193
Mutation Research, 57 (1978) 193-200 @ Elsevier/North-Holland Biomedical Press
CAFFEINE CONCENTRATIONS IN MICE PLASMA AND TESTICULAR TISSUE AND THE EFFECT OF CAFFEINE ON THE DOMINANT LETHAL TEST
H.U. AESCHBACHER,
H. MILON
Nest16 Products Technical Assistance CH-1350 Orbe (Switzerland) (Received 1 August 1977) (Revision received 2 January 1978) (Accepted 9 January 1978)
and H.P. WURZNER Company
Limited,
Biological
Laboratory,
Summary
Large groups of male Swiss mice received per OSon average 100 mg caffeine per kg body weight per day for 1 or 8 weeks. The dominant lethal test was designed to achieve maximum sensitivity in order to detect any possible mutagenic effect. No mutagenic induction of dominant lethals, pre-implantation egg loss or depression of the fertility of females, caused by caffeine at the dose levels administered were observed. The half life of caffeine, which was between 2.5 and 3 h, was similar in plasma and testicular tissue. It was concluded that caffeine did not accumulate in the testicular tissue of mice. The maximum concentration of caffeine found was below 10 Erg/g testicular tissue, which is about a 100 times lower than concentrations that cause chromosome aberrations in cultured mammalian cells.
Introduction
The possibility, that caffeine might produce mutagenic effects is a much investigated and reviewed subject [1,7-g]. The general conclusion of these reviews and investigations is, that the various mutagenic effects observed with caffeine in in vitro systems, could never be confirmed in in vivo mammalian tests. Studies on the various mechanisms of caffeine mutagenicity [ 71, might illuminate some aspects which would explain the absence of a mutagenic effect of caffeine in the living animal. Two main differences between in vitro and in vivo mutagenicity test systems do not allow for ready extrapolation of these two systems. Both the number of individuals exposed to the test substance and the actual exposure level at the
194
target cell are much lower in in vivo tests. These differences have led to the criticism that in vivo tests are not very sensitive. We have tried to investigate these problems by increasing the number of animals for the dominant lethal test and by determining the peak concentrations of caffeine present in the target organ. Experimental For the dominant lethal test and the determination of caffeine levels in plasma and testicules, male SPF Swiss mice CD-1 obtained from Charles River, Saint-Aubin-les-Elbeuf, France, were used. The animals were kept under conventional conditions. Temperature and relative humidity were maintained at 22°C and 55% respectively. An artificial day/night light cycle of 12 h was applied. The mice were maintained on a standard commercial diet and fed ad libitum (NAFAG, No. 185/194 Gossau, Switzerland). Caffeine was given either in their drinking water or by gavage. During the treatment period 5 males were kept together per Makrolon III cage on movable racks (48 places) and were then separated for mating. Dominant lethal test Two assays were performed in order to investigate both short-term treatment and long-term treatment covering all stages of spermatogenesis. In the first assay 3 groups were set up. The first group, a control consisting of 50 male mice at an initial age of about 10 weeks received by gavage tap water for 5 consecutive days. The second group of also 50 male mice received 90 mg caffeine per kg body weight per day by the same route of administration. The third group, representing a positive control group of 10 males, was injected with 0.125 mg of trenimon/kg body weight once i.p. Each of the males of all groups was then mated with 3 approximately ll-week-old, virgin females, at the first, 3rd and 6th week after the end of the treatment period. In the second assay, two groups were used. The first group, consisting of 150 male mice (8 weeks old), consumed an average of 112 mg caffeine per kg body weight per day for 8 weeks in their drinking water. The second group of 150 male mice received tap water only, for the same period of time. The caffeine solutions were freshly prepared each day by dissolving caffeine in warm sterilized tap water. Liquid consumption was recorded daily and body weights once a week. On this basis, the above-mentioned average intake of caffeine was calculated. At the first and third week after the treatment period, each male was mated with two approximately ll-week-old untreated females during 4 days. The latter mating was used to confirm results of the first one. For both assays, all females were killed 13 days after the mid-time of the mating period and scored for dead and living implants. The fertility was evaluated by the number of pregnant females in each group. For statistical evaluation of mutagenic induction, a recently described method was used [ 21. The chi-square test (corrected) was chosen to check for possible effects of the treatments on the fertility. Determination of caffeine in plasma and testicles The caffeine concentration in testicles and plasma of mice was determined
