day: Statistically significant decreases in mean daily food consumption were observed in female rats for the test period overall (Days 2-29) and during week 3, respectively. These findings were not dose-related and thus not considered treatment-related.

(Please refer to table 7 in the "Any other information on results incl. tables" section.)

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: an increase in mean food efficiency for the overall test period (Days 2-29) was seen in females and correlated with increased body weight gain for the same test period. Since week 4 control group values for food efficiency were notably lower than historical control values for rats of the same age, these findings were not considered treatment-related or adverse and considered not to be of toxicological importance.

(Please refer to table 8 in the "Any other information on results incl. tables" section.)

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

150 mg/kg bw/day: Red cell distribution width was slightly increased in females compared to control. Since there were no corresponding red cell mass findings, the statistically significant change in mean haematology results was not considered to be adverse or treatment-related.

(Please refer to table 9 in the "Any other information on results incl. tables" section.)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

150 and 1000 mg/kg bw/day: creatinine was statistically significantly decreased in males compared to control. Due to the absence of a dose-response relationship and the small magnitude of the change it is not considered treatment-related or adverse.

(Please refer to table 10 in the "Any other information on results incl. tables" section.)

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: absolute adrenal weights were significantly increased in females compared to controls.

150 mg/kg bw/day: both absolute and relative ovarian weights and kidney-to-brain weight ratios were significantly decreased in females compared to controls.

Since there were no microscopic correlates observed for these findings, they were determined to be toxicologically insignificant.

(Please refer to table 11, 12 and 13 in the "Any other information on results incl. tables" section.)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: a lung adhesion in 1 female had no corresponding microscopic finding.

400 mg/kg bw/day: an epididymal mass (2.0 x 2.0 mm) from 1 male was histologically confirmed as a small sperm granuloma.

Control: 1 male exhibited a small appearing right testis and epididymis which were confirmed microscopically as mild unilateral tubular atrophy and intratubular cellular debris, respectively.

All these incidental findings were considered unrelated to treatment and, as such, toxicologically insignificant. All other macroscopic changes were observed at comparable incidences in the control and treated groups, and/or commonly observed in untreated rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: minimal focal lobular atrophy in the pancreas of 1/10 males. Slight focal fibrosis in the salivary glands in 1/10 females These observations were considered
unrelated to treatment due to their isolated occurrences, focal distribution and their common occurrence in untreated rats.

All other microscopic changes were of comparable incidence and severity in the test substance-treated and control animals, and are commonly observed in untreated laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 4: Mean weekly dermal irritation score

Group		1	2	3	4
Dose Level (mg/kg/day)		0	150	400	1,000
Number Of Animals In Group		10	10	10	10
Males
Day	3	0.0/0.0	0.0/0.0	0.0/0.0	0.1/0.0
	10	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	17	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	24	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	28	0.0/0.0	0.0/0.0	0.1/0.0	0.1/0.0
Females
Day	3	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	10	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	17	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	24	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0
	28	0.0/0.0	0.0/0.0	0.0/0.0	0.0/0.0

 

Table 5: Mean daily body weight (g)

Group		1	2	3	4
Dose Level (mg/kg/day)		0	150	400	1,000
Number Of Animals In Group		10	10	10	10
Males
Day
Initial (Day 1)	Mean	255.8	257.4	255.7	255.8
	SD	7.4	6.4	6.1	6.5
8	Mean	284.0	279.8	278.8	282.8
	SD	11.9	12.7	9.5	9.4
15	Mean	308.2	300.6	300.5	303.6
	SD	15.2	14.4	10.6	12.6
21	Mean	319.2	314.6	311.2	313.5
	SD	14.9	17.1	12.9	14.1
28	Mean	336.2	331.2	324.2	339.6
	SD	14.1	21.1	11.8	19.0
Females
Initial (Day 2)	Mean	184.7	183.1	182.1	183.6
	SD	6.3	6.2	4.9	6.7
8	Mean	198.1	195.0	199.8	199.0
	SD	9.2	6.1	8.0	7.8
15	Mean	207.3	205.2	206.9	206.6
	SD	12.1	4.5	10.2	6.8
21	Mean	222.6	216.9	215.8	225.3
	SD	10.8	7.6	9.1	9.3
28	Mean	222.8	220.3	222.9	233.6*
	SD	7.4	8.9	8.7	8.3

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test.

