Registration Dossier
Registration Dossier
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EC number: 202-653-9 | CAS number: 98-29-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
An extended one-generation study in rats (OECD test guideline n°443 - basic design) is available with the registered substance 4-tert-butylpyrocatechol (TBC) (Charles River, 2021, reliability 1). TBC has been administered to rats by dietary. The dose levels of TBC used in this study are 0, 300, 1000 and 3125 ppm. No reproductive, or developmental toxicity was observed in rats based on this study up to the highest tested dose (3125 ppm). TBC had no effect on the integrity and performance of the adult male and female rat reproductive systems (no effect on gonadal function, estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation).
Therefore, it can be concluded that 4-tert-butylpyrocatechol does not induce effect on reproduction/fertility up to the dose level of 3125 ppm.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- Description only of the Palatability study (part of the reproductive screening test - DRF for the extended one generation study).
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 29 October 2019 - 20 November 2020.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: No guideline, but conducted following good laboratory practices.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- The 14-Day Palatability study is included in the Reproduction/Developmental Toxicity Screening test by dietary performed as a DRF for the extended one-generation study.
The objective of the palatability study was to determine whether the feeding of 4-tert-butylpyrocatechol up to 12500 ppm (corresponding to approximately 1000 mg/kg/day) for 14 days via diet was tolerated by Wistar Han rats and to select dose levels for the Dose Range finding (the Reproduction/Developmental toxicity screening test). No guidelines were applicable as this study was intended for dose level selection only.
The aim of this study is to investigate the palatability of diet prepared, the extend of local effects due to the corrosivity of the test item and to select the suitable high dose for the reprotoxic screening test. In this study, the test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 14 days. 6250, 9350 and 12500 ppm were selected as dose levels. A control group was added in order to compared all groups (with acetone), since acetone was used during preparation of the diets. Three females per group were dosed. Only mortality, clinical signs, body weight and food consumption were evaluated in this study. All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and collected. The stomach was collected and all gross lesions were recorded. Gross lesions were not retained and histopathological examination was not performed. - GLP compliance:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- 4-tert-butylpyrocatechol (CAS n°: 98-29-3)
- Source: manufacturer
- Purity: higher than 98%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: Homogeneity and stability of the diet under test conditions was demonstrated in the analytical method development and validation study (Test Facility Reference No. 20224318). Stability in diet: Stability for at least 8 days in the freezer (closed containers) followed by 1 day at room temperature under normal laboratory light conditions (open containers) is confirmed over the concentration range 100 to 15000 ppm, Project 20224318. Stability for at least 3 weeks in the freezer (≤- 15°C) under normal laboratory light conditions is confirmed over the concentration range 100 to 15000 ppm, Project 20224318.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING: PREPARATION OF DIET CONTAINING TEST ITEM:
The test item was dissolved in acetone and pre-mixed with a small amount (~1-2 kg) of powder feed for one hour (± 5 minutes). An equal amount of acetone was used for all groups (Groups 1-4). The amount of acetone was based on the amount of test item used in the highest dose group (Group 4, 1:1 ratio w/w). Subsequently, this pre-mix was mixed for approximately 10 minutes with the remaining required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared at least biweekly for use at room temperature for a maximum of 1 day. If not used on the day of preparation, prepared diets were kept in the freezer (≤-15ºC) for a maximum of 3 weeks prior to use (stability for 3 weeks in the freezer (≤- 15°C) was confirmed under Test Facility Reference No. 20224318). Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 1 day during the respective food consumption measurement interval. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl:WI(Han)
- Details on species / strain selection:
- The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River
Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants. - Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females: nulliparous and non-pregnant females.
- Age at study initiation: Females were 8-9 weeks old (Group 1) and 16 to 18 weeks old (Group 2 to 4).
- Weight at study initiation: Females weighed between 167 and 244 g at initiation of administration.
- Housing: On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
- Diet (e.g. ad libitum): Prepared diets were provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: No data.
- A health inspection was performed before the initiation of administration.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. For animal welfare reasons, animals were provided with wooden sticks (Swedish aspend wood, Bioservices, Uden, The Netherlands). Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The actual daily mean temperature during the study period was 22°C.
- Humidity (%): Actual daily mean relative humidity of 50 to 53%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle was maintained. - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
The test item was dissolved in acetone and pre-mixed with a small amount (~1-2 kg) of powder feed for one hour (± 5 minutes). An equal amount of acetone was used for all groups (Groups 1-4). The amount of acetone was based on the amount of test item used in the highest dose group (Group 4, 1:1 ratio w/w). Subsequently, this pre-mix was mixed for approximately 10 minutes with the remaining required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared at least biweekly for use at room temperature for a maximum of 1 day. If not used on the day of preparation, prepared diets were kept in the freezer (≤-15ºC) for a maximum of 3 weeks prior to use ((stability for 3 weeks in the freezer (≤- 15°C) was confirmed under Test Facility Reference No. 20224318). Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 1 day during the respective food consumption measurement interval. - Details on mating procedure:
- Not applicable.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 14 days of treatment.
- Frequency of treatment:
- Daily.
- Dose / conc.:
- 6 250 ppm (nominal)
- Dose / conc.:
- 9 350 ppm (nominal)
- Dose / conc.:
- 12 500 ppm (nominal)
- No. of animals per sex per dose:
- 3 females per group.
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- - Mortality: Twice daily throughout the study.
- Clinical observations: Once daily up to the day prior to necropsy.
- Body weights: On Day 1 prior to first administration and on Days 5 and 10 and 14.
- Food consumption: Daily. - Postmortem examinations (parental animals):
- All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and collected. The stomach was collected and all gross lesions were recorded. Gross lesions were not retained and histopathological examination was not performed.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Clinical appearance: no findings in the control group and at the low dose level (6250 ppm). At 9350 ppm, piloerection was observed at Day 5 to Day 8. At 12500 ppm, piloerection was observed at Day 5 to Day 9.
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality was observed at any dose level.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Control group: Normal. The body weight of group 1 animals is lower due to the younger age of these animals (the additional control group added).
- 6250 ppm TBC (group 2): Slightly reduced body weight gain was noted between Day 1-5 of treatment. Between Days 5-10 and 10-14 of treatment normal body
weight (gain) was noted for all animals.
- 9350 ppm TBC(group 3): Slightly reduced body weight gain was noted between Day 1-5 of treatment. Between Days 5-10 and 10-14 of treatment, slight body
weight gain was noted for 1/3 animals and no or a reduction of body weight gain was observed for 2/3 animals.
- 12500 ppm TBC (group 4): Overall, slightly reduced or no body weight gain was noted between Days 1-5 and 5-10 of treatment. Between Day 10-14 of
treatment, slight body weight gain was noted in 2/3 animals and a reduction of body weight gain in 1/3 animals. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- - Control group: Normal.
- 6250 ppm TBC (group 2): After the first day of administration a decreased food consumption was noted, which recovered from Day 2-3 onwards. Fluctuations in food consumption were noted throughout the study period.
- 9350 ppm TBC (group 3): After the first day(s) of administration a decreased food consumption was noted, which recovered from Day 3-4 onwards. Food
consumption was lower compared to group 4. Fluctuations in food consumption were noted throughout the study period.
