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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
Study period:
from 10 september 2008 to 15 september 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study without any deviations
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd.; Laboratory Animal Services; Wölferstrasse 4; 4414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 288 to 334 g; Females: 185 to 209 g
- Fasting period before study: no
- Housing: individually (except during paring period) in Makrolon type-3 cages with wire mesh tops and sterilized standard
softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Randomization: yes, Computer-generated random algorithm. In addition body weights (recorded on the day of allocation)
were taken into consideration in order to ensure similar mean body weights in all groups.
- Diet: Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
was available ad libitum (batch no. 77/07).
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimation period: yes 6 days, under test conditions after health examination. Only animals without any visible signs
of illness will be used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): There was a 12-hour fluorescent light / 12-hour dark cycle with music during the light period.

IN-LIFE DATES: From: september 10th, 2008 to january 31th 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The dose formulations were prepared daily using the test item as supplied by the Sponsor.
PMP was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle (highly purified water) was added (w/v).
Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining
vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of each concentration
were taken prior to dosing for analysis of concentration and homogeneity. Samples of each concentration were taken from the middle
only to confirm stability (4 hrs and 7 days).
The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure developed at Harlan Laboratories.
The formulations were found to be in the acceptable range of nominal concentration, homogeneous and stable when kept 4 hours and 7 days at
room temperature.
Duration of treatment / exposure:
Paramethoxyphenol was administered to male rats for at least 28 days and to
female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1
generation reached day 4 post partum.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150 and 300 mg/kg bw/day
Basis:
other: nominal concentrations
No. of animals per sex per dose:
10 males and 10 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection: The dose levels were selected by the Sponsor, based on the results of the range finding study B96131
the summary of this range finding study is as follows:

The purpose of this study was to select suitable dosages to be used in the subsequent combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test in the rat.
Four groups of 3 males and 3 females were treated by gavage with Paramethoxyphenol once daily. Males were treated over a 14-day
pre-pairing period and during the pairing period and up to one day before necropsy (for a total of at least 28 days of treatment).
Females were treated throughout the pre-pairing, pairing and gestation period up to day 13 post coitum.

The following dose levels were applied:
Group 1: 0 mg/kg body weight/day (control group)
Group 2: 100 mg/kg body weight/day
Group 3: 300 mg/kg body weight/day
Group 4: 1000 mg/kg body weight/day
Group 5: 500 mg/kg body weight/day

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body
weight was used. Control animals were dosed with the vehicle alone (highly purified water).

The following results were obtained:

Mortality and General Tolerability
At 1000 mg/kg bw/day, all males and females were noted to have a strong reaction to the
treatment with the test item after three days of treatment. All animals were noted to have ventral
recumbency, rhythmical muscle twitching, ruffled fur and were limp. Therefore they were
sacrificed for ethical reasons and it was decided to add an additional group at 500 mg/kg bw/day.
At 500 mg/kg/ bw/day, during the whole treatment period all males and females had reduced
activity. Rhythmical muscle twitching, ventral recumbency and animals limp were observed
immediately after application until early afternoon in males and in females.
At 300 mg/kg bw/day, ruffled fur and reduced activity were noted in males and females until
day 8 of the pre-pairing period.
At 100 mg/kg bw/day, no effects were observed.

Food Consumption and Body Weights
At 500 mg/kg bw/day, mean food consumption and mean body weight, mean body weight
gain were reduced in males and females during the pre-pairing period. In males, mean body
weight remained reduced until the end of the study.
At 300 mg/kg bw/day, food consumption was lower in females during the first week of the prepairing
period and mean body weight gain was reduced in males during the pre-pairing period .
At 100 mg/kg bw/day, no change in mean food consumption and mean body weight were observed.

Reproduction Data
There was no test-item effect on precoital time, fertility, number of implantation and pre- and
post-implantation losses.

Macroscopical Findings
At 1000 mg/kg bw/day, a reduction of the spleen size was noted in all males and females.

Conclusion
Based on the results of this dose range-finding study, dose levels of 50, 150 and 300 mg/kg
bw/day were considered applicable for the subsequent dose toxicity study with reproduction/
developmental toxicity screening test.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside
the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions,
and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling
as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION (g/animal/day):
Males: Pre-pairing period days 1-4, 4-8, 8-11 and 11-14 and weekly after pairing periods
Females: Pre-pairing period days 1-4, 4-8, 8-11 and 11-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.
No food consumption was recorded during the pairing period.

OTHER:
Viability / Mortality: twice daily

FUNCTIONAL OBSERVATION BATTERY (FOB):
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were
performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily
dose administration. Animals were observed for the following:
a) Cage-side observations: unusual body movements, abnormal behavior and posture as well
as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing, responsiveness to sharp noise, paw pinch, gait
evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability
(Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151
(FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and
the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATION
Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males (randomly selected) from each group.
Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all
animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water
ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HEMATOLOGY
The following hematology parameters were determined:
Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean
corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, total Leukocyte count,
Differential leukocyte count: Platelet count
Coagulation: Prothrombin time (= Thromoplastin time), Activated partial Thromoplastin time.

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides,
Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Creatine kinase, Sodium,
Potassium, Chloride, Calcium, Phosphorus, Protein (total), Albumin, Globulin and Albumin/Globulin ratio.
Sacrifice and pathology:
SACRIFICE
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects.
Dams were sacrificed on day 5 post partum.
Dams which birth did not occur on the expected date (day 21 post coitum) were sacrificed on day 25 post coitum.