 

Table 6: Mean daily body weight gains (g)

Group		1	2	3	4
Dose Level (mg/kg/day)		0	150	400	1,000
Number Of Animals In Group		10	10	10	10
Males
Day
1-8	Mean	4.0	3.2	3.3	3.9
	SD	0.8	1.0	0.9	0.9
8-15	Mean	3.5	3.0	3.1	3.0
	SD	0.7	0.6	0.5	0.6
15-21	Mean	1.6	2.0	1.5	1.4
	SD	0.7	0.6	0.6	0.6
21-28	Mean	2.4	2.4	1.9	3.7
	SD	0.7	0.7	0.7	3.2
Total(1-28)	Mean	2.9	2.6	2.4	3.0
	SD	0.3	0.5	0.3	0.7
Females
2-9	Mean	1.9	1.7	2.5	2.2
	SD	0.9	1.3	1.0	1.0
9-16	Mean	1.3	1.5	1.0	1.1
	SD	1.0	0.6	1.2	1.3
16-23	Mean	2.2	1.7	1.3	2.7
	SD	1.3	0.5	0.8	0.7
23-29	Mean	0.0	0.6	1.2	1.4*
	SD	1.2	1.1	1.0	1.4
Total(2-29)	Mean	1.4	1.4	1.5	1.8**
	SD	0.2	0.4	0.3	0.3

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test.

** Statistically significant difference from control at p < 0.01 by Dunnett’s Multiple Comparisons test.

 

Table 7: Mean daily food consumption (g)

Group		1	2	3	4
Dose Level (mg/kg/day)		0	150	400	1,000
Number Of Animals In Group		10	10	10	10
Males
Day
1-8	Mean	22.2	21.5	21.5	21.8
	SD	1.5	1.2	1.3	1.0
8-15	Mean	24.1	23.6	23.6	24.2
	SD	1.2	1.6	1.1	0.8
15-21	Mean	20.3	20.4	19.8	20.1
	SD	0.8	1.3	1.0	1.1
21-28	Mean	24.7	23.5	22.5	24.2
	SD	2.9	1.7	2.5	1.0
Total(1-28)	Mean	22.8	22.3	21.9	22.6
	SD	1.o	1.3	1.2	0.8
Females
2-9	Mean	17.1	16.0	16.6	17.0
	SD	1.1	0.9	1.3	1.0
9-16	Mean	18.3	17.6	17.9	17.4
	SD	1.6	1.1	1.7	1.3
16-23	Mean	19.3	18.4	18.0*	19.4
	SD	1.5	1.0	0.6	0.8
23-29	Mean	18.5	17.3	17.9	19.7
	SD	2.6	1.1	1.0	1.6
Total (2-29)	Mean	18.3	17.4*	17.6	18.4
	SD	0.9	0.7	0.8	0.7

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test.

 

Table 8: Mean daily food efficiency

Group		1	2	3	4
Dose Level (mg/kg/day)		0	150	400	1,000
Number Of Animals In Group		10	10	10	10
Males
Day
1-8	Mean	0.18	0.15	0.15	0.18
	SD	0.03	0.04	0.03	0.03
8-15	Mean	0.14	0.13	0.13	0.12
	SD	0.03	0.03	0.02	0.02
15-21	Mean	0.08	0.10	0.08	0.07
	SD	0.03	0.03	0.03	0.03
21-28	Mean	0.10	0.10	0.08	0.16
	SD	0.03	0.03	0.03	0.15
Total(1-28)	Mean	0.13	0.12	0.11	0.13
	SD	0.01	0.02	0.01	0.03
Females
2-9	Mean	0.11	0.10	0.15	0.13
	SD	0.05	0.07	0.05	0.05
9-16	Mean	0.07	0.08	0.05	0.06
	SD	0.05	0.03	0.07	0.08
16-23	Mean	0.11	0.09	0.07	0.14
	SD	0.06	0.03	0.04	0.03
23-29	Mean	0.01	0.03	0.06	0.07
	SD	0.07	0.06	0.05	0.07
Total (2-29)	Mean	0.07	0.08	0.08	0.10*
	SD	0.01	0.03	0.01	0.02

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test.