195
by gas chromatography after chloroform extraction. 50 ~1 of chloroform was added to 50 ~1 of plasma and 10 ,ul of citrate buffer in a 0.3 ml Reacti-Vial. The two testicles were hand-homogenized with 600 ~.tl of phosphate buffer and transferred to a conical centrifuge tube. After rinsing with 200 ~1 of phosphate buffer, 600 ~1 of chloroform was added. Then both tubes were whirl mixed for 30 set and centrifuged at 1000 rpm for 10 min for plasma and at 1500 rpm for 15 min for testicle homogenates. 2 ~1 of the chloroform layer was carefully withdrawn with a syringe and injected onto the gas chromatograph column. The gas chromatographic conditions (Perkin Elmer 3920) were as follows: the 1.8 m long, 2 mm i.d. glass column was packed with 3% SE-30 on Chromosorb-W AW-DMCS 80-100 mesh. The temperatures used were 190°C for the column, 250°C for the injector and detector. As gas carrier N2 + HCOOH vapours was used. Flame ionisation was used as detector and a Hewlett-Packard 3380A Integrator served for quantitation. No internal standard was used but standard solutions were injected at the beginning and end of each serie. The extraction coefficients were evaluated by determination of known amounts of caffeine added to plasma or testicles. These coefficients were found to be 93% for plasma and 90% for testicles. For the determination of caffeine concentrations in plasma and testicles two assays were set up. The first assay was carried out to establish the half-life of caffeine in mouse plasma and testicles. A total of 28 mice, 4-6 animals per group, were given a single dose of 100 mg caffeine per kg body weight by gavage. At 0.5, 1, 1.5, 3 and 5 h after administration of caffeine, the animals were anaesthesized with chloroform and blood was taken from the orbital venous plexus. Plasma was prepared and frozen immediately, then animals were killed and the testicles removed and frozen. With each group, two untreated control animals were used to check the blank values. In the second assay the maximum level of caffeine present in plasma and testicular tissue during longterm treatment was investigated. In this trial, 60 male mice of about 40 g body weight received caffeine in their drinking water for 2 weeks. The caffeine solution was made up fresh each day. According to the body weight and the daily liquid consumption, the average caffeine intake was calculated and found to be 125 mg caffeine/kg body weight. A group of 24 male control mice remained untreated. From 10 treated animals and 4 controls blood was drawn under chloroform anesthesia every 4 h during the last 24 h of the treatment period. Plasma was prepared at once and frozen. Immediately after each blood taking, the animals were killed and the testicles removed, frozen and used for caffeine determination. Results
Dominant lethal test In a first assay male mice were treated with caffeine for 5 consecutive days. No statistically significant mutagenic induction nor significant depression of the fertility was found (Table 1). In the trenimon-treated group however, a significant mutagenic induction and depression of fertility was observed. The increase was noted only for the first and third week matings, the sixth week mating being again non significant (Table 1). For all groups, a significant increase in
196 TABLE
1
DOMINANT
LETHAL
TEST
-SHORT
TERM
TREATMENT
Control water Mating Number Number Number I;~limits Number
I week after treatment of pregnant females of living implants of dead implants
Mating Number Number Number BtIimits Number
3 weeks after treatment of pregnant females per group of living implants of dead implants
of total
of total
per group
implants/female
implants/female
Mating 6 weeks after treatment Number of pregnant females per group Number of living implants Number of dead implants I; f limits Number of total implants/female
127 1609 116 0.068 13.5
138 1761 156 0.078 13.9
tap
i 0.016
126 1589 124 0.076 13.6
(90 mg/
t 0.016
142 1831 140 t 0.015
147 1857 169 0.083 13.8
Caffeine kg/day)
f 0.016
0.071 13.9
143 1792 175 0.089 13.7
f 0.014
t 0.015
In each group (control and treated with caffeine) 50 male mice were mated The groups treated with trenimon consisted of 10 males and 30 females each. animals received the test substance (or water for controls) by gavage during estimated from the betabinomial test with 96% confidence intervals. Significance limits are given asp < 0.06 = *, p < 0.001 = ***.
