Short-term toxicity study in rats dosed with menthone

Toxicology Letters, 32 (1986) 147- 152

147

Elsevier

TOXLett.

1602

SHORT-TERM

(Peppermint

TOXICITY

oil; menthone;

C. MADSEN,

G. WURTZEN

of Toxicology

Institute

STUDY IN RATS DOSED WITH MENTHONE

(Received

February

(Revision

received

(Accepted

April

rat; encephalopathy)

and J. CARSTENSEN

National Food Agency,

24th, April

19, Merkh@

Bygade, DK-2860 &borg

~~en~ark~

1986) 2&h,

1986)

3Oth, 1986)

SUMMARY Menthone,

a component

of peppermint

at dose levels of 0, 200, 400 and 800 mg/kg was reduced

to 400 mg/kg

ed a dose-dependent activity

b.w./day,

b.w. in the female group

decrease

and bilirubin.

oil, was given p-0. to groups

in creatinine

The relative

content

respectively,

receiving

of 10 male and 10 female

for 28 days.

the highest dose. Analyses

and a dose-dependent

in the white matter

level for menthone

in this study

of the cerebellum

of the two highest

was lower than 200 mg/kg

Cyst-like

rats

19 days the dose of plasma

increase in alkaline

weights of liver and spleen were increased.

histopathologically

After

show-

phosphatase

spaces were seen

dose groups.

The no-effect

b.w./day.

1NTRODUCTlON

Menthone is a component of peppermint oil. The content in peppermint oil is 1530%. Vacuolisation of hepatocytes was found in short-term toxicity studies in rats with peppermint oil and its components pulegone and menthole. Histopathological changes in the cerebellum consisting of cyst-like spaces were seen in the peppermint oil and pulegone study [1,2]. Menthone has a chemical structure similar to that of pulegone and menthole (Fig. 1). A subacute toxicity study on menthone in rats was performed to elucidate to what extent the toxicity of menthone contributes to the toxicity of peppermint oil.

MATERIALS

AND

METHODS

Materials Menthone racemic 97%, purum, Fluka; vehicle; soy bean oil, food grade. 0378-4274/86/~

03.50

0 Elsevier

Science

Publishers

B.V. (Biomedical

Division)

148

Animals 4-week-old Wistar, SPF rats, male and female, were obtained from Mollegaards Breeding Centre Ltd., Ejby (DK-4623 L. Skensved). Diet The rats were fed a pelleted diet (Chow 101, Institute of Toxicology, National Food Agency). Food and acidified water (citric acid, pH 3.5) were given ad lib. Experimental The animals were kept in stainless steel wire cages (2 per cage) at 23’C + l”C, relative humidity 60% f 5%, air change 6-8 times/h, and electric light from 21.~-09.00 h. Four groups of 10 male and 10 female rats were given 0, 200, 400 and 800 mg menthone/kg b.w./day, respectively, by gavage, diluted in soy bean oil. As the female rats in the highest dose group showed serious weakening after 19 days of dosing the dose was reduced to 400 mgikg. The average doses for the female groups were 0, 200, 400 and 671 mg/kg b.w./day. Clinical, clinicochemical and hematological examinations were performed as described by Thorup et al. [l]. In addition, reticulocytes in whole blood were examined and the concentration of bilirubin [3] and the activity of alkaline phosphatase [4] were determined in plasma of blood samples obtained on the last day of the dosing period. The rats were killed after 4 weeks of dosing by exsanguination in COz-anaesthesia. A thorough autopsy was performed and the following organs were excised and weighed: kidneys, adrenals, spleen, heart, liver and brain. Samples from these organs and from lung, aorta, mesenterial lymph node, thymus, stomach, jejunum, colon, thyroid, parathyroid, pancreas, testis, ovary, uterus, spinal cord and ischiatic nerve were fixed in 1OVobuffered formalin, prepared for light microscopy and stained with haematoxylin-eosin (all), Per1 (liver) and PAS (liver). Frozen sections were prepared from the liver and stained with Oil Red 0. Statistical methods Body weights, food and water consumption were compared by means of Student’s t-test. Analysis of variance was performed on haematoiogical parameters, clinicochemical parameters and absolute and relative organ weights. The mean values were compared by the method of Tukey [5].