 

Table 9: Mean haematology and coagulation values

Dose Level (mg/kg/day)		Control	150	400	1000
Number Of Animals In Group		9^2	10	10	10
Males
Parameter
Hemoglobin Cone. (g/dL)	Mean	16.1	16.0	16.3	16.3
	SD	0.3	0.5	0.5	0.6
Hematocrit (%)	Mean	45.8	45.9	46.2	46.8
	SD	1.1	1.7	1.5	1.6
Erythrocyte Count (RBC, X 106/µL)	Mean	8.70	8.77	8.71	8.74
	SD	0.30	0.36	0.42	0.37
Platelet Count (x 103/µL)	Mean	1038	936	1005	901
	SD	154	157	158	191
Total Leukocyte Count (WBC, X 103/µL)	Mean	10.77	11.72	11.30	12.05
	SD	2.16	1.95	1.77	1.71
Mean Corpuscular Volume (fL)	Mean	52.7	52.4	53.1	53.6
	SD	1.0	1.4	1.4	1.5
Mean Corpuscular Hemoglobin (pg)	Mean	18.5	18.3	18.7	18.7
	SD	0.6	0.5	0.4	0.5
Mean Corpuscular Hemoglobin Concentration (g/dL)	Mean	35.1	34.9	35.2	34.8
	SD	0.5	0.3	0.4	0.6
Red Cell Distribution Width(%)	Mean	11.4	11.6	11.5	11.6
	SD	0.3	0.4	0.3	0.3
Absolute Reticulocyte Count (x 103/µL)	Mean	181.5	184.0	181.1	190.9
	SD	21.4	15.8	23.4	16.7
Absolute Neutrophils (x 103/µL)	Mean	1.43	1.79	1.48	1.63
	SD	0.31	0.33	0.30	0.43
Absolute Lymphocytes (x 103/µL)	Mean	8.84	9.29	9.22	9.80
	SD	1.84	1.59	1.69	1.58
Absolute Monocytes (x 103/µL)	Mean	0.20	0.26	0.23	0.24
	SD	0.05	0.07	0.07	0.06
Absolute Eosinophils (x 103/µL)	Mean	0.16	0.21	0.21	0.21
	SD	0.04	0.07	0.09	0.07
Absolute Basophils (x 103/µL)	Mean	0.08	0.10	0.09	0.09
	SD	0.03	0.03	0.03	0.03
Absolute Large Unstained Cells (x 103/µL)	Mean	0.07	0.07	0.07	0.08
	SD	0.02	0.02	0.02	0.02
Coagulation parameters
Number Of Animals In Group		8^2	9^2	9^2	8^2
Prothrombin time (seconds)	Mean	13.0	12.8	13.3	13.4
	SD	0.9	0.9	1.1	0.6
Activated Partial Thromboplastin time (seconds)	Mean	16.6	18.7	16.4	16.3
	SD	2.5	8.2	2.3	2.3
Females
Number of animals evaluated		7^2	10	10	10
Hemoglobin Cone. (g/dL)	Mean	15.7	15.7	15.8	15.8
	SD	0.4	0.3	0.5	0.5
Hematocrit (%)	Mean	43.3	43.6	43.7	44.0
	SD	1.3	1.0	1.3	1.4
Erythrocyte Count (RBC, X 106/µL)	Mean	8.33	8.34	8.35	8.30
	SD	0.13	0.23	0.31	0.27
Platelet Count (x 103/µL)	Mean	886	940	997	821
	SD	183	186	271	167
Total Leukocyte Count (WBC, X 103/µL)	Mean	7.45	7.76	8.17	7.57
	SD	2.45	1.45	1.84	2.04
Mean Corpuscular Volume (fL)	Mean	52.0	52.3	52.4	53.1
	SD	1.7	1.5	1.0	1.1
Mean Corpuscular Hemoglobin (pg)	Mean	18.9	18.8	18.9	19.1
	SD	0.4	0.5	0.3	0.3
Mean Corpuscular Hemoglobin Concentration (g/dL)	Mean	36.3	36.0	36.0	36.0
	SD	0.5	0.3	0.4	0.6
Red Cell Distribution Width(%)	Mean	10.8	11.4*	11.2	11.3
	SD	0.2	0.6	0.2	0.4
Absolute Reticulocyte Count (x 103/µL)	Mean	176.2	204.4	211.9	218.0
	SD	45.1	66.4	30.3	29.7
Absolute Neutrophils (x 103/µL)	Mean	1.60	1.41	1.38	1.43
	SD	0.64	0.77	0.45	0.44
Absolute Lymphocytes (x 103/µL)	Mean	5.48	5.97	6.39	5.79
	SD	1.80	0.93	1.58	1.69
Absolute Monocytes (x 103/µL)	Mean	0.11	0.12	0.12	0.11
	SD	0.06	0.05	0.05	0.06
Absolute Eosinophils (x 103/µL)	Mean	0.17	0.16	0.16	0.13
	SD	0.05	0.07	0.04	0.04
Absolute Basophils (x 103/µL)	Mean	0.07	0.08	0.08	0.07
	SD	0.05	0.05	0.04	0.05
Absolute Large Unstained Cells (x 103/µL)	Mean	0.03	0.02	0.04	0.03
	SD	0.02	0.02	0.02	0.02
Coagulation parameters
Number of animals evaluated		10	9^2	9^2	7^2
Prothrombin time (seconds)	Mean	14.7	14.8	14.9	14.9
	SD	0.3	0.4	0.5	0.8
Activated Partial Thromboplastin time (seconds)	Mean	14.9	14.8	15.1	15.9
	SD	1.3	2.1	1.3	3.4

