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Historical control data for hematology parameters obtained from toxicity studies performed on different Wistar rat strains: Acceptable value ranges, definition of severity degrees, and vehicle effects

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Abstract

The physiological and health status of control animals may vary. Due to this variation, it is important to define acceptable ranges of control hematology parameters to gain a better understanding of adverse and non-adverse effects of test substances. After generating historical control data for two Wistar rat strains (RccHan™:WIST and Crl:WI(Han)) from different breeders, the data sets were statistically analyzed using Minitab®. After noticing that single outliers can affect the study control data set, the respective outliers were verified relative to the available histopathology findings, for example, inflammatory pulmonary lesions following vehicle aspiration or spontaneous sperm granuloma affecting the health status and hematology data of the respective animals. Such data points were excluded from the control data set. Comparing both data sets, it was obvious that different blood sampling and anesthesia methods as well as strain differences may result in slightly different values. After excluding the outliers, a data set from animals with presumably good health status was generated to define acceptable ranges and severity degrees. To evaluate effects, possibly influencing hematology parameters and defined acceptable ranges, selected vehicles and different study types were observed.
Article
Historical control data for hematology
parameters obtained from toxicity
studies performed on different Wistar
rat strains: Acceptable value ranges,
definition of severity degrees, and
vehicle effects
Marlies de Kort
1
, Klaus Weber
2
, Bjo
¨rn Wimmer
3
,
Katharina Wilutzky
1
, Patricia Neuenhahn
1
, Philip Allingham
1
,
and Anne-Laure Leoni
1
Abstract
The physiological and health status of control animals may vary. Due to this variation, it is important to define acceptable
ranges of control hematology parameters to gain a better understanding of adverse and non-adverse effects of test
substances. After generating historical control data for two Wistar rat strains (RccHan:WIST and Crl:WI(Han)) from
different breeders, the data sets were statistically analyzed using Minitab
. After noticing that single outliers can affect the
study control data set, the respective outliers were verified relative to the available histopathology findings, for example,
inflammatory pulmonary lesions following vehicle aspiration or spontaneous sperm granuloma affecting the health status
and hematology data of the respective animals. Such data points were excluded from the control data set. Comparing both
data sets, it was obvious that different blood sampling and anesthesia methods as well as strain differences may result in
slightly different values. After excluding the outliers, a data set from animals with presumably good health status was
generated to define acceptable ranges and severity degrees. To evaluate effects, possibly influencing hematology para-
meters and defined acceptable ranges, selected vehicles and different study types were observed.
Keywords
Historical control data, hematology, toxicity studies, rat, Wistar strains, acceptable ranges, alterations, severity degrees,
vehicle effects
Date received: 30 March 2020; accepted: 5 May 2020
Introduction
Toxicity studies can only be interpreted when appropriate
reference values established from healthy control animals
are available. Specific reference intervals are needed for
each animal species being tested. Many physiological para-
meters vary with age, sex, housing, food/water consump-
tion, circadian rhythm, increased or decreased activity,
stress, or sexual cycle.
1
BSL BIOSERVICE Scientific Laboratories Munich GmbH, Munich,
Germany
2
AnaPath GmbH, Liestal, Switzerland
3
Individual Contributor, Salzburg, Austria
Corresponding author:
Marlies de Kort, BSL BIOSERVICE Scientific Laboratories Munich GmbH,
Behringstrasse 6/8, 82152 Planegg/Munich, Germany.
Email: mkort@bioservice.com
Toxicology Research and Application
Volume 4: 1–32
ªThe Author(s) 2020
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Toxicology Research and Application
The use of historical control data in preclinical studies is
discussed in detail by Keenan et al.,
1
whereas this publica-
tion, like several others,
2–5
relates solely to proliferative
lesions. Nevertheless, the base recommendations as sum-
marized by the Historical Control Data Working Group of
the Society of Toxicologic Pathology are also valid for
historical control data other than proliferative lesions.
When adapted, these recommendations
1
also easily apply
to data other than pathology data and consist mainly of:
the use of the concurrent control group as the most
relevant comparator to determine treatment-related
effects;
the estimation of the potential impact of design-
related parameters (e.g. laboratory, species/strain,
administration route, vehicle, feed, feeding prac-
tices, study duration, housing) on study outcome,
which underlies the appropriate selection of studies
for the use of control data;
standardized diagnostic practices, including sam-
pling procedures, use of instruments, and application
of diagnostic criteria, which can impact study and
control data;
use of control data from the laboratory that con-
ducted the study under review, which will likely
be more comparable than control data compiled
from several laboratories;
careful evaluation of published control data, which
might be helpful in the interpretation of study data;
and
presenting control data as a range of incidences or
percentages, mean, and standard deviation and/or
minimum/maximum value ranges for any parameter.
Furthermore, recommendations for the evaluation of
clinical pathology data have been published.
6,7
The evaluation of hematology parameters is standar-
dized by several ICH (M3 R2 2009),
8
OECD (407/2008,
9
408/2018
10
), and EPA (2000)
11
guidelines for toxicity
studies. Standard parameters include red blood cell
(RBC), hemoglobin (HB), hematocrit (HCT), total and
differential leukocyte count (white blood cell (WBC)),
mean corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglobin con-
centration (MCHC), reticulocyte (RET), and platelet
count (PLT).
However, additional parameters such as erythrocyte
indices methemoglobin (MET-HB) and Heinz body
(HEINZBOD) may be included in the evaluation as appro-
priate to the testing of specific test items. Furthermore,
there is no indication in the guidelines for an approach
using absolute and/or relative differential leukocyte count
values. Moreover, RET values can be split further into the
maturity indices using the fluorescence behavior of these
cells. Hence, there are different outlines for control data
compilations in different laboratories.
Scarce data are published on historical control data for
hematology parameters from toxicity studies performed in
rodents.
12–14
In some circumstances, the published data
cannot be used for comparisons due to missing methodo-
logical data, for example, animal ages or study lengths
(e.g. data published by Said and Abiola
15
).
Material and methods
RccHan:WIST
General: Control data for hematology parameters were col-
lected at RCC Ltd/Harlan Laboratories Ltd, Switzerland,
over a 6-year period from 2002 to 2007. The animals ori-
ginated from a single production site (RCC Ltd/Harlan
Laboratories Ltd, Fu
¨llinsdorf, Switzerland) thus minimiz-
ing the influence of breeding and environment-related vari-
ables. Data were collected from control animals at ages
from 7 weeks up to 71 weeks (including dose-range
finding studies up to carcinogenicity studies); 5062 males
and 5046 females are included in this survey.
Some hematology parameters (HEINZBOD, neutrophil
(NEUT), and MET-HB) were compiled from fewer animals
and studies because they were outside the range of standard
laboratory evaluations. All studies were performed in com-
pliance with the Swiss Ordinance relating to Good Labora-
tory Practice (SWISS GLP, 2005
16
) based on the OECD
Principles of Good Laboratory Practice (OECD, 1997) and/
or the standard operating procedures of the testing labora-
tory. The studies were performed under permission of the
ethical committee and Swiss authorities. RCC Ltd/Harlan
Laboratories Ltd, Switzerland was accredited by AAA-
LAC. Clinical laboratory data were generated at RCC Ltd
(Fu
¨llinsdorf, Switzerland) in an accredited laboratory in
accordance with the ISO/IEC 17025 standard under accred-
itation number STS 085 from the Swiss Accreditation
Service.
The data described below were obtained from various
types of repeated-dose toxicity studies (i.e. feeding,
gavage, dermal, inhalation, or intravenous administration).
Animals were housed individually or in groups of three or
five (depending on the administration route) in Makrolon
cages with wire mesh tops and standardized softwood bed-
ding (Lignocel, Schill AG, Muttenz, Switzerland). Rats had
free access to standard rat maintenance diet 3433 (Provimi
Kliba AG, Kaiseraugst, Switzerland) and to tap water. The
animal facility was air-conditioned with 10–15 air changes
per hour, temperatures of 22C+3C, and relative humid-
ity of 30–70%. A 12-h fluorescent light/dark cycle was
maintained with music during the light period.
The evaluations were generally performed during the
pretest and at the end of the toxicity study prior to necropsy
in fasted animals that had free access to water. Experienced
technicians withdrew blood with heparin-coated glass
capillaries from the retro-orbital plexus of animals under
light isoflurane anesthesia (5%isoflurane/95%oxygen)
2Toxicology Research and Application
into tri-potassium-ethylenediaminetetraacetic acid-coated
anticoagulant tubes for complete blood cell count evalua-
tion. Sampling was performed early in the working day
prior to necropsy to reduce biological variations caused
by circadian rhythms.
Hematology evaluation methods: Commercially available test
methods (flow cytometry) based on the ADVIA 120 hema-
tology system (Bayer AG, now Siemens AG; including the
reagents) were used to determine hematological para-
meters, and in-house procedures based on published scien-
tific procedures
17,18
were used for microscopy, blood
smear preparation, and staining (differential WBC count,
red cell morphology, and HEINZBOD counts). MET-HB
determination (spectroscopy) was based on a commercially
available test method in conjunction with an OSM-3
(Radiometer) hemoximeter.
Crl:WI(Han)
General: Control data on hematology parameters were col-
lected at BSL BIOSERVICE Scientific Laboratories
Munich GmbH (BSL), over a 10-year period from 2010
to 2019. The animals derived from a controlled, full-
barrier maintained breeding system (Charles River, Sulz-
feld, Germany) thus minimizing the influence of breeding
and environment-related variables. The present data were
collected from control animals at ages of 8–12, 13–18, and
19–40 weeks (including dose-range finding, subacute toxi-
city, and repeated-dose toxicity studies—OECD and
pharma on the one hand and implantation and reprotoxicol-
ogy studies on the other); 1636 males and 1596 females are
included in this survey.
All studies were performed in compliance with OECD
Principles of Good Laboratory Practice
19
(revised in 2018)
and the testing laboratory’s standard operating procedures.
In accordance with German animal protection law, the
study types were reviewed and accepted by local authori-
ties. Furthermore, the studies were subjected to an ethical
review process and were authorized by the Bavarian animal
welfare administration. Since 2014, studies were performed
in an AAALAC-accredited laboratory.
The data described below were obtained from dose-
range finding, repeated-dose toxicity, reprotoxicology, and
implantation studies with various administration routes
(i.e. feeding, gavage, dermal, intravenous, subcutaneous,
or intramuscular). Animals were housed individually or
in groups of two to five (depending on the administration
route) in type III/H (approximately 800 cm
2
) or type IV
(approximately 1800 cm
2
) cages (most commonly indivi-
dually ventilated) on Altromin sawdust bedding. Rats had
free access to standard rat maintenance diet 1324 (Altro-
min) and to acidified water. The animal facility was air
conditioned with 10–15 air changes per hour, temperatures
of 22C+3C, and relative humidity of 55%+10%.
There was a 12-h artificial light/dark cycle. Makrolon
or
cardboard tunnels, wood bricks, and sizzle material were
used as environmental enrichment.
Blood sampling was generally performed at the end of
the toxicity study prior to necropsy in overnight-fasted ani-
mals that had free access to water. Experienced technicians
withdrew blood, with uncoated cannulas (length: 40 mm
and diameter: 0.9 mm) from the abdominal aorta of animals
under ketamine–xylazine anesthesia into EDTA-coated
anticoagulant tubes for complete blood cell count evalua-
tion. Sampling was performed early in the working day
prior to necropsy to reduce biological variations caused
by circadian rhythms.
Hematology evaluation methods: Commercially available
test methods (flow cytometry) based on the ADVIA 120
hematology system (Bayer AG, now Siemens AG; includ-
ing the reagents) were used to determine hematological
parameters.
