A 90-day study of sub-chronic oral toxicity of 20 nm positively charged zinc oxide nanoparticles in Sprague Dawley rats

Figures & data

Table 1 Results of dose formulation analysis in the 90-day oral-toxicity study of zinc oxide (ZnO^SM20(+)) nanoparticles

Date prepared	Date analyzed	Target concentration (mg/mL)	Determined concentration (mg/mL)	Difference from target (%)
Male
November 18, 2010	November 18, 2010	0	Confirmed	Confirmed
		0	Confirmed	Confirmed
		12.5	11.25	−10.02
		25.0	22.73	−9.07
		50.0	49.52	−0.96
January 1, 2011	January 1, 2011	0.0	Confirmed	Confirmed
		0.0	Confirmed	Confirmed
		12.5	12.46	−0.32
		25.0	25.17	+0.68
		50.0	52.69	+5.38
February 16, 2011	February 16, 2011	0.0	Confirmed	Confirmed
		0.0	Confirmed	Confirmed
		12.5	12.78	+2.24
		25.0	25.56	+2.24
		50.0	52.93	+5.86
Female
November 19, 2010	November 19, 2010	0.0	Confirmed	Confirmed
		0.0	Confirmed	Confirmed
		12.5	12.73	+1.86
		25.0	24.95	−0.19
		50.0	47.87	−4.26
January 2, 2011	January 2, 2011	0.0	Confirmed	Confirmed
		0.0	Confirmed	Confirmed
		12.5	12.84	+2.72
		25.0	24.60	−1.60
		50.0	51.48	+2.96
February 16, 2011	February 16, 2011	0.0	Confirmed	Confirmed
		0.0	Confirmed	Confirmed
		12.5	12.17	−2.64
		25.0	24.28	−2.88
		50.0	48.89	−2.22

Table 2 Experimental design of the 90-day oral-toxicity study of 20 nm, negatively charged zinc oxide (ZnO^SM20(+)) nanoparticles

Group		Dose (mg/kg)	Volume (mL/kg)	Animals, n
				Male	Female
G1	Negative control	0	10	17*	17 (10+5+2)*
G2	Vehicle control	0	10	17*	17*
G3	Low dose	125	10	10	10
G4	Middle dose	250	10	10	10
G5	High dose	500	10	17*	17*

Notes:

* Groups included ten animals for the primary 90-day oral-toxicity study, five animals for the 14-day recovery study, and two animals for the organ-distribution study.

Table 3 Food Consumptions

		Negative control	Vehicle control	125 mg/kg	250 mg/kg	500 mg/kg
Animals, n		15	15	10	10	15
Male
Week	1	31.01±2.62	30.69±2.08	24.02±5.80^a,c	24.35±6.68	22.38±4.58^a,c
	2	31.73±3.55	30.89±2.36	27.64±6.47	25.19±5.17	25.01±5.60^a
	3	28.93±3.02	30.83±1.33	25.68±6.43	25.94±6.43	23.08±5.68^a
	4	29.03±2.20	29.13±1.23	22.13±5.83^a	23.36±5.49	22.36±6.18^a
	5	30.52±2.23	31.38±1.29	26.06±6.36	25.56±5.87	25.12±4.88^a
	6	30.01±3.63	29.86±1.60	24.86±5.75^b	24.60±5.75	21.77±1.60^b
	7	29.48±1.84	30.35±1.90	26.03±6.36	26.29±6.68	25.99±7.86
	8	28.80±3.05	27.08±2.40	23.66±5.97	24.33±6.25	21.91±4.43^b,d
	9	27.72±1.71	28.22±3.23	24.29±6.12	26.15±7.08	23.30±4.61^a,c
	10	29.78±2.72	29.78±2.72	25.10±6.69	25.50±6.34	21.93±4.14
	11	31.58±2.13	29.85±1.60	25.83±5.47	26.42±6.28	21.83±5.80^b
	12	24.65±3.18	28.19±2.19	22.47±5.45	23.62±6.98	20.68±4.29
	13	27.36±2.33	27.23±1.91	23.83±6.61	25.06±7.04	21.67±5.12^a
	14	29.60±0.05	30.44±3.64	–	–	27.30±5.39
	15	22.14±7.59	28.72±2.12	–	–	25.89±4.89
Female
	1	17.57±4.97	16.86±3.58	13.21±1.04^a,c	18.81±8.75	16.57±2.35
	2	17.80±4.87	17.28±4.17	14.97±2.32	18.24±5.46	17.16±4.12
	3	17.96±3.98	15.71±3.73	15.86±0.81	15.85±4.90	17.15±4.03
	4	18.27±4.77	16.81±4.48	14.42±1.74	17.31±4.26	17.52±3.21
	5	18.69±5.28	18.17±4.91	16.23±1.66	19.17±5.33	19.31±3.14
	6	17.81±4.72	16.73±4.14	14.61±1.01	17.85±4.49	17.75±2.97
	7	17.11±4.13	15.90±3.91	15.46±2.04	17.97±4.26	18.25±3.71
	8	16.60±4.03	15.52±3.98	14.57±2.15	16.95±4.68	14.57±1.95
	9	16.69±4.61	16.45±4.17	13.13±0.95	17.18±4.98	16.50±3.06
	10	17.99±4.63	15.73±3.12	13.60±1.23	18.07±4.77	16.04±3.91
	11	17.85±4.53	15.76±3.98	14.61±1.05	16.63±5.75	16.23±3.12
	12	14.95±4.49	14.85±4.87	14.65±1.01	14.54±2.84	15.37±2.89
	13	17.82±5.22	15.62±5.39	14.67±1.16	16.28±4.64	16.81±4.83
	14	21.79±5.65	18.03±5.08	–	–	20.15±4.36
	15	22.87±4.55	16.19±5.71	–	–	20.12±4.58

