Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 14 may 2008 to 14 oct 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
but not relevant for the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyrocatechol
EC Number:
204-427-5
EC Name:
Pyrocatechol
Cas Number:
120-80-9
Molecular formula:
C6H6O2
IUPAC Name:
pyrocatechol

Test animals

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. In addition body weights (recorded on the day of allocation) will be 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. 31/08).
- 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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, and were stable at least one week.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of post coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical part was conducted at Harlan Laboratories Ltd., Itingen, Switzerland under GLP compliant conditions to verify the identity of the test item Catechol administered and to determine the content, homogeneity and stability of application formulations.
Several application formulations were prepared and representative analytical samples were collected and dispatched to the analytical laboratories internally. The test item concentrations were determined by HPLC coupled to a UV/VIS detector and quantified with the area under the peak.
The identity of Catechol was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogeneous distribution of Catechol in distilled water was demonstrated. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
In conclusion, the results obtained within this analytical part confirm the correct preparation and storage of application formulations during the conduct of this study.
Duration of treatment / exposure:
Males: minimum 4 weeks
Females: approximately 7 weeks
Frequency of treatment:
Daily
Details on study schedule:
Mating, Gestation and Lactation:
During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
a) The daily vaginal smear was sperm positive, or
b) A copulation plug was observed.
The day of mating was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, this female was paired with a male of the same group which had already mated successfully. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 80 and 160 mg /kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 males and 10 females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (Harlan Laboratories Study B96186, non-GLP).
Positive control:
No

Examinations

Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- The sex ratio of the pups was recorded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter size, live births, still births and any gross anomalies.

GROSS EXAMINATION OF DEAD PUPS: yes

Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
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).

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: Ovaries

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 inlation 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.
Postmortem examinations (offspring):
SACRIFICE
Pups were sacrificed on day 4 post partum.

GROSS NECROPSY
Pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

HISTOPATHOLOGY / ORGAN WEIGTHS: no observation
Statistics:
• 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: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Macroscopical findings:
In males, no test item-related findings were noted.
In group 4, one male was noted to have an enlargement of liver and of mandibular lymph node and dark-red foci on the stomach. Another male was noted to have red foci on the left side of thymus.
In group 3, reddish foci on the thymus were noted in two males.
In group 2, pelvic dilatation was noted in three males, enlarged mandibular lymph node was noted in one male, dark-red foci were observed on the lungs of one male and on thymus of another male.
In group 1, one male was noted to have isolated dark red foci on the right cranial lung lobe and dark red nodules on the right perirenal adipose tissue. Dark red foci were noted on the thymus of another male.
In absence of any dose dependency, these findings were not considered to be test item-related.
In females, no test item-related findings were noted. The watery cyst on the left ovary in one female in group 2 was considered of incidental nature.

Histopathology findings:
Microscopically, the following findings were recorded as possible treatment-related:
Liver: Diffuse hepatocellular hypertrophy was recorded at minimal severity in three males and two females of group 4.
Stomach: An increased incidence and severity of squamous hyperplasia was recorded in the forestomach of animals of both sexes in groups 3 and 4.
Other Findings: In the kidneys, the severity of hyaline droplets was slightly increased in males of group 4. All these droplets were negative for PAS stain and were considered to be an alpha-2 microglobulin, which is a native protein in male rat. Therefore, they were considered to be an incidental increase of a spontaneous lesion.
In the spleen, there was a slightly increase in severity of extramedullary hemopoiesis in animals of both sexes in groups 2 - 4. Since a dose-dependency of this finding was not clear, it was considered to be most likely incidental.
The remainders of findings recorded were within the range of normal background lesions, which may be recorded in animals of this strain and age.
Sperm Staging: The treatment with the test item did not reveal effects on the completeness of stages or cell populations. There was no indication for maturation arrest, reabsorption of sperm or any other degenerative type. All findings recorded are within the range of normal background alterations.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
histopathological examination
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: due to the squamous hyperplasia in the stomach.
Dose descriptor:
NOEL
Remarks:
reproduction/developmental
Effect level:
160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No Observed effect.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Findings at First Litter Check and during Lactation
The mean number of pups at first litter check and on day 4 post partum was not affected by the treatment with the test item. The sex ratio was also not affected.

Pup Weights to Day 4 Post Partum
Pup weight development was not affected by the treatment with the test item.

Macroscopical Findings
At necropsy of pups, no test item-related findings were noted.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Reproduction and breeding data: Summary of performance

 

Groups

(mg/kg/day)

1

(0)

2

(30)

3

(80)

4

(160)

Female numbers

41-50

51-60

61-70

71-80

Number of females, which died before the

scheduled necropsy (A)

0

0

0

1

Number of females paired

10

10

10

9

Number of females mated (B)

8

10

9

9

Number of pregnant females

8

10

10

9

Number of females which did not

deliver any pups (C)

/

/

1

/

Number of females, which lost their litters (D)

/

/

/

1

Number of females which reared their pups until day 4 post partum

8

10

9

8

 

(A) = Female No. 71 died spontaneously on day 14 of the pre-pairing period

(B) = Female Nos. 45 and48 ingroup 1 did not mate and were not pregnant (no corpora lutea and no implantation sites were noted)

(C) = Female no.61 ingroup 3 had no mating noted but was pregnant (implantation sites noted at necropsy)

(D) = Female No. 77 lost its litter (two pups) on day 2 post partum

Applicant's summary and conclusion

Conclusions:
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.
The NOEL for reproduction/developmental toxicity was considered to be 160 mg/kg/day, the highest dose tested without observed effect.
Executive summary:

The purpose of this study (OECD 422, Harlan 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. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Catechol 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.

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: 80 mg/kg body weight/day

Group 4: 160 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:

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.

In pups, the mean number of pups at first litter check and on day 4 post partum was not affected by the treatment with the test item. The sex ratio was also not affected, and weight development was not affected by the treatment with the test item. At necropsy of pups, no test item-related findings were noted.

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.

The NOEL for reproduction/developmental toxicity was considered to be 160 mg/kg/day, the highest dose tested without observed effect.

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