* statistically significant difference from control at p <0.05 by Dunnett/Tamhane-Dunnett.

2 Measurements for all animals were not performed due to a clotted sample.

 

Table 10: Mean clinical biochemistry data

Dose Level (mg/kg/day)		Control	150	400	1000
Number Of Animals In Group		10	4-92	6-92	7-102
Males
Parameter
Aspartate Aminotransferase (U/L)	Mean	88	94	90	90
	SD	12	6	10	12
Alanine Aminotransferase (U/L)	Mean	43	43	41	43
	SD	9	4	4	4
Sorbital Dehydrogenase (U/L)	Mean	2.0 2	0.8	1.0	1.7
	SD	2.0	0.5	1.0	1.4
Alkaline Phosphatase (U/L)	Mean	130	139	136	147
	SD	22	19	21	19
Total Bilirubin (mg/dL)	Mean	0.13	0.13	0.13	0.12
	SD	0.02	0.02	0.02	0.01
Blood Urea Nitrogen (mg/dL)	Mean	16	17	16	16
	SD	2	2	1	3
Creatinine (mg/dL)	Mean	0.32	0.28*	0.29	0.27*
	SD	0.02	0.03	0.02	0.03
Cholesterol (mg/dL)	Mean	78	70	73	74
	SD	12	10	13	13
Triglycerides (mg/dL)	Mean	20	16	20	18
	SD	2	2	5	4
Glucose (mg/dL)		104	112	107	111
		8	7	5	12
Total Protein (g/dL)	Mean	6.2	6.2	6.1	6.2
	SD	0.2	0.2	0.2	0.2
Albumin (g/dL)	Mean	3.4	3.3	3.3	3.4
	SD	0.2	0.2	0.2	0.2
Number of animals evaluated		10	6-102	8-102	7-102
Globulin (g/dL)	Mean	2.8	2.9	2.8	2.8
	SD	0.2	0.2	0.1	0.2
Calcium (mg/dL)	Mean	10.0	9.9	10.0	10.2
	SD	0.3	0.2	0.3	0.4
Inorganic Phosphorus (mg/dL)	Mean	7.5	7.5	7.5	7.8
	SD	0.4	0.3	0.6	0.4
Sodium (mmol/L)	Mean	141.0	138.1	138.7	140.4
	SD	4.3	3.0	2.8	4.3
Potassium (mmol/L)	Mean	5.23	5.10	5.32	5.13
	SD	0.13	0.39	0.59	0.36
Chloride (mmol/L)	Mean	101.9	101.0	101.3	102.1
	SD	2.7	2.0	1.8	2.1
Females
Number of animals evaluated		7^2	10	10	10
Aspartate Aminotransferase (U/L)	Mean	105	97	98	101
	SD	17	17	19	33
Alanine Aminotransferase (U/L)	Mean	40	39	42	40
	SD	5	6	6	10
Sorbital Dehydrogenase (U/L)	Mean	8.1	9.7	7.3	9.2
	SD	3.5	2.5	3.1	2.4
Alkaline Phosphatase (U/L)	Mean	108	116	105	118
	SD	24	30	18	20
Total Bilirubin (mg/dL)	Mean	0.14	0.14	0.13	0.14
	SD	0.02	0.01	0.02	0.01
Blood Urea Nitrogen (mg/dL)	Mean	21	22	21	22
	SD	2	3	3	4
Creatinine (mg/dL)	Mean	0.40	0.40	0.38	0.37
	SD	0.04	0.03	0.03	0.03
Cholesterol (mg/dL)	Mean	80	79	78	72
	SD	15	14	9	16
Triglycerides (mg/dL)	Mean	21	24	20	21
	SD	4	5	3	5
Glucose (mg/dL)	Mean	98	96	101	103
	SD	7	12	9	10
Total Protein (g/dL)	Mean	6.5	6.5	6.6	6.6
	SD	0.3	0.2	0.3	0.3
Albumin (g/dL)	Mean	3.6	3.5	3.5	3.6
	SD	0.2	0.1	0.1	0.2
Number of animals evaluated		10	10	10	10
Globulin (g/dL)	Mean	3.0	3.0	3.1	3.1
	SD	0.2	0.2	0.3	0.2
Calcium (mg/dL)	Mean	10.5	10.5	10.6	10.6
	SD	0.2	0.3	0.3	0.4
Inorganic Phosphorus (mg/dL)	Mean	6.9	6.8	6.9	7.0
	SD	0.6	0.5	0.5	0.7
Sodium (mmol/L)	Mean	141.5	138.4	138.7	140.6
	SD	3.5	3.6	3.6	2.3
Potassium (mmol/L)	SD	4.65	4.54	4.46	4.41
	Mean	0.41	0.24	0.50	0.30
Chloride (mmol/L)	SD	102.3	100.6	101.4	102.6
		2.9	1.8	1.9	1.6

