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Short term toxicity study in rats dosed with pulegone and menthol
Toxicology Letters, 19 (1983) 207-210
207
Elsevier
SHORT
TERM TOXICITY
STUDY
IN RATS DOSED
WITH PULEGONE
AND MENTHOL (Pulegone;
menthol;
food flavoring;
I. THORUP,
G. WURTZEN,
toxicity;
J. CARSTENSEN
encephalopathy;
rats)
and P. OLSEN
Institute of Toxicology, National Food Institute, 19 Merkhnj Bygade, DK 2860 Snborg (Denmark) (Received
June 29th,
(Accepted
July 9th,
1983) 1983)
SUMMARY Pulegone
and menthol,
administered kg body creatinine
by gavage
wt./day,
components
respectively.
content,
lowered
in the white matter
of peppermint
oil, were investigated
for 28 days at 0, 20, 80, 160 mg pulegone At the two highest
terminal
doses,
body weight and caused
of cerebellum.
For menthol
relative liver weights and vacuolisation
in rats. The substances
pulegone
induced
histopathological
at all dose levels a significant
of hepatocytes
was found.
were
and 0, 200, 400, 800 mg menthol/ atonia,
decreased
blood
changes
in the liver and
increase
in absolute
No sign of encephalopathy
and
was observ-
ed in rats given menthol. The no effect
level for pulegone
was 20 mg/kg
body
wt./day
and for menthol
~200
mg/kg
body
wt./day.
INTRODUCTION
Neuropathy has been induced in rats given peppermint oil [I]. Peppermint oil consists of several compounds [2]. The substances responsible for the toxic effect have not yet been identified. 2 short-term studies were conducted with pulegone and menthol, which constitute l-3% and 35-50% of peppermint oil, respectively. At one time pulegone was used as an ineffective abortifacient and may provoke convulsions in man [3, 41. A few years ago intoxication and deaths in women were reported [5, 61. In 1972 it was recommended that its use as a food flavour be prohibited [3] and the Committee of Experts on Flavourings of the Council of Europe proposed that its presence in food be limited [7]. The results of one study on menthol occur at ingestion of about 2 mg/kg
03784274/83/$
03.00 0 Elsevier
in man suggested that adverse effects may body wt./day, while other evidence from
Science Publishers
B.V.
208
human exposure showed that adverse effects are unlikely to occur from 0.2 mgikg body wt./day. Menthol did not cause any toxic effects in rats dosed up to 200 mglkg body wt./day for 5 weeks. Based upon this observation the Joint FAO/WHO Expert Committee on Food Additives (JECFA) allocated an acceptable daily intake (ADI) at 0.2 mg/kg body wt./day (8). MATERIALS
AND METHODS
Materials. Pulegone puriss. 99%, Fluka 82569 and (- )-menthol puriss. 99‘70, Fluka 63660. Animals, diet and experiment. Strain, diet, caging and environmental conditions were as described by Thorup et al. [I). Groups of 10 male and 10 female rats were given 0, 20, 80 or 160 mg pulegone and 0, 200, 400, or 800 mg menthol/kg body wt./day, respectively, by gavage, diluted with soybeain oil. ClinicaI, clinical chemical and pathological examinations were performed by the method of Thorup et al. [l]. In addition, reticulocytes in whole blood were examined in the menthol study and for rats dosed with pulegone the concentrating ability of the kidney was measured on urine by its osmolality. Urine was examined for presence of blood, ketones, glucose, and proteins by Ames Labstix @. Student’s t-test was performed on all parameters for males and females separately and analyses of variance were applied to organ weights. P
AND
DISCUSSION
After a few days of dosing with pulegone, the rats showed dose-dependent atonia. Both compounds induced significantly increased water consumption at the highest dose. The kidney function test on pulegone-treated rats revealed no abnormality.
TABLE RATS
I GIVEN
PULEGONE
AND
MENTHOL
ORALLY
FOR
28
DAYS.
MICROSCOPIC
CHANGES.
10 male and mg/ky
10 females
per group.
body wt./day
Vacuolisation of hepatocytes Channes in cerebellum
Pulegone
-.--
Menthol
0
20
80
I 606
0
200
400b
800”
0 0
0 0
10 4
15 13
0 0
4 0
5 0
4 n
209
TABLE RATS
II GIVEN
PLASMA FEMALES mg/kg
PULEGONE
(8 MALES
ORALLY
AND 8 FEMALES
FOR
28 DAYS.
PER GROUP)
(10 MALES
AND
IN 10
Pulegone 0
Weight
CONCENTRATION GAIN
PER GROUP).
body wt./day
Creatinine
CREATININE
AND WEIGHT
pmol/l
gain g
The values are means aMean of I rats.
f
20
80
160
Females
40 f
2
28 + 2
34 + 6a
< 10
Males
39 f
3
36 + 1
14 + 3b
< 10
Females
42 f
I
38 + 6
26 f
6
Males
II
8
15 k 8
46 f
8
f
13 + 7 -2
+ 6’
S.E.M.
bMean of 4 rats; 4 rats
< 10 pmol/l.