Statistical analysis
General: The values for each of the reported parameters
were grouped by sex and age range derived from study type
and duration. The number of animals, the respective mean,
standard deviation, minimum and maximum were reported
for all parameters. The data evaluated from Harlan Labora-
tories Ltd includes animals at ages of 7 weeks up to
71 weeks
13
in six different age-groups (7, 8–12, 13–
18, 19–40, 41–70, and 71 weeks). Outliers were detected
during the respective studies. No statistical outlier test was
done with Minitab
(version 18, Minitab, LLC) because
only summarized data was available, meaning no individ-
ual data and medians were available.
The data collected at BSL was evaluated including ani-
mals at ages of 8 to 40 weeks in three different age-groups
(8–12, 13–18, and 19–40 weeks) to permit comparison of
the two different data sets. All parameters were analyzed
statistically using Minitab
(version 18, Minitab, LLC).
Individual parameters for each of the three age-groups were
checked for parametric/nonparametric distribution using
the Anderson–Darling normality test to exclude all outliers
before further analysis. All statistically identified outliers
were then assigned to the respective study and were exam-
ined on available histopathology finding.
Comparison of different study types: A data set for reproduc-
tion toxicity studies (OECD 422) and implantation studies
(ISO 10993) was generated to consider differences between
various study types with animals at the ages of 19–40
weeks using variance analysis.
Vehicle effects: Further statistical data analysis was per-
formed on the data set. Data from repeated-dose toxicity
studies (oral route) from BSL at the ages of 8–12 and 19–40
weeks were analyzed to see if different vehicles may have
an impact on hematological parameters by applying
Bonett’s and Levene’s tests for variance analysis at a
de Kort et al. 3
significance level of 95%.pValues below 0.05 were
treated as significantly different.
Comparison of different rat strains: Both data sets as well
those collected from other related published data were
compared, whereas Crl:WI(Han) and RccHan:WIST
were statistically analyzed in addition.
BSL and RCC Ltd/Harlan Laboratories Ltd, Switzer-
land, summarized data were compared in Minitab
with a
two-sample t-test at a significance level of 95%.pValues
below 0.05 were treated as significantly different. As the
true values for the data sets were unknown, a possible
offset between both laboratories was calculated by com-
paring the means of all three age-groups against each
other.
Results
Setting acceptable ranges and grading deviations
For statistical correctness and data comparability, the
acceptable ranges of both data sets were defined as mean
value +2 standard deviations. In some cases, acceptable
ranges started at 0.0%or 0.0 G/L, what does not mean that
0.0 values are really acceptable as it can be seen in the case
of relative high fluorescence reticulocyte (HRET) and
absolute NEUT values (see Tables 7 and 8).
The degrees of variation (decrease or increase) were
estimated for three severity degrees (slight ¼grade 1, mod-
erate ¼grade 2, severe ¼grade 3) using the mean and the
minimum and maximum ranges obtained from control ani-
mal values. The severity degrees were defined random, for
example, in RBC, HCT, red cell volume distribution width
(RDW), MCV, MCH, MCHC, hemoglobin concentration
distribution width (HDW), RET, low fluorescence reticu-
locyte (LRET), HRET, and WBC, as an increase or
decrease of 10%(slight), 50%(moderate), and 70%
(severe) referring to the respective acceptable ranges. For
these parameters, the division of severity into 10%,50%,
and 70%was chosen because they can be correlated with
pathological findings, for example, a deviation of 10%in
HCT or HB usually has no effect in the histopathological
examination. But a deviation of more than 10%becomes
visible as either extramedullary hemopoiesis or bone mar-
row atrophy, or embolism. A deviation of 70%and more is
usually associated with severe damage or even death. In the
differential leukocyte count, the acceptable range for eosi-
nophil (EOS), basophil (BASO) and monocyte (MONO)
started at 0.0%or 0.0 G/L. Therefore, only increasing
severity degrees were defined, as deviations of 10%to
20%is already extremely unusual for these parameters. The
severity degree settings are necessary to interpret slight,
moderate, and severe effects of test substances.
The acceptable ranges and grading deviations for
RccHan:WIST Rats are presented in Tables 1 to 15 and
for Crl:WI(Han) in Tables 16 to 19.
Values obtained from RccHan:WIST rats
As a standard evaluation, the following parameters are
included: RBC, HB, HCT, total and differential leukocyte
count (WBC), MCV, MCH, MCHC, RET, large unstained
cell (LUC) and PLT.
13
Additional parameters, such as ery-
throcyte indices MET-HB and HEINZBOD, were evalu-
ated depending on the type of the toxicity study or test
substance. Values were available from the following age-
groups: 7 weeks, 8–12 weeks, 13–18 weeks, 19–40
weeks, 41–70 weeks, and 71 weeks.
RBC. RBC mean values from 8.32 to 8.88 T/L in males and
7.58 to 8.05 T/L in females were considered normal ranges
for animals at ages from 8 to 71 weeks. In males and
females of ages 7 weeks, the mean values were 7.27 and
7.25, respectively. Therefore, acceptable ranges were lower
in young (6.4–8.1 T/L in both sexes) compared to older
animals (7.0–9.8 T/L in males vs 6.5–8.9 T/L in females),
whereas the ranges for females were lower than for males
(see also Table 1).
HB. Mean HB values were also slightly lower in animals at
ages 7 weeks compared to older animals (males and
females: 9.1 mmol/L, with an acceptable range of 8.5–9.7
mmol/L). The acceptable data ranges were not significantly
different between sexes at ages between 8 and 71 weeks
(males, 8.3–11.1 mmol/L; females, 8.3–10.7 mmol/L) with
mean values ranging from 9.7 to 10.0 mmol/L in males and
from 9.5 to 9.7 mmol/L in females (see also Table 1).
HCT. Relative HCT showed mean values that were compa-
rable and very stable between sexes and ages, whereas the
mean values in males were higher compared to their female
counterparts. The acceptable ranges were defined in males
as 39.0–51.0%, and in females as 37.0–49.0%(see
Table 2).
RDW. The mean values for the relative RDW in both sexes
were comparable at different ages. Slightly lower mean
values were noted in females (12.2–14.3%) than in males
(13.1–15.1%). The acceptable ranges for animals 7 weeks
of age were considered 9.1–19.1%for males and 10.1–
15.3%for females. In older animals, the acceptable ranges
were estimated as slightly lower for females (5.3–23.3%)
than for males (6.5–23.3%) (see also Table 2).
MCV. The mean MCV value in males at 7 weeks of age
was 59.8 fL with an acceptable range from 55.0 to 64.6 fL,
which was slightly higher compared to older animals with
mean values ranging from 50.7 to 54.7 fL. In females, the
mean MCV value at 7 weeks of age was 58.6 fL with an
acceptable range from 55.0 to 62.2 fL, which was slightly
higher than the mean values ranging from 53.6 to 56.8 fL in
older females. The acceptable ranges established for males
from 8 weeks onward were from 45.9 to 59.9 fL and in their
female counterparts from 48.8 to 63.0 fL (see also Table 3).
4Toxicology Research and Application
Table 1. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
RBC (T/L)
M
7 57 7.27 0.40 6.34 8.50 6.4–8.1
>10% >10% >50% >50% >70% >70%
8–12 1932 8.32 0.47 6.53 10.59
7.0–9.8
13–18 881 8.55 0.44 6.91 9.98
19–40 1598 8.88 0.46 6.15 10.39
41–70 283 8.84 0.42 7.70 10.11
71 311 8.36 0.64 3.64 9.71
F
7 56 7.25 0.38 6.44 7.93 6.4–8.1
8–12 1812 7.98 0.41 5.87 9.62
6.5–8.9
13–18 904 7.93 0.41 6.71 9.40
19–40 1645 8.05 0.41 6.48 9.34
41–70 292 7.87 0.40 6.61 9.06
71 337 7.58 0.53 5.03 8.87
HB (mmol/L)
M
7 57 9.1 0.3 8.4 10.3 8.5–9.7
>10% >10% >50% >50% >70% >70%
8–12 1932 9.9 0.5 8.0 11.5
8.3–11.1
13–18 881 10.0 0.4 8.4 11.3
19–40 1598 10.0 0.4 6.3 11.2
41–70 283 9.7 0.4 8.8 11.3
71 198 9.7 0.7 5.3 10.9
F
7 56 9.1 0.3 8.3 9.8 8.5–9.7
8–12 1812 9.6 0.4 6.2 11.1
8.3–10.7
13–18 904 9.7 0.4 8.4 10.9
19–40 1645 9.6 0.4 7.5 11.2
41–70 292 9.5 0.4 8.0 10.5
71 218 9.5 0.6 6.1 10.8
RBC: red blood cell; HB: hemoglobin; STD: standard deviation; N: number of animals included in determination.
Table 2. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
HCT (%)
M
7 57 43.0 2.0 39.0 50.0
39.0–51.0
>10% >10% >50% >50% >70% >70%
8–12 1932 45.0 2.0 33.0 54.0
13–18 881 45.0 2.0 36.0 52.0
19–40 1598 45.0 2.0 29.0 52.0
41–70 283 46.0 2.0 40.0 50.0
71 198 45.0 3.0 27.0 51.0
F
7 56 43.0 2.0 37.0 48.0
37.0–49.0
8–12 1812 43.0 2.0 28.0 50.0
13–18 904 43.0 2.0 35.0 49.0
19–40 1645 43.0 2.0 33.0 51.0
41–70 292 43.0 2.0 35.0 50.0
71 218 43.0 3.0 29.0 51.0
RDW (%)
M
7 57 14.1 2.5 11.4 24.1 9.1–19.1
>10% >10% >50% >50% >70% >70%
8–12 1932 13.1 3.3 10.0 31.4
6.5–23.3
13–18 861 14.0 3.4 10.1 32.8
19–40 1544 15.1 4.1 11.1 33.5
41–70 263 13.8 3.2 10.9 31.0
71 198 14.9 3.6 11.0 30.6
F
7 56 12.7 1.3 10.4 17.0 10.1–15.3
8–12 1812 12.7 3.4 9.4 32.5
5.3–23.3
13–18 885 14.1 3.4 10.1 33.5
19–40 1598 13.3 3.6 10.0 30.5
41–70 272 12.2 3.0 9.9 30.5
71 218 14.3 4.5 10.5 29.8
HCT: hematocrit; RDW: red cell volume distribution width; STD: standard deviation; N: number of animals included in determination.
de Kort et al. 5
MCH. In general, mean MCH values were slightly higher in
females at ages >13 weeks than in males. The highest val-
ues were noted in the youngest animals (7 weeks). The
mean value in young males was 1.26 fmol/L compared to
older animals with mean values ranging from 1.10 to
1.20 fmol/L. Although in females at 7 weeks of age, the
mean value of 1.25 fmol/L was higher than in females
between 8 and 70 weeks of age (1.20–1.22 fmol/L), the
animals at ages 71 weeks showed a similar mean value
of 1.25 fmol/L. The acceptable range was established at
1.1–1.4 fmol/L for both sexes at 7 weeks of age compared
to older animals for which it ranged from 1.0 to 1.4 fmol/L
in both sexes (see also Table 3).
MCHC. The mean MCHC values in young animals at
7 weeks of age were slightly lower than in older animals
(males, 21.01 mmol/L vs. mean values ranging from 21.34
to 22.23 mmol/L; females, 21.35 mmol/L vs. mean values
ranging from 21.84 to 22.36 mmol/L). The acceptable
range was established at 19.1–24.4 for males and at 19.7–
24.3 mmol/L for females at all ages (see Table 4).
HDW. HDW values were also slightly lower in young ani-
mals at ages 7 weeks when compared to older animals
(males, 1.29 mmol/L vs. mean values ranging from 1.60 to
1.79 mmol/L; females, 1.19 mmol/L vs. mean values rang-
ing from 1.34 to 1.48 mmol/L). In general, the values were
lower in females than in males. The acceptable ranges were
set at 0.9–1.7 mmol/L for males and 0.8–1.6 for females at
ages 7 weeks, whereas for animals at ages 8 weeks
acceptable ranges were 1.1–2.2 and 1.0–1.9 mmol/L for
males and females, respectively (see also Table 4).