* statistically significant difference from control at p <0.05 by Dunnett/Tamhane-Dunnett.

 

Table 11: mean Organ weights

Dose Level (mg/kg/day)		Control	150	400	1000
Number Of Animals In Group		10	10	10	10
Males
Organ
Adrenals (paired)	Mean	0.082	0.068	0.066	0.063
	SD	0.027	0.013	0.016	0.012
Brain	Mean	2.01	2.00	1.98	2.00
	SD	0.14	0.13	0.13	0.12
Heart	Mean	1.18	1.14	1.17	1.23
	SD	0.09	0.10	0.11	0.07
Kidneys (paired)	Mean	2.63	2.56	2.46	2.54
	SD	0.30	0.26	0.23	0.19
Liver	Mean	9.58	9.05	9.05	9.19
	SD	0.90	0.76	0.65	0.72
Spleen	Mean	0.76	0.73	0.69	0.73
	SD	0.06	0.09	0.08	0.07
Thymus	Mean	0.490	0.464	0.455	0.435
	SD	0.095	0.081	0.046	0.077
Epididymides (paired)	Mean	1.295	1.315	1.321	1.315
	SD	0.123	0.154	0.096	0.095
Testes (paired)	Mean	3.59	3.53	3.57	3.71
	SD	0.39	0.33	0.17	0.22
Females
Adrenals (paired)	Mean	0.073	0.071	0.077	0.084*
	SD	0.007	0.009	0.011	0.011
Brain	Mean	1.84	1.89	1.87	1.92
	SD	0.09	0.09	0.06	0.18
Heart	Mean	0.89	0.89	0.85	0.88
	SD	0.07	0.05	0.07	0.05
Kidneys (paired)	Mean	1.70	1.59	1.73	1.77
	SD	0.11	0.16	0.17	0.13
Liver	Mean	6.43	6.32	6.42	6.79
	SD	0.61	0.58	0.42	0.40
Spleen	Mean	0.59	0.58	0.61	0.62
	SD	0.07	0.09	0.04	0.06
Thymus	Mean	0.348	0.348	0.370	0.367
	SD	0.053	0.054	0.048	0.068
Ovaries (paired)	Mean	0.162	0.130*	0.145	0.156
	SD	0.035	0.020	0.022	0.024
Uterus	Mean	0.438	0.381	0.453	0.583
	SD	0.140	0.072	0.109	0.350

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test

 