Trenimon (0.12 kg i.p. once)
16 *** 52 46 0.462 5.7
23 * 171 58 0.259 9.9
29 362 36 0.086 13.6
mg/
f > ***
f 0.064
***
f 0.035
with 150 untreated females. Except for trenirnon, all the 5 days. 9 values are given as
fertility with time after the last treatment, was observed. In the second assay, male mice were treated with caffeine for 8 weeks thereby exposing the whole cycle of spermatogenesis to caffeine. In this longterm assay no mutagenic induction whatsoever nor any significant depression of the fertility could be observed (Table 2). In both assays, the average number of total implants per female was the same for females mated to male8 receiving caffeine and females mated to untreated males. A pre-implantation egg loss caused by caffeine can therefore be excluded. Other possible disturbances like non-fertilization caused by caffeine were also unlikely, since caffeine did not alter the fertility rate. Caffeine levels in plasma and testicles of mice When male mice received a single dose of 100 mg of caffeine per kg body weight, the maximum level of caffeine was reached 1.5 h after application in both plasma and testicles. Disappearence of caffeine followed a similar pattern in plasma and testicular tissue (Figs. 1 and 2). From the values obtained, a halflife of about 2.6 h in plasma and 2.9 h in testicle8 was estimated. Therefore, considering the standard error of the mean the half-lives of caffeine in plasma and testicular tissue are comparable. The half-lives of caffeine agree well with the results of Burg [4] who used 14C-labelling. In the second assay, the maxi-
197 TABLE
2
DOMINANT
LETHAL
TEST
- LONG
TERM
TREATMENT Control
Mating
1 week
after
3 weeks
after
Caffeine
(112
mg/kg/day)
treatment
Number of pregnant females Number of living implants Number of dead implants li * limits Total implants/female Mating
tap water
per group
261
266
3450
3461
218
218
0.059
f 0.008
f 0.010
0.060
13.7
14.4
treatment
Number pf pregnant females Number of living implants Number of dead implants I; f limits Total implants/female
per group
273 3486 248
270 3331 264 0.067
0.089 13.3
f 0.009
13.6
In each of the two groups. 150 males were mated test substances in the drinking water for 8 weeks. test with 95% confidence intervals.
with 300 untreated fi values are given
f 0.010
females. The males received their as estimated from the betabinomial
mum caffeine level in plasma and testicles over a 24-h period was determined. The highest peaks occurred between midnight and 4 a.m. At 4 p.m. no caffeine could be detected (Tables 3 and 4). The maximum level of caffeine in testicles never exceeded 10 E.cg/gtissue. The results obtained over the entire 24 h were again similar for plasma and testicles. An accumulation of caffeine in testicular tissue or plasma can be excluded since caffeine could not be detected in any of the samples at 4 p.m. The results in Tables 3 and 4 are given as single values because, in some samples only traces of caffeine could be detected (limits of the method) and because it was of interest to note the highest individual exposure levels.
%50a 3
I
t ‘5 25E ” lo01
’
’
’
0.5
1.0
1.5
3.0
I Time
(h)
5.0
-
Fig. 1. Plasma Figs.
1 and 2, Caffeine
per OS.
05 Fig.
levels
in mice
plasma
and testicles
1.0
’ 15
3.0
I Time
(h)
5D
2. Testicles.
after
a single
dose
of 100 mg/kg
body
weight
198 TABLE
3
PLASMA
CONCENTRATION
(pg
caffeine/ml)
16 h
20 h
Oh
4h
+ + -
3.8 2.1 + 2.4 1.1 + +
5.5 + -
+
+ + -
-
-
+
-
-
0.8
12 h -~.---
6h + -
TABLE
+ 3.5
-
__--~
1.9
-
2.5 +
+
4
TESTICULAR ---ah
CONCENTRATION 12h
(pg
caffeine/g)
16 h
20 h
Oh
4h
-
3.3 1.2 2.7
1.2 3.9 + 5.4
8.7 6.6 6.6 + +
+ + -
+ + + +
+ +
+ +
-
-
-
+
-
+ + +
-
-
3.0 -
4.5 -
+ 7.5
+ + + +
+ -
Caffeine level in mouse plasmaand testicles (Table 4) after and ad libitum ingestion of 125 mg/kg b.w./day in drinking water for 2 weeks. Each value represents a different mouse. In both tables, the values from the same animal carry the same number since plasma and testicles used were from the same animal. The results were obtained from 60 mice receiving an average of 125 mg of caffeine/kg/day for two weeks. During the last day of treatment, 10 treated animals and 4 untreated controls were killed every 4 h. Negative results were obtained for all control animals. Plasma, - = CO.1 pg/ml (not detectable), + = between 0.1 and 0.5 pg/ml (traces). Testicles, - =
Mutation Research, 57 (1978) 193-200 @ Elsevier/North-Holland Biomedical Press
CAFFEINE CONCENTRATIONS IN MICE PLASMA AND TESTICULAR TISSUE AND THE EFFECT OF CAFFEINE ON THE DOMINANT LETHAL TEST
H.U. AESCHBACHER,
H. MILON
Nest16 Products Technical Assistance CH-1350 Orbe (Switzerland) (Received 1 August 1977) (Revision received 2 January 1978) (Accepted 9 January 1978)
and H.P. WURZNER Company
Limited,
Biological
Laboratory,
Summary
Large groups of male Swiss mice received per OSon average 100 mg caffeine per kg body weight per day for 1 or 8 weeks. The dominant lethal test was designed to achieve maximum sensitivity in order to detect any possible mutagenic effect. No mutagenic induction of dominant lethals, pre-implantation egg loss or depression of the fertility of females, caused by caffeine at the dose levels administered were observed. The half life of caffeine, which was between 2.5 and 3 h, was similar in plasma and testicular tissue. It was concluded that caffeine did not accumulate in the testicular tissue of mice. The maximum concentration of caffeine found was below 10 Erg/g testicular tissue, which is about a 100 times lower than concentrations that cause chromosome aberrations in cultured mammalian cells.