HEINZBOD. Several compounds can oxidize HB to MET-
HB, which causes precipitation and/or formation of
HEINZBODs in the erythrocytes.
20–24
Normally in healthy
rats, approximately 1%of oxyhemoglobin is metabolized
into MET-HB. As no HEINZBODs were found in the
RccHan:WIST control groups in animals at ages of 8 to
70 weeks, the acceptable range is deemed to be 0.0 (see
Table 5). HEINZBODs were evaluated by special staining
when preparing slides only if changes in the MET-HB
levels were seen in treated animals.
RET. RET values were significantly higher in young ani-
mals at ages of 7 weeks compared to older animals.
Between 8 and 12 weeks of age, the values were still higher
than in animals at ages from 13 weeks onward. At ages
more than 12 weeks, lower variation was noted between
animals of different ages; however, mean values in males
were lower than in females.
Absolute numbers showed a mean value of 426 G/L and
acceptable range of 290–562 G/L in males at 7 weeks of
age and a mean value of 224 G/L (acceptable range of
Table 3. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MCV (fL)
M
7 57 59.8 2.4 54.2 64.2 55.0–64.6
>10% >10% >50% >50% >70% >70%
8–12 1932 54.7 2.6 45.8 64.4
45.9–59.9
13–18 881 53.1 2.6 46.0 60.5
19–40 1598 50.7 2.4 43.5 61.8
41–70 283 51.7 2.4 43.7 60.6
71 198 53.1 2.9 45.3 63.9
F
7 56 58.6 1.8 55.3 62.6 55.0–62.2
8–12 1812 54.0 2.4 46.4 61.5
48.8–63.0
13–18 904 54.7 2.7 46.5 63.6
19–40 1645 53.6 2.4 45.8 63.3
41–70 292 55.3 2.3 47.4 61.3
71 218 56.8 3.1 49.8 78.9
MCH
(fmol/L)
M
7 57 1.26 0.06 1.12 1.41 1.1–1.4
>10% >10% >50% >50% >70% >70%
8–12 1932 1.20 0.06 0.10 1.44
1.0–1.4
13–18 881 1.17 0.06 1.00 1.39
19–40 1598 1.13 0.06 0.97 1.39
41–70 283 1.10 0.05 0.98 1.33
71 198 1.15 0.06 0.92 1.33
F
7 56 1.25 0.04 1.18 1.35 1.1–1.4
8–12 1812 1.20 0.05 1.05 1.37
1.0–1.4
13–18 904 1.22 0.06 1.07 1.42
19–40 1645 1.20 0.05 0.99 1.41
41–70 292 1.21 0.06 1.07 1.40
71 218 1.25 0.07 1.09 1.55
MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; STD: standard deviation; N: number of animals included in determination.
6Toxicology Research and Application
Table 4. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MCHC
(mmol/L)
M
7 57 21.01 0.92 19.42 23.04
19.1–24.4
>10% >10% >50% >50% >70% >70%
8–12 1932 21.90 1.06 19.85 28.43
13–18 881 22.05 0.89 19.81 25.04
19–40 1598 22.23 1.05 19.66 25.33
41–70 283 21.34 0.93 20.10 24.24
71 198 21.70 0.95 18.64 24.17
F
7 56 21.35 0.80 20.24 23.31
19.7–24.3
8–12 1812 22.23 0.93 19.68 25.28
13–18 904 22.36 0.93 20.08 27.41
19–40 1645 22.33 0.95 19.96 25.09
41–70 292 21.84 1.02 20.32 24.40
71 218 21.98 1.03 19.60 24.15
HDW
(mmol/L)
M
7 57 1.29 0.19 1.02 1.70 0.9–1.7
>10% >10% >50% >50% >70% >70%
8–12 1932 1.60 0.20 1.08 2.25
1.1–2.2
13–18 861 1.70 0.19 1.19 2.35
19–40 1544 1.79 0.20 1.25 2.45
41–70 263 1.61 0.22 1.23 2.16
71 198 1.60 0.17 1.21 2.02
F
7 56 1.19 0.17 0.97 1.53 0.8–1.6
8–12 1812 1.48 0.17 1.01 2.08
1.0–1.9
13–18 885 1.45 0.14 1.03 1.94
19–40 1589 1.46 0.15 1.08 1.92
41–70 272 1.34 0.15 1.08 1.95
71 218 1.43 0.16 1.11 1.97
MCHC: mean corpuscular hemoglobin concentration; HDW: hemoglobin concentration distribution width; STD: standard deviation; N: number of
animals included in determination.
Table 5. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
HEINZBOD
(%)
M
8–12 265 0.0 0.0 0.0 0.0
0.0
0.1–0.2%
0.2–0.5%
>0.5%
13–18 90 0.0 0.0 0.0 0.0
19–40 230 0.0 0.0 0.0 0.0
41–70 10 0.0 0.0 0.0 0.0
F
8–12 274 0.0 0.0 0.0 0.0
13–18 90 0.0 0.0 0.0 0.0
19–40 295 0.0 0.0 0.0 0.0
41–70 30 0.0 0.0 0.0 0.0
RET (G/L)
M
7 57 426 68 324 682 290–562
>10% >10% >50% >50% >70% >70%
8–12 1912 224 46 71 452 132–316
13–18 881 200 34 108 325
55–299
19–40 1598 176 34 53 510
41–70 283 168 28 92 269
71 198 177 61 50 682
F
7 56 339 69 191 553 201–477
8–12 1793 221 41 101 493 139–303
13–18 904 194 42 97 385
70–306
19–40 1645 186 43 81 768
41–70 292 184 41 64 385
71 218 188 59 87 713
HEINZBOD: Heinz body; RET: reticulocyte; STD: standard deviation; N: number of animals included in determination.
de Kort et al. 7
Table 6. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
RET (%)
M
7 57 5.90 0.94 4.00 9.20 4.0–7.8
>10% >10% >50% >50% >70% >70%
8–12 1912 2.90 0.60 0.70 4.00 1.7–4.1
13–18 881 2.30 0.39 1.30 4.00
0.6–3.6
19–40 1598 2.00 0.39 0.60 6.10
41–70 283 1.90 0.32 1.10 3.20
71 198 2.10 0.72 0.60 13.50
F
7 56 4.70 0.96 2.70 7.70 2.7–6.7
8–12 1793 2.80 0.52 1.30 6.50 1.7–3.9
13–18 904 2.50 0.54 1.20 4.90
0.9–4.1
19–40 1645 2.30 0.53 0.90 10.10
41–70 292 2.30 0.51 0.80 5.20
71 218 2.50 0.78 1.10 13.80
LRET (%)
M
7 57 44.8 11.5 24.5 67.7 21.8–67.8
>10% >10% >50% >50% >70% >70%
8–12 1912 57.1 13.0 23.7 91.4
30.7–84.3
13–18 861 59.0 11.2 28.9 85.6
19–40 1544 59.7 12.3 32.6 89.1
41–70 263 56.5 12.9 33.6 89.0
71 198 64.8 13.2 19.2 86.2 38.4–91.2
F
7 56 47.9 10.3 23.4 67.0 27.3–68.5
8–12 1793 55.7 12.3 19.9 90.1
29.4–81.1
13–18 885 57.3 10.8 28.8 86.0
19–40 1589 56.3 12.4 15.5 86.6
41–70 272 50.8 10.7 32.4 77.9
71 218 58.9 14.7 22.1 85.5 29.5–88.3
RET: reticulocyte; LRET: low fluorescence reticulocyte; STD: standard deviation; N: number of animals included in determination.
Table 7. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MRET (%)
M
7 57 34.5 4.6 20.6 40.8
19.3–44.5
5–20% >10% >20% >50% >70% >70%
8–12 1912 32.5 5.8 8.1 44.6
13–18 861 32.4 5.4 13.7 43.5
19–40 1544 30.9 5.6 10.0 42.5
41–70 263 31.9 6.3 10.7 41.5
71 198 27.1 5.2 12.9 39.1 16.7–37.5
F
7 56 35.2 4.4 19.7 40.6
21.1–44.7
8–12 1793 33.0 5.5 9.1 48.0
13–18 885 33.7 5.5 13.5 46.0
19–40 1589 32.3 5.6 12.6 47.0
41–70 272 32.8 4.3 21.0 43.5
71 218 29.4 6.4 13.8 42.7 16.6–42.2
HRET (%)
M
7 57 20.7 14.5 2.0 54.9 0.0–49.7
>10% >10% >50% >50% >70% >70%
8–12 1912 10.4 10.2 0.4 56.6
0.0–33.4
13–18 861 8.6 8.3 0.4 48.0
19–40 1544 9.4 8.8 0.3 42.0
41–70 263 11.6 8.3 0.2 37.8
71 198 8.0 12.7 0.6 67.4
F
7 56 16.9 12.7 2.1 56.9 0.0–42.3
8–12 1793 11.3 10.9 0.2 61.4
0.0–41.9
13–18 885 9.1 8.3 0.2 44.6
19–40 1589 11.4 11.1 0.1 70.7
41–70 272 16.5 10.9 0.9 40.7
71 218 11.7 15.1 0.5 62.7
MRET: medium fluorescence reticulocyte; HRET: high fluorescence reticulocyte; STD: standard deviation; N: number of animals included in
determination.
8Toxicology Research and Application
Table 9. RccHan:WIST (Harlan Laboratories Ltd)
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
NEUT (%)
M
7 25 10.5 3.4 6.0 17.1 3.7–17.3
5–20% >10% >20% >50% >70% >70%
8–12 896 14.5 5.1 3.9 51.2 4.3–24.7
13–18 379 15.9 4.2 5.4 32.0
19–40 761 20.9 5.5 6.8 42.4 9.9–31.9
41–70 63 25.0 6.8 12.1 50.8 11.4–38.6
71 55 33.1 9.6 18.2 68.9 13.9–52.3
F
7 24 10.4 4.8 3.7 27.5 0.8–20.0
8–12 816 15.4 6.6 4.4 63.8 2.2–28.6
13–18 416 14.3 5.2 3.8 49.5
19–40 793 19.7 6.5 6.0 69.6 6.7–32.7
41–70 64 24.8 9.3 11.8 61.0 6.2–43.4
71 60 33.8 10.6 10.9 66.0 12.6–55.0
EOS (G/L)
M
7 52 0.07 0.04 0.02 0.26
0.0–0.3
— >10% — >50% — >70%
8–12 1927 0.08 0.04 0.00 0.41
13–18 881 0.09 0.04 0.00 0.38
19–40 1598 0.12 0.05 0.03 0.84
41–70 283 0.13 0.06 0.04 0.52
71 311 0.12 0.06 0.01 0.63
F
7 51 0.07 0.05 0.03 0.30
0.0–0.2
8–12 1805 0.07 0.04 0.00 0.46
13–18 904 0.07 0.03 0.00 0.41
19–40 1645 0.07 0.03 0.01 0.41
41–70 292 0.08 0.03 0.03 0.22
71 337 0.09 0.04 0.02 0.32
EOS: eosinophil; NEUT: neutrophil; STD: standard deviation; N: number of animals included in determination.
