Journal List > Lab Anim Res > v.26(2) > 1053599

TOOLS
Kobayashi, Pillai, Michael, Cherian, and Ohnishi: Determining NOEL/NOAEL in Repeated-dose Toxicity Studies, When the Low Dose Group Shows Significant Difference in Quantitative Data
Original Article

Lab. Anim. Res. 2010;26(2):133-137.

Published online: 17 June 2010

DOI: https://doi.org/10.5625/lar.2010.26.2.133

Determining NOEL/NOAEL in Repeated-dose Toxicity Studies, When the Low Dose Group Shows Significant Difference in Quantitative Data

Katsumi Kobayashi1, ∗, K. Sadasivan Pillai2, Mathews Michael2, K.M. Cherian2, Mariko Ohnishi1

1National Institute of Technology and Evaluation (NITE), Tokyo, Japan

2Frontier Lifeline, International Centre for Cardio-Thoracic and Vascular Diseases, Chennai, India

∗Corresponding author: Katsumi Kobayashi, National Institute of Technology and Evaluation (NITE), 49-10, Nishihara-nichome, Shibuya-ku, Tokyo 151-1166, Japan Tel: +81-3-3481-1735 Fax: +81-3-3481-1950 E-mail: kobayashi-katsumi@nite.go.jp

Received 20 January 201026 April 2010       Accepted 14 May 2010

Copyright © 2010 Korean Association for Laboratory Animal Science

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In repeated-dose 28-day oral toxicity study design, the low dose is fixed as the no observed effect level (NOEL). But, in practice the low dose usually shows significant difference in few measurable items in most of the studies. We investigated 109 of repeated-dose 28-day oral toxicity studies in rats conducted according to the Chemical Substance Control Law, Japan and examined the measurable items (functional observational battery, urinalysis, hematology, blood chemistry and absolute and relative organ weights) of the low dose group which showed a statistical significant difference (P<0.05) compared to the respective control groups. The investigation revealed that, 205/12,167 (1.6%) measurable items showed a significant difference in the low dose groups. The significant difference shown by urinalysis was high (3.3%), followed by clinical chemistry parameters, hematology, relative organ weights and absolute organ weights (1.8-1.1%). We conclude from the investigation that the low dose may be considered as NOEL, if the significant difference of measurable items of it is about 2% (maximum <5%), compared to the control. However, due consideration may be given to the clinical relevance of the items that showed a significant difference.

Keywords: Repeated-dose toxicity study, statistical significant difference, rats, low dose, NOEL/NOAEL

REFERENCES

Kobayashi K.., Pillai K.S.., Sakuratani Y.., Abe T.., Kamata E.., Hayashi M.2008. Evaluation of statistical tools used in short-term repeated dose administration toxicity studies with rodents. J. Toxicol. Sci. 33(1):97–104.
crossref
Kobayashi K.., Sakuratani Y.., Abe T.., Nishikawa S.., Yamada J.., Hirose A.., Kamata E.., Hayashi M.2010. Relation between statistics and treatment-related changes obtained from toxicity studies in rats: if detected a significant difference in low or middle dose for quantitative values, this change is considered as incidental change? J. Toxicol. Sci. 35(1):79–85.
crossref
Ministry of Health, Labour and Welfare. 2009. http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPage.jsp.
NITE. 2007. http://www.safe.nite.go.jp/kasinn/pdf/28test.pdf.
OECD. 1995. Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Repeated Dose 28-Day Oral Toxicity Study in Rodents. No. 407, p. 8, OECD, Paris.
OECD. 2000. Organization for Economic Cooperation and Development. Guidance Notes for Analysis and Evaluation of Repeat-Dose Toxicity Studies. OECD Environment, Health and Safety Publications Series on Pesticides No. 10. ENV/JM/MONO/2000 (18), p.87. OECD, Paris.