‘Mean
of 9 rats.
Weight gain was significantly reduced in the highest (20%) and intermediate (10%) dosage groups for rats given pulegone. Furthermore, the blood creatinine showed a dose-dependent decrease, significant at the highest dose. An increased number of neutrophile granulocytes was found at the highest dose in both studies. At autopsy all rats in the highest group dosed with pulegone showed markely distended and atonic stomachs packed with food. Significant decreases in terminal body weight and organ weights were found for rats dosed with pulegone. The relative organ weights were not therefore taken into consideration. However, doserelated vacuolisation of hepatocytes in the zone around the central vein was induced by pulegone (Table I). Liver cell necrosis had been demonstrated when pulegone was given i.p. to mice [9]. The decrease in blood creatinine (Table II) reflects the lower total muscle mass [lo] and might be related to the observed liver change. Pulegone
treated
rats revealed
dose-related
alterations
in the brain (Table
I). The
changes were seen at the two highest doses and appeared as cyst-like spaces in the white matter of the cerebellum, resembling those found in rats dosed peppermint oil [l]. No decrease in body weight was induced by menthol, but a significant increase in absolute and relative organ weight was seen for the liver (males: all doses; females: high and intermediate dose). Observation of vacuolisation of hepatocytes, was not dose-related (Table I) and may reflect adaptation [ 111. The rats given menthol did not show any sign of alteration in the brain. This investigation indicates that pulegone might play a significant role in the pathogenesis of encephalopathy in rats given peppermint oil. The no effect level in these 28 day gavage studies was 20 mg/kg body wt./day for pulegone and was not established but was < 200 mg/kg body wt./day for menthol.
210
REFERENCES
1 I. Thorup, permint 2 D.
G. Wurtzen, Oil, Toxicol.
Morrison
J. Carstensen Lett.,
Smith
and
Characterization
of Essential
Chromatographic
Analysis,
3 Martindale. don,
and P. Olsen, Short Term Toxicity
Study in Rats Dosed Pep-
19 (1983) 211-215. L.
Levi,
Essential
Oils
Oils. Determination Agric.
Food
The Extra Pharmacopoeia.
Treatment
of
of Geographical
Chem.,
Compositional
Origins
Data
of Peppermint
for
the
Oils by Gas
9 (1961) 230-244.
28th Edition,
N.W.
Blacow,
The Pharmaceutical
Press, Lon-
1982, p. 682.
4 F. Grundschober, 5 J.B. Sullivan,
Literature B.H.
and hepatotoxicity, 6 P. Gunby,
Plant
review of pulegone,
Rumack,
H. Thomas,
J. Am. Med. Assoc., known
for centuries
R.G.
Perfumer Peterson
and flavorist., and P. Bryson,
4 (1979) 15-17. Pennyroyal
oil poisoning
242 (1979) 2873-2874.
still causes
problems
today,
J. Am. Med. Assoc.,
241 (1979)
2246-2247. 7 Council
of Europe,
Strasbourg, 8 JECFA,
Joint FAO/WHO
tain Food 9 W.P.
Additives
Gordon,
toxicity
Flavouring
substances
and
natural
sources
Committee
on Food Additives,
of
flavourings,
Maisonneuve,
1981, p. 53.
A.J.
Expert
Toxicological
Evaluation
of Cer-
(1976) Series No. 10, pp. 64-69. Forte,
of pennyroyal
R.J. McMurtry,
oil and its constituent
J. Gal and S.D. Nelson, terpenes
in the mouse,
Hepatotoxicity
and pulmonary
Toxicol.
Pharmacol.,
Appl.
65
(1982) 413-424. 10 J.E.
Schutte,
creatinine: 11 T. Balazs,
J.C.
Longhurst,
an accurate Development
measure
F.A.
Graffney,
of total striated
of tissue resistance
B.C.
Bastian
and
C.G.
muscle mass, J. Appl.
to toxic effects
of chemicals,
Blomquist,
Physiol.,
Totalplasma
51 (1981) 762-766.