Table 8. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
WBC
(G/L)
M
7 57 5.64 1.42 3.48 9.44 2.8–8.5
>10% >10% >50% >50% >70% >70%
8–12 1932 7.19 1.63 1.79 15.50
3.2–10.513–18 881 6.80 1.52 2.61 14.42
19–40 1598 6.22 1.49 2.56 13.10
41–70 283 5.69 1.37 2.62 10.61 2.9–8.6
71 311 5.76 1.40 2.53 13.79
F
7 56 4.49 0.99 2.73 7.20 2.5–6.5
8–12 1810 5.00 1.33 0.81 11.83
1.4–7.713–18 904 4.49 1.16 1.69 9.39
19–40 1645 3.64 1.08 1.35 9.51
41–70 292 3.26 0.89 1.35 7.21 0.9–6.8
71 337 3.83 1.46 1.40 14.97
NEUT
(G/L)
M
7 25 0.57 0.29 0.22 1.19 0.0–1.2
>10%
>10%
>50%
>50%
>70%
>70%
8–12 896 1.02 0.37 0.29 4.52
0.0–2.8
13–18 379 1.10 0.33 0.37 2.69
19–40 761 1.31 0.45 0.41 4.65
41–70 63 1.41 0.67 0.59 5.39
71 55 2.03 1.36 0.83 9.51 0.0–4.8 5–10% >10% >50%
F
7 24 0.43 0.24 0.13 1.25 0.0–1.0
8–12 816 0.74 0.34 0.22 5.82
0.0–1.6 >10% >50% >70%
13–18 416 0.65 0.24 0.20 2.04
19–40 793 0.69 0.25 0.25 2.11
41–70 64 0.85 0.37 0.36 2.22
71 60 1.45 0.96 0.46 5.25 0.0–3.4
WBC: white blood cell; NEUT: neutrophil; STD: standard deviation; N: number of animals included in determination.
de Kort et al. 9
Table 10. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
EOS (%)
M
7 52 1.3 0.7 0.5 4.3
0.0–3.9
— >10% — >50% — >70%
8–12 1927 1.2 0.5 0.0 4.5
13–18 881 1.4 0.6 0.0 5.2
19–40 1598 1.9 0.7 0.5 9.7
41–70 283 2.3 0.8 0.9 7.3
71 311 2.1 0.8 0.6 7.9
F
7 51 1.6 1.2 0.6 7.5
0.0–4.6
8–12 1805 1.4 0.9 0.0 19.0
13–18 904 1.6 0.8 0.0 10.5
19–40 1645 2.1 1.0 0.5 14.5
41–70 292 2.5 0.9 1.0 7.0
71 337 2.4 1.1 0.6 7.0
BASO
(G/L)
M
7 52 0.02 0.01 0.01 0.05
0.0–0.1 — >10% — >50% — >70%
8–12 1927 0.04 0.03 0.00 0.33
13–18 881 0.03 0.03 0.00 0.19
19–40 1598 0.03 0.02 0.00 0.19
41–70 283 0.02 0.01 0.00 0.08
71 311 0.03 0.02 0.00 0.10
F
7 51 0.01 0.01 0.00 0.06
8–12 1805 0.02 0.02 0.00 0.16
13–18 904 0.02 0.02 0.00 0.16
19–40 1645 0.01 0.01 0.00 0.10
41–70 292 0.01 0.01 0.00 0.05
71 337 0.03 0.02 0.00 0.10
EOS: eosinophil; BASO: basophil; STD: standard deviation; N: number of animals included in determination.
Table 11. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
BASO (%)
M
7 52 0.3 0.2 0.1 0.9
0.0–1.4
— >10% — >50% — >70%
8–12 1927 0.6 0.4 0.0 3.4
13–18 881 0.5 0.4 0.0 3.4
19–40 1598 0.4 0.3 0.0 2.9
41–70 283 0.3 0.2 0.0 1.2
71 311 0.6 0.2 0.1 1.4
F
7 51 0.3 0.2 0.0 1.1
0.0–1.5
8–12 1805 0.5 0.4 0.0 2.5
13–18 904 0.4 0.4 0.0 2.4
19–40 1645 0.4 0.3 0.0 2.5
41–70 292 0.2 0.2 0.0 1.2
71 337 0.7 0.4 0.0 2.2
LYMPH
(G/L)
M
7 52 4.66 1.10 3.05 7.98 2.4–6.9
5–10% >10% >10% >50% >50% >70%
8–12 1927 5.83 1.47 1.27 13.56
2.1–8.813–18 881 5.41 1.34 1.91 12.78
19–40 1598 4.64 1.23 0.96 10.40
41–70 283 3.82 0.97 1.73 7.07 1.8–5.8
71 311 3.54 0.77 1.52 6.37
F
7 51 3.76 0.87 2.24 6.12 2.0–5.5
8–12 1805 4.01 1.20 0.46 10.68
0.9–6.513–18 904 3.63 1.07 0.80 8.62
19–40 1645 2.77 0.93 0.38 6.56
41–70 292 2.28 0.71 0.70 4.73 0.5–4.0
71 337 2.25 0.87 0.62 11.06
BASO: basophil; STD: standard deviation; LYMPH: lymphocyte; N: number of animals included in determination.
10 Toxicology Research and Application
Table 12. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
LYMPH
(%)
M
7 52 85.5 3.9 76.6 91.7 77.7–93.3
>5–10% >10% >10% >50% >50% >70%
8–12 1927 80.8 5.2 45.7 92.9 69.5–91.2
13–18 881 79.3 4.9 55.3 92.4
19–40 1598 74.3 5.9 24.9 89.5 62.5–86.1
41–70 283 67.3 6.9 41.1 82.6 44.0–81.1
71 311 62.4 9.2 26.2 83.7
F
7 51 84.1 5.2 66.1 92.6 73.7–94.5
8–12 1805 79.6 6.7 30.0 93.1 66.2–93.0
13–18 904 80.3 6.2 44.1 93.1
19–40 1645 75.4 7.0 22.0 90.3 61.4–89.4
41–70 292 69.6 7.4 31.8 84.1 39.6–84.4
71 337 59.6 10.0 15.5 87.9
MONO
(G/L)
M
7 52 0.10 0.05 0.02 0.27
0.0–0.3
— >10% — >50% — >70%
8–12 1927 0.13 0.05 0.03 0.45
13–18 881 0.13 0.05 0.02 0.36
19–40 1598 0.14 0.05 0.03 0.60
41–70 283 0.16 0.07 0.03 0.44
71 311 0.16 0.07 0.02 0.41
F
7 51 0.08 0.04 0.02 0.17
0.0–0.3
8–12 1805 0.09 0.04 0.01 0.32
13–18 904 0.08 0.03 0.00 0.19
19–40 1645 0.07 0.03 0.02 0.40
41–70 292 0.09 0.04 0.02 0.28
71 337 0.12 0.06 0.02 0.56
STD: standard deviation; LYMPH: lymphocyte; MONO: monocyte; N: number of animals included in determination.
Table 13. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MONO (%)
M
7 52 1.8 0.7 0.6 3.9
0.4–3.2
<0.005 >10% >50% >70%
8–12 1927 1.8 0.6 0.5 5.0
13–18 881 1.9 0.6 0.4 4.8
19–40 1598 2.2 0.7 0.6 10.5
0.8–4.641–70 283 2.8 0.9 0.7 6.3
71 311 2.7 0.9 0.7 5.7
F
7 51 1.8 0.7 0.6 3.7
0.4–3.28–12 1805 1.7 0.6 0.4 7.9
13–18 904 1.8 0.6 0.0 5.5
19–40 1645 2.0 0.7 0.5 6.9
0.6–5.141–70 292 2.6 0.8 1.0 6.9
71 337 3.1 1.0 1.1 8.8
LUC (G/L)
M
7 52 0.04 0.02 0.01 0.12
0.0–0.2 — >10% >50% >70%
8–12 1927 0.06 0.03 0.00 0.31
13–18 881 0.06 0.04 0.00 0.34
19–40 1598 0.05 0.04 0.00 0.57
41–70 283 0.05 0.03 0.01 0.22
71 311 0.05 0.03 0.01 0.18
F
7 51 0.03 0.02 0.01 0.07
8–12 1805 0.04 0.02 0.00 0.24
13–18 904 0.04 0.02 0.00 0.24
19–40 1645 0.03 0.02 0.00 0.14
41–70 292 0.02 0.01 0.00 0.11
71 337 0.04 0.02 0.00 0.15
STD: standard deviation; MONO: monocyte; LUC: large unstained cell; N: number of animals included in determination.
de Kort et al. 11
132–316 G/L) in males at ages between 8 and 12 weeks
compared to males at ages 13 weeks with mean values
ranging from 168 to 200 G/L (acceptable range of 55–299
G/L) (see also Table 5).
For relative values, males at 7 weeks of age showed a
mean value of 5.9%with an acceptable range of 4.0 to 7.8%
and males between 8 and 12 weeks of age had a mean value
of 2.9%and an acceptable range of 1.7–4.1%(compared to
older animals, 13 weeks and onward, with values ranging
from 1.9%to 2.3%and an acceptable range of 0.6–3.6%)
(see also Table 6).
Absolute numbers in females 7 weeks showed a mean
value of 339 G/L (acceptable range of 201–477 G/L), and a
mean value of 221 G/L (acceptable range of 139–303 G/L)
in females at ages between 8 and 12 weeks compared to
females at ages of 13 weeks and older whose mean values
ranged from 184 to 194 G/L (acceptable range of 70–306
G/L) (see also Table 5).
Table 14. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
LUC (%)
M
7 52 0.8 0.4 0.2 1.7
0.0–1.9
— >10% — >50% — >70%
8–12 1927 0.9 0.4 0.0 3.9
13–18 881 0.9 0.4 0.0 3.5
19–40 1598 0.8 0.5 0.1 8.0
41–70 283 0.8 0.4 0.1 3.2
71 311 0.9 0.5 0.2 3.4
F
7 51 0.7 0.3 0.3 1.8
0.0–1.7
8–12 1805 0.8 0.4 0.0 5.1
13–18 904 0.8 0.4 0.0 5.1
19–40 1645 0.8 0.4 0.0 4.7
41–70 292 0.7 0.4 0.0 2.2
71 337 0.9 0.4 0.0 2.6
PLT (G/L)
M
7 57 1234 150 825 1492
702–1534
5–10% >10% >10% >50% >50% >70%
8–12 1932 1041 144 119 1638
13–18 881 976 137 113 1607
19–40 1598 912 120 412 1405
564–133241–70 283 919 115 661 1268
71 198 948 192 516 1875
F
7 56 1285 176 856 1761
753–16378–12 1812 1074 149 186 1588
13–18 904 1023 135 645 1498
19–40 1645 963 133 400 1698
517–122941–70 292 898 136 223 1471
71 218 863 173 287 1828
STD: standard deviation; LUC: large unstained cell; PLT: platelet count; N: number of animals included in determination.
Table 15. RccHan:WIST (Harlan laboratories Ltd).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MET-HB (%)
M
7 30 0.9 0.1 0.7 1.0
0.4–1.4 — >10% — >50% — >70%
8–12 1280 0.9 0.2 0.3 1.4
13–18 543 0.9 0.2 0.3 1.3
19–40 634 0.9 0.2 0.0 1.5
41–70 49 0.8 0.2 0.5 1.2
F
7 30 0.9 0.1 0.7 1.0
8–12 1279 0.9 0.2 0.3 1.4
13–18 559 1.0 0.2 0.4 1.4
19–40 644 0.9 0.2 0.0 1.4
41–70 49 0.8 0.2 0.5 1.2
MET-HB: methemoglobin; STD: standard deviation; N: number of animals included in determination.
12 Toxicology Research and Application
For relative values, females at 7weeks of age showed a
mean value for 4.7%with an acceptable range of 2.7–6.7%
and females between 8 and 12 weeks of age had a mean
value of 2.8%with an acceptable range of 1.7–3.9%(com-
pared to older animal values ranging from 2.3%to 2.5%
with an acceptable range of 0.9–4.1%; see also Table 6).