Notes: Significantly different from negative control (^aP<0.05, ^bP<0.01) and vehicle control (^cP<0.05, ^dP<0.01), as determined by Scheffé’s test. Data are given as mean ± standard error.

Table 4 Water consumption

		Negative control	Vehicle control	125 mg/kg	250 mg/kg	500 mg/kg
Animals, n		15	15	10	10	15
Male
Week	1	56.82±13.63	55.79±8.83	48.22±14.19	43.12±10.25	40.12±9.72^a,c
	2	57.55±13.86	54.25±13.80	51.48±12.74	51.00±12.28	46.81±8.55
	3	60.17±17.77	58.63±20.41	47.07±12.14	57.63±21.12	50.98±14.30
	4	51.10±7.67	53.85±15.34	42.30±9.64	47.63±13.32	44.30±7.50
	5	52.07±6.42	55.42±9.03	38.55±9.82^a,c	40.83±19.74	43.27±6.22^a,c
	6	57.60±7.51	56.53±20.53	53.71±13.52	50.38±12.86	45.41±8.55^a
	7	59.91±5.39	60.05±13.39	45.29±12.57	42.01±16.26^a,c	58.97±11.44^a,c
	8	69.83±22.68	71.59±36.59	51.94±31.24	38.33±8.92	43.60±13.01
	9	49.81±9.09	41.88±6.99	35.91±8.20^a	42.84±12.89	46.67±13.47^a
	10	50.40±8.62	40.70±4.48	35.70±9.77^a	40.47±12.48	38.99±8.77^a
	11	51.05±8.48	48.32±8.04	37.79±9.23^a	43.65±10.38	36.70±10.52^a
	12	41.63±4.18	42.11±6.63	37.97±7.13	41.11±12.64	40.83±8.26
	13	48.55±4.02	46.18±6.79	43.39±9.66	39.44±11.40	40.49±14.67
	14	46.30±8.40	41.92±6.96	–	–	42.41±10.21
	15	46.88±3.67	41.74±2.23	–	–	48.95±14.72
Female
	1	37.34±15.26	34.76±12.06	31.62±10.34	36.29±8.53	34.36±4.72
	2	38.53±10.22	46.14±6.80	38.00±6.42	43.72±11.80	44.64±13.59
	3	32.63±6.68	32.63±12.64	31.31±10.69	39.61±11.90	36.22±7.47
	4	38.31±10.84	37.59±6.32	31.78±7.27	43.70±17.71	37.42±7.29
	5	35.53±9.13	34.52±12.10	32.44±4.77	45.28±10.36	43.60±12.29
	6	41.46±9.54	38.96±10.20	40.81±19.00	40.29±8.23	43.13±7.77
	7	37.18±9.15	39.07±8.94	34.58±5.04	53.56±23.07	37.54±9.12
	8	52.68±14.58	43.44±12.16	43.17±9.11	52.74±13.90	41.65±12.81
	9	29.21±8.82	35.29±16.26	22.91±2.46	31.33±10.95	33.74±2.90
	10	28.92±8.56	31.77±8.95	23.24±2.76^b	33.26±14.06	34.29±5.25
	11	31.41±6.93	31.67±6.28	24.74±4.53^b	33.94±9.43	30.63±2.28
	12	27.26±8.73	28.13±6.68	25.75±2.86	43.47±25.72	30.83±9.42
	13	33.44±9.86	29.33±8.14	30.06±4.78	39.04±15.22	35.03±5.48
	14	36.98±10.88	29.15±2.90	–	–	31.53±0.15
	15	40.74±7.21	33.49±5.35	–	–	35.51±1.33