Introduction
The possibility, that caffeine might produce mutagenic effects is a much investigated and reviewed subject [1,7-g]. The general conclusion of these reviews and investigations is, that the various mutagenic effects observed with caffeine in in vitro systems, could never be confirmed in in vivo mammalian tests. Studies on the various mechanisms of caffeine mutagenicity [ 71, might illuminate some aspects which would explain the absence of a mutagenic effect of caffeine in the living animal. Two main differences between in vitro and in vivo mutagenicity test systems do not allow for ready extrapolation of these two systems. Both the number of individuals exposed to the test substance and the actual exposure level at the
194
target cell are much lower in in vivo tests. These differences have led to the criticism that in vivo tests are not very sensitive. We have tried to investigate these problems by increasing the number of animals for the dominant lethal test and by determining the peak concentrations of caffeine present in the target organ. Experimental For the dominant lethal test and the determination of caffeine levels in plasma and testicules, male SPF Swiss mice CD-1 obtained from Charles River, Saint-Aubin-les-Elbeuf, France, were used. The animals were kept under conventional conditions. Temperature and relative humidity were maintained at 22°C and 55% respectively. An artificial day/night light cycle of 12 h was applied. The mice were maintained on a standard commercial diet and fed ad libitum (NAFAG, No. 185/194 Gossau, Switzerland). Caffeine was given either in their drinking water or by gavage. During the treatment period 5 males were kept together per Makrolon III cage on movable racks (48 places) and were then separated for mating. Dominant lethal test Two assays were performed in order to investigate both short-term treatment and long-term treatment covering all stages of spermatogenesis. In the first assay 3 groups were set up. The first group, a control consisting of 50 male mice at an initial age of about 10 weeks received by gavage tap water for 5 consecutive days. The second group of also 50 male mice received 90 mg caffeine per kg body weight per day by the same route of administration. The third group, representing a positive control group of 10 males, was injected with 0.125 mg of trenimon/kg body weight once i.p. Each of the males of all groups was then mated with 3 approximately ll-week-old, virgin females, at the first, 3rd and 6th week after the end of the treatment period. In the second assay, two groups were used. The first group, consisting of 150 male mice (8 weeks old), consumed an average of 112 mg caffeine per kg body weight per day for 8 weeks in their drinking water. The second group of 150 male mice received tap water only, for the same period of time. The caffeine solutions were freshly prepared each day by dissolving caffeine in warm sterilized tap water. Liquid consumption was recorded daily and body weights once a week. On this basis, the above-mentioned average intake of caffeine was calculated. At the first and third week after the treatment period, each male was mated with two approximately ll-week-old untreated females during 4 days. The latter mating was used to confirm results of the first one. For both assays, all females were killed 13 days after the mid-time of the mating period and scored for dead and living implants. The fertility was evaluated by the number of pregnant females in each group. For statistical evaluation of mutagenic induction, a recently described method was used [ 21. The chi-square test (corrected) was chosen to check for possible effects of the treatments on the fertility. Determination of caffeine in plasma and testicles The caffeine concentration in testicles and plasma of mice was determined
195