Relative values for LRET (young stages) were signifi-
cantly lower in animals of both sexes at ages 7 weeks
compared to older animals. In animals at 71 weeks of age,
the mean values tended to increase again. In young males at
7 weeks of age, the mean value was 44.8%(acceptable
range of 21.8–67.8%) compared to animals between 8 and
70 weeks of age with mean values ranging from 56.5%to
59.7%(acceptable range of 30.7–84.3%) with slightly
increased mean values in males at ages of 71 weeks and
older at 64.8%. In females at 7 weeks of age, the mean
value was 47.9%(acceptable range of 27.3–68.5%) com-
pared to females aged 8 to 70 weeks with mean values
ranging from 50.8%to 57.3%(acceptable range of 29.4–
81.1%) and females older than 70 weeks with a mean value
of 58.9%. In general, the female ranges, except at
7 weeks of age, are lower than their male counterparts
(see also Table 6).
Medium fluorescence reticulocytes (MRET) showed
similar values for animals from both sexes at ages 7to
70 weeks (males had mean values from 30.9%to 34.5%
and an acceptable range of 19.3 44.5%; females had means
ranging from 32.3%to 35.2%and an acceptable range of
21.1–44.7%). In animals older than 70 weeks, the mean
value was 27.1%for males and 29.4%for females, demon-
strating a reduction with increasing age. Therefore, the
acceptable ranges for males and females 71 weeks old
were set at 16.7–37.5 and 16.6–42.2%, respectively (see
also Table 7).
The mean values for mature, HRETs varied in wide
ranges. In males, there were mean values from 8.0%to
20.7%and in females, there were mean values between
9.1%and 16.9%. Due to the wide variation, the acceptable
ranges were set from 0.0%to 33.4%for males and 0.0%to
41.9%for females at ages from 8 weeks onward and from
0.0%to 49.7%for males and 0.0%to 42.3%for females at
ages 7 weeks (see also Table 7).
WBC. The WBC values for males were slightly higher than
for their female counterparts, whereas the mean values for
both sexes increased significantly from 8 to 12 weeks com-
pared to younger animals. Thereafter, mean values showed
a reduction with increasing age whereas the values
increased again in animals at 71 weeks and older. The mean
values for males ranged from 5.64 to 7.19 G/L with an
acceptable range from 2.8 to 8.6 G/L at ages of 7 and
from 41 weeks onward, and 3.2 to 10.5 G/L at ages between
8 and 40 weeks. The mean values for females ranged from
3.26 to 5.0 G/L with an acceptable range from 0.9 to
6.8 G/L in females at ages of 7 and from 41 weeks
onward, and 1.4 to 7.7 G/L at ages between 8 and 40 weeks
(see also Table 8).
NEUT. Absolute NEUT numbers were lowest in animals
aged 7 weeks and highest in animals at 71 weeks. In
males, the mean value at 7 weeks was 0.57 G/L (accep-
table range of 0.0–1.2 G/L) and at 71 weeks was 2.03 G/L
(acceptable range of 0.0–4.8 G/L). In males from 8 to
70 weeks old, the mean value ranged from 1.02 to 1.41
G/L (acceptable range of 0.0–2.8 G/L). In females, the
mean value at 7 weeks was 0.43 G/L (acceptable range
of 0.0–1.0 G/L) and in animals 71 weeks was 1.45 G/L
(acceptable range of 0.0–3.4 G/L). In females of ages from 8
to 70 weeks, the mean values ranged from 0.65 to 0.85 G/L
(acceptable range of 0.0–1.6 G/L) (see also Table 8).
Relative NEUT values increased with age, with mean
values ranging from 10.5%to 33.1%in males and from
10.4%to 33.8%in females, across the different age-
groups. The acceptable ranges are different for different
age-groups, starting from 0.8–20.0%in animals at
7 weeks of age up to 12.6–55.0%in animals at ages
71 weeks (see also Table 9).
EOS. Mean absolute values for EOS ranged from 0.07 to
0.13 G/L with an acceptable range of 0.0–0.3 G/L in males
and from 0.07 to 0.09 G/L in females with an acceptable
range of 0.0–0.2 for all ages (see Table 9).
Relative EOS values were slightly higher in animals at
ages from 19 weeks onward compared to younger animals.
In males from 7 to 18 weeks, the mean values were from
1.2%to 1.4%, and in older animals between 1.9%and
2.3%. In females from 7to18weeks,themeanwas
1.4%to 1.6%, and in older animals from 2.1%to 2.5%.
The acceptable ranges were established as 0.0–3.9%for
males and 0.0–4.6%for females (see Table 10).
BASO. The absolute mean number of BASO was low in both
sexes at all ages with no significant difference between
females and males. The acceptable range was 0.0 to 0.1
G/L for both sexes and all ages (see Table 10). Similarly,
the acceptable relative value range was established at 0.0–
1.4%for males and at 0.0–1.5%for females (see Table 11).
LYMPH. The absolute mean values decreased slightly at
ages at and above 19 weeks in both sexes. For males
7 weeks old, the mean value was 4.66 G/L, from 8 to
40 weeks, between 4.64 and 5.83 G/L, and in older males,
between 3.54 and 3.82 G/L. For females at ages of
7 weeks, the mean value was 3.76 G/L, from 8 to
40 weeks of age, between 2.77 and 4.01 G/L, and in older
females, between 2.25 and 2.28 G/L. The acceptable
range for males at ages of 7 weeks was established at
2.4–6.9 G/L, for ages between 8 and 40 weeks, at 2.1–8.8
G/L, and for older males, at 1.8–5.8 G/L. The acceptable
range for females at ages of 7 weeks was established at
2.0–5.5 G/L, for ages from 8 to 40 weeks, at 0.9–6.5, and
for older females, at 0.5–4.0 G/L (see also Table 11).
de Kort et al. 13
Relative lymphocyte (LYMPH) values behaved simi-
larly. For males at ages of 7 weeks, the mean value was
85.5%; between 8 and 40 weeks, from 74.3%to 80.8%; and
between 62.4 and 67.3%in older males. In females at ages
of 7 weeks, the mean value was 84.1%; from 8 to
40 weeks, between 75.4 and 80.3%; and between 59.6 and
69.6%in older females (see also Table 12).
MONO. The absolute mean values were slightly higher in
males (0.10–0.16 G/L) than in females (0.07–0.12 G/L).
There were no differences between female and male ranges
at different ages, however, an increase in both sexes can be
seen for older animals. The acceptable range was set at 0.0–
0.3 G/L for males and females (see Table 12).
The relative MONO values showed a minor increase in
animals aged 19 weeks and older. In males aged 7to
18 weeks, the mean value ranged from 1.8%to 1.9%
(acceptable range of 0.4–3.2%). In older males, the mean
values ranged from 2.2%to 2.8%(acceptable range of
0.8–4.6%). In females aged 7 to 18 weeks, the mean value
ranged between 1.7%and 1.8%(acceptable range of 0.4–
3.2%), and in older females, the mean values ranged from
2.0%to 3.1%(acceptable range of 0.6–5.1%)(seealso
Table 13).
LUC. LUC mean values ranged from 0.02 to 0.06 G/L for
both sexes and all age-groups. Therefore, the acceptable
range was established at 0.0 to 0.2 G/L for both sexes (see
Table 13).
Relative LUC mean values ranged from 0.8%to 0.9%
for males, with an acceptable range of 0.0–1.9%.For
females, the relative mean values were between 0.7%and
0.9%, with an acceptable range of 0.0–1.7%(see also
Table 14).
PLT. The absolute mean values were slightly higher in both
sexes at ages from 7 weeks to 18 weeks compared to
mean values from older animals. In males, at ages from
7 to 18 weeks, the mean values ranged from 976 to
1234 G/L (acceptable range of 702–1534 G/L), and in older
males, the mean values ranged from 912 to 948 G/L (accep-
table range of 564–1332 G/L). In females at ages from
7 to 18 weeks, the mean values ranged from 1023 to
1285 G/L (acceptable range of 753–1637 G/L), and in older
females, the mean values ranged from 863 to 963 G/L
(acceptable range of 517–1229 G/L) (see also Table 14).
MET-HB. Mean relative MET-HB values ranged from 0.8%
to 1.0%for both sexes and all age-groups. The acceptable
range was established as 0.4%to 1.4%(see Table 15).
Values obtained from Crl:WI(Han) rats
Obtained outliers were examined on available histopathol-
ogy data.
25
Therefore, outliers were associated with histo-
pathology findings like, for example, inflammatory
pulmonary lesions following vehicle aspiration or
spontaneous sperm granuloma. After excluding all statisti-
cally identified outliers, the following values from the
standard parameter list were evaluated: RBC, HB, HCT,
WBC, MCV, MCH, MCHC, RET, LUC, and PLT. Values
were only available for the 8–12, 13–18, and 19–40 week
age-groups.
RBC. Mean RBC values in animals at ages of 8–40 weeks
ranged from 8.51 to 9.07 T/L (acceptable range of 7.1–10.3
T/L) for males and were somewhat lower for females, 8.01
to 8.26 T/L (acceptable range of 6.9–9.3 T/L; see
also Table 16).
HB. Mean HB values in animals at ages of 8–40 weeks
ranged from 9.9 to 10.0 mmol/L (acceptable range of
8.6–11.1 mmol/L) for males and were slightly lower for
females, 9.3 to 9.4 mmol/L (acceptable range of 8.1–10.5
mmol/L; see also Table 16).
HCT. Relative HCT values were slightly higher in males
(mean values ranging from 47.4%to 47.6%; acceptable
range of 40.8–54.1%) than in females (mean values ranging
from 44.1%to 44.4%; acceptable range of 38.2–49.9%; see
also Table 16).
RBC values for all age-groups were higher in males than
in females. While the female RBC values were fairly con-
stant, values in males increased with age. In contrast, HB
and HCT values were constant throughout all age-groups
but were higher in males than in females (Figure 1).
MCV. The mean MCV values in animals between 8 and 40
weeks old were similar in males and females (males, 52.5
to 55.4 fL; females, 53.5 to 55.0 fL), as were the acceptable
ranges for males, 48.4 to 60.7 fL, and for females, 49.5 to
59.6 fL (see Table 16).
MCH. The MCH acceptable ranges in 8- to 40-week-old
animals were the same for both sexes, reaching from 1.0
to 1.3 fmol/L (see Table 17).
MCHC. Mean MCHC values were similar for both sexes
(males, 20.81 to 21.04 mmol/L; females, 21.02 to 21.25
mmol/L). The acceptable ranges were slightly different
between sexes, 19.1 to 22.7 mmol/L and 19.1 to 23.0
mmol/L, respectively, for males and females (see
Table 17).
MCV, MCH, and MCHC values were generally slightly
higher in females than in males. Females and males at ages
between 8 and 12 weeks showed rather constant MCV and
MCH values but varied at higher ages, whereas the MCHC
values remained constant throughout all age-groups
(Figure 2).
