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Description of key information

One study has been choosen has a key study, the OECD 422 study performed in 2009. Reduction of body weught and local effect were observed based on this study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 10 sept 2008 to 14 oct 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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: 287 to 339 g; Females: 181 to 218 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages (22 x 37 = 814 cm2, height 15 / 18 cm) 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. 21/08).
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimation period: yes 7 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 12-hour fluorescent light / 12-hour dark cycle with music during the light period.

IN-LIFE DATES: From: 22 sept 2008 to end october 2008
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
Catechol was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle 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 brown glass beakers.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories Study B96186, dose formulations were stable for at least one week.
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 about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (3.5 hrs and 7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. R. Geissbühler (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. The following acceptance criteria was applied to analytical results: sample contents should be within a range of ±20% of nominal. Formulations were considered homogeneous if the maximum deviation from mean calculated from top, middle and bottom samples was not more than 15%. The results obtained from storage stability samples should not deviate more than 10% from time-zero reference (content or mean of homogeneity samples). Analyzed samples were not discarded without written consent from the study director.
Duration of treatment / exposure:
Males: minimum 4 weeks
Females: approximately 7 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 30, 80 and 160 mg/kg bw/day
Basis:
actual ingested
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, after the results of a dose range-finding study.
The summary of the dose-range finding study is the following:

The purpose of this study (Harlan study B96186, 2009) 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 with Catechol.
Four groups of 3 males and 3 females were treated by gavage with Catechol 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: 30 mg/kg body weight/day
Group 3: 90 mg/kg body weight/day
Group 4: 180 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 (distilled water).
The following results were obtained:

Mortality and General Tolerability
At 180 mg/kg/day, one male was found dead at the beginning of the pairing period. Beforehand, tremor was observed for the whole pre-pairing period. At necropsy, no abnormal findings were noted.
At 180 mg/kg/day, tremor was noted in males and females during the pre-pairing period, in one male during the pairing period and in females until the beginning of the gestation period. At 90 mg/kg/day tremor was also noted in males and females during the pre-pairing period.

Food Consumption and Body Weights
At 180 mg/kg, food consumption was reduced in males at the onset of the treatment period.
Body weight and body weight gain were also decreased at the onset of the treatment.

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

Macroscopical Findings
No test-item related findings were noted.

CONCLUSION
Based on the results of this dose range-finding study with Catechol, dose levels of 30, 80 and 160 mg/kg/day were considered applicable for the subsequent dose toxicity study with reproduction/ developmental toxicity screening test.
Positive control:
No
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). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

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: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1-8, 8-14 and 14-16; 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 (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) 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 (one minute evaluation), 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, Albumin/Globulin ratio.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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 and pups 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.
Statistics:
• 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 not 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.
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
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Details on results:
Mortality
At 160 mg/kg/day, during the pre-pairing period one male and one female were found dead on day 3 and 14, respectively, and during the pairing period one male was found dead on day 10.
All animals were noted to have tremor after each administration. All animals were also noted to have hind legs stretched out, ventral recumbency and circle movements few days before or on the same day when death occurred. At macroscopical examination, a dark-red discoloration of lungs in the female and of thymus in one male was observed. Congestion and edema of the lung were recorded histologically, but the exact cause of death of the above-mentioned decedents could not be established from the tissues and organs examined.

General Tolerability
At 160 mg/kg/day, all males and females were noted to have tremor after each application for the entire duration of the treatment. Chromodacryorrhea, hind legs stretched out, ventral recumbency and turning circle were observed on one single day in three males during the pre-pairing period.
One female was also noted to have hind legs stretched out, ventral recumbency and turning circle on day 9 of the pre-pairing period. All these clinical signs were considered to be test item-related, although some occurred occasionally.

Functional Observational Battery
None of the parameters investigated gave any indication of a test item-related effect.

Food Consumption
At 160 mg/kg, in males and females food consumption was statistically significantly higher between days 8 - 14 of the pre-pairing period. This was considered to be likely due to the treatment with the test item as it was observed in both males and females. A general higher food consumption was noted in females for almost the whole treatment period.

Body Weights
In males and females, body weight and body weight gain were not considered to be affected by the treatment with the test item.

Clinical Laboratory Investigations
At 160 mg/kg/day, in males the level of total bilirubine was statistically significantly increased.
The decrease of the activity of creatine kinase was not considered to be adverse.
At 80 mg/kg/day, in males the statistically significantly higher activity of alanine aminotransferase was within the range of historical control data and thus not considered to be test item-related.

Reproduction and Breeding Data
Mean precoital time, conception rate, fertility and gestation indexes were not affected by the treatment with the test item.
Implantation rate and post-implantation loss were not affected by the treatment with the test item.

Organ Weights
At 160 mg/kg/day, liver weights were statistically significantly increased in males and females. This was considered to be of metabolic nature since only hepatocellular hypertrophy and no liver injury was recorded during the histopathological examination. Statistically significant increase of absolute weight of kidneys was also observed in males and females. In males, this was caused by the slight increased severity of hyaline droplets, which were considered to be an incidental increase of a spontaneous lesion. The relative kidney weights for males and females were not statistically different than for the control group.