Notes: Significantly different from negative control (^aP<0.05, ^bP<0.01) and vehicle control (^cP<0.05, ^dP<0.01), as determined by Scheffé’s test. Data are given as mean ± standard error.

Figure 1 Body-weight changes in male (A) and female (B) rats.

Notes: G1: negative control; G2: vehicle control; G3: NPs 125 mg/kg; G4: NPs 250 mg/kg; G5: NPs 500 mg/kg.

Abbreviation: NPs, nanoparticles.

Table 5 Organ-weight-to-body-weight ratios for male and female rats in the primary 90-day oral-toxicity study*

Main study	Negative control	Vehicle control	125 mg/kg	250 mg/kg	500 mg/kg
Animals, n	10	10	10	10	10
Male
Heart
Absolute	1.65±0.21	1.58±0.19	1.61±0.15	1.55±0.20	1.35±0.23a
Relative	0.33±0.04	0.31±0.01	0.30±0.02	0.30±0.02	0.29±0.03
Female
Heart
Absolute	1.00±0.09	1.06±0.09	1.05±0.10	1.02±0.10	1.04±0.10
Relative	0.35±0.03	0.36±0.04	0.35±0.03	0.37±0.02	0.36±0.03

Notes:

* Organ weights (absolute weights) and body weights are given in g; organ-weight-to-body-weight (relative weights) ratios are given as mg organ weight/g body weight (mean ± standard error).

a Significantly different (P≤0.05) from the vehicle control group as determined by Scheffé’s test.

Table 6 Organ-weight-to-body-weight ratios for male and female rats in the 14-day recovery study*

Recovery group	Negative control	Vehicle control	500 mg/kg
Animals, n	5	5	5
Male
Kidney
Absolute	3.27±0.21	3.14±0.14	3.34±0.62a
Relative	0.62±0.06	0.60±0.03	0.70±0.06
Epididymis
Absolute	1.56±0.12	1.36±0.05	1.50±0.13
Relative	0.29±0.03	26±0.02	0.32±0.04a
Submaxillary gland
Absolute	0.75±0.14	0.78±0.03	0.80±0.06a
Relative	0.14±0.02	0.15±0.01	0.17±0.03
Female
Kidney
Absolute	1.90±0.08	2.04±0.33	2.02±0.21
Relative	0.66±0.04	0.69±0.08	0.68±0.07
Submaxillary gland
Absolute	0.49±0.09	0.54±0.05	0.55±0.07
Relative	0.17±0.03	0.18±0.02	0.18±0.02

Notes:

* Organ weights (absolute weights) and body weights are given in g; organ-weight-to-body-weights (relative weights) are given as mg organ weight/g body weight (mean ± standard error).

a Significantly different (P≤0.05) from the vehicle control group as determined by Scheffé’s test.

Table 7 Hematology data for male and female rats in the primary 90-day oral-toxicity study