Toxicol.,
(1974) 247-255.
207
Elsevier
SHORT
TERM TOXICITY
STUDY
IN RATS DOSED
WITH PULEGONE
AND MENTHOL (Pulegone;
menthol;
food flavoring;
I. THORUP,
G. WURTZEN,
toxicity;
J. CARSTENSEN
encephalopathy;
rats)
and P. OLSEN
Institute of Toxicology, National Food Institute, 19 Merkhnj Bygade, DK 2860 Snborg (Denmark) (Received
June 29th,
(Accepted
July 9th,
1983) 1983)
SUMMARY Pulegone
and menthol,
administered kg body creatinine
by gavage
wt./day,
components
respectively.
content,
lowered
in the white matter
of peppermint
oil, were investigated
for 28 days at 0, 20, 80, 160 mg pulegone At the two highest
terminal
doses,
body weight and caused
of cerebellum.
For menthol
relative liver weights and vacuolisation
in rats. The substances
pulegone
induced
histopathological
at all dose levels a significant
of hepatocytes
was found.
were
and 0, 200, 400, 800 mg menthol/ atonia,
decreased
blood
changes
in the liver and
increase
in absolute
No sign of encephalopathy
and
was observ-
ed in rats given menthol. The no effect
level for pulegone
was 20 mg/kg
body
wt./day
and for menthol
~200
mg/kg
body
wt./day.
INTRODUCTION
Neuropathy has been induced in rats given peppermint oil [I]. Peppermint oil consists of several compounds [2]. The substances responsible for the toxic effect have not yet been identified. 2 short-term studies were conducted with pulegone and menthol, which constitute l-3% and 35-50% of peppermint oil, respectively. At one time pulegone was used as an ineffective abortifacient and may provoke convulsions in man [3, 41. A few years ago intoxication and deaths in women were reported [5, 61. In 1972 it was recommended that its use as a food flavour be prohibited [3] and the Committee of Experts on Flavourings of the Council of Europe proposed that its presence in food be limited [7]. The results of one study on menthol occur at ingestion of about 2 mg/kg
03784274/83/$
03.00 0 Elsevier
in man suggested that adverse effects may body wt./day, while other evidence from
Science Publishers
B.V.
208
human exposure showed that adverse effects are unlikely to occur from 0.2 mgikg body wt./day. Menthol did not cause any toxic effects in rats dosed up to 200 mglkg body wt./day for 5 weeks. Based upon this observation the Joint FAO/WHO Expert Committee on Food Additives (JECFA) allocated an acceptable daily intake (ADI) at 0.2 mg/kg body wt./day (8). MATERIALS
AND METHODS
Materials. Pulegone puriss. 99%, Fluka 82569 and (- )-menthol puriss. 99‘70, Fluka 63660. Animals, diet and experiment. Strain, diet, caging and environmental conditions were as described by Thorup et al. [I). Groups of 10 male and 10 female rats were given 0, 20, 80 or 160 mg pulegone and 0, 200, 400, or 800 mg menthol/kg body wt./day, respectively, by gavage, diluted with soybeain oil. ClinicaI, clinical chemical and pathological examinations were performed by the method of Thorup et al. [l]. In addition, reticulocytes in whole blood were examined in the menthol study and for rats dosed with pulegone the concentrating ability of the kidney was measured on urine by its osmolality. Urine was examined for presence of blood, ketones, glucose, and proteins by Ames Labstix @. Student’s t-test was performed on all parameters for males and females separately and analyses of variance were applied to organ weights. P
AND
DISCUSSION
After a few days of dosing with pulegone, the rats showed dose-dependent atonia. Both compounds induced significantly increased water consumption at the highest dose. The kidney function test on pulegone-treated rats revealed no abnormality.
TABLE RATS
I GIVEN
PULEGONE
AND
MENTHOL
ORALLY
FOR
28
DAYS.
MICROSCOPIC
CHANGES.
10 male and mg/ky
10 females
per group.
body wt./day
Vacuolisation of hepatocytes Channes in cerebellum
Pulegone
-.--
Menthol
0
20
80
I 606
0
200
400b
800”
0 0
0 0
10 4
15 13
0 0
4 0
5 0
4 n
209
TABLE RATS
II GIVEN
PLASMA FEMALES mg/kg
PULEGONE
(8 MALES
ORALLY
AND 8 FEMALES
FOR
28 DAYS.
PER GROUP)
(10 MALES
AND
IN 10
Pulegone 0
Weight
CONCENTRATION GAIN
PER GROUP).
body wt./day
Creatinine
CREATININE
AND WEIGHT
pmol/l
gain g
The values are means aMean of I rats.
f
20
80
160
Females
40 f
2
28 + 2
34 + 6a
< 10
Males
39 f
3
36 + 1
14 + 3b
< 10
Females
42 f
I
38 + 6
26 f
6
Males
II
8
15 k 8
46 f
8
f
13 + 7 -2
+ 6’
S.E.M.
bMean of 4 rats; 4 rats
< 10 pmol/l.