- 12500 ppm TBC (group 4): After the first day(s) of administration a decreased food consumption was noted, which recovered from Day 4-5 onwards.
Fluctuations in food consumption were noted throughout the study period. - Food efficiency:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Conclusions:
- Based on the results of this palatability study, selected dose levels for the reproductive screening test were 3125, 6250 and 12500 ppm.
- Executive summary:
A palatability study was conducted to examine whether the feeding of 4-tert-butylpyrocatechol up to 12500 ppm (corresponding to approximately 1000 mg/kg/day) was tolerated by Wistar Han rats and to select dose levels for
the Reproductive screening test. No guidelines were applicable as this study was intended for dose level selection only. The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 14 days. 6250, 9350 and 12500 ppm were selected as dose levels. A control group was added in order to compare all groups, since acetone was used during preparation of the diets. Four Groups of 3 female rats per group were performed.
Based on the results of this palatability study, selected dose levels for the Reproduction/developmental toxicity screening test were 3125, 6250 and 12500 ppm.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 29 October 2019 - 20 November 2020.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Similar to OECD 421 guideline but without GLP compliance (DRF).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- 4-tert-butylpyrocatechol (CAS N°: 98-29-3)
- Source: manufacturer .
- Purity: higher than 98%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light in tightly closed container.
- Stability and homogeneity of the test material in diet under test conditions (e.g. in the exposure medium) and during storage: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20224318) demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability for at least 8 days in the freezer (closed containers) followed by 1 day at room temperature under normal laboratory light conditions (open containers) is confirmed over the concentration range 100 to 15000 ppm, Project 20224318. Stability for at least 3 weeks in the freezer (≤- 15°C) under normal laboratory light conditions is confirmed over the concentration range 100 to 15000 ppm, Project 20224318. Homogeneity: Duplicate sets of samples (approximately 5 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. After finalization of the study report, backup samples were discarded. The diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
TREATMENT OF TEST MATERIAL PRIOR TO TESTING : Preparation of Diet Containing Test Item:
The test item was dissolved in acetone and pre-mixed with a small amount (~1-2 kg) of powder feed for one hour (± 5 minutes). An equal amount of acetone was used for all groups (Groups 1-4). The amount of acetone was based on the amount of test item used in the highest dose group (Group 4, 1:1 ratio w/w). Subsequently, this pre-mix was mixed for approximately 10 minutes with the remaining required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared at least biweekly for use at room temperature for a maximum of 1 day. If not used on the day of preparation, prepared diets were kept in the freezer (≤-15ºC) for a maximum of 3 weeks prior to use (stability for 3 weeks in the freezer (≤- 15°C) was confirmed under Test Facility Reference No. 20224318). Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 1 day during the respective food consumption measurement interval. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl: WI(Han).
- Details on species / strain selection:
- The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River
Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to
be susceptible to the effects of reproductive toxicants. - Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: Yes.
- Age at study initiation: At initiation of administration, males were 10-11weeks old and females were 13-15 weeks old.
- Weight at study initiation: At initiation of administration, males weighed between 296 and 341 g and females weighed between 209 and 250 g.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, 600 x 330 x 180 mm (length x width x height). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, 380 x 220 x 180 mm (length, width, height). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, 600 x 330 x 180 mm (length x width x height) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, 380 x 220 x 180 mm (length x width x height). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, 380 x 220 x 180 mm (length x width x height). Pups were housed with the dam. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 11 days prior to start of the pretest period (females) or 9 days before the commencement of administration (males).
- A health inspection was performed before the initiation of administration.
- Animal identification: Prior to start of the pretest period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pretest period, reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by earmark and tattoo. Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
- Selection, assignment, replacement and diposition of animals: A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of administration by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
- Animals were randomly assigned to groups at arrival. Males and females were randomized separately.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. For animal welfare reasons, animals were
provided with wooden sticks (Swedish aspend wood, Bioservices, Uden, The Netherlands). Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 18 to 24°C were maintained. The actual daily mean temperature during the study period was 21 to 22°C.
- Humidity (%): Target humidity of 40 to 70% were maintained. The actual daily mean relative humidity of 48 to 54%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained. - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Remarks:
- In diet with acetone.
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The oral route of administration via dietary inclusion was selected because this is a common route of administration, which is recommended by the Regulatory Authorities for this type of study and as administration via oral gavage resulted in high toxicity due to corrosivity of the test item.
DIET PREPARATION:
The test item was dissolved in acetone and pre-mixed with a small amount (~1-2 kg) of powder feed for one hour (± 5 minutes). An equal amount of acetone was used for all groups (Groups 1-4). The amount of acetone was based on the amount of test item used in the highest dose group (Group 4, 1:1 ratio w/w). Subsequently, this pre-mix was mixed for approximately 10 minutes with the remaining required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared at least biweekly for use at room temperature for a maximum of 1 day. If not used on the day of preparation, prepared diets were kept in the freezer (≤-15ºC) for a maximum of 3 weeks prior to use (stability for 3 weeks in the freezer (≤- 15°C) was confirmed under Test Facility Reference No. 20224318, for exceptions, see Appendix 8). Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 1 day during the respective food consumption measurement interval.
VEHICLE
- Justification for use and choice of vehicle (if other than water): acetone (recommended in the OECD guideline).
ADMINISTRATION OF TEST MATERIAL:
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values. The same diets remained in the food hopper for a maximum of one day. On the day of weighing the remaining diet in the food hopper was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper. - Details on mating procedure:
- After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Concentration analysis: Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or
equal to ± 20% for diet of target concentration. After finalization of the study report, backup samples were discarded. Analyses were performed using a
validated analytical procedure (Test Facility Reference No. 20224318). Diet preparation samples were collected for analysis at Day 1 and day 22. The first day
test diets were available to the animals was designated as Day 1.
The concentrations analyzed in the diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). - Duration of treatment / exposure:
- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of two weeks prior to mating and during the
mating period. Females were exposed for 51 to 56 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to
conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day of scheduled necropsy. Females which failed to deliver
were treated for 41 to 44 days. The first day test diets were available to the animals was designated as Day 1. - Frequency of treatment:
- Daily.
- Dose / conc.:
- 3 125 ppm (nominal)
- Dose / conc.:
- 6 250 ppm (nominal)
- Dose / conc.:
- 12 500 ppm (nominal)
- No. of animals per sex per dose:
- 10 animals per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
The doses were selected based on the results of the 14-day palatability study with dietary administration of the test item to rats.
- Parental animals: Observations and examinations:
- MORTALITY/MORIBUNDITY CHECKS: Yes.
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
CLINICAL OBSERVATIONS: Yes.
- Time schedule: Clinical observations were performed once daily, beginning prior to the first administration of the test item and lasting throughout the administration periods up to the day prior to necropsy.
- The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
BODY WEIGHT: Yes.
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Animal Nos. 42, 43 and 72 were inadvertently weighed extra on Post Coitum Day 24 (Animal No. 72) and PND 12 (Animal Nos. 42 and 43). A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was quantitatively measured daily, except for males and females which were housed together for mating and for females without evidence of mating.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- Relative Food Consumption was calculated against the body weight for scheduled intervals and test Item Intake was alculated as concentration of test item in diet (ppm) against relative food consumption.
WATER CONSUMPTION: Yes.