RESULTS

The female rats in the highest dose group showed, after 19 days of dosing, signs of toxic effects. They had pale mucous membranes and showed signs of pain. To

149

C”,

/\ CH,

A

CH,

CH,

CH,

CH,

MENTHONE

PULEGONE

Fig.

=‘a

CH,

A

CH,

MENTHOLE

1.

prevent

deaths

in this group,

the dose was reduced

to 400 mg/kg.

4 animals

died

during the study due to accidental intratracheal dosing. Food consumption was significantly reduced in the highest dose male group during the entire dosing period. In the females food consumption was significantly reduced in all dose groups within the first 2 weeks and in the highest dose group also in the 3rd week. The reduced food consumption was accompanied by a decrease in body weight gain.

TABLE BODY

1 WEIGHT

AND

RELATIVE

800a mg MENTHONE/kg

ORGAN

b.w./DAY

mg/kg

OF RATS

DOSED

WITH

0, 200, 400 AND

bw/day

0 Number

WEIGHTS

FOR 28 DAYS

200

400

800a

of animals

female

10

9

8

10

male

10

10

9

10

Body

weight (g)

female

187.

+

18

176

*

13

168

*9

166

f

11**

male

286

+ 29

258

f

21

243

+ 25

234

f

26**

Kidneys

(mg/g)

female

7.08

f

0.44

7.58

f

0.52

7.68

f

0.48

7.16

f

0.42*

female

2.73

f

male

2.36

+

0.36

3.15

f

0.42

3.39

f

0.79

3.53

f

0.48*

0.29

2.47

+

0.27

3.13

+

0.52

3.10

f

0.52***

Spleen (mg/g)

Liver (mg/g) female

31.14

&

1.10

34.37

*

2.01

37.53

*

1.51

40.21

f

2.14***

male

31.46

+

2.18

34.28

I?- 2.61

37.19

f

1.67

42.40

+

2.42***

female

9.84

+

0.74

10.44

?

0.84

10.73

*

0.59

10.91

+

0.92*

male

6.84

+

0.46

7.14

*

0.91

7.93

?z

0.62

8.35

+

0.56***

Brain (mg/g)

Means

k SD (* P
a Dose reduced

to 400

** PcO.01;

mg/kg

*** P
in the female

group

on day 19.

150 TABLE II CLINICAL CHEMICAL PARAMETERS

IN RATS DOSED WITH MENTHONE (n = &‘sex/group)

mg/kg bwiday 0

200

Creatinine” (pmol/l)

Female Male

68.8 64.2

Alkalineb phosphatase

Female 159 Male 260

+ +

7.6 6.3

t 38 + 55

62.9 66.1 225 363

400 I rt

1.3 6.8

z!z 68 rt 178

59.4 59.6 211 440

800’ + t

5.5 6.5

+ 64” + 120e

50.5 49.2 346 451

t z!z

7.6 *** 5.5 ***

+ 149** It: 144**

(U/l) B~lirubinb Female (mgi 100ml)” Male

0.19 2 0.13 *

0.06 0.06

0.23 f 0.20 f

0.06 0.06

0.30 f 0.26 +

0.19 0.05

0.35 +0.44 +

0.14*** 0.19***

Results are mean rtr SD. (** PcO.01; *** P
The terminal body weights in both males and females were decreased. The decrease was dose-dependent and statistically significant (Table I). Blood examination showed a dose-dependent decrease of creatinine concentration and a dosedependent increase of both alkaline phosphatase activity and bilirubin content in blood plasma (Table II). There was a statistically significant dose-related increase of the relative weight of the kidneys, spleen, liver and brain of the female groups and of the spleen, liver and brain of the male ‘groups (Table I). Histological examination revealed dose-related alterations in the brain (Table II). Cyst-like spaces appeared scattered in the white matter of the cerebellum in the two highest dosed groups (Fig. 2). To confirm this the slides were reexamined with the numbers blinded. The findings resembled the changes found in rats dosed with peppermint oil [I]. TABLE III NUMBER OF ANIMALS WITH HISTOLOGICAL CHANGES IN THE CEREBELLUM AFTER ADMINISTRATION OF MENTHONE FOR 28 DAYS In parentheses: the number of animals per group. mg/kg bw/day 0