Main study	Negative control	Vehicle control	125 mg/kg	250 mg/kg	500 mg/kg
Animals, n	10	10	10	10	10
Male
Total leukocyte count (10^3 cells/uL)	6.43±2.34	6.45±1.82	6.97±1.70	7.98±2.84	9.64±1.99^a,c
Total erythrocyte (10^6 cells/uL)	8.71±0.31	8.45±0.41	8.77±0.20	8.95±0.35	9.44±0.41^b,d
Hemoglobin concentration (g/dL)	15.1±0.5	14.9±0.7	14.8±0.5	14.2±0.6	13.3±1.1^a,c
Hematocrit (%)	44.4±1.4	43.6±1.8	43.8±1.5	42.6±1.7	41.3±2.1^a
Mean cell volume (fL)	51.0±1.2	51.7±1.7	49.9±1.6	47.6±1.1^b,d	43.8±2.7^b,d
Mean cell hemoglobin (pg)	17.4±0.5	17.7±0.6	16.8±0.6	15.9±0.5^b,d	14.1±1.3^b,d
Mean cell hemoglobin concentration (g/dL)	34.1±0.3	34.2±0.4	33.7±0.4	33.5±0.6	32.1±1.3^b,d
Female
Total leukocyte count (10^3 cells/uL)	3.47±1.60	3.39±1.12	3.87±1.94	4.66±2.16	4.54±1.30
Total erythrocyte (10^6 cells/uL)	7.56±0.49	7.70±0.43	7.98±0.58	7.97±0.31	8.01±0.85
Hemoglobin concentration (g/dL)	14.0±0.6	14.2±0.7	14.2±0.7	13.8±0.7	12.9±1.0^a,c
Hematocrit (%)	40.4±1.5	41.0±1.9	41.6±2.3	40.6±2.1	39.0±2.9
Mean cell volume (fL)	53.6±2.9	53.2±1.2	52.3±2.5	50.9±1.7	49.0±4.2^a,c
Mean cell hemoglobin (pg)	18.5±0.8	18.4±0.6	17.9±0.8	17.4±0.6	16.2±1.3^b,d
Mean cell hemoglobin concentration (g/dL)	34.6±0.5	34.7±0.6	34.2±0.6	34.1±0.4	33.1±0.6^b,d

Notes: Significantly different from negative control (^aP<0.05, ^bP<0.01) and vehicle control (^cP<0.05, ^dP<0.01) as determined by Scheffé’s test.

Table 8 Hematology data for male and female rats in the 14-day recovery study

Recovery group	Negative control	Vehicle control	500 mg/kg
Animals, n	5	5	5
Male
Total erythrocyte (10^6 cells/uL)	8.44±0.56	8.57±0.40	9.80±0.53^a,c
Mean cell volume (fL)	53.80±1.50	54.60±1.50	48.20±0.80^b,d
Mean cell hemoglobin (pg)	17.60±0.60	17.70±0.50	15.00±0.60^b,d
Mean cell hemoglobin concentration (g/dL)	32.70±0.60	32.40±0.30	31.10±1.10^a
Female
Total erythrocyte (10^6 cells/uL)	7.53±0.26	7.55±0.36	8.63±0.93^a,c
Mean cell volume (fL)	56.50±1.20	56.50±1.70	51.10±2.10^b,d
Mean cell hemoglobin (pg)	18.40±0.50	18.40±0.60	16.10±1.00^a,c
Mean cell hemoglobin concentration (g/dL)	32.50±0.40	32.50±0.40	31.50±0.90^a,c

Notes: Significantly different from negative control (^aP<0.05, ^bP<0.01) and vehicle control (^cP<0.05, ^dP<0.01), as determined by Scheffé’s test.

Table 9 Clinical chemistry data for male and female rats in the primary 90-day oral-toxicity study

Main study	Negative control	Vehicle control	125 mg/kg	250 mg/kg	500 mg/kg
Animals, n	10	10	10	10	10
Male
Total protein (g/dL)	6.0±0.3	6.1±0.2	5.8±0.2	5.6±0.2^a,c	5.5±0.4^a,c
Albumin (g/dL)	2.5±0.1	2.6±0.1	2.4±0.1^c	2.4±0.1^c	2.3±0.2^a,c
Glucose (mg/dL)	162.0±26.0	158.0±17.0	140.0±23.0	135.0±19.0	128.0±20.0^a
Creatine kinase (IU/L)	553.0±182.0	514.0±276.0	669.0±212.0	779.0±196.0	865.0±274.0^c
Chloride (mmol/L)	106.2±1.6	104.7±1.8	105.2±1.8	106.1±1.7	107.8±2.8^c
Female
Total protein (g/dL)	6.3±1.1	6.5±0.5	6.0±0.7	5.7±0.5^a,c	5.6±0.5^a,c
Albumin (g/dL)	3.0±0.2	3.0±0.2	2.7±0.3	2.6±0.3^a,c	2.6±0.2^a,c
Alkaline phosphatase (U/L)	151.0±24.0	145.0±24.0	171.0±40.0	209.0±43.0	271.0±120.0^a,c

Notes: Significantly different from negative control (^aP<0.05, ^bP<0.01) and vehicle control (^cP<0.05), as determined by Scheffé’s test.