RET. For relative RET values, again there were similarities
between sexes and among all age-groups (males, 1.69 to
2.44%; females, 2.05 to 2.30%) with acceptable ranges of
0.6–4.3%for males and 1.0–3.5%for females. The RET
14 Toxicology Research and Application
Table 16. Crl:WI(Han) (BSL).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
RBC (T/L)
M
8–12 721 8.51 0.67 6.09 10.41
7.1–10.3
>10% >10% >50% >50% >70% >70%
13–18 259 8.99 0.56 7.20 10.38
19–40 433 9.07 0.62 6.88 10.47
F
8–12 684 8.01 0.54 6.08 9.68
6.9–9.313–18 250 8.26 0.49 6.60 9.72
19–40 432 8.04 0.53 6.12 9.56
HB (mmol/L)
M
8–12 719 9.9 0.6 7.4 11.8
8.6–11.1
>10% >10% >50% >50% >70% >70%
13–18 256 10.0 0.6 8.2 11.9
19–40 433 10.0 0.6 8.4 12.0
F
8–12 685 9.3 0.6 7.3 11.1
8.1–10.513–18 250 9.4 0.5 8.1 10.9
19–40 433 9.3 0.5 7.7 11.2
HCT (%)
M
8–12 721 47.4 3.3 36.0 57.4
40.8–54.1
>10% >10% >50% >50% >70% >70%
13–18 259 47.5 2.8 38.6 55.1
19–40 434 47.6 3.2 35.5 56.3
F
8–12 684 44.1 2.9 34.1 52.8
38.2–49.913–18 249 44.4 2.4 37.0 53.0
19–40 433 44.1 2.9 33.1 55.2
MCV (fL)
M
8–12 448 55.4 2.6 49.9 63.6
48.4–60.7
>10% >10% >50% >50% >70% >70%
13–18 222 52.6 1.8 47.4 57.7
19–40 430 52.5 2.0 47.8 58.2
F
8–12 423 55.0 2.3 49.5 62.9
49.5–59.613–18 212 53.5 2.0 49.2 60.3
19–40 434 54.9 2.0 49.9 61.1
STD: standard deviation; RBC: red blood cell; HB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; BSL: BSL BIOSERVICE Scientific
Laboratories Munich GmbH; N: number of animals included in determination.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
female 6,08 9,68 3,58 0,225
male 6,09 10,41 3,5 9 0,222
Sex Min Max G P
Grubbs ' Te s t
malefemale
10,5
10,0
9,5
9,0
8,5
8,0
7,5
7,0
6,5
female 6,60 9,72 3,37 0,166
male 7,20 10,38 3,21 0,306
Sex Min Max G P
Grubbs ' Te st
Sex
RBC [T/L
]
Outlier Plot of RBC [T/L] vs Sex
Gro up ed Ag e = 13 -18 we ek s
malefemale
11
10
9
8
7
6
female 6,12 9,56 3,63 0,111
male 6,88 10,47 3,55 0,155
Sex Min Max G P
Grubbs ' Te s t
Sex
RBC [T/ L]
Outlier Plot of RBC [T/L] vs Sex
Grouped Age = 19-40 weeks
female 7,33 11,12 3,52 0,279
male 7,39 11,80 3,93 0,056
Sex Min Max G P
Grubbs ' Te s t
malefemale
12
11
10
9
8
female 8,14 10,87 2,87 0,956
male 8,2011,923,450,123
Sex Min Max G P
Grubbs ' Te st
Sex
HB [mmol/L]
Outlier Plot o f HB [mmo l/L] vs Sex
Group ed Ag e = 13- 18 w ee ks
malefemale
12
11
10
9
8
female 7,70 11,24 3,69 0,089
male 8,45 12,05 3,71 0,079
Sex Min Max G P
Grubbs ' Te s t
Sex
HB [mmo l/ L]
Outlier Plo t of HB [mmol/ L] vs Sex
Gro up ed Ag e = 19 -4 0 w ee ks
female 34,10 52,80 3,46 0,349
male 36,00 57,40 3,46 0,366
Sex Min Max G P
Grubbs ' Te s t
malefemale
55
50
45
40
35
female 37,00 53,00 3,60 0,065
male 38,60 55,10 3,19 0,334
Sex Min Max G P
Grubbs ' Te s t
Sex
HCT [%]
Outlier Plot o f HCT [%] vs Sex
Group ed Ag e = 13- 18 w ee ks
malefemale
55
50
45
40
35
30
female 33,10 55,20 3,82 0,050
male 35,50 56,30 3,81 0,052
Sex Min Max G P
Grubbs ' Te s t
Sex
HCT [%]
Outlier Plot o f HCT [%] vs Sex
Grouped Age = 19-40 weeks
Figure 1. Grubbs’ Outlier Test performed for RBC, HB, and HCT. RBC: red blood cell; HB: hemoglobin; HCT: hematocrit.
de Kort et al. 15
(a)
(b)
(c)
(d)
(e)
(f)
(g) (h) (i)
malefe male
64
62
60
58
56
54
52
50
female 49,50 62,90 3,48 0,195
male 49,90 63,60 3,11 0,799
Sex Min Max G P
Grubbs ' Te s t
Sex
MCV [fL]
Outlier Plot o f MCV [fL] vs Sex
Group ed Ag e = 08 -12 we eks
malefe male
62
60
58
56
54
52
50
48
46
female 49,20 60,30 3,45 0,099
male 47,40 57,70 2,98 0,585
Sex Min Max G P
Grubbs ' Te s t
Sex
MCV [fL]
Outlier Plot o f MCV [fL] vs Sex
Grouped Age = 13-18 weeks
malefe male
62
60
58
56
54
52
50
48
46
female 49,90 61,10 3,09 0,818
male 47,80 58,20 2,86 1,000
Sex Min Max G P
Grubbs' Test
Sex
MCV [fL]
Outli er Plo t o f MCV [fL] vs Sex
Grouped Age = 19-40 weeks
malefem ale
1,3 5
1,3 0
1,2 5
1,2 0
1,15
1,10
1,0 5
1,0 0
female 1,02 1,32 3,03 0,952
male 1,01 1,30 2,88 1,000
Sex Min Max G P
Grubbs ' Te s t
Sex
MCH [fmol]
Outlier Plo t of MCH [fm ol] vs Sex
Group ed Ag e = 08 -12 we eks
malefem ale
1,3 0
1,2 5
1,2 0
1,15
1,10
1,0 5
1,0 0
female 1,02 1,27 2,71 1,000
male 1,01 1,24 2,66 1,000
Sex Min Max G P
Grubbs' Test
Sex
MCH [fmol]
Outlier Plot o f MCH [fmol] vs Sex
Grouped Age = 13-18 weeks
malefem ale
1,3 5
1,3 0
1,2 5
1,2 0
1,15
1,10
1,0 5
1,0 0
fema le 1,0 4 1,3 4 3 ,5 8 0 ,13 1
male 0,97 1,29 3,74 0,069
Sex Min Max G P
Grubbs ' Te s t
Sex
MCH [fmol]
Outlier Plot of MCH [fmol] vs Sex
Grouped Age = 19-40 weeks
malefemale
24
23
22
21
20
19
18
female 18,94 23,47 2,75 1,000
male 18,01 22,79 3,19 0,585
Sex Min Max G P
Grubbs ' Te s t
Sex
MCHC [mmol/L]
Outlier Plot o f MCHC [mmol/ L] vs Sex
Group ed Ag e = 08 -12 we eks
malefe male
24
23
22
21
20
19
female 19,62 23,10 2,52 1,000
male 18,75 23,41 3,18 0,286
Sex Min Max G P
Grubbs ' Te s t
Sex
MCHC [mmol/ L]
Outlie r Plot o f MCHC [m mol/ L] vs Se x
Grouped Age = 13-18 weeks
malefemale
25
24
23
22
21
20
19
18
female 18,38 24,34 3,39 0,279
male 18,44 23,66 3,16 0,623
Sex Min Max G P
Grubbs' Test
Sex
MCHC [mmol/ L]
Outlie r Plot o f MCHC [m mol/ L] vs Se x
Grouped Age = 19-40 weeks
Figure 2. Grubbs’ Outlier Test performed for MCV, MCH, and MCHC. MCV: mean corpuscular volume; MCH: mean corpuscular
hemoglobin; MCHC: mean corpuscular hemoglobin concentration.
Table 17. Crl:WI(Han) (BSL).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MCH
(fmol/L)
M
8–12 438 1.15 0.05 1.01 1.30
1.0–1.3
>10% >10% >50% >50% >70% >70%
13–18 220 1.11 0.05 1.01 1.24
19–40 421 1.10 0.05 0.98 1.29
F
8–12 412 1.16 0.05 1.02 1.32
1.0–1.313–18 211 1.14 0.05 1.02 1.27
19–40 426 1.15 0.05 1.04 1.34
MCHC
(mmol/L)
M
8–12 438 20.81 0.88 18.01 22.79
19.1–22.7
>10% >10% >50% >50% >70% >70%
13–18 220 21.04 0.75 18.75 23.41
19–40 420 20.93 0.87 18.44 23.66
F
8–12 412 21.08 0.87 18.94 23.47
19.1–23.013–18 211 21.25 0.73 19.62 23.10
19–40 425 21.02 0.98 18.38 24.34
RET (%)
M
8–12 350 2.44 0.90 0.20 5.26
0.6–4.3
>10% >10% >50% >50% >70% >70%
13–18 198 1.96 0.38 1.15 3.00
19–40 402 1.69 0.39 0.30 3.04
F
8–12 323 2.30 0.58 0.30 4.17
1.0–3.513–18 188 2.17 0.46 1.13 3.62
19–40 405 2.05 0.52 0.10 3.63
WBC (G/L)
M
8–12 720 6.21 2.01 0.56 14.00
1.7–10.3
>10% >10% >50% >50% >70% >70%
13–18 258 5.34 1.69 0.78 10.05
19–40 434 4.35 1.32 0.92 8.52
F
8–12 685 3.74 1.58 0.39 9.66
0.4–6.913–18 247 3.29 1.35 0.80 7.04
19–40 431 2.31 0.94 0.36 5.39
MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RET: reticulocyte; WBC: white blood cell; BSL: BSL
BIOSERVICE Scientific Laboratories Munich GmbH; STD: standard deviation; N: number of animals included in determination.
16 Toxicology Research and Application
count decreased with increasing age. The decrease through
age-groups was more significant in males than females (see
also Table 17).
WBC. The mean WBC values were higher in males (4.35–
6.21 G/L, acceptable range of 1.7–10.3 G/L) than in
females (2.31–3.74 G/L, acceptable range of 0.4–6.9
G/L). Similar to the RET count, the WBC values decreased
in older animals, again more significantly in males than in
females (see also Table 17).
NEUT. The mean relative values for NEUT were higher in
males (13.2–20.7%)thaninfemales(13.017.1%) with
acceptable ranges at ages between 8 and 18 weeks of
3.7%to 31.0%in males and 3.5–25.9%in females. The
acceptable range for animals between 19 and 40 weeks of
age increased for both males (7.7–33.6%)andfemales
(5.6–28.6%; see also Table 18).
Figure 3 demonstrates that WBC and RET decreased
with increasing age in both sexes, whereas RET values
were slightly higher and WBC values were significantly
lower in females compared to their male counterparts. In
contrast, the NEUT values were higher in males than in
females and varied more the older the animals were. How-
ever, the values were very similar between animals of both
sexes at ages between 8 and 12 weeks.
EOS. The mean relative EOS values were similar for males
(0.4–0.5%) and females (0.5 to 0.6%) with acceptable
ranges of 0.0–1.1%for males and 0.0–1.6%for females
(see Table 18).
BASO. The mean relative BASO values were exactly the
same for males and females (0.1–0.2%) at all ages, decreas-
ing in older individuals, with acceptable ranges of 0.0–
0.5%for males and 0.0–0.4%for females (see Table 18).
LYMPH. Relative LYMPH values were higher in females
than in males, reaching 75.5%to 82.7%and 79.5%to
82.8%in males and females, respectively, with acceptable
ranges of 64.8–92.6%for males and 68.5–93.8%for
females at ages between 8 and 18 weeks. The acceptable
ranges for animals at ages between 19 and 40 weeks
decreased for both males (62.5–88.5%)andfemales
(67.0–92.0%; see also Table 18).
MONO. Mean relative MONO values were similar for
males (2.4–2.6%) and females (2.2–2.7%) with respective
acceptable ranges of 0.0–5.3 and 0.0–0.6%for males and
females. MONO values were constant throughout all age-
groups (see also Table 19).