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
- Oestrous cyclicity (parental animals):
- Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females
beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. - Litter observations:
- Mortality/Moribundity Checks – F1-Generation:
Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical Observations – F1-Generation:
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
Body Weights – F1-Generation:
Live pups were weighed individually on PND 1, 4, 7 and 13.
Sex – F1-Generation:
Sex was externally determined for all pups on PND 1 and 4.
Culling – F1-Generation:
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable. - Postmortem examinations (parental animals):
- SACRIFICE:
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver with evidence of mating (Nos. 41, 68 and 72): Post-coitum Day 25.
All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0 females were not fasted.
GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other
suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the
number of corpora lutea was recorded in addition.
HISTOPATHOLOGY / ORGAN WEIGHTS
- The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated:
Epididymis / gland, coagulation / gland, parathyroid / gland, prostate / gland, seminal vesicle / gland, thyroid / liver / ovaries / testes.
- Tissue collection and preservation: Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated:
Cervix / epididymides / gland, coagulation / gland mammary / gland, parathyroid / gland, prostate / gland, seminal vesicle / gland, thyroid / gross lesions - masses / liver / ovaries / stomach / testes / uterus.
- Histology: The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin: All animals: Gross lesions-masses / All animals of Groups 1-4: Stomach.
- Histopathology: All tissues as defined under Histology were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist. - Postmortem examinations (offspring):
- SACRIFICE AND EXAMINATION:
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection
were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
Except for a few missing pups, one pup died during the course of the study. This pup was examined externally and sexed (both externally and internally).
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded.
Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup) and was preserved in 10% buffered formalin. - Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1%
or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics
number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3
observations.
The following pairwise comparisons were made: Group 2 vs. Group 1.
Group 3 vs. Group 1.
Group 4 vs. Group 1.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted
using Fisher’s exact test whenever the overall test is significant. - Reproductive indices:
- For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual
values of F0-females, the remaining group values were calculated from the total number in each group.
- Mating index (%): Number of females mated / Number of females paired x 100
- Number of days between initiation of cohabitation and confirmation of mating
- Fertility index (%): Number of pregnant females / Number of females mated x 100
- Gestation index (%): Number of females with living pups on Day 1 / Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.
- Post-implantation survival index (%): Total number of offspring born / Total number of uterine implantation sites x 100 - Offspring viability indices:
- For each group, the following calculations were performed:
- Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born x 100
- Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100
- Viability index (%): Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering x 100
- Lactation index (%): Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling) X 100 - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- In females at 12500 ppm, treatment related clinical signs were observed and consisted of hunched posture and piloerection. Hunched posture was noted during Weeks 2 and 3 of treatment in several females and piloerection was noted on multiple days during the entire treatment period. In combination with the effects on
body weights, these clinical observations at 12500 ppm were considered to be adverse and related to the substance.
No clinical signs were noted for all males and females at 3125 and 6250 ppm during the observation period.
See tables of results in the corresponding attached document in the field below 'Attached background material'. - Mortality:
- no mortality observed
- Description (incidence):
- No mortality occurred during the study period.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In males treated at 6250 and 12500 ppm, body weight and body weight gain were dose-dependently decreased. When compared to the concurrent control
group, statistically significant reduced body weight gains were observed at 6250 ppm and at 12500 ppm from Day 8 onwards, most markedly at 12500 ppm
during the first week of treatment where males showed on average a body weight loss of 8% of their initial body weight. The reduced weight gain resulted instatistically significantly lower mean body weights during the entire treatment period at 6250 and 12500 ppm in males (up to 11% and 18%, respectively,
when compared to controls). At 3125 ppm, body weight gain of males was slightly lower during the mating period compared to the concurrent control group,
reaching statistical significance on Day 15.
In females, a dose-dependently reduced body weight gain was observed at 6250 and 12500 ppm when compared to concurrent controls throughout the
treatment period. During the first week of administration, body weight loss of 9% of their initial body weight was observed at 12500 ppm. The reduced body weight gain resulted in statistically significantly lower mean body weights during the entire treatment period at 6250 and 12500 ppm compared to controls (up to 18% and 27% during lactation, respectively). At 3125 ppm, body weight gain of females was similar to concurrent controls during the premating and the
first week of pregnancy, but slightly lower from the second week of pregnancy onwards and during lactation. Mean body weights were statistically
significantly lower during throughout the treatment period (up to 9% during lactation) when compared to controls.
The reduction in body weight (gain) during the first week of treatment was accompanied by a reduced food consumption, which recovered dose dependentlyover all groups in time. Therefore, the decrease in body weight (gain) during the first week of treatment was most probably related to an unpleasant taste of
the test item and not adverse (palatability effect). At 6250 and 12500 ppm, body weight (gain) of both sexes remained dose-dependently lower throughout
the entire treatment period (for females this included the post-coitum and lactation period) when compared to concurrent controls. Based on the magnitude of the change in males and females treated at 6250 and 12500 ppm (up to 11% and 18% in males and up to 18% and 27% during lactation in females,
respectively) the reductions in mean body weight were considered to be adversely related to treatment with the test item. At 3125 ppm, mean body weight
(gain) of males and females was slightly lower than concurrent controls at some intervals during the treatment period. As the reductions in mean body weight were modest (below 15% reduction) statistically significant on a few time intervals only, they were considered not to be toxicologically relevant.
See tables of results in the corresponding attached document in the field below 'Attached background material'. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- After the first days of administration, a decreased food consumption was noted in males and females of all test groups, which recovered from Day 2-3 onwards at 3125 ppm, from Day 3-4 at 6250 ppm and from Day 4-5 onwards at 12500 ppm. This reduced food consumption during the first days of administration was
considered to be a palatability effect. In males at 6250 and 12500 ppm, relative food consumption recovered to similar levels of the control group from Days 4-5
and Days 7-8 onwards, respectively, for the remaining treatment period. In females at 12500 ppm, (relative) food consumption recovered from Day 11-12
after start treatment onwards, however, during post-coitum and lactation, relative food consumption was statistically significantly decreased in females treated
at 12500 ppm (up to 31% and 42%, respectively, compared to concurrent controls). In females treated at 6250 ppm, relative food consumption was reduced
during post-coitum and reduced at some time points during the lactation period (up to 30% and 28%, respectively, compared to concurrent controls). In females
treated at 3125 ppm, food consumption was slightly lower at some time intervals during the treatment period.
Mean test article intake over the study period was indicated in table 1 in the field below 'Any other information on results incl. tables'.
See also the tables of results in the corresponding attached document in the field below 'Attached background material'. - Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings after treatment with 4-tert-butylpyrocatechol were noted in the stomach of the males and females of all test item treated
groups and are summarized in Table 2 in the field below 'Any other information on results incl. tables'. See also tables in the corresponding attached document in the field below ''Attached background material'. Hyperkeratosis and squamous cell hyperplasia of the forestomach were observed from 3125 ppm. These
forestomach lesions were regarded to be a local response to dietary administration of the test item, however, since ulcerations/erosions were present in treated
animals only (from 3125 ppm in males), these changes were therefore considered to be adversely related to the test-item. - Reproductive function: oestrous cycle:
- effects observed, treatment-related
- Description (incidence and severity):
- Length and regularity of the estrous cycle were considered to be affected by treatment with the test item at 12500 ppm. In all groups, a normal length and
regularity of the estrous cycle was observed before treatment (pretest). However, after start of treatment, all females at 12500 ppm, except for one female with a regular cycle (No. 75), showed irregular cycles, appeared to be acyclic or cycles were unable to be determined.
