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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 March 1983 - 15-17 June 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guideline 408 and under GLP conditions. Report is well-documented.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
number of animals in high-dose group (n=12) due to recovery experiment (n=6)
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): Ethylhexyl Methoxycinnamate, 2-ethylhexyl-p-methoxy cinnamate
- Physical state: Liquid
- Stability under test conditions: 1 year

Test animals

Species:
rat
Strain:
other: Füllinsdorf Albino SPF (outbred)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of biological and medical research, Füllinsdorf, Switzerland
- Age at study initiation: about 7 weeks
- Weight at study initiation:
Males: about 162 gr.
Females: about 136 gr.
- Housing: 2 rats per stainless-steel wire-mesh cage
- Diet (e.g. ad libitum): Ad libitum, powdered complete rodent breeding diet
- Water (e.g. ad libitum): Ad libitum, tap water
- Acclimation period: minimum 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 20-25
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): powdered complete rodent breeding diet (NAFAG 850).
The feed mixture was prepared weekly by mixing the test compound with powdered diet for 10-15 min. in a mixing machine.

VEHICLE
- No vehicle used (unchanged diet)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of active ingredient found after preparation of the mixture corresponds well to the added concentration (88-110% of nominal).
The standard deviation of the feed mixtures is at less than 6.3%.
Duration of treatment / exposure:
minimum 90 days
Frequency of treatment:
continuous
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 450, 1000 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
12
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on the results of a preliminary dose-range finding study, which covered the dose-levels of 175, 350 and 700 mg/kg/day, the dosing schedule for the 13-week study was chosen.
- Rationale for animal assignment (if not random): At random
- Rationale for selecting satellite groups: None selected
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked:
Morbidity/mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly for each group and sex.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated from the average feed intake.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At weeks 7 and 12
- Dose groups that were examined: Control and high-dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At weeks 2, 6 and 14
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 6/sex/dose
- Parameters checked:
Red Blood Cells (RBC)
Haemoglobin (HB)
Mean Corpuscular Volume (MCV)
Packed Cel]. Volume (PCV)
Mean Corpuscular Haemoglobin (MCH)
Mean Corpuscular Haemoglobin Concentration (MCHC)
Reticulocytes (RETI) Method of Dacie
White Blood Cells (WBC)
Differential Cell Count:
- Polymorphonuclear Neutrophil (SEG)
- Juvenile Neutrophil (BAND)
- Lymphocyte (LYMPH)
- Monocyte (MONO)
- Eosinophil (EOS)
- Basophil (BASO)
- Immature White Blood Cell (IMMAT WBC)
- Immature Lymphocyte (IMMAT LYMPH)
- Plasma Cell (PLASM CELL)
- Nucleated Red Blood Cell (NRBC)
Thrombocytes (THROM)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At weeks -1, 6, 14 and 19
- Animals fasted: No data
- How many animals: No data
- Parameters checked:
Total bilirubin
Urea
Glucose
Cholesterol
Serum-Sodium
Serum-Potassium
Serum-Total-Proteins
Serum-Electrophoresis
Alkaline phosphatase
Aspartate aminotransferase
Alanine aminotransferase
Glutamate dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: At weeks 4 and 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during collection
- Parameters checked:
Color
Volume in ml
Specific gravity
pH
Protein content
Glucose content
Occult blood
Bilirubin, Urobilinogen
Ketone Bodies
Microscopic composition
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to full gross necropsy

ORGAN WEIGHTS:
Brain, heart, liver, kidneys, testes, ovaries, adrenal glands were weight wet (paired organs = weight of both organs).

