Evaluation of the effects of dark period shortening on CD-1 mice
in chronic toxicology studies
Debra Farrell 1, Raluca Kubaszky 1 and Roy Forster 2
CIToxLAB North America, Laval, Quebec, Canada; 2 CiToxLAB France, 27005 Evreux, France
The source (Charles River, St-Constant, Québec), age (6 weeks at start of treatment, starting
weight (refer to graphs), environmental conditions (21+/-3°C, 30-70% RH, 10-15 air changes/
hour), water supply (UV-reverse osmosis treated, ad libitum), diet (Teklad Certified Rodent
Diet no. 2018C, ad libitum), housing (individual wire-mesh) and enrichment (Nylabones and
Cozee pads) were identical for both studies.
Parameters monitored included clinical observations, body weight, body weight gain and food
intake, measured weekly for the first 13 weeks and monthly thereafter. In the carcinogenicity
study mice, palpable masses were examined after Week 26 and ophthalmological examination
and white cell differential counts assessed at 12 months and beyond. At the completion of each
experiment, macroscopic and microscopic pathology examinations were performed.
Results
Throughout the period of comparison there were no untoward clinical signs in control animals
of either study, nor were there any differences in the type and frequency of findings observed
between the 2 studies, giving no indication of an effect of the altered light cycle. Beyond
the 13-week period of comparison, the signs observed in the carcinogenicity mice were of
the type commonly seen in aging laboratory-housed mice. At ophthalmological evaluations
conducted during Weeks 52 and 104, incidental findings noted included superficial punctate
keratopathy, cataract, ectopic pupil, persistent pupillary membrane, focal/punctate anterior,
posterior cortical, capsular or subcapsular white opacities, microphthalmia, iris degeneration
or keratitis. These findings were observed at the pretreatment evaluation, had low incidence
or were considered spontaneous, incidental findings expected for the CD-1 mouse aging population evaluated.
38
6.5
36
6
34
32
30
28
26
5.5
5
4.5
4
24
3.5
22
7
8
9
10
11
12
13
13 - 14
6
12 - 13
5
11 - 12
4
10 - 11
3
9 - 10
2
8-9
1
7-8
0
6-7
-1
5-6
-2
4-5
3
20
3-4
At the start of each study the mice were 6 weeks of age and the body weights were consistent
with the supplier’s growth curves. In the 13 week study the mice received a once daily (in the
morning) oral gavage of sterile water at a dose volume of 5 mL/kg. The light cycle for the
13-week study was a standard 12 light: 12 dark, commencing with light at 07:00. In the carcinogenicity study the mice received twice daily (approximately 12 hours apart) oral gavage of
sterile water at a dose volume of 5 mL/kg/dose. As a result of the twice daily dosing, the daily
light cycle for the carcinogenicity study was altered to approximately 14 hours light and 10
hours dark, with minor occasional excursions from the light cycle up to 17 hours light, where
the morning light cycle commenced at 07:00.
7
2-3
Body weight, food intake and clinical observation data from vehicle control groups of male
and female age-matched CD-1 mice (CRL: CD-1 (1-CR)) from a carcinogenicity study (N=110/
sex) and a 13-week study (N-15/sex) were compared in order to ascertain what, if any, impact
might arise from a 2-hour prolongation of the light cycle on the carcinogenicity study.
40
1-2
Materials and methods
Graph 2: group mean food consumption
-1 to 1
The standard lighting regimen for regulatory toxicology studies in rodents considered suitable
for normal animal activity, neuroendocrine regulation and for decreased risk of possible retinal
damage due to prolonged exposure to light, is typically 12 hours light and 12 hours dark with
occasional interruptions for infrequent activities (i.e. toxicokinetic sampling). The purpose
of the current assessment is to compare data collected from a study with a shortened dark
period (10 hours dark, from 9 pm to 7 am, and 14 hours light, from 7 am to 9 pm) with a study
performed under standard lighting conditions, (12/12 hours light/dark). The study with shortened dark period was a carcinogenicity study with twice daily oral gavage administration,
while the study with standard lighting conditions was a 13 week study with once daily oral
gavage dosing. The comparison is made for data over the first 13 weeks of the studies. Both
studies were fully GLP compliant and all other conditions of animal housing, care and use were
similar between the two studies.
Graph 1: group mean body weights
Mean FC (g/animal/day)
Introduction
Mean BW in grams
1
Week
Week
Control 14h light/ 10h dark females
Control 14h light/ 10h dark males
Control 14h light/ 10h dark females
Control 14h light/ 10h dark males
Control 12h light/ 12h dark males
Control 12h light/ 12h dark females
Control 12h light/ 12h dark males
Control 12h light/ 12h dark females
Body weight progression (Graph 1) was very consistent between the two studies with males
and females each showing an essentially identical progression, versus their respective comparator, throughout the period of comparison. Beyond the 13-week timepoint the body weight
trajectory continued for the light-reduced carcinogenicity control mice.
Throughout the period of comparison the food intake (Graph 2) of the 10 hour-dark carcinogenicity control mice was comparable to that of the animals on the normal light cycle. If
anything, males of the 10 hour-dark group tended to have a slightly elevated, but consistent
intake in comparison to the other group of males. Beyond the 13-week timepoint food intake
tended to increase in the aging carcinogenicity control mice, and to a similar proportion in
males and females.
At histopathology evaluation, there were no microscopic findings considered related to the
2-hour shortening of the light cycle. In the carcinogenicity control mice examined either at
scheduled termination, or pre-terminally euthanized or found dead, ocular findings including
hyperkeratosis, keratitis, ulcer or mineralization were observed with low incidence in both male
and female animals. In the Harderian glands, incidental findings consisted of observations such
as, but not limited to, glandular degeneration, pigment accumulation, dilatation and/or chronic
inflammation. No macroscopic or microscopic changes of the optic nerve were recorded,
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with the exception of small left optic nerve in 1/110 carcinogenicity control female mice, not
confirmed at histopathological evaluation.
Discussion and conclusion
Data collected from a study with a shortened dark cycle (14/10 hours light/dark) were
compared with a study performed under standard lighting conditions, (12/12 hours light/
dark).
The comparison covered the first 13 weeks of treatment, and post-mortem investigations. There were no differences in body weight evolution or food consumption.
Possible effects on the eyes were evaluated by ophthalmological examinations, ocular
necropsy findings and histopathology of the eyes; there was no indication of findings
related to the difference in dark period.