At 3125 and 6250 ppm, most females had regular cycles of 4 to 5 days. An irregular cycle was also noted for female No. 66 at 6250 ppm (with normal litter).
Given this single occurrence this did not indicate a relation with treatment.
See tables of results in the corresponding attached document in the field below 'Attached background material'. - Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- - Mating index was considered not to be affected by treatment with the test item. All females showed evidence of mating.
- Precoital time was not affected by treatment with the test item. Despite the test item related effect on the length and regularity of the estrous cycle in
females at 12500 ppm, all females showed evidence of mating within four days.
- The mean number of implantation sites at necropsy was statistically significantly lower at 12500 ppm when compared to controls (9.2 vs 12.9, respectively). The individual values of 5/10 females were below the range of the concurrent controls (considered to be adversly related to treatment with the test item at
this dose level). A trend in a lower number of implantation sites was also noted in females treated at 6250 ppm compared to controls. Since no statistical
significance was achieved and the individual values remained within the range of the concurrent controls, this was considered not to be toxicologically
relevant. At 3125, the number of implantation sites was considered not to be affected by treatment with the test item.
- Fertility index was considered not to be affected by treatment. Except for one female of the control group (No. 41), one female of the 6250 ppm group
(No. 68) and one female of the 12500 group (No. 72), all females were pregnant. The fertility indices were 90%, 100%, 90% and 90% for the control, 3125,
6250 and 12500 ppm groups, respectively. This incidental case of non-pregnancy, without related histopathology changes in reproductive organs, was
considered to be unrelated to treatment.
- Gestation Index and Duration: gestation index and duration of gestation were considered not to be affected by treatment with the test item. The gestation
indices were 100% for all groups.
- Parturition/Maternal Care: No signs of difficult or prolonged parturition were noted among the pregnant females.
- Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
- Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91%, 92%, 94% and 93%
for the control, 3125, 6250 and 12500 ppm groups, respectively.
See tables of results in the corresponding attached document in the field below ''Attached background material'. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Remarks on result:
- other:
- Remarks:
- Systemic effects: Adverse reduction in mean body weight and decreased food consumption was observed in males and females at 6250 ppm and 12500 ppm. 3125 ppm in the diet corresponds to mean daily test item intake levels of 193 mg/kg b.w./day in males, and 273 mg/kg b.w./day in females.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- < 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- gross pathology
- histopathology: non-neoplastic
- Remarks on result:
- other: Local effects (forestomach): Hyperkeratosis and squamous cell hyperplasia of the forestomach were observed from 3125 ppm. Since ulcerations/erosions were present treated animals only (from 3125 ppm in males), these changes were therefore considered to be
- Remarks:
- Local effects (forestomach): Hyperkeratosis and squamous cell hyperplasia of the forestomach were observed from 3125 ppm. Since ulcerations/erosions werepresent in treated animals only (from 3125 ppm in males), these changes were therefore considered to be adversely related to the test-item.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 6 250 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Reduced number of implantation sites.
- Remarks on result:
- other:
- Remarks:
- Based on the reduction of the mean number of implantation sites at 12500 ppm. 6250 ppm in the diet corresponds to mean daily test item intake levels of 371 mg/kg b.w./day in males, and 507 mg/kg b.w./day in females.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs occurred among pups that were considered to be related to treatment with the test item.
The nature and incidence of the clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. - Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- - Litter size: Litter size was decreased in females treated at 12500 ppm, which was considered due to the decreased number of implantation sites in this
group.
- Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with
the test item. The live birth indices were 100% for all groups.
- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by
treatment with the test item. Viability indices were 98%, 98%, 100% and 100% for the control, 3125, 6250 and 12500 ppm groups, respectively.
One pup of the control group was found dead on PND 3, and one and two pups of the control and 3125 ppm group, respectively, were missing on PND 3.
Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in lactation index of 100% for all
groups.
See tables of results in the corresponding attached document in the field below 'Attached background material'. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A decrease in pup body weights was observed in all treatment groups in a dose-related manner, which aggravated during lactation. This was considered to be
related to treatment with the test item. At 12500 ppm, mean pup body weights were statistically significantly decreased on PND 4, 7 and 13. At birth on PND 1,
mean pup body weight was also decreased (~10% when compared to concurrent controls), although no statistical significance was achieved. Furthermore,
mean pup body weights were statistically significantly lower on PND 4 for male pups at 6250 ppm and on PND 7 and 13 for all pups at 3125 and 6250 ppm. This
resulted in a decreased mean combined pup body weight of 15%, 25% and 40% on PND 13 for the 3125, 6250 and 12500 ppm groups, respectively, when
compared to concurrent controls.
The magnitude of this change was considered to represent an adverse effect on pup development and occurred in the presence of a reduced food consumption of the dams.
See tables of results in the corresponding attached document in the field below 'Attached background material'. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
- Other effects:
- no effects observed
- Description (incidence and severity):
- - Sex Ratio: Sex ratio was considered not to be affected by treatment with the test item. At 6250 ppm, the sex ratio of the pups was statistically significantly higher for male than female pups (60% male pups compared to 44% male pups in the control group). Since no dose-related trend was observed and it was within the normal range, this was considered not related to treatment with the test item.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- < 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Remarks on result:
- other:
- Remarks:
- Decrease in pup body weights in all treatment groups. 3125 ppm in the diet corresponds to mean daily test item intake levels of 193 mg/kg b.w./day in males, and 273 mg/kg b.w./day in females.
- Conclusions:
- Considering the information in this dose range finder, the following dose levels were selected for the extended one-generation reproductive toxicity study in rat by diet: 300, 1000 and 3125 ppm.
- Executive summary:
In this dose range finder study for the extended one-generation reproductive toxicity study, Wistar Han rats were treated with 4-tert-butylpyrocatechol by daily dietary administration at dose levels of 3125, 6250 and 12500 ppm. These dose levels correspond to 193, 371 and 677 mg test item / kg body weight for males and to 273, 507 and 854 mg test item/ kg body weight for females, respectively. The rats of the control group received standard powder rodent diet. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 51 to 56 days, including the day of necropsy). Females that failed to deliver were treated for 41 to 44 days.
P0 Results: Parental toxicity was observed in all treatment groups. Females treated at 12500 ppm showed hunched posture during Weeks 2 and 3 of treatment and piloerection on multiple days during the entire treatment period. In combination with the effects on body weights, these clinical observations were considered to be adverse. During the first week of treatment, a body weight loss up to 9% was observed in both males and females at 12500 ppm and a markedly reduced body weight gain was observed in males and females at 6250 ppm. This reduction in body weight (gain) during the first week of treatment was accompanied by a reduced food consumption, which recovered dose dependently over all groups in time. Therefore, the decrease in body weight (gain) during the first week of treatment was most probably related to an unpleasant taste of the test item and not adverse (palatability effect). At 6250 and 12500 ppm, body weight (gain) of both sexes remained dose-dependently lower throughout the entire treatment period (for females this included the post-coitum and lactation period) when compared to concurrent controls. Based on the magnitude of the change in males and females treated at 6250 and 12500 ppm (up to 11% and 18% in males and during lactation in females, respectively) the reductions in mean body weight were considered to be adversely related to treatment with the test item. At 3125 ppm, mean body weight (gain) of males and females was slightly lower than concurrent controls at some intervals during the treatment period. As the reductions in mean body weight were modest (below 15% reduction) statistically significant on a few time intervals only, they were considered not to be toxicologically relevant.