Table 12: Mean organ body weight ratios

Dose Level (mg/kg/day)		Control	150	400	1000
Number Of Animals In Group		10	10	10	10
Males
Organ
Adrenals (paired)	Mean	0.270	0.228	0.225	0.21
	SD	0.085	0.039	0.056	0.039
Brain	Mean	6.67	6.74	6.79	6.73
	SD	0.50	0.32	0.35	0.50
Heart	Mean	3.90	3.85	3.99	4.12
	SD	0.30	0.42	0.34	0.22
Kidneys (paired)	Mean	8.72	8.63	8.41	8.52
	SD	0.91	0.67	0.69	0.51
Liver	Mean	31.74	30.50	30.94	30.85
	SD	2.50	1.59	1.40	1.49
Spleen	Mean	2.53	2.47	2.37	2.45
	SD	0.17	0.24	0.22	0.13
Thymus	Mean	1.616	1.559	1.556	1.457
	SD	0.262	0.227	0.118	0.246
Epididymides (paired)	Mean	4.297	4.438	4.534	4.415
	SD	0.437	0.521	0.455	0.183
Testes (paired)	Mean	11.93	11.87	12.22	12.48
	SD	1.44	0.74	0.57	0.62
Females
Adrenals (paired)	Mean	0.371	0.361	0.392	0.418
	SD	0.034	0.046	0.061	0.063
Brain	Mean				9.48
	SD	9.35	9.68	9.48	1.04
Heart	Mean	0.44	0.33	0.52	4.36
	SD	4.52	4.57	4.32	0.32
Kidneys (paired)	Mean	0.28	0.28	0.26	8.74
	SD	8.63	8.12	8.78	0.70
Liver	Mean	0.37	0.60	0.80	33.52
	SD	32.69	32.33	32.57	1.95
Spleen	Mean	2.29	2.52	1.67	3.04
	SD	3.00	2.97	3.08	0.31
Thymus	Mean	0.33	0.39	0.20	1.816
	SD	1.779	1.776	1.882	0.358
Ovaries (paired)	Mean	0.301	0.245	0.262	0.769
	SD	0.823	0.667*	0.736	0.122
Uterus	Mean	0.165	0.104	0.102	2.874
	SD	2.246	1.941	2.288	1.749

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test

 

 

Table 13: Mean organ to brain weight ratios

Dose Level (mg/kg/day)		Control	150	400	1000
Number Of Animals In Group		10	10	10	10
Males
Organ
Adrenals (paired)	Mean	0.041	0.034	0.033	0.031
	SD	0.013	0.007	0.009	0.005
Heart	Mean	0.58	0.57	0.59	0.61
	SD	0.03	0.05	0.04	0.04
Kidneys (paired)	Mean	1.31	1.28	1.24	1.27
	SD	0.11	0.05	0.11	0.09
Liver	Mean	4.76	4.53	4.56	4.60
	SD	0.31	0.31	0.26	0.36
Spleen	Mean	0.38	0.37	0.35	0.37
	SD	0.03	0.03	0.03	0.04
Thymus	Mean	0.243	0.231	0.230	0.218
	SD	0.044	0.032	0.020	0.040
Epididymides (paired)	Mean	0.645	0.659	0.668	0.659
	SD	0.061	0.079	0.067	0.059
Testes (paired)	Mean	1.79	1.76	1.80	1.86
	SD	0.20	0.14	0.11	0.143
Females
Adrenals (paired)	Mean	0.040	0.037	0.041	0.044
	SD	0.004	0.005	0.006	0.007
Heart	Mean	0.48	0.47	0.46	0.46
	SD	0.04	0.03	0.04	0.02
Kidneys (paired)	Mean	0.93	0.84*	0.93	0.93
	SD	0.07	0.06	0.08	0.08
Liver	Mean	3.51	3.34	3.45	3.56
	SD	0.32	0.26	0.26	0.30
Spleen	Mean	0.32	0.31	0.33	0.32
	SD	0.04	0.04	0.03	0.04
Thymus	Mean	0.190	0.184	0.199	0.192
	SD	0.027	0.026	0.026	0.036
Ovaries (paired)	Mean	0.088	0.069**	0.078	0.082
	SD	0.019	0.012	0.010	0.012
Uterus	Mean	0.238	0.200	0.243	0.306
	SD	0.070	0.032	0.060	0.181

* Statistically significant difference from control at p < 0.05 by Dunnett’s Multiple Comparisons test

** Statistically significant difference from control at p < 0.01 by Dunnett’s Multiple Comparisons test

Table 14: Range of dermal doses (g)

Dose group (mg/kg bw/day	Control	150	400	1,000
Number of animals (males)	10	10	10	10
Days
1-7	0	0.042-0.045	0.11-0.12	0.27-0.29
8-14	0	0.044-0.051	0.12-0.13	0.30-0.33
15-20	0	0.047-0.055	0.12-0.14	0.31-0.35
21-27	0	0.049-0.058	0.13-0.15	0.32-0.37
28	0	0.053-0.062	0.13-0.15	0.34-0.42
Number of animals (females)	10	10	10	10
Days
1-7	0	0.030-0.034	0.077-0.084	0.19-0.21
8-14	0	0.031-0.035	0.082-0.095	0.21-0.23
15-20	0	0.034-0.036	0.084-0.097	0.21-0.24
21-27	0	0.035-0.039	0.089-0.100	0.23-0.26
28	0	0.036-0.041	0.095-0.100	0.24-0.27