LUC. The mean relative values for LUCs were similar in
both sexes but increased minimally throughout the
Table 18. Crl:WI(Han) (BSL).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
NEUT (%)
M
8–12 485 13.2 4.7 4.1 31.3 3.7–31.0
>5–20% >10% >20% >50% >70% >70%
13–18 221 17.6 6.7 4.2 40.0
19–40 428 20.7 6.5 1.7 44.1 7.7–33.6
F
8–12 451 13.0 4.8 3.1 29.5 3.5–25.9
13–18 206 14.7 5.6 5.3 32.7
19–40 426 17.1 5.7 5.0 38.6 5.6–28.6
EOS (%)
M
8–12 472 0.4 0.3 0.0 1.4
0.0–1.1
>10%
>50%
>70%
13–18 215 0.4 0.3 0.0 1.5
19–40 425 0.5 0.3 0.0 1.5
F
8–12 440 0.6 0.5 0.0 2.4
0.0–1.613–18 206 0.5 0.4 0.0 1.8
19–40 413 0.5 0.4 0.0 2.0
BASO (%)
M
8–12 488 0.2 0.1 0.0 0.7
0.0–0.5
>10%
>50%
>70%
13–18 217 0.2 0.1 0.0 0.5
19–40 430 0.1 0.1 0.0 0.5
F
8–12 446 0.2 0.1 0.0 0.5
0.0–0.413–18 206 0.2 0.1 0.0 0.5
19–40 426 0.1 0.1 0.0 0.4
LYMPH (%)
M
8–12 484 82.7 4.9 65.8 92.8 64.8–92.6
>5–10% >10% >10% >50% >50% >70%
13–18 221 77.9 6.5 56.2 88.7
19–40 427 75.5 6.5 52.1 87.3 62.5–88.5
F
8–12 453 82.8 5.5 62.3 93.9 68.5–93.8
13–18 207 80.9 6.2 59.7 91.7
19–40 426 79.5 6.2 56.0 93.3 67.0–92.0
NEUT: neutrophil; EOS: eosinophil; BASO: basophil; LYMPH: lymphocyte; BSL: BSL BIOSERVICE Scientific Laboratories Munich GmbH; STD: standard
deviation; N: number of animals included in determination.
de Kort et al. 17
age-groups. The values ranged in both sexes from 0.2%to
0.4%with acceptable ranges of 0.0–1.1%for males and
0.0–1.0%for females (see Table 19).
PLT. Mean platelet values are relatively similar in males
(691–858 G/L) and females (720–846 G/L) with acceptable
ranges of 439–1217 G/L for males and 500–1156 G/L for
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
malefemale
14
12
10
8
6
4
2
0
female 0,39 9,66 3,75 0,112
male 0,56 14,00 3,88 0,070
Sex Min Max G P
Grubbs ' Te s t
Sex
WBC [G/L]
Outlier Plot o f WBC [G/L] vs Sex
Grouped Age = 08-12 weeks
malefe male
10
8
6
4
2
0
female 0,80 7,04 2,77 1,000
male 0,78 10,05 2,79 1,000
Sex Min Max G P
Grubbs ' Te s t
Sex
WBC [G/L
]
Outlier Plot o f WBC [G/L] vs Sex
Grouped Age = 13-18 weeks
malefemale
9
8
7
6
5
4
3
2
1
0
female 0,36 5,39 3,28 0,419
male 0,92 8,52 3,15 0,672
Sex Min Max G P
Grubbs ' Te s t
Sex
WBC [G/L]
Outli er Plo t o f WBC [G/ L] vs Se x
Gro up ed Ag e = 19 -4 0 w ee ks
malefemale
5
4
3
2
1
0
female 0,30 4,17 3,47 0,153
male 0,20 5,26 3,15 0,541
Sex Min Max G P
Grubbs' Test
Sex
RET [%]
Outlier Plot of RET [%] vs Sex
Gro up ed Ag e = 0 8- 12 w ee ks
malefemale
3,5
3,0
2,5
2,0
1,5
1,0
female 1,13 3,62 3,19 0,236
male 1,15 3,00 2,75 1,000
Sex Min Max G P
Grubbs' Test
Sex
RE
T [%]
Outlier Plot o f RET [%] vs Sex
Grouped Age = 13-18 weeks
malefemale
4
3
2
1
0
female 0,10 3,63 3,73 0,069
male 0,30 3,04 3,59 0,118
Sex Min Max G P
Grubbs ' Te s t
Sex
RET [%]
Outlier Plot of RET [%] vs Se x
Grouped Age = 19-40 weeks
malefemale
35
30
25
20
15
10
5
0
female 3,10 29,50 3,47 0,216
male 4,10 31,30 3,80 0,062
Sex Min Max G P
Grubbs ' Te s t
Sex
NEUT [%]
Outlier Plot of NEUT [%] vs Sex
Grouped Age = 08-12 weeks
malefemale
40
30
20
10
0
female 5,30 32,70 3,23 0,225
male 4,2040,003,350,152
Sex Min Max G P
Grubbs' Test
Sex
NEUT [%]
Outlier Plot of NEUT [%] vs Sex
Grouped Age = 13-18 weeks
malefemale
50
40
30
20
10
0
female 5,00 38,60 3,75 0,068
male 1,70 44,10 3,62 0,112
Sex Min Max G P
Grubbs' Test
Sex
NEUT [%]
Outlier Plot of NEUT [%] vs Sex
Grouped Age = 19-40 weeks
Figure 3. Grubbs’ Outlier Test performed for WBC, RET, and NEUT. WBC: white blood cell; RET: reticulocyte; NEUT: neutrophil.
Table 19. Crl:WI(Han) (BSL).
Parameter Sex
Age in
weeks NMean STD Min Max
Acceptable
range +
2 STD
Grade 1 Grade 2 Grade 3
Decrease Increase Decrease Increase Decrease Increase
MONO (%)
M
8–12 463 2.6 1.3 0.8 7.6
0.0–5.3
<0.005 >10%
>50%
>70%
13–18 193 2.4 0.9 0.8 5.3
19–40 420 2.4 1.0 0.9 5.9
F
8–12 447 2.7 1.6 0.6 8.7
0.0–6.013–18 196 2.6 1.2 0.8 6.6
19–40 431 2.2 0.9 0.7 5.3
LUC (%)
M
8–12 229 0.3 0.2 0.0 1.0
0.0–1.1
>10%
>50%
>70%
13–18 102 0.3 0.2 0.0 1.0
19–40 325 0.4 0.3 0.0 1.5
F
8–12 207 0.2 0.2 0.0 0.9
0.0–1.013–18 97 0.3 0.2 0.0 0.9
19–40 322 0.4 0.3 0.0 1.3
PLT (G/L)
M
8–12 717 858 179 183 1365
439–1217
>5–10% >10% >10% >50% >50% >70%
13–18 258 768 140 309 1178
19–40 434 691 125 217 1094
F
8–12 682 846 155 236 1352
500–115613–18 250 805 135 387 1188
19–40 432 720 109 302 1105
MONO: monocyte; LUC: large unstained cell; PLT: platelet count; BSL: BSL BIOSERVICE Scientific Laboratories Munich GmbH; STD: standard
deviation; N: number of animals included in determination.
18 Toxicology Research and Application
females. PLT decreased with increasing age, somewhat
more in males than in females (see also Table 19).
For further information, please see supplementary data
Figures S1 and S2.
As already mentioned, all hematology parameters
(except MET-HB) were measured with the ADVIA 120
Hematology System. The ADVIA 120 is an instrument
with a well-established clinical track record and years of
successful use in a large number of laboratories worldwide.
It was also compared to newer systems like the ADVIA
2120 by Harris et al.
26
Overall, both systems showed a very
high similarity, thus the confidence intervals of both sys-
tems are comparable.
27
Within BSL, a small stability study with 19 untreated
animals was done, the results showed that the samples
remain stable for 72 h at 2–8.
Comparison of parameters from different study types
Selected parameters have been summarized and compared
for Crl:WI(Han) rats in Figures 4 to 6.
A comparison of repeated-dose toxicity studies (OECD
408)
10
with reproduction (OECD 422)
28
and implantation
(ISO 10993)
29
studies revealed several differences in
hematology parameters.
After variance analysis, almost every parameter among
males from repeated-dose and reproduction toxicity studies
resulted in unexpected high variation. Males at the end of
reproduction toxicity studies (OECD 422) were about 18–
19 weeks old. Animals at that age were included in age-
groups 13–18 and 19–40 weeks for better comparability
because those at the end of repeated-dose toxicity (OECD
408) and implantation studies were older than 19 weeks. A
second variance analysis comparing reproduction study
males at the age of 18–19 weeks with repeated-dose toxi-
city study males at the age of 13–18 weeks only showed
variation in MCHC and WBC.
Reproduction study females compared to repeated-dose
toxicity study females showed differences in almost every
parameter except RBC, HCT, HB, and MCH, as expected.
Implantation study males were rather similar to males in
repeated-dose toxicity studies, whereas females showed
more variances, especially in the differential leukocyte
count.
In repeated-dose studies with animals at ages from 19 to
40 weeks, NEUT values were slightly higher in males than
in females. In contrast, in reproductive toxicity studies, the
NEUT ranges were higher in females than in males. The
male NEUT values in reprotoxicity studies were lower
compared to implantation and repeated-dose studies due
to their real age of 18–19 weeks, that means, these animals
were at the lower limit of their age-group. When comparing
the mean values of reprotoxicity study males with repeated-
dose study males, no significant difference was noted.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
malefema le
50
40
30
20
10
0
female 5,00 38,60 3,75 0,068
male 1,70 44,10 3,62 0,112
Sex Min Max G P
Grubbs ' Te st
Sex
NEUT [%]
Outlier Plot of NEUT [%] vs Sex
Grouped Age = 19-40 weeks
malefe male
60
50
40
30
20
10
female 13,20 55,40 3,19 0,143
male 7,40 30,10 3,16 0,142
Sex Min Max G P
Grubbs' Test
Sex
NEUT [%]
Outlier Plot of NEUT [%] vs Sex
Grouped Age of Animals = 19-40 Weeks
malefem ale
70
60
50
40
30
20
10
0
female 9,50 6 8,60 3 ,37 0,053
male 6,50 46,10 3,2 0 0,106
Sex Min Max G P
Grubbs ' Te st
Sex
NEUT [%]
Outlier Plot of NEUT [%] vs Sex
Grouped Age = 19-40 weeks
malefemale
90
80
70
60
50
female 56,00 93,30 3,76 0,063
male 52,10 87,30 3,62 0,115
Sex Min Max G P
Grubbs ' Te s t
Sex
L
YMPH [%]
Outlier Plo t of LYMPH [%] vs Sex
Grouped Age = 19-40 weeks
malefemale
90
80
70
60
50
40
female 41,40 83,70 3,04 0,244
male 67,40 90,50 2,92 0,335
Sex Min Max G P
Grubbs ' Te s t
Sex
LYMP H [ %]
Outlier Plo t of LYMPH [%] vs Sex
Group ed Age of An imals = 19- 40 We eks
malefemale
90
80
70
60
50
40
30
20
10
female 19,10 86,50 3,32 0,065
male 49,50 88,50 3,25 0,086
Sex Min Max G P
Grubbs ' Te s t
Sex
LYMP H [ %]
Outli er Plo t o f LYMPH [%] vs S ex
Grouped Age = 19-40 weeks
malefemale
4
3
2
1
0
female 0,10 3,63 3,73 0,069
male 0,30 3,04 3,59 0,118
Sex Min Max G P
Grubbs ' Te s t
Sex
RET [%]
Outlier Plot o f RET [%] vs Sex
Grouped Age = 19-40 weeks
malefemale
6
5
4
3
2
1
0
female 0,63 5,51 3,18 0,149
male 0,95 2,31 2,51 1,000
Sex Min Max G P
Grubbs ' Te s t
Sex
RET [%]
Outli er Plo t o f RET [%] vs S ex
Group ed Age of An imals = 19- 40 We eks
OECD 422
OECD 422
OECD 422
Implantation Studies
ISO 10993
Implantation Studies
ISO 10993
OECD 408
OECD 408
OECD 408
Figure 4. Grubbs’ Outlier Test comparison of NEUT, LYMPH, and RET for three different study types. Left column, repeated-dose
studies (OECD 408); middle column, reproduction toxicity studies (OECD 422); right column, implantation studies (ISO 10993).