Test item-related morphologic alterations following the administration of 4-tertbutylpyrocatechol for at least 28 days to rats, were present in the stomach of males and females starting at a dose of 3125 ppm. These morphologic alterations consisted of a combination of hyperkeratosis, squamous cell hyperplasia of the forestomach (correlating to macroscopic finding of irregular surface) and in a few animals ulcerations/erosion of forestomach or glandular stomach at the limiting ridge. These forestomach lesions were regarded to be a local response to dietary administration of the test item, however, since ulcerations/erosions were present in treated animals only (from 3125 ppm in males), these changes were therefore considered to be adversely related to the test-item.
Higher relative liver weights were recorded for males treated at 3125, 6250 and 12500 ppm. Since no microscopic examination on liver was performed, morphologic correlation could not be evaluated. However, as the increase in liver weight was of a minor degree, these changes were considered not to represent adverse effects of treatment with the test item.
Reproductive results: Normal length and regularity of the estrous cycle was disturbed in nearly all females after start treatment at 12500 ppm. Despite the disturbance in estrous cycle during premating, all females showed evidence of mating within 4 days after start mating. It therefore was considered that the cohabitation with a male stimulated the females to a reset of their estrous cycle. Disturbance of the estrous had no effect on the mating performance and fertility as all female delivered offspring. However, females treated at 12500 ppm showed a decrease in the mean number of implantations sites compared to controls, which was considered to be adversely related to treatment with the test item. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices and precoital time).
Developmental results: Litter size was decreased in females treated at 12500 ppm, which was considered due to the decreased number of implantation sites in this group. A dose-dependent, treatment-related effect was noted on pup body weights in all treatment groups. Pup body weights (both sexes) were reduced on PND 1 and PND 4 (12500 ppm group only) and on PND 7 and 13 (at 3125, 6250 and 12500 ppm), resulting in 15%, 25% and 40% lower mean body weight on PND 13, respectively, compared to concurrent controls. The magnitude of this change was considered to represent an adverse effect on pup development and occurred in the presence of a reduced food consumption of the dams. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, macroscopic examination).
In conclusion, based on the results of this dose range finder study for the extended one-generation reproductive toxicity study, the following no-observed-adverse-effect levels (NOAEL) of 4-tert-butylpyrocatechol were established:
- Parental NOAEL (systemic toxicity): 3125 ppm (corresponds to mean daily test item intake levels of 193 mg/kg b.w./day in males, and 273 mg/kg b.w./day in females), based on decreased body weight and food consumption in males and females at 6250 ppm (corresponds to mean daily test item intake levels of 371 mg/kg b.w./day in males, and 507 mg/kg b.w./day in females) and 12500 ppm (corresponds to mean daily test item intake of 677 mg/kg b.w./day in males and 854 mg/kg b.w./day in females).
- Parental NOAEL (local toxicity): < 3125 ppm (corresponds to mean daily test item intake levels of 193 mg/kg b.w./day in males, and 273 mg/kg b.w./day in females), based on histopathological forestomach lesions in males.
- Reproduction NOAEL: 6250 ppm (corresponds to mean daily test item intake levels of 371 mg/kg b.w./day in males, and 507 mg/kg b.w./day in females), based on reduced number of implantation sites at 12500 ppm.
- Developmental NOAEL < 3125 ppm (corresponds to mean daily test item levels of 193 mg/kg b.w./day in males, and 273 mg/kg b.w./day in females), based on reduced pup body weight at 3125, 6250 and 12500 ppm and reduced litter size at 12500 ppm in males.
For the extended one-generation reproductive toxicity study, the total duration of exposure will be longer than in the preliminary reproductive toxicity study; there will be a 10-week premating period as opposed to the 2-week premating period. It is therefore expected that the effects on body weight and food consumption may become more severe after a longer treatment period. In addition, during the current study, F1-animals will be weaned at PND 21 and treated afterwards. Given the effects observed in the F0-animals and the pup body weights, dose levels of 6250 and 12500 ppm were not considered appropriate for the extended one-generation reproductive toxicity study. Considering this information, the following dose levels were selected for the extended one-generation reproductive toxicity study in rat: 300, 1000 and 3125 ppm.
- Endpoint:
- extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 April 2020 to 01 July 2021.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
- Version / remarks:
- June, 2018.
- Deviations:
- yes
- Remarks:
- The achieved dietary concentrations of 4-tert-butylpyrocatechol were not always within the acceptance criterion of 80 – 120 %.
- GLP compliance:
- yes
- Justification for study design:
- SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS::
- Premating exposure duration for parental (P0) animals : 10 weeks prior to mating.
- Basis for dose level selection :The dose levels were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test).
- Inclusion/exclusion of extension of Cohort 1B: No extension of the Cohort 1B. The substance is not wide dispersive, there is no exposure to consumers and clear conclusion on reprotoxicity can be done based on this study without F2 generation.
- Termination time for F2 : Not applicable.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : No inclusion of Cohorts 2A and 2B since the substance does not induce neurotoxic effect.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: No inclusion of Cohort 3 since the substance does not induce immunotoxic effect.
- Route of administration : The oral route of administration via dietary inclusion was selected since it is a common route of administration, which is recommended by the Regulatory Authorities for this type of study and as administration via oral gavage resulted in high toxicity due to corrosivity of the test item. - Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- 4-tert-butylpyrocatechol (CAS N° 98-29-3).
- Source: manufacturer.
- Purity: higher than 98%.
- No correction needed.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light in tightly closed containers.
- Stability and homogeneity of the test material in the diet under test conditions and during storage: (1) Stability in diet: Stability for at least 8 days in the freezer (closed containers) followed by 1 day at room temperature under normal laboratory light conditions (open containers) is confirmed over the concentration range 100 to 15000 ppm, Test Facility Study n° 20224318. Stability for at least 3 weeks in the freezer (≤-15°C) under normal laboratory light conditions is confirmed over the concentration range 100 to 15000 ppm, Test Facility Study n° 20224318. (2) Homogeneity analysis: Duplicate sets of samples (approximately 5 g accurately weighed) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%. After acceptance of the analytical results, backup samples were discarded.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stability in diet: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20224318) demonstrated that the test item was stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20224318.
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl: WI(Han).
- Details on species / strain selection:
- The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing and for reproduction and developmental toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, and toxicants.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P-Generation: At initiation of administration, animals were 6 -7 weeks old.
- Weight at study initiation: P-Generation: At initiation of administration, animals weighed between 173 and 219 g (males) and between 111 to 146 g (females).
- Fasting period before study: No.
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; 60x33x18 cm or type 2000P; 61x43.5x21.5 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; 38x22x18 cm). During the post-mating phase, males were housed in Macrolon type IV or type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, 38x22x18 cm). During the lactation phase, females will be housed in Macrolon plastic cages (type III, 38x22x18 cm). Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort Surplus) or until weaning on PND 21 (Cohorts 1A, 1B, 1C).