‘Mean
of 9 rats.
Weight gain was significantly reduced in the highest (20%) and intermediate (10%) dosage groups for rats given pulegone. Furthermore, the blood creatinine showed a dose-dependent decrease, significant at the highest dose. An increased number of neutrophile granulocytes was found at the highest dose in both studies. At autopsy all rats in the highest group dosed with pulegone showed markely distended and atonic stomachs packed with food. Significant decreases in terminal body weight and organ weights were found for rats dosed with pulegone. The relative organ weights were not therefore taken into consideration. However, doserelated vacuolisation of hepatocytes in the zone around the central vein was induced by pulegone (Table I). Liver cell necrosis had been demonstrated when pulegone was given i.p. to mice [9]. The decrease in blood creatinine (Table II) reflects the lower total muscle mass [lo] and might be related to the observed liver change. Pulegone
treated
rats revealed
dose-related
alterations
in the brain (Table
I). The
changes were seen at the two highest doses and appeared as cyst-like spaces in the white matter of the cerebellum, resembling those found in rats dosed peppermint oil [l]. No decrease in body weight was induced by menthol, but a significant increase in absolute and relative organ weight was seen for the liver (males: all doses; females: high and intermediate dose). Observation of vacuolisation of hepatocytes, was not dose-related (Table I) and may reflect adaptation [ 111. The rats given menthol did not show any sign of alteration in the brain. This investigation indicates that pulegone might play a significant role in the pathogenesis of encephalopathy in rats given peppermint oil. The no effect level in these 28 day gavage studies was 20 mg/kg body wt./day for pulegone and was not established but was < 200 mg/kg body wt./day for menthol.
210
REFERENCES
1 I. Thorup, permint 2 D.
G. Wurtzen, Oil, Toxicol.
Morrison
J. Carstensen Lett.,
Smith
and
Characterization
of Essential
Chromatographic
Analysis,
3 Martindale. don,
and P. Olsen, Short Term Toxicity
Study in Rats Dosed Pep-
19 (1983) 211-215. L.
Levi,
Essential
Oils
Oils. Determination Agric.
Food
The Extra Pharmacopoeia.
Treatment
of
of Geographical
Chem.,
Compositional
Origins
Data
of Peppermint
for
the
Oils by Gas
9 (1961) 230-244.
28th Edition,
N.W.
Blacow,
The Pharmaceutical
Press, Lon-
1982, p. 682.
4 F. Grundschober, 5 J.B. Sullivan,
Literature B.H.
and hepatotoxicity, 6 P. Gunby,
Plant
review of pulegone,
Rumack,
H. Thomas,
J. Am. Med. Assoc., known
for centuries
R.G.
Perfumer Peterson
and flavorist., and P. Bryson,
4 (1979) 15-17. Pennyroyal
oil poisoning
242 (1979) 2873-2874.
still causes
problems
today,
J. Am. Med. Assoc.,
241 (1979)
2246-2247. 7 Council
of Europe,
Strasbourg, 8 JECFA,
Joint FAO/WHO
tain Food 9 W.P.
Additives
Gordon,
toxicity
Flavouring
substances
and
natural
sources
Committee
on Food Additives,
of
flavourings,
Maisonneuve,
1981, p. 53.
A.J.
Expert
Toxicological
Evaluation
of Cer-
(1976) Series No. 10, pp. 64-69. Forte,
of pennyroyal
R.J. McMurtry,
oil and its constituent
J. Gal and S.D. Nelson, terpenes
in the mouse,
Hepatotoxicity
and pulmonary
Toxicol.
Pharmacol.,
Appl.
65
(1982) 413-424. 10 J.E.
Schutte,
creatinine: 11 T. Balazs,
J.C.
Longhurst,
an accurate Development
measure
F.A.
Graffney,
of total striated
of tissue resistance
B.C.
Bastian
and
C.G.
muscle mass, J. Appl.
to toxic effects
of chemicals,
Blomquist,
Physiol.,
Totalplasma
51 (1981) 762-766.
Toxicol.,
(1974) 247-255.