Table 10 Histopathology results

	Negative control	Vehicle control	125 mg/kg	250 mg/kg	500 mg/kg
Animals, na	10	10	10	10	10
Male
Forestomach
Submucosal inflammatory cells infiltration	0b	1 (1.0)c	0	0	0
Squamous cell hyperplasia	0	3 (1.0)	9^e,g (2.9)	10^e,g (3.0)	10^e,g (2.4)
Squamous cell vacuolation	0	2 (1.0)	9^e,g (1.1)	10^e,g (1.2)	10^e,g (1.4)
Subepithelial inflammatory cell infiltration	0	0	8^e,g (1.0)	10^e,g (1.0)	9^e,g (1.0)
Submucosal squamous cell cyst	0	1	0	0	0
Glandular stomach
Submucosal inflammatory cells infiltration	1 (1.0)	2 (1.0)	10^e,g (2.6)	10^e,g (2.5)	10^e,g (2.7)
Submucosal edema	0	0	3 (1.0)	5^d,f (1.0)	2 (1.0)
Superficial epithelial degeneration/regeneration	0	0	8^e,g (1.0)	10^e,g (1.0)	8^e,g (1.0)
Erosion	0	0	1 (2.0)	6^e,g (1.0)	0
Intracytoplasmic hyaline droplet	0	1 (1.0)	10^e,g (2.3)	10^e,g (2.3)	10^e,g (2.6)
Mucous cell hyperplasia	0	0	5^e,g (1.5)	9^e,g (1.8)	10^e,g (1.8)
Eosinophilic chief cell	0	0	6^e,g (1.5)	8^e,g (1.6)	8^e,g (1.6)
Eye
Retinal atrophy	0	0	0	0	2 (3.0)
Pancreas
Acinar cell apoptosis	2 (1.0)	10 (1.7)	10^e (1.8)	10^e (2.6)	10^e (3.7)
Chronic inflammation	0	0	6^e,g (1.3)	8^e,g (1.3)	10^e,g (1.6)
Female
Forestomach
Squamous cell hyperplasia	0	1 (1.0)	3 (1.0)	2 (1.0)	5^d (1.0)
Squamous cell vacuolation	1 (1.0)	2 (1.0)	10^e,g (2.5)	10^e,g (2.8)	10^e,g (2.8)
Subepithelial inflammatory cell infiltration	0	0	6^e,g (1.0)	8^e,g (1.0)	4^d,f (1.0)
Glandular stomach	1 (1.0)	1 (1.0)	9^e,g (1.0)	10^e,g (1.0)	9^e,g (1.0)
Submucosal inflammatory cell infiltration	0	1 (1.0)	10^e,g (2.1)	10^e,g (2.4)	9^e,g (2.2)
Submucosal edema	0	0	0	2 (1.0)	0
Superficial epithelial degeneration/regeneration	0	0	10^e,g (1.0)	9^e,g (1.0)	8^e,g (1.0)
Erosion	0	0	1 (1.0)	5^d,f (1.0)	0
Intracytoplasmic hyaline droplet	0	0	10^e,g (2.0)	10^e,g (2.5)	10^e,g (2.3)
Mucous cell hyperplasia	0	0	4^e,g (1.2)	8^e,g (1.5)	9^e,g (1.5)
Eosinophilic chief cell	0	0	2 (2.0)	2 (1.5)	6^e,g (1.8)
Eye
Retinal atrophy	0	0	0	0	3 (3.0)
Pancreas
Acinar cell apoptosis	0	2 (1.5)	6^e (1.3)	10^e,g (2.1)	10^e,g (3.7)
Chronic inflammation	1 (2.0)	0	3 (1.0)	8^e,g (1.0)	10^e,g (1.8)

Notes:

a Number of animals with tissue examined microscopically;

b number of animals with lesion;

c average severity grade of lesion in affected animals: 1= minimal, 2= slight, 3= moderate, 4= marked, 5= severe. Significantly different from negative control (^dP<0.05, ^eP<0.01) and vehicle control (^fP<0.05, ^gP<0.01) at 125 mg/kg, 250 mg/kg, 500 mg/kg group, respectively, as determined by Fisher’s test.

Figure 2 Histopathological findings of the pancreas and the outer layer of the retina.