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. Animals were separated during designated procedures/activities. Each cage were clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): Prepared powder diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals were free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed (without the test item) was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Animal identification: Prior to start of the treatment period, each F0-animal was identified using a chip. F1-pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet. From weaning (PND 21) onwards, the animals of Cohorts 1A, 1B and 1C were identified using a chip, which was implanted between PND 18-20. The animals of Cohort Surplus were identified by tail mark.
- Acclimation period: The F0-animals were allowed to acclimate to the Test Facility toxicology accommodation for 14 days before the commencement of administration.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.
- Veterinary care: Veterinary care was available throughout the course of the study and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments were documented in the study records. A health inspection was performed before the initiation of administration.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The actual daily mean temperature during the study period was 20 to 22°C (target temperatures 18 to 24°C).
- Humidity (%): The actual daily mean relative humidity during the study was 44 to 75% (target humidity 40 to 70%). The values that were outside the targeted range occurred occasionally for 20 days with a maximum of 75% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.
IN-LIFE DATES: From: 06 May 2020 to 27 November 2020 - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
The test item was dissolved in acetone and pre-mixed with a small amount (~1-2 kg) of powder feed for one hour (± 5 minutes). An equal amount of acetone was used for all groups (Groups 1-4). The amount of acetone was based on the amount of test item used in the highest dose group (Group 4, 1:1 ratio w/w). Subsequently, this pre-mix was mixed for approximately 10 minutes with the remaining required amount of powder diet. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared at least biweekly for use at room temperature for a maximum of 1 day. If not used on the day of preparation, prepared diets were kept in the freezer (≤-15ºC) for a maximum of 3 weeks prior to use (stability for 3 weeks in the freezer (≤- 15°C)) was confirmed under Test Facility Study No. 20224318). Any remaining food left after filling the food hoppers may be stored at room temperature for a maximum of 1 day for supplementing food during the respective food consumption measurement interval.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake has been estimated based on the body weight and food consumption values. The same diets remain in the food hopper for a maximum of one day. On the day of weighing the remaining diet in the food hopper, diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag. - Details on mating procedure:
- - M/F ratio per cage: Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating.
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which the female that had not shown evidence of mating (No. 170) was separated from the cohabitated male.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
Once mating has occurred, the males and females were separated. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Sample Collection and Analysis:
Diet preparation samples were collected for analysis as indicated below:
Food batches prepared for week 1 of treatment (a) (08 May 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 3 of treatment (a) (19 May 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 6 of treatment (a) (11 Jun 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 11 of treatment (a) (15 Jul 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 14 of treatment (a) (05 Aug 2020) (C): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 15 of treatment (a) (11 Aug 2020) (d): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 16 of treatment (a) (18 Aug 2020) (e) : All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for week 21 of treatment (a) (23 Sep 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
Food batches prepared for Week 25 of treatment (a) (28 Oct 2020): All groups for concentration analyses* / Groups 2 and 4 for homogeneity*
(a) The food batches prepared for these respective periods (Week X of treatment), analysis date included.
* The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations will be averaged and utilized as the concentration
results.
(c) Diets prepared for lactation period ‘Days 1-3’ of F0-females.
(d) Diets prepared for lactation period ‘Days 4-6’ of F0-females.
(e) Diets prepared for lactation period ‘from Day 7 onwards’ of F0-females.
All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. For samples from diets prepared for Week 3 of treatment, UVVis spectra from 200-900 nm were recorded for one sample of each group. The spectra were
recorded after extraction, filtration and further dilution if required. These spectra were compared with spectra for blank extraction solvent and with spectra
for two calibration solutions. Residual samples were discarded after completion of the sample analysis.
Analyses described below were performed using a validated analytical procedure (Test Facility Study No. 20224318):
Concentration analysis: Duplicate sets of samples (approximately 5 g accurately weighed) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample
concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were
discarded. - Duration of treatment / exposure:
- The duration of treatment period was the following:
- F0 males: 11 to 12 weeks (including 10 weeks pre-mating).
- F0 females: 16 to 18 weeks (including 10 weeks pre-mating).
- F0 females which failed to deliver or had a total litter loss: 13 to 15 weeks.
- F1 animals (Cohort 1A): 10 to 11 weeks.
- F1 animals (Cohort 1B): 12 to 13 weeks.
- F1 animals (Cohort 1C): 4 to 6 weeks.
- F1 animals (Cohort Surplus): not applicable. - Frequency of treatment:
- Daily.
- Details on study schedule:
- Age at mating of the mated animals in the study: [...] weeks
SELECTION, ASSIGNMENT, REPLACEMENT AND DISPOSITION OF ANIMALS:
F0-Animals were randomly assigned to groups at arrival. Males and females were randomized separately. Animals in poor health were not assigned to groups. At least upon receipt of the animals, a health inspection was performed and any assigned animals considered unsuitable for use in the study was replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
After initiation of administration, study animals may be replaced during the replacement period with alternate animals in the event of accidental injury, non-test item-related health issues, or similar circumstances. The alternate animals may be used as replacements on the study within 1 to 3 days. The disposition of all animals were documented in the study records.
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4.
- Selection of F1 generation:
On PND 21, pups from available litters per group were selected and assigned according to the following schedule. In total, 20 females with 8 live pups/litter were selected (if possible). Litters were selected and approved by the Study Director as per Test Facility SOPs. The selected litter numbers were the following:
- Cohort 1A (Reproductive toxicity): 20 males and 20 females. Age at necropsy: PND 89 - 95.
- Cohort 1B (Reproductive toxicity): 20 males and 20 females. Age at necropsy: higher or equal to PND 97.
- Cohort 1C (Reproductive toxicity): 20 males and 20 females. Age at necropsy: after vaginal patency/Balanopreputial separation positive.
- Surplus (Tyroid hormones): 10 males and 10 females (one pup (male or female) per litter and representative of 20 litters in total). Age at necropsy: PMD 22 - 24.- Dose / conc.:
- 0 ppm (nominal)
- Dose / conc.:
- 300 ppm (nominal)
- Remarks:
- Anticipated dose level: 30 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 11-14 mg/kg/day for males and 14-17 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 19 mg/kg/day for males and 20 mg/kg/day for females. - Dose / conc.:
- 1 000 ppm (nominal)
- Remarks:
- Anticipated dose level: 100 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 42-50 mg/kg/day for males and 51-59 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 65 mg/kg/day for males and 68 mg/kg/day for females. - Dose / conc.:
- 3 125 ppm (nominal)
- Remarks:
- Anticipated dose level: 300 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 144-172 mg/kg/day for males and 176-194 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 221 mg/kg/day for males and 230 mg/kg/day for females. - No. of animals per sex per dose:
- F0 animals: 25 animals per sex and per dose.
F1 animals:
- Cohort 1A: 20 animals per sex and per dose.
- Cohort 1B: 20 animals per sex and per dose.
- Cohort 1C: 20 animals per sex and per dose.
