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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD) which was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): p-Methylanisole
- Physical state: liquid, yellowish
- Analytical purity: > 99 %
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: K. Thomae GmbH, Biberach/Riss, Germany
- Average weight at study initiation: males ca. 184 g, females ca. 148 g
- Age at study initiation: 42 days
- Housing: singly in type DK III stainless steel wire cages (Becker & Co., Castrop-Rauxel, Germany)
- Diet: Kliba rats/mice/hamsters maintenance diet, 343 meal (Klingentalmühle AG, Kaiseraugst, Switzerland), ad libitum throughout the study
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Before the start of the administration period the animals were distributed according to weight among the individual test groups separated by sex.
Individual animals of test groups 1- 3 received the corresponding doses daily from Monday - Friday by gavage in varying concentrations, depending on the latest body weight determination. The animals of the vehicle control group received the vehicle by gavage.
The administration volume used in the study was 5 ml/kg body weight. The test substance was administered as a solution. The doses were prepared in the first two weeks daily and from the 15 day on once a week. The test substance was weighed and diluted to the appropriate volume with olive oil. The doses were dissolved using a magnetic stirring apparatus.
At the end of the administration period the animals were fasted for about 16 hours (withdrawal of food) and sacrificed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Investigations to characterize the test substance in respect of purity and homogeneity were carried out before the start of the study.
Analytical check of the stability of p-methylanisole in the vehicle over a period of 5 days was performed before the start of the study. Gaschromatography was used to determine the content of p-methylanisole in the undiluted test substance and in the applied doses.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw in olive oil DAB 9
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS:
Food consumption:
The food consumption over a period of 7 days was determined weekly and calculated as mean food consumption in grams per animal and day.

Body weight data:
The body weight was determined before the start of the administration period in order to randomize the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter in weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as the body weight change.

Clinical observations:
The general state of health of the animals was checked twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and public holidays). Furthermore the animals were checked for the appearance of clinical signs before and after each test substance administration.

Mortality:
A check was made for dead or moribund animals twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and public holidays).

CLINICAL CHEMISTRY AND HEMATOLOGY:
Blood was taken from the retroorbital venous plexus in the morning from non-fasted, not anesthetized animals. The blood samplings and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer using a random number generator. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results.
The results of the clinicochemical and hematological examinations are expressed in units of the International System (SI). The following examinations were carried out in 5 animals per test group and sex.

Hematological examinations:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (S Plus model, by Coulter, Krefeld, Germany):
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
Differential blood count was evaluated visually.

Clotting analyses:
The clotting analyses were carried out using a ball coagulometer (KC 10 model, by Amelung, Lemgo, Germany).
The following parameter was determined:
- thromboplastin time (Hepato Quick's test)

Clinicochemical examinations:
An automatic analyzer (Hitachi 737; by Boehringer, Mannheim, Germany) was used to examine the clinicochemical parameters.
The following parameters were determined:
Enzymes
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
- serum-gamma-glutamyltransferase
Blood chemistry
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol
- magnesium
Sacrifice and pathology:
The animals were sacrificed by decapitation under CO2 anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The weight of the animals as well as the weights of liver, kidneys, adrenal glands, testes, thymus and spleen from all animals sacrificed at scheduled dates was determined.
Subsequently the following organs or tissues were fixed in 4 % formaldehyde solution:
liver, heart, stomach, gross lesions, kidneys, thymus, testes, spleen, brain, mesenteric lymph nodes, adrenal glands, salivary glands (Glandula madibularis/sublingualis), all organs with gross lesions.
After the organs had been fixed, processing, the examination by light microscopy and the evaluation of findings was performed.
Statistics:
Clinical data, clinical chemistry and hematology (except diff. blood count): Kruskal-Wallis-h-test (non-parametric ANOVA); if p-values were =< 0.05, pairwise comparison of each dose group with the control group was performed using the Mann-Whitney-U-test.
Organ weights: KRUSKAL-WALLIS-h test . if p-values were =< 0.05, pairwise comparison of each dose group with the control group was performed using WILCOXON-Test.

Results and discussion

Results of examinations

Details on results:
The administration of p-Methylanisole to male and female Wistar rats for 4 weeks at doses of 100, 300 and 1000 mg/kg body weight led to following
substance-induced findings :

Group 3 (1000 mg/kg body weight group):
- all male and female rats showed clinical signs such as salivation, ataxia and tremor after gavageing, but (with one exception) not on the following day; one male rat showed laboured respiration
- increase in cholesterol in the females
- significantly increased mean absolute and relative liver weights in male and female rats
- diffuse hypertrophy of the hepatocytes and single cell necrosis of hepatocytes in all male and female rats
- significantly decreased mean absolute and relative spleen weights in male rats without a morphological counterpart by light microscopy
- significantly increased mean absolute and relative kidney weights in female rats without a morphological counterpart by light microscopy
- decreased mean relative thymus weight in males without a morphological counterpart by light microscopy
- focal hyperplasia and hyperkeratosis of the mucosa of the forestomach in one male rat

Group 2 (300 mg/kg body weight group):
- some male and female animals showed salivation after gavage of the test substance
- significantly decreased mean absolute spleen weights in male rats without a morphological counterpart by light microscopy.

Group 1 (100 mg/kg body weight group):
- no substance related changes


The clinical symptoms (such as salivation, ataxia, tremor) were observed only after gavageing of the test substance, but not on the following day, with the exception of one female which showed salivation on day 28 in the morning also. These signs are most probably a consequence of the irritating potential of the test substance and are thus only a local, but not a systemic toxic effect. This is confirmed by the fact, that in one male animal of the highest dose group hyperkeratosis and focal hyperplasia were seen in the forestomach. Also the increased food consumption of the animals might be a compensatory reaction on the irritation of the forestomach.

No abnormalities were discovered by light microscopy that might explain the altered weights of spleen, thymus or kidneys.
The other microscopic lesions reported were either single observations or they were equally distributed over the treated and control animals.

Effect levels

Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no substance related changes.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean food consumption of the animals

Mean Food Consumption (g/animal/day)

day 7

day 14

day 21

day 28

males

0 mg/kg bw

23.3

24.0

23.5

23.7

100 mg/kg bw

23.3

23.7

22.9

23.9

300 mg/kg bw

22.6

22.9

21.6

22.6

1000 mg/kg bw

22.5

24.3

25.4

26.4

females

0 mg/kg bw

16.7

15.3

15.0

16.3

100 mg/kg bw

17.1

15.7

15.6

15.9

300 mg/kg bw

15.3

14.8

14.8

15.6

1000 mg/kg bw

16.7

19.2

19.0

20.6

Table 2: Mean body weight of the animals

Mean body weight (g)

day 0

day 7

day 14

day 21

day 28

males

0 mg/kg bw

185.2

233.4

277.3

308.9

337.9

100 mg/kg bw

182.9

232.0

275.0

302.6

333.3

300 mg/kg bw

184.3

227.0

264.1

289.0

315.7

1000 mg/kg bw

185.0

229.2

274.7

304.1

333.0

females

0 mg/kg bw

147.6

164.2

180.7

189.8

202.8

100 mg/kg bw

148.5

167.1

183.6

193.4

207.3

300 mg/kg bw

147.0

160.8

173.1

188.0

200.2

1000 mg/kg bw

148.8

169.6

187.2

208.2

225.9

Applicant's summary and conclusion