Table 1.
Group composition in Chemical Substance Control Law Guideline of Japan (NITE, 2007)
Group, dosage Number of animal/sex
Dosing period for 28 days Recovery period for 14 days
Control 10 5
Low 5 -
Medium 5 -
High 10 5
Table 2.
Items of quantitative data examined
Measurement data Items examined
Functional observational battery Grip strength, hindlimb foot splay, motor activity
Urinalysis Urine volume, specific gravity, osmotic pressure, sodium, potassium, chloride
Hematology White blood cell, differential leukocyte ratio, red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet, reticulocyte, prothrombin time, activated partial thromboplastin time, etc.
Blood chemistry Total protein, albumin/globulin ratio, total cholesterol, glucose, blood urea nitrogen, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, triglyceride, inorganic phosphate, calcium, sodium, potassium, chloride, etc.
Absolute and relative organ weights Brain, thyroid, thymus, heart, lungs, liver, spleen, adrenal glands, testes, ovaries, etc.
Table 3.
Statistics analysis tools used for the repeated dose 28-day oral toxicity studies in rats
Analytical tools Bartlett'shomogenous test
ANOVA
Dunnetts's multiple comparison test
Scheffé's multiple comparison test
Duncan's multiple range test
Kruskal-Wallis's H test
Nonparametric type Dunnett's test
Nonparametric type Scheffé's test
Steel test
etc.
Table 4.
Number of items showed a significant difference (P<0.05) from control group in each study
Study No. Number of item Number of items showed a significant differencea
Low dose Medium dose High dose Top dose
1 120 0 (0.0) 2 (1.7) 4 (3.3) 10 (11)
2 104 6 (5.7) 5 (4.8) 4 (3.8) -
3 108 1 (0.9) 2 (1.8) 3 (2.7) -
4 128 2 (1.5) 10 (7.8) 23 (18) -
5 98 4 (4.8) 10 (10) 17 (17) -
6 120 0 (0.0) 4 (3.3) 10 (8.3) 24 (20)
7 146 2 (1.3) 6 (4.1) 42 (28) -
8 118 1 (0.8) 1 (0.8) 0 (0.0) 1 (0.8)
9 118 1 (0.8) 2 (1.6) 27 (22) -
10 116 1 (0.8) 10 (8.6) 26 (22) -
11 116 2 (1.7) 10 (8.6) 26 (22) -
12 156 3 (1.9) 11 (7.0) 36 (23) -
13 106 2 (1.8) 5 (4.7) 30 (28) -
14 100 7 (7.0) 11 (11) 23 (23) -
15 100 2 (2.0) 2 (2.0) 2 (2.0) -
16 128 3 (2.3) 10 (7.8) 44 (34) -
17 96 5 (5.2) 0 (0.0) 4 (4.1) -
18 118 2 (1.6) 0 (0.0) 1 (0.8) 4 (3.3)
19 114 0 (0.0) 1 (0.8) 1 (0.8) -
20 102 4 (3.9) 1 (0.9) 6 (5.8) -
21 94 3 (3.1) 6 (6.3) 2 (2.1) -
22 128 0 (0.0) 2(1.5) 23 (17) -
23 136 3 (2.2) 7 (5.1) 11 (8.0) -
24 76 1 (1.3) 2 (2.6) 5 (6.5) 21 (27)
25 110 2 (1.8) 5 (4.5) 17 (15) -
26 118 0 (0.0) 0 (0.0) 0 (0.0) 4 (3.3)
27 116 0 (0.0) 2 (1.7) 9 (7.7) 23 (19)
28 100 2 (2.0) 4 (4.0) 37 (37) -
29 158 5 (3.1) 12 (7.5) 31 (19) -
30 120 0 (0.0) 2 (1.6) 10 (8.3) -
31 134 0 (0.0) 0 (0.0) 4 (2.9) 16 (11)
32 126 1 (0.7) 6 (4.7) 12 (9.5) 30 (23)
33 106 3 (2.8) 1 (0.9) 23 (21) -
34 128 0 (0.0) 3 (2.3) 3 (2.3) 38 (29)
35 118 0 (0.0) 9 (7.6) 24 (20) 40 (33)
36 54 0 (0.0) 2 (3.7) 9 (16) -
37 106 0 (0.0) 2 (1.8) 6 (5.6) -
38 122 1 (0.8) 5 (4.0) 44 (36) -
39 100 6 (6.0) 17 (17) 27 (27) -
40 104 1 (0.9) 3 (2.8) 7 (6.7) -
41 104 2 (1.9) 4 (3.8 9 (8.6) -
42 106 3 (2.8) 1 (0.9) 25 (23) -
43 151 2 (1.3) 9 (2.9) 31 (20) -
44 106 2 (1.8) 9 (8.4) 3 (2.8) -
45 112 3 (2.6) 9 (8.0) 18 (16) -
46 86 0 (0.0) 5 (5.8) 14 (16) 30 (34)