by gas chromatography after chloroform extraction. 50 ~1 of chloroform was added to 50 ~1 of plasma and 10 ,ul of citrate buffer in a 0.3 ml Reacti-Vial. The two testicles were hand-homogenized with 600 ~.tl of phosphate buffer and transferred to a conical centrifuge tube. After rinsing with 200 ~1 of phosphate buffer, 600 ~1 of chloroform was added. Then both tubes were whirl mixed for 30 set and centrifuged at 1000 rpm for 10 min for plasma and at 1500 rpm for 15 min for testicle homogenates. 2 ~1 of the chloroform layer was carefully withdrawn with a syringe and injected onto the gas chromatograph column. The gas chromatographic conditions (Perkin Elmer 3920) were as follows: the 1.8 m long, 2 mm i.d. glass column was packed with 3% SE-30 on Chromosorb-W AW-DMCS 80-100 mesh. The temperatures used were 190°C for the column, 250°C for the injector and detector. As gas carrier N2 + HCOOH vapours was used. Flame ionisation was used as detector and a Hewlett-Packard 3380A Integrator served for quantitation. No internal standard was used but standard solutions were injected at the beginning and end of each serie. The extraction coefficients were evaluated by determination of known amounts of caffeine added to plasma or testicles. These coefficients were found to be 93% for plasma and 90% for testicles. For the determination of caffeine concentrations in plasma and testicles two assays were set up. The first assay was carried out to establish the half-life of caffeine in mouse plasma and testicles. A total of 28 mice, 4-6 animals per group, were given a single dose of 100 mg caffeine per kg body weight by gavage. At 0.5, 1, 1.5, 3 and 5 h after administration of caffeine, the animals were anaesthesized with chloroform and blood was taken from the orbital venous plexus. Plasma was prepared and frozen immediately, then animals were killed and the testicles removed and frozen. With each group, two untreated control animals were used to check the blank values. In the second assay the maximum level of caffeine present in plasma and testicular tissue during longterm treatment was investigated. In this trial, 60 male mice of about 40 g body weight received caffeine in their drinking water for 2 weeks. The caffeine solution was made up fresh each day. According to the body weight and the daily liquid consumption, the average caffeine intake was calculated and found to be 125 mg caffeine/kg body weight. A group of 24 male control mice remained untreated. From 10 treated animals and 4 controls blood was drawn under chloroform anesthesia every 4 h during the last 24 h of the treatment period. Plasma was prepared at once and frozen. Immediately after each blood taking, the animals were killed and the testicles removed, frozen and used for caffeine determination. Results
Dominant lethal test In a first assay male mice were treated with caffeine for 5 consecutive days. No statistically significant mutagenic induction nor significant depression of the fertility was found (Table 1). In the trenimon-treated group however, a significant mutagenic induction and depression of fertility was observed. The increase was noted only for the first and third week matings, the sixth week mating being again non significant (Table 1). For all groups, a significant increase in
196 TABLE
1
DOMINANT
LETHAL
TEST
-SHORT
TERM
TREATMENT
Control water Mating Number Number Number I;~limits Number
I week after treatment of pregnant females of living implants of dead implants
Mating Number Number Number BtIimits Number
3 weeks after treatment of pregnant females per group of living implants of dead implants
of total
of total
per group
implants/female
implants/female
Mating 6 weeks after treatment Number of pregnant females per group Number of living implants Number of dead implants I; f limits Number of total implants/female
127 1609 116 0.068 13.5
138 1761 156 0.078 13.9
tap
i 0.016
126 1589 124 0.076 13.6
(90 mg/
t 0.016
142 1831 140 t 0.015
147 1857 169 0.083 13.8
Caffeine kg/day)
f 0.016
0.071 13.9
143 1792 175 0.089 13.7
f 0.014
t 0.015
In each group (control and treated with caffeine) 50 male mice were mated The groups treated with trenimon consisted of 10 males and 30 females each. animals received the test substance (or water for controls) by gavage during estimated from the betabinomial test with 96% confidence intervals. Significance limits are given asp < 0.06 = *, p < 0.001 = ***.