Histopathological Examinations
At 160 mg/kg/day, diffuse hepatocellular hypertrophy was recorded at minimal severity in three males and two females. This finding correlated with significantly increased absolute weight of the liver as well as with macroscopical findings consisting of enlargement. But, there were no further indicators of liver injury, hence, this lesion was considered to be of metabolic nature and of adaptive character.
At 80 and 160 mg/kg/day, incidence and severity of squamous hyperplasia in the stomach were increased in animals of both sexes. This lesion represents a localized stomach reaction to a repeatedly gavages irritant test material.
The treatment with the test item did not reveal any effects on the completeness of stages or cell populations of the testes.
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Clinical signs; body weight; food consumption;
Critical effects observed:
not specified
Conclusions:
The NOEL (No Observed Effect Level) was considered to be 30 mg/kg/day, based on clinical observations and body weight.
Executive summary:

The purpose of this study (Harlan study B96197, 2009), was to generate information concerning the effects of Catechol on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time.

The test item was administered orally by gavage once daily at dose levels of 30, 80 and 160 mg/kg/body weight/day, for approximately 28 days for males and at maximum 52 days for females. The following observations were made:

 

Mortality

At 160 mg/kg/day, during the pre-pairing period one male and one female were found dead on day 3 and 14, respectively, and during the pairing period one male was found dead on day 10.

All animals were noted to have tremor after each administration. All animals were also noted to have hind legs stretched out, ventral recumbency and circle movements few days before or on the same day when death occurred. At macroscopical examination, a dark-red discoloration of lungs in the female and of thymus in one male was observed. Congestion and edema of the lung were recorded histologically, but the exact cause of death of the above-mentioned decedents could not be established from the tissues and organs examined.

 

General Tolerability

At 160 mg/kg/day, all males and females were noted to have tremor after each application for the entire duration of the treatment. Chromodacryorrhea, hind legs stretched out, ventral recumbency and turning circle were observed on one single day in three males during the pre-pairing period.

One female was also noted to have hind legs stretched out, ventral recumbency and turning circle on day 9 of the pre-pairing period. All these clinical signs were considered to be test item-related, although some occurred occasionally.

 

Functional Observational Battery

None of the parameters investigated gave any indication of a test item-related effect.

 

Food Consumption

At 160 mg/kg, in males and females food consumption was statistically significantly higher between days 8 - 14 of the pre-pairing period. A general higher food consumption was noted in females for almost the whole treatment period.

 

Body Weights

In males and females, body weight and body weight gain were not considered to be affected by the treatment with the test item.

 

Organ Weights

At 160 mg/kg/day, liver weights were statistically significantly increased in males and females.

This was considered to be of metabolic nature since only hepatocellular hypertrophy and no liver injury was recorded during the histopathological examination.

 

Histopathological Examinations

At 160 mg/kg/day, diffuse hepatocellular hypertrophy was recorded at minimal severity in three males and two females. This finding correlated with significantly increased absolute weight of the liver as well as with macroscopical findings consisting of enlargement. But, there were no further indicators of liver injury, hence, this lesion was considered to be of metabolic nature and of adaptive character.

At 80 and 160 mg/kg/day, incidence and severity of squamous hyperplasia in the stomach were increased in animals of both sexes. This lesion represents a localized stomach reaction to a repeatedly gavages irritant test material.

The treatment with the test item did not reveal any effects on the completeness of stages or cell populations of the testes.

 

Based on the findings noted at histopathological examination the general NOEL (No Observed Effect Level) was considered to be 30 mg/kg/day, due to the squamous hyperplasia in the stomach. This lesion represents a localized stomach reaction to a repeatedly gavage irritant test material.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Species:
rat

Additional information

Only one study with reliability 1 was selected as key study.

The purpose of this study (Harlan, 2009), was to determine dose levels of Catechol suitable for use in a subsequent combined repeated dose toxicity screening test in the rat.

The test item was administered orally by gavage once daily at dose levels of 30, 90 and 180 mg/kg/body weight/day to 3 males and 3 females. 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.

One male at dose level of 180 mg/kg/day was found dead at the beginning of the pairing period. Since no macroscopical findings were noted and the clinical sign was tremor as the other animals, it was not clear if the causes of this death were due to the treatment with Catechol.

Treatment with the test item at 90 and 180 mg/kg/day caused tremor in males and females during the pre-pairing period. At 180 mg/kg/day, this effect was prolonged in one male during the pairing period and in all females until the beginning of the gestation period. At 180 mg/kg, food consumption and body weight gain were decreased in males at the onset of the treatment.

The relevant reproduction data such as number of corpora lutea, pre- and postimplantation loss were not affected by the treatment with the test item.

The general NOEL was considered to be 30 mg/kg/day.

Based on the results of this dose range-finding study, dose levels of 30, 80 and 160 mg/kg/day were considered applicable for the subsequent dose toxicity study with reproduction/ developmental toxicity screening test.

The following results were obtained:

In parent animals, in males and females, body weight and body weight gain were not considered to be affected by the treatment with the test item.

At 160 mg/kg/day, liver weights were statistically significantly increased in males and females. This was considered to be of metabolic nature since only hepatocellular hypertrophy and no liver injury was recorded during the histopathological examination.

At 80 and 160 mg/kg/day, incidence and severity of squamous hyperplasia in the stomach were increased in animals of both sexes. This lesion represents a localized stomach reaction to a repeatedly gavages irritant test material.

Based on the findings noted at histopathological examination the general NOEL (No Observed Effect Level) was considered to be 30 mg/kg/day, due to the squamous hyperplasia in the stomach.

Other available studies, by different route of application had reliability 3, and were not taken into account for assessment.

Justification for classification or non-classification

Considering the nature of the effects observed after oral exposure in animal’s study: squamous hyperplasia in the stomach which results from a local effect of irritant material and the value of NOEL = 30 mg/kg/day.

and according to classification criteria of EC regulation 1272/2008 the catechol should not be classified for repeated STOT.