- Cohort Surplus: 10 animals per sex and per dose. - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) (see DRF feed) with dietary exposure of 4-tert-butylpyrocatechol in rats and in an attempt to produce graded responses to the test item. During this study, Wistar Han rats received 0, 3125, 6250 and 12500 ppm by dietary administration for at least 28 days. No mortalities were observed during the treatment period. In the current study, the total duration of exposure was longer than in the preliminary reproductive toxicity study; there was be a 10-week premating period as opposed to the 2-week premating period. It was therefore expected that the effects on body weight and food consumption observed in the preliminary study may become more severe after a longer treatment period. In addition, during the current study, F1-animals were weaned at PND 21 and treated afterwards. Given the effects observed in the F0-animals and the pup body weights in the preliminary study, dose levels of 6250 and 12500 ppm were not considered appropriate for the current study. Considering this information, a dose level 3125 ppm was selected as high dose. Low and mid dose levels were selected to be 300 and 1000 ppm, respectively.
- Rationale for animal assignment (if not random): F0-Animals were randomly assigned to groups at arrival. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0 animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.
- Administration of test materials:
The test item was administered to the appropriate animals by inclusion in the diet ad libitum. F0-males and females were treated up to and including the day before scheduled necropsy.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, spilled diet from the food hopper or when they commence eating for themselves.
From weaning onwards (PND 21), F1-animals of Cohorts 1A received diet containing test item up to and including the day before scheduled necropsy. The F1-animals of Cohort 1B and 1C received diet containing test item until the day of necropsy. The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were housed with their dams until necropsy and therefore had access to the diet containing test item at the concentration provided to the dams.
The first day test diets containing test item were available to the animals was designated as Day 1.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The same diets remained in the food hopper for a maximum of one day. Daily the remaining diet in the food hopper was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag. - Parental animals: Observations and examinations:
- F0-GENERATION:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least twice daily, in the morning and at the end of the working day, throughout the study.
- Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least once daily, beginning during the first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy.
- Procedure: Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.
ARENA OBSERVATIONS: Yes.
- Time schedule: Once before the first administration of the test item and at weekly intervals during the treatment period.
- Procedure: Clinical observations were conducted in a standard arena.
BODY WEIGHT: Yes.
- Time schedule for examinations: Males and females were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 postcoitum and during lactation on PND 1, 4, 7, 14 and 21.
- Procedure: Animals were weighed individually. A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- Time schedule and procedure: Food consumption of animals was quantitatively measured once daily throughout the study from Day 1 of administration onwards except for males and females which were housed together for mating and for females without evidence of mating. Measurements were conducted at approximately the same time.
WATER CONSUMPTION: Yes.
- Time schedule for examinations: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.
OTHER:
- Laboratory evaluations: F0-Animals (selected animals - 10/sex/group): The following parameters were examined: Hematology, coagulation, clinical chemistry, thyroid hormone and urinalysis. For more details and tables see the field below 'Any other information on materials and methods incl. tables'.
- General Reproduction data: From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. For more details see the field below 'Any other information on materials and methods incl. tables'. - Oestrous cyclicity (parental animals):
- F0 GENERATION:
- Time schedule: Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was
observed. Vaginal lavage was continue for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was also be taken to determine the stage of estrus.
- Procedure: Estrous stages were determined by examining the cytology of vaginal lavage samples. - Sperm parameters (parental animals):
- F0 GENERATION:
For all males, the following assessments were performed:
- Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy.
- Sperm motility and progressive motility were assessed from all samples.
- Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded. Evaluation has been performed for all samples.
- One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples. - Litter observations:
- IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS - F1-GENERATION UNTIL WEANING (PND 21):
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes.
Culling on PND 4. To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, were not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring (pups until weaning (PND 21)):
- Mortality/moribundity: Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pup 2 of Litter No. 103 was sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
- Clinical observations: Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
- Body weight: Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort Surplus, a terminal weight was recorded on the day of scheduled necropsy.
- Sex ratio: Sex was externally determined for all pups on PND 1, 4 and 13.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/Nipple retention: All male pups in each litter were examined for the number of areola/nipples on PND 13.
GROSS EXAMINATION OF DEAD PUPS:
Stillborn pups and pups found dead between birth and PND 13 will be sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology.
If possible, defects or cause of death were evaluated.
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.
OTHER:
- Thyroid hormone. F1 culled pups (2 pups per litters). Time point(s): PND 4. For more details and tables see the field below 'Any other information on materials and methods incl. tables'.
IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS - F1-GENERATION FROM WEANING (PND 21) ONWARDS:
F1 GENERATION FROM WEANING (PMD 21): Cohorts 1A; 1B and 1C (60 animals/sex/group):
The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort Surplus and spare F1-animals as these were terminated on/before PND 24.
CAGE SIDE OBSERVATIONS: Yes, cohorts 1A, 1B and 1C.
- Time schedule: Twice daily throughout the study.
- Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
DETAILED CLINICAL OBSERVATIONS: Yes, cohorts 1A, 1B and 1C.
- Time schedule: At least once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- Procedure: Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.
ARENA OBSERVATIONS: Yes, cohorts 1A, 1B and 1C.
- Time schedule: Once on the day of weaning and thereafter at weekly intervals during the treatment period.
- Procedure: Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.
BODY WEIGHT: Yes, cohorts 1A, 1B and 1C.
- Time schedule for examinations: Weekly from weaning onwards. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.
- Procedure: Animals were individually weighed.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, cohorts 1A, 1B, 1C.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- Time schedule and procedure: Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy. For animals of Cohort 1A, food consumption was measured up to the day prior to scheduled necropsy. For animals of Cohorts 1B and 1C, food consumption was measured up to and including the day of necropsy.
WATER CONSUMPTION: Yes, cohorts 1A, 1B, 1C.
- Time schedule for examinations: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.
OTHER:
LABORATORY EVALUATIONS:
- Laboratory evaluations: F1-animals (cohort 1A animals - selected animals (10/sex/group)): The following parameters were examined: Hematology, coagulation, clinical chemistry, thyroid hormone and urinalysis. For more details and tables, see the field below 'Any other information on materials and methods incl. tables'. Time point(s): on the day of scheduled necropsy.
- Thyroid hormones for F1-animals - Cohorts Surplus (All surplus pups). For more details and tables, see the field below 'Any other information on materials and methods incl. tables'. Time point(s): PND 22-24.
VAGINAL PATENCY (Cohorts 1A, 1B and 1C):
- Time schedule: Daily for all females from PND 25 onwards until vaginal patency was present.
- Procedure: Vaginal patency (vaginal opening) was monitored by visual inspection of the vaginal area. Body weight was recorded on the day of acquisition of vaginal patency.
BALANOPREPUTIAL SEPARATION (Cohorts 1A, 1B and 1C):
- Time schedule: Daily for all males from PND 35 onwards, until balanopreputial separation was present.
- Procedure: Balanopreputial separation (prepuce opening) was monitored by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.
STAGE OF ESTRUS DETERMINATION (Cohorts 1A, 1B and 1C):
- Time schedule: On the day of scheduled necropsy, a vaginal lavage was be taken.
- Procedure: Estrous stages were determined by examining the cytology of vaginal lavage samples.
ESTROUS CYCLE DETERMINATION (Cohort 1A only):
- Time schedule: During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.
- Procedure: Both periods: Estrous stages were determined by examining the cytology of vaginal lavage samples.