200

400

800”

0 (9) 1 (8) 7 (10) 0 (10) 3 (9) 5 (10) -a In the female group the dose was reduced to 400 m&kg after 19 days of dosing. Females Males

0 (10) 0 (10)

151

Fig. 2. Cyst-like paraffin

section.

spaces scattered Object

all over the white matter

x 25. Magnification

of the cerebellum.

Hematoxylin-eosin

-stained

x 300.

DISCUSSION

The reduction of food consumption general toxic effect of the substance.

and body weight is considered

a result of a

The dose-related increase of the relative brain weight is due to growth depression. In a study on growth retardation Feron [6] showed that non-toxic retarded growth lasting for 4 weeks results in a marked increase in relative brain weight and a slight decrease in relative kidney, liver and spleen weight. The increase of relative kidney (female), spleen and liver weight thus reflects a true increase as a function of dosing and indicates a toxic effect of the substance. An adverse effect on the liver is also seen in the marked dose-related increase of bilirubin and alkaline phosphatase. These findings were not accompanied by light microscopic changes in the liver and possibly express subcellular changes resulting in intrahepatic cholestasis [7]. The creatinine content in blood plasma reflects the total muscle mass. A decrease in blood creatinine might be related to biochemical changes in the liver. These findings resemble the results in the pulegone study [2]. The encephalopathy induced in the rats dosed with menthone at 400 or 800 mg/kg b.w./day was confined to the white matter of the cerebellum. The lesions were cystlike spaces, which caused no cellular reaction in the adjacent tissue. The spaces were not surrounded by a membrane and did not seem to occur intracellularly. The

152

pathomorphological picture indicated that the observed cyst-like spaces were present between unbroken myelin sheets. The encephalopathy produced no clinical symptoms behavioural

that could be observed tests were performed.

by a general

clinical

inspection.

No special

In the peppermint oil study [l] a dose of 40 mg/kg b.w. resulted in cyst-like spaces in the cerebellum. Comparing the effective levels in the menthone and peppermint oil studies indicates that menthone is not the substance being primarily responsible for the effect in the cerebellum seen in the peppermint oil study. The increase in liver weight and bilirubin content of plasma was statistically significant even in the lowest dose group. On the basis of these findings a no-effect level cannot be established. A likely no-effect level is less than 200 mg/kg b.w./day.

REFERENCES I. Thorup, permint

G. Wtirtzen, oil, Toxicol.

I. Thorup, pulegone

G. Wiirtzen, and menthole,

L. Jendrassik and bilirubins, Biochem. Deutschen L. Brandum

J. Carstensen Toxicol.

and P. Olsen,

Lett.,

fur Klinische

in biologischen

and J.D.

Monrad,

V. Feron,

A. de Groot,

condition

of growth

Zimmermann,

and P. Olsen, Short term toxicity

study in rats dosed with pep-

19 (1983) 211-215.

retardation, Hepatotoxicity,

photometrische

Chemie,

Statistik

Metoden

Z. Klin. Chem.

II, Den private

study

Cosmet.

Appleton

Toxicol., Century

zur

in rats dosed

Bestimmung

von Methoden

with

des

Blut-

zur Bestimmung

von

Klin. Biochem.,

ingeniorfond,

and H. Til, An evaluation

Food

term toxicity

Standardisierung

Fliissigkeiten,

M. Spanjers

Short

19 (1983) 207-210.

P. Grof, Vereinfachte Z., 297 (1938) 81-89.

Gesellschaft

Enzymaktivitaten

H.J.

J. Carstensen Lett.,

10 (1972) 181-192.

Kobenhavn,

of the criterion

‘organ

11 (1973) 85-94.

Crofts,

New York,

1978.

1982. weight’

under