Trenimon (0.12 kg i.p. once)
16 *** 52 46 0.462 5.7
23 * 171 58 0.259 9.9
29 362 36 0.086 13.6
mg/
f > ***
f 0.064
***
f 0.035
with 150 untreated females. Except for trenirnon, all the 5 days. 9 values are given as
fertility with time after the last treatment, was observed. In the second assay, male mice were treated with caffeine for 8 weeks thereby exposing the whole cycle of spermatogenesis to caffeine. In this longterm assay no mutagenic induction whatsoever nor any significant depression of the fertility could be observed (Table 2). In both assays, the average number of total implants per female was the same for females mated to male8 receiving caffeine and females mated to untreated males. A pre-implantation egg loss caused by caffeine can therefore be excluded. Other possible disturbances like non-fertilization caused by caffeine were also unlikely, since caffeine did not alter the fertility rate. Caffeine levels in plasma and testicles of mice When male mice received a single dose of 100 mg of caffeine per kg body weight, the maximum level of caffeine was reached 1.5 h after application in both plasma and testicles. Disappearence of caffeine followed a similar pattern in plasma and testicular tissue (Figs. 1 and 2). From the values obtained, a halflife of about 2.6 h in plasma and 2.9 h in testicle8 was estimated. Therefore, considering the standard error of the mean the half-lives of caffeine in plasma and testicular tissue are comparable. The half-lives of caffeine agree well with the results of Burg [4] who used 14C-labelling. In the second assay, the maxi-
197 TABLE
2
DOMINANT
LETHAL
TEST
- LONG
TERM
TREATMENT Control
Mating
1 week
after
3 weeks
after
Caffeine
(112
mg/kg/day)
treatment
Number of pregnant females Number of living implants Number of dead implants li * limits Total implants/female Mating
tap water
per group
261
266
3450
3461
218
218
0.059
f 0.008
f 0.010
0.060
13.7
14.4
treatment
Number pf pregnant females Number of living implants Number of dead implants I; f limits Total implants/female
per group
273 3486 248
270 3331 264 0.067
0.089 13.3
f 0.009
13.6
In each of the two groups. 150 males were mated test substances in the drinking water for 8 weeks. test with 95% confidence intervals.
with 300 untreated fi values are given
f 0.010
females. The males received their as estimated from the betabinomial
mum caffeine level in plasma and testicles over a 24-h period was determined. The highest peaks occurred between midnight and 4 a.m. At 4 p.m. no caffeine could be detected (Tables 3 and 4). The maximum level of caffeine in testicles never exceeded 10 E.cg/gtissue. The results obtained over the entire 24 h were again similar for plasma and testicles. An accumulation of caffeine in testicular tissue or plasma can be excluded since caffeine could not be detected in any of the samples at 4 p.m. The results in Tables 3 and 4 are given as single values because, in some samples only traces of caffeine could be detected (limits of the method) and because it was of interest to note the highest individual exposure levels.
%50a 3
I
t ‘5 25E ” lo01
’
’
’
0.5
1.0
1.5
3.0
I Time
(h)
5.0
-
Fig. 1. Plasma Figs.
1 and 2, Caffeine
per OS.
05 Fig.
levels
in mice
plasma
and testicles
1.0
’ 15
3.0
I Time
(h)
5D
2. Testicles.
after
a single
dose
of 100 mg/kg
body
weight
198 TABLE
3
PLASMA
CONCENTRATION
(pg
caffeine/ml)
16 h
20 h
Oh
4h
+ + -
3.8 2.1 + 2.4 1.1 + +
5.5 + -
+
+ + -
-
-
+
-
-
0.8
12 h -~.---
6h + -
TABLE
+ 3.5
-
__--~
1.9
-
2.5 +
+
4
TESTICULAR ---ah
CONCENTRATION 12h
(pg
caffeine/g)
16 h
20 h
Oh
4h
-
3.3 1.2 2.7
1.2 3.9 + 5.4
8.7 6.6 6.6 + +
+ + -
+ + + +
+ +
+ +
-
-
-
+
-
+ + +
-
-
3.0 -
4.5 -
+ 7.5
+ + + +
+ -
Caffeine level in mouse plasmaand testicles (Table 4) after and ad libitum ingestion of 125 mg/kg b.w./day in drinking water for 2 weeks. Each value represents a different mouse. In both tables, the values from the same animal carry the same number since plasma and testicles used were from the same animal. The results were obtained from 60 mice receiving an average of 125 mg of caffeine/kg/day for two weeks. During the last day of treatment, 10 treated animals and 4 untreated controls were killed every 4 h. Negative results were obtained for all control animals. Plasma, - = CO.1 pg/ml (not detectable), + = between 0.1 and 0.5 pg/ml (traces). Testicles, - =