47 104 2 (1.9) 0 (0.0) 7 (6.7) -
48 70 0 (0.0) 1 (1.4) 0 (0.0) -
49 50 0 (0.0) 2 (4.0) 9 (18) 30 (60)
50 74 0 (0.0) 1 (1.3) 0 (0.0) -
51 132 3 (2.2) 5 (3.7) 9 (6.8) -
52 142 12 (8.4) 11 (7.7) 7 (4.9) -
53 94 1 (1.0) 2 (2.1) 2 (2.1) -
54 116 5 (4.3) 7 (6.0) 3 (2.5) -
55 98 7 (7.1) 3 (3.0) 23 (23) -
56 104 6 (5.7) 12 (11) 21 (20) -
57 88 2 (2.2) 1 (1.1) 6 (6.8) -
58 98 0 (0.0) 0 (0.0) 8 (8.1) -
59 100 0 (0.0) 0 (0.0) 1 (1.0) 8 (8.0)
60 104 2 (1.9) 8 (7.6) 37 (35) 58 (55)
61 96 1 (1.0) 7 (7.2) 16 (16) -
62 128 1 (0.7) 3 (2.3) 25 (19) -
63 138 2 (1.4) 6 (4.3) 49 (35) -
64 128 1 (0.7) 1 (0.7) 12 (9.3) -
65 130 0 (0.0) 3 (2.3) 9 (6.9) 33 (25)
66 104 0 (0.0) 6 (5.7) 42 (40) -
67 94 6 (6.3) 3 (3.1) 4 (4.2) -
68 106 3 (1.8) 3 (1.8) 3 (1.8) -
69 110 0 (0.0) 0 (0.0) 2 (1.8) 21 (19)
70 122 0 (0.0) 2 (1.6) 4 (3.2) 0 (0.0)
71 162 1 (0.6) 2 (1.2) 9 (5.5) -
72 136 1 (0.7) 3 (2.2) 18 (13) -
73 104 1 (0.9) 0 (0.0) 1 (0.8) 4 (3.8)
74 106 0 (0.0) 0 (0.0) 7 (6.6) 19 (25)
75 112 7 (6.2) 1 (0.8) 1 (0.8) -
76 118 1 (0.8) 3 (2.5) 6 (5.0) 13 (11)
77 90 0 (0.0) 0 (0.0) 2 (2.2) 14 (15)
78 116 0 (0.0) 4 (3.4) 2 (1.7) 5 (4.3)
79 106 1 (0.9) 0 (0.0) 4 (3.7) 8 (7.5)
80 62 2 (3.2) 2 (3.2) 2 (3.2) 3 (4.8)
81 96 1 (1.0) 1 (1.0) 0 (0.0) -
82 124 3 (2.4) 3 (2.4) 30 (24) 53 (42)
83 110 1 (0.9) 3 (2.7) 13 (11) -
84 100 1 (1.0) 1 (1.0) 2 (2.0) -
85 102 1 (0.9) 2 (1.9) 2 (1.9) 14 (13)
86 120 4 (3.3) 12 (10) 15 (12) 29 (24)
87 162 1 (0.6) 4 (2.4) 16 (9.8) -
88 134 3 (2.2) 1 (0.7) 3 (2.2) 17 (12)
89 90 1 (1.1) 2 (2.2) 2 (2.2) -
90 108 1 (0.9) 2 (1.8) 1 (0.9) -
91 100 2 (2.0) 2 (2.0) 4 (4.0) -
92 92 0 (0.0) 0 (0.0) 0 (0.0) 2 (2.1)
93 110 2 (1.8) 5 (4.5) 21 (19) 33 (30)
94 100 0 (0.0) 0 (0.0) 1 (1.0) -
95 104 1 (0.9) 1 (0.9) 1 (0.9) -
96 104 5 (4.8) 11 (10) 22 (21) -
97 98 4 (4.0) 5 (5.1) 19 (19) -
98 114 0 (0.0) 0 (0.0) 8 (7.0) 43 (37)
99 118 3 (2.5) 3 (2.5) 0 (0.0) -
100 88 1 (1.1) 1 (1.1) 1 (1.1) -
101 118 3 (2.5) 3 (2.5) 12 (10) -
102 120 1 (0.8) 0 (0.0) 3 (2.5) 18 (15)
103 116 0 (0.0) 1 (0.8) 1 (0.8) 14 (12)
104 150 1 (0.6) 6 (4.0) 15 (10) -
105 134 5 (3.7) 7 (5.2) 14 (10) -
106 100 0 (0.0) 0 (0.0) 6 (6.0) 11 (11)
107 136 1 (0.7) 1 (0.7) 4 (2.9) -
108 130 2 (1.5) 4 (3.0) 16 (12) 31 (23)
109 116 1 (0.8) 1 (0.8) 2 (1.7) 9 (7.7)
Number of study 109 109 109 109 37
Total 12,167 205 414 1318 731
Mean 112 1.9 3.8 12.1 20
Maximum 162 8.4% 17% 40% 60%
Minimum 50 0.0% 0.0% 0.0% 0.0%
Mode - 0.0% - - -

a Values in parentheses are percent of items.

Table 5.
Number of quantitative data showed a significant difference (P<0.05) from control group
Quantitative data examined Number of items examined Number of items showed a significant differencea
Low dose medium dose High dose Top dose
FOB 68 1 (1.4) 3 (4.4) 8 (11) 1/10 (10)
Urinalysis 392 13 (3.3) 30 (7.6) 81 (20) 40/96 (40)
Hematology 3,586 56 (1.5) 106 (2.9) 318 (8.8) 176/1198 (14)
Clinical chemistry 4,285 79 (1.8) 163 (3.8) 455 (10) 267/1426 (18)
Absolute organ weight 1,928 22 (1.1) 44 (2.2) 188 (9.7) 103/672 (15)
Relative organ weight 1,908 34 (1.7) 68 (3.5) 268 (14) 144/672 (21)
Total 12,167 205 (1.6) 414 (3.4) 1318 (10) 731/4074 (17)

a Values in parentheses are percent of items.

TOOLS
Similar articles