GROSS NECROPSY
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens
of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®). All P generation animals were exsanguinated.
Dead pups, except those excessively cannibalized, were examined macroscopically.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death
occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a
solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

HISTOPATHOLOGY:
The following tissues from all parental males were preserved in Bouin’s fixative or in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative).

Ovaries were preserved in neutral phosphate buffered 4% formaldehyde solution.

In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the
following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions, Brain, Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus,
Thyroids, and parathyroids if possible, Trachea and lungs (preserved by inhalation with fixative and then immersion), Uterus (with vagina),
Urinary bladder, Lymph nodes (mesenterial, mandibular), Peripheral nerve (sciatic) and Bone marrow.

ORGAN WEIGHTS:
The testes and epididymides of all parental males were weighed as pairs.

In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from
any adherent tissue, as appropriate, and their wet weight taken:
Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Liver, Thymus, Spleen.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and
reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance
estimate was applied if the variables could be assumed to follow a normal distribution
for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopical findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
- CLINICAL SIGNS AND MORTALITY

All the animals survived until the scheduled necropsy.
Males: At 300 mg/kg bw/day all males were noted to have decreased activity for the entire
treatment period. Salivation was observed during the second half of the pre-pairing period and
occasionally in two males during the pairing period.
Females:
At 300 mg/kg bw/day all females were noted to have reduced activity. Salivation was
also noted starting on day 10 of the gestation period onwards. In three females, ruffled fur and difficulty in delivery at the
end of the gestation period were noted.

At 150 mg/kg bw/d, in two females ruffled fur and difficulty in delivery at the end of the gestation period were also noted.
Since these effects occured in two females and no other clinical signs were observed, these symptoms were not considered
to be adverse.

- BODY WEIGHT
Males: At 300 mg/kg bw/day mean body weight and mean body weight gain were statistically significantly reduced
during the pre-pairing period.
At 50 and 150 mg/kg bw/d, mean body weight and mean body weight gain were not affected by the substance.

Females: At 300 mg/kg bw/day mean body weight and mean body weight gain were statistically significantly reduced
during the whole pre-pairing period.
At 150 mg/kg bw/d, the body weight gain was occasionally statistically significantly reduced during the pre pairing period and
no effects were observed for body weight.
At 50 mg/kg bw/d, no effects were observed.

- FOOD CONSUMPTION
Males: At 300 mg/kg bw/day mean food consumption was reduced during the whole pre-pairing
period. At 150 mg/kg bw/day, mean food consumption was transiently lower between days 4 and
8 but it was not considered to be adverse since it affected marginally the body weight.
Females: At 300 mg/kg bw/day mean food consumption was reduced during the pre-pairing
period. At 150 mg/kg bw/day, mean food consumption was lower during the first week of the
pre-pairing period, since mean body weight gain was marginally affected and it recovered
afterwards, this was not considered to be adverse.
At 50 mg/kg bw/d, no effects were observed for both females and males.

- Organ Weights
Relative kidney weights were statistically significantly increased in groups 2, 3 and 4 and
relative liver weight was statistically significantly higher in males in group 4. In absence of any
histopathological findings and taking into consideration the historical control data, this increase
was not considered to be test item-related.
No test item-related effects were noted in organ weights of females.

- Macroscopical Findings
The findings noted during the macroscopical examination did not give any indication of a test
item-related effect.
Males
In groups 3 and 4, dark-red foci were noted on the thymus in two males each and on the stomach
of one male in group 2. A dilation of the right brain ventricle was observed in one male in group 1.
Females
In group 4, one female was noted to have the right uterine horn dilated. In group 3, moderate
alopecia on the cervical region or on the body was observed two females.

- Histopathology Findings
No microscopic findings, considered to distinguish treated rats from controls, were noted.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs (salivation and reduced activity), decrease of food consumption and body weight
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs (salivation and reduced activity), decrease of food consumption and body weight

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the clinical signs and reduced food consumption observed at 300 mg/kg bw/day, a general NOAEL was established at 150 mg/kg bw/day.
Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item

Paramethoxyphenol RNCAS 150-76-5 to rats over approximately 28 days. Paramethoxyphenol was administered in highly purified water as vehicle at dosages of 50, 150, and 300 mg/kg body weight/day, and controls received the vehicle only. Paramethoxyphenol was administered to male rats

for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached

day 4 post partum.

Administrations at 300 mg/kg bw/day caused a reduction of the activity and signs of discomfort in all animals for the entire treatment period.

Ruffled fur and difficulty in delivery occurred in three females. Food consumption, body weight and body weight gain were reduced for the whole pre-pairing period in males and females. All these effects were attributed to the treatment.

 

At 150 mg/kg bw/day, the transient lower food consumption noted in males and females was not considered to be adverse since it affected

marginally the body weight or body weight gain and recovered afterwards. At this dose level, two females were noted to have ruffled fur and

difficulty in delivery. Since these effects occured in two females and no other clinical signs were observed, these symptoms were not

considered to be adverse.

 

The parameters investigated during the functional observational battery did not indicate any test item-related effect.

 

The assessment of hematology and clinical biochemistry data and histopathology examination did not reveal any test item-related effects.

The organ weights were also not affected by the treatment with the test item.

 

Based on the clinical signs and reduced food consumption and body weight/body weight gain observed at 300 mg/kg bw/day,

a general NOAEL was established at 150 mg/kg bw/day.