NEUT: neutrophil; LYMPH: lymphocyte; RET: reticulocyte.
de Kort et al. 19
Therefore, NEUT values in males are deemed to have
remained relatively constant throughout the different study
types. Finally, in implantation studies, higher variation was
noted for females, whereas the NEUT values in males were
slightly lower than in their female counterparts (Figure 4).
In repeated-dose toxicity studies, LYMPH values
shifted toward lower values with increasing age (higher
shift in males than in females). In young animals
(8–12 weeks), there were no differences for LYMPH in
either sex. By contrast, LYMPH values decreased in
(a)
(b)
(c)
(d)
(e)
(f)
malefema le
25
24
23
22
21
20
19
18
female 18,38 24,34 3,39 0,279
male 18,44 23,6 6 3, 16 0,62 3
Sex Min Max G P
Grubbs ' Te s t
Sex
MCHC [mmol/ L]
Outlier Plot o f MCHC [mmol/ L] vs Sex
Grouped Age = 19-40 weeks
malefemale
22
21
20
19
18
female 17,95 21,30 2,46 1,000
male 18,13 22,29 2,82 0,480
Sex Min Max G P
Grubbs ' Te s t
Sex
MCHC [mmol/L]
Outlier Plot of MCHC [mmol/L] vs Sex
Gro up ed Ag e o f Ani ma ls = 19 -4 0 We e ks
malefemale
24
23
22
21
20
19
female 19,38 23,60 2,70 0,559
male 19,13 23,04 2,37 1,000
Sex Min Max G P
Grubbs' Test
Sex
MCHC [mmol/L
]
Outlier Plot of MCHC [mmol/L] vs Sex
Gro up ed Ag e = 19 -4 0 w ee ks
malefemale
9
8
7
6
5
4
3
2
1
0
female 0,36 5,39 3,28 0,419
male 0,92 8,52 3,15 0,672
Sex Min Max G P
Grubbs ' Te s t
Sex
WBC [G/L]
Outli er Plo t o f WBC [G/ L] vs Se x
Grouped Age = 19-40 weeks
malefemale
8
7
6
5
4
3
2
1
0
female 0,57 6,99 2,88 0,431
male 1,75 7,69 2,52 1,000
Sex Min Max G P
Grubbs ' Te s t
Sex
WBC [G/L]
Outli er Plo t o f WBC [G/ L] vs Se x
Gro up ed Ag e o f Ani ma ls = 19 -4 0 We e ks
malefemale
9
8
7
6
5
4
3
2
1
0
female 0,32 4,72 2 ,66 0,674
male 1,49 8 ,70 3, 17 0,124
Sex Min Max G P
Grubbs' Test
Sex
WBC [G/L
]
Outli er Plo t o f WBC [G/ L] vs Se x
Gro up ed Ag e = 19 -4 0 w ee ks
OECD 408
OECD 422
OECD 422
Implantation Studies
ISO 10993
Implantation Studies
ISO 10993
OECD 408
Figure 6. Grubbs’ Outlier Test comparison of MCHC and WBC for three different study types. Left column, repeated-dose studies
(OECD 408); middle column, reproduction toxicity studies (OECD 422); right column, implantation studies (ISO 10993). MCHC: mean
corpuscular hemoglobin concentration; WBC: white blood cell.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
malefemale
11
10
9
8
7
6
female 6,12 9,56 3,63 0,111
male 6,88 10,47 3 ,55 0,155
Sex Min Max G P
Grubbs ' Te s t
Sex
RBC [T/ L]
Outlie r Plot o f RBC [T/L] vs Se x
Group ed Ag e = 19- 40 we eks
malefemale
11
10
9
8
7
6
5
female 5,58 8,62 3,28 0,099
male 7,31 10,47 3,05 0,214
Sex Min Max G P
Grubbs ' Te st
Sex
RBC [T/L]
Outlier Plot o f RBC [T/L] vs Sex
Group ed Ag e o f Anim als = 19 -4 0 W eeks
malefemale
10,5
10,0
9,5
9,0
8,5
8,0
7,5
7,0
female 7,41 9,62 2,94 0,255
male 7,6410,532,880,331
Sex Min Max G P
Grubbs ' Te s t
Sex
RBC [T/L]
Outli er Plo t o f RBC [T/L] vs Sex
Gro up ed Ag e = 19 -4 0 w ee ks
malefemal e
12
11
10
9
8
female 7,70 11,24 3,69 0,089
male 8,45 12,05 3 ,71 0,079
Sex Min Max G P
Grubbs ' Te s t
Sex
HB [mmo l/ L]
Outli er Plo t o f HB [ mmo l/ L] vs Se x
Grouped Age = 19-40 weeks
malefemale
12
11
10
9
8
7
female 7,27 9,69 3,01 0,268
male 8,01 11,55 3,37 0,064
Sex Min Max G P
Grubbs ' Te st
Sex
HB [mmo l/ L
]
Outlie r Plot o f HB [m mol/ L] vs Sex
Group ed Ag e o f Anim als = 19 -4 0 W eeks
malefemale
11,5
11,0
10, 5
10, 0
9,5
9,0
8,5
8,0
female 8,01 11,30 3,16 0,126
male 8,82 11,36 2,73 0,581
Sex Min Max G P
Grubbs ' Te s t
Sex
HB [mmol/L]
Outlier Plot o f HB [mmo l/L] vs Sex
Group ed Ag e = 19- 40 we eks
malefemale
55
50
45
40
35
30
female 33,10 55,20 3,82 0,050
male 35,50 56,30 3,81 0,052
Sex Min Max G P
Grubbs' Test
Sex
HCT [%]
Outlier Plot o f HCT [%] vs Sex
Group ed Ag e = 19- 40 we eks
malefemale
55
50
45
40
35
30
female 31,90 52,50 3,44 0,054
male 38,90 55,90 3,16 0,143
Sex Min Max G P
Grubbs ' Te s t
Sex
HCT [%
]
Outli er Plo t o f HCT [%] vs Se x
Group ed Age of An imals = 19- 40 We eks
malefemale
55
50
45
40
35
female 37,20 52,20 2,97 0,248
male 41,20 53,20 2,28 1,000
Sex Min Max G P
Grubbs ' Te s t
Sex
HCT [%
]
Outli er Plo t o f HCT [%] vs Se x
Gro up ed Ag e = 19 -4 0 w ee ks
OECD 422
OECD 422
OECD 422
Implantation Studies
ISO 10993
Implantation Studies
ISO 10993
Implantation Studies
ISO 10993
OECD 408
OECD 408
OECD 408
Figure 5. Grubbs’ Outlier Test comparison of RBC, HB, and HCT for three different study types. Left column, repeated-dose studies
(OECD 408); middle column, reproduction toxicity studies (OECD 422); right column, implantation studies (ISO 10993). RBC: red
blood cell; HB: hemoglobin; HCT: hematocrit.
20 Toxicology Research and Application
females from reproduction and implantation studies com-
pared to females from repeated-dose studies, whereas at
first glance, male LYMPH values in reprotoxicology stud-
ies seemed to be higher. However, due to their real age as
mentioned above, male LYMPH values can be said to be
comparable for all the different study types analyzed
(Figure 4).
Generally speaking, RET values varied in high ranges in
all studies. Comparing RET values of reprotoxicology with
repeated-dose toxicity studies, a clear indication of
increased RET values was noted in females in reprotoxi-
cology studies (Figure 4).
RBC, HB, and HCT values were somewhat invariable
among the different study-type categories. To confirm this
suspicion, a variance analysis was done, verifying that
RBC, HB, and HCT values of all three study types are
comparable (Figure 5).
MCHC values decreased slightly in females and males
from reproduction studies compared to repeated-dose stud-
ies, whereas no changes were noted in implantation studies
(Figure 6).
WBC values showed significant differences between
sexes in reprotoxicology studies compared to repeated-
dose studies and were not comparable, based on variance
analysis, whereas WBC values from repeated-dose toxicity
and implantation study animals were comparable
(Figure 6).
Number, mean values, and standard deviations are sum-
marized in Table 20. Other hematology parameters were
not compared. For further information, see supplementary
data Figures S3 to S5.
Vehicle effects
The following vehicles were used in repeated-dose toxicity
studies performed with Crl:WI(Han) rats: aqua ad injectio-
nem, control diet, corn oil and dried corn oil, cotton seed
oil, dimethyl sulfoxide (DMSO) in water, DMSO/polyethy-
lene glycol 400 (PEG400) 1:5, hydroxyethyl cellulose,
methylcellulose, NaCl 0.9%, paraffinum liquidum, poly-
ethylene glycol 300 (PEG300), PEG400, and sesame oil.
Most data were available for studies performed with
aqua ad injectionem (water) and corn oil. Therefore, vehi-
cle effects were statistically evaluated only for these vehi-
cles (variance analysis, Levene’s test,
30
Bonett’s test
31
).
For other vehicles, the sample size was insufficient to per-
form statistical analyses (Figures 7 to 14).
No significant differences regarding WBC were estab-
lished for either of the most commonly used vehicles, aqua
ad injectionem or corn oil. No differences were noted for
males or females at ages of 8–12 or 19–40 weeks. However,
there were clear increases in males and females at ages of
8–12 and 19–40 weeks using paraffinum liquidum and
decreases using DMSO (Figures 7 and 8). Using DMSO/
PEG400 1:5, WBC increased in males at ages of 8 to
12 weeks and decreased in females at the same ages; using
dried corn oil, WBC increased in males and decreased in
females at ages between 19 and 40 weeks.
Further, no significant differences were noted in
LYMPH values when using the most common vehicles,
aqua ad injectionem and corn oil. However, values were
seen to decrease when using the vehicle NaCl 0.9%in
males at the ages of 8–12 or 19–40 weeks. Using PEG400,
the values increased at ages between 8 and 12 weeks in
Table 20. Number, mean values, and STDs of RBC, HB, HCT, MCHC, RET, WBC, NEUT, and LYMPH for three different study types:
Repeated-dose studies (OECD 408), reproduction toxicity studies (OECD 422), and implantation studies (ISO 10993).
Parameter Sex
Age in
weeks
Repeated dose (OECD 408) Reprotox (OECD 422) Implantation (ISO 10993)
NMean STD NMean STD NMean STD
RBC (T/L) M 19–40 433 9.07 0.62 115 9.07 0.58 100 9.18 0.54
F 19–40 432 8.04 0.53 124 7.19 0.49 95 8.28 0.46
HB (mmol/L) M 19–40 433 10.0 0.6 115 9.8 0.5 106 10.0 0.5
F 19–40 433 9.3 0.5 123 8.6 0.5 102 9.5 0.6
HCT (%) M 19–40 434 47.6 3.2 115 48.7 3.1 106 47.2 2.6
F 19–40 433 44.1 2.9 125 43.6 3.4 101 44.6 2.6
MCHC (mmol/L) M 19–40 420 20.93 0.87 116 20.20 0.74 96 21.09 0.83
F 19–40 425 21.02 0.98 122 19.69 0.71 94 21.29 0.86
RET (%) M 19–40 402 1.69 0.39 116 1.65 0.28
F 19–40 405 2.05 0.52 125 2.86 0.83
WBC (G/L) M 19–40 434 4.35 1.32 116 4.87 1.24 105 4.41 1.36
F 19–40 431 2.31 0.94 125 3.60 1.18 98 2.25 0.93
NEUT (%)