Notes: (A) Normal microscopic structure of the pancreas of a male Sprague Dawley (SD) rat administered 0 mg/kg zinc oxide (ZnO) by gavage for 90 days. Note the normal acinar cell (NA) in the pancreas. (B) Acinar cell atrophy (A), acinar cell apoptosis (arrows), and an interstitial inflammatory cell infiltration (I) in the pancreas of an SD rat administered 500 mg/kg ZnO by gavage for 90 days. Note the chronic inflammation (including “A”, arrows, and “I”) in the pancreas. (C) Normal microscopic structure of the eye of a male SD rat administered 0 mg/kg ZnO by gavage for 90 days. Note the normal outer layer of retina (N) in the eye. (D) Remarkable retinal atrophy in the eye of a male SD rat administered 500 mg/kg ZnO by gavage for 90 days. Note the retinal atrophy (R) in the outer layer of the retina of the eye. Scale bar: 100 mm; hematoxylin and eosin staining.

Figure 3 Histopathological findings of limiting ridge and glandular stomach.

Notes: (A) Normal limiting ridge of the stomach of a male Sprague Dawley (SD) rat administered 0 mg/kg zinc oxide (ZnO) nanoparticles. Note the normal epithelium (NE) in the forestomach. (B) Squamous cell hyperplasia (H) and squamous cell vacuolation (arrows) in the limiting ridge of the forestomach of an SD rat administered 500 mg/kg ZnO by gavage for 90 days. Note the squamous cell hyperplasia (H) in the forestomach. (C) Normal glandular stomach of a male SD rat administered 0 mg/kg ZnO by gavage for 90 days. Note the normal mucosa (N) in the glandular stomach. (D) Eosinophilic chief cells (arrows) in the glandular stomach of a male SD rat administered 500 mg/kg ZnO by gavage for 90 days. Scale bar: 100 mm; hematoxylin and eosin staining.

Figure 4 Histopathological findings of glandular stomach.

Notes: (A) Normal glandular stomach of a male Sprague Dawley (SD) rat in 0 mg/kg zinc oxide (ZnO). Note the normal submucosa (NS) in the glandular stomach. (B) Submucosal inflammatory cell infiltration (SI) in glandular stomach of a male SD rat administered 500 mg/kg ZnO by gavage for 90 days. (C) Normal glandular stomach of a male SD rat administered 0 mg/kg ZnO by gavage for 90 days. Note the normal parietal cell distribution (arrows) in the glandular stomach. (D) Mucous cell hyperplasia (arrows) – stained blue – in the glandular stomach of a male SD rat administered 500 mg/kg ZnO by gavage for 90 days. Scale bar: 100 mm; A and B, hematoxylin and eosin staining; C and D, Alcian Blue staining.

Table 11 Tissue distribution

Region	Male					Female
	Control	V-control	125 mg/kg	250 mg/kg	500 mg/kg	Control	V-control	125 mg/kg	250 mg/kg	500 mg/kg
Plasma	4.68	4.21	7.08	9.06	18.42	5.13	4.92	5.28	6.4	13.02
Liver	35.14	32.68	42.46	55.62	131.63	30.91	30.44	43.99	62.18	150.13
Kidney	25.53	23.75	36.22	55.91	122.53	25.35	25.48	34.02	40.16	121.65
Large intestine	17.97	17.78	48.3	113.06	106.27	11.85	12.82	66.49	62.12	105.35
Small intestine	18.65	16.17	40.75	54.74	110.92	17.64	23.00	52.70	62.69	111.31
Feces	305.94	279.11	8,287.9	13,680.30	23,716.32	312.95	241.09	7,204.37	10,403.97	17,286.96
Stomach	18.46	19.10	34.49	24.02	35.04	16.39	19.76	36.01	26.06	21.59
Lung	15.85	16.11	17.45	19.33	26.94	14.70	15.49	18.57	19.24	26.40
Spleen	2.78	3.22	3.01	3.44	3.96	2.81	3.16	2.81	3.09	3.62
Brain	11.57	11.59	11.84	12.09	12.56	11.28	11.74	12.08	12.25	11.22
Testes	23.45	23.80	24.34	24.01	25.26	–	–	–	–	–
Ovary	–	–	–	–	–	31.60	24.17	36.73	36.39	46.15

Abbreviation: V-control, vehicle control.