HISTOPATHOLOGY: Yes
All gross lesions
Pituitary
Thyroids
Salivary glands
Thymus
Adrenal glands
Lungs with bronchi
Trachea
Heart
Aorta
Liver
Esophagus
Stomach
Duodenum
Je junum
Ileum
Caecum
Colon
Rectum
Pancreas
Mesenteric lymph node
Spleen
Kidneys
Urinary bladder
Testes
Epididymis
Prostate
Seminal vesicle
Ovaries
Uterus
Brain
Nervus ischiadicus
Eyes
Bone
Bone marrow
Muscle
Skin
Mammary glands
Other examinations:
Not relevant
Statistics:
Results of hematology and blood chemistry: Dunn-test
Body and organ weights: Growth rate, adjusted organweights and Jonckheere- and U-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Only observation in connection with the treatment was the occurrence of soiled tails especially in high-dose groups.
No mortalities related to the treatment with the test compound occurred during the study.

CLINICAL CHEMISTRY
None of the effects found were significantly or treatment related. For some observed effects individual values remained within the normal ranges for rats.

ORGAN WEIGHTS
There was a slight increase of the absolute and the allometrically adjusted weights of kidneys and livers of treated males, but only the weights of the kidneys at the high-dose were statistically significant different from those of the controls. After recovery no weight difference was found, which indicates most probably that the weight difference reflects the adaptation to the eliminatory task. For females, no effects were found.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopical changes related to the treatment were confined to the livers. They consisted of the reduction of the glycogen content, especially in high-dose animals, which was accompanied by the shrinkage of hepatocytes in some males and females and a slight non-significant increase of the phagocytized iron positive material in the Kupffer cells of mid and high-dose females. After the recovery period, there was no obvious difference between the livers of high-dose and control animals.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs; organ weights; histopathology
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Ethylhexyl methoxycinnamate was well tolerated at any dose-level under the conditions of this 13-week oral feeding study in rats. A NOAEL of 450 mg/kg/day was established. Based on these results, ethylhexyl methoxycinnamate does not need to be classified as toxic after repeated exposure based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI 67/548/EEC.
Executive summary:

Ethylhexyl Methoxycinnamate, an UV-B absorber used in sunscreens was orally administered to rats (12/sex/group) at the doses 0 (control), 200, 450 and 1000 mg/kg/day, 7 days per week for at least 13 weeks. At the termination of the treatment, half of the controls and of the high-dose rats were allowed 5 additionally weeks on normal diet prior to sacrifice.

Body weight, feed consumption and signs of toxicity were recorded weekly. Ophthalmoscopy and urine analysis were performed twice during the study. Blood chemical and hematological investigations were carried out at the beginning, during and at the end of the treatment period. An additional blood chemical investigation was performed after the recovery period. Post mortem investigations comprised full autopsy, organ weight determinations and histological examination.

The feed intake and the body weight development of treated animals were similar to those of controls. No symptoms indicative of pathologic conditions, ophthalmological abnormalities or mortalities as consequence of the treatment with the test compound were recorded during the study. Laboratory investigations in high-dose females (1000 mg/kg/day) revealed an increase of the plasma activity of GLDH which was reversed after the recovery period. The absolute as well as the allometrically adjusted weights of the kidneys were slightly increased in males of the high-dose group. No deviations of the weights were found after the recovery period, thus indicating an adaptive change. The glycogen content of the livers in high-dose animals was reduced and in 5 of 12 animals it was accompanied by slight shrinkage of the hepatocytes. In females of mid and high-dose group the amount of the iron positive material phagocytized by Kupffer cells was slightly increased. These conditions were reversed after the recovery period.

There was no obvious effect related to the treatment, which was detectable by the hematological, blood chemical and urine parameters at the mid- (450 mg/kg/day) and low-dose (200 mg/kg/day) levels. A slight increase of the iron positive material phagocytized by the Kupffer cells was observed in mid-dose females.

It is concluded that the treatment with Ethylhexyl Methoxycinnamate was well tolerated at any dose level and that under the conditions of this study only minor and reversible changes occur at the doselevel of 1000 mg/kg/day, whereas the dose of 450 mg/kg/day does not induce any adverse effect in the rat. Based on these results, ethylhexyl methoxycinnamate does not need to be classified as toxic after repeated exposure based on the criteria outlined in regulation 1272/2008/EC and 67/548/EEC.