The estrous cycle data of the first period is not reported. Data were retained in the raw data.
SPERM ANALYSIS (Cohort 1A only): For descriptions and tables, see the field below 'Any other information on materials and methods incl. tables'.
SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS (Cohort 1A only): For descriptions and table (table 18), see the field below 'Any other information on materials and methods incl. tables'.
- Postmortem examinations (parental animals):
- SACRIFICE:
F0 Generation:
Terminal procedures are summarized in the table 11 (see table 11 in the field below ' Anyother information on materials and methods inc. tables').
All animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies are summarized below:
- Males which sire: After successful mating and a minimum of 10 weeks of treatment.
- Males which failed to sire: At the end of the mating period and after a minimum of 10 weeks of treatment.
- Females which delivered: Lactation Day 23-25.
- Females which failed to deliver: With evidence of mating: Post-coitum Days 26-27 (N°108, 117, 124, 130, 141, 144, 151, 158, 184, 187, 192 and 195). Without evidence of mating: Approximately 27 days after the last day of the mating period (N° 170).
- Females with total litter loss (N° 125 and 173): within 24 hours after the last pup was found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.
GROSS NECROPSY:
F0 Generation:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
ORGAN WEIGHTS
F0 Generation:
The organs identified for weighing in the Tissue Collection and Preservation table (see table 12 in the field below ' Anyother information on materials and methods inc. tables') were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
HISTOLOGY / HISTOPATHOLOGY
Tissue collection and preservation:
Representative samples of the tissues identified in the Tissue Collection and Preservation table (see table 12 in the field below ' Anyother information on materials and methods inc. tables') were collected from all animals and preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.
For females which fail to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) have to be fixed (if applicable), but not have to be examined histopathologically in first instance.
The tissues indicated in Table 12 (see field 'Anyother information on materials and methods incl. tables') were prepared for microscopic examination and weighed, respectively.
Tissues were processed at Charles River Laboratories Frederick. Tissues in the Tissue Collection and Preservation table (see table 12 in the field below ' Anyother information on materials and methods inc. tables') from a selection of the F0-animals identified in the Terminal Procedures table (see table 11) were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).
All tissues as defined under Histology from a selection of the F0-animals identified in the Terminal Procedures table (see table 12 in the field below ' Anyother information on materials and methods inc. tables) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation will be communicated to the Study Director; tissues will be evaluated and reported. Any additional stains or evaluations, if deemed necessary by the pathologist, will be added by study plan amendment following discussion with the Study Director and in consultation with the Sponsor.
Terminal procedures, tissue collection/preservation and organ weights for F0-animals are summarized in the field below 'Any other information on materials and methods incl. tables', in tables 11 and 12. - Postmortem examinations (offspring):
- TERMINAL PROCEDURES - F1-GENERATION UNTIL WEANING:
- Unscheduled deaths (F1-Generation):
The pup sacrificed in extremis on PND 7 was euthanized by an intraperitoneal injection of sodium pentobarbital.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
- Culled Pups (PND 4) (F1 Generation):
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood were collected for the F1-generation, if possible.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
In addition, the stomach was collected and preserved in 10% buffered formalin at necropsy for 10 selected animals/sex/group. These animals were selected and approved in advance by the Study Director in the study files. Histology and histopathology were performed on all collected stomachs.
TERMINAL PROCEDURES - F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C, cohort suplus and Spare F1-animals):
Terminal procedures for F1 - generation are summarized in the table 13 (See the field below 'Any other information on materials and methods incl. tables').
SACRIFICE:
Spare F1-animals which are not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.
For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
GROSS NECROPSY:
COHORT 1A:
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 14 in the field below 'Any other information on materials and methods incl. tables'.
COHORT 1B:
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 15 in the field below 'Any other information on materials and methods incl. tables'.
COHORT 1C:
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded.
The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 16 in the field below 'Any other information on materials and methods incl. tables'.
COHORT SURPLUS:
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. For data collection and preservation, see table 17 in the field below 'Any other information on materials and methods incl. tables'.
On PND 22, blood samples (1.0 mL) were collected between 8.00 and 11.30 a.m. from all animals by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).
ORGAN WEIGHT:
COHORT 1A:
The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table (see table 14 in the field below 'Any other information on materials and methods incl. tables') were weighed and collected.
COHORT 1B:
The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table (see table 15 in the field below 'Any other information on materials and methods incl. tables') were weighed and collected.
COHORT 1C:
No organ weights for this cohort.
COHORT SURPLUS:
The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table (see table 17 in the field below 'Any other information on materials and methods incl. tables') were weighed and collected.
HISTOLGY / HISTOPATHOLOGY:
Histology: Tissues were processed at Charles River Laboratories Frederick. Tissues in the Tissue Collection and Preservation tables (see tables 14 to 17 in the field 'Any other information on materials and methods incl. tables') from a selection of the F1-animals identified in the Terminal Procedures tables (see table 13) were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).
Histopathology: All tissues as defined under Histology from a selection of the F1-animals identified in the Terminal Procedures tables (see stables 14 to 17) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
A peer review on the histopathology data was performed by a second pathologist, during which full tissue review was performed of at least five animals/sex/group of the highest dose with 50% or greater survival/sex. Remaining tissues were reviewed according to standard operating procedures.
Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported
COHORT 1A:
In addition to the procedures described above, for Cohort 1A animals of Groups 1 and 4, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the cohort 1A animals of group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea. - Statistics:
- STATIISTICS
DATA COLLECTION IN TOXDATA
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1or 5% levels. Numerical data collected on scheduled occasions were analyzed according to sex and occasion. Descriptive statistics number, mean and
standard deviation were reported when possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate.
The following pairwise comparisons were made:
Group 2 vs Group 1
Group 3 vs Group 1
Group 4 vs Group 1.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test)
Non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test)
Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenthe overall test is significant.
DATA COLLECTED IN PROVANTIS
Inferential methods: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided
tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are the following:
Group 2 vs Group 1
Group 3 vs Group 1
Group 4 vs. Group 1
Parametric/non-Parametric method was used for analysis of Hematology, Coagulation and Clinical variables.Parametric/non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted usingDunnett’s or Dunn’s test, respectively.
Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest. - Reproductive indices:
- Reproduction variables:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual
values of F0-females, the remaining group values were calculated from the total number in each group.
- Mating index males (%) = Number of males mated / Number of males paired * 100.
- Mating index females (%) = Number of females mated / Number of females paired * 100.
- Precoital time = number of days between initiation of cohabitation and confirmation of mating.
- Fertility index males (%) = Number of pregnent females / Number of males mated * 100.
- Fertility index females (%) = Number of pregnent females / Number of females mated * 100.
- Gestion index (%) = Number of females with living pups on Day 1/ Number of pregnant females.
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition. - Offspring viability indices:
Developmental variables:
For each group, the following calculations were performed:
- Post-implantation survival index (%) = Total number of offspring born / Total number of uterine implantation sites * 100.
(Post-implantation survival index will be expressed as 100% when the number of offspring exceeds the number of implantation sites recorded).
- Live birth index (%) = Number of live offspring on day 1 after littering / Total number of offspring born * 100.
- Percentage live males at first litter check (%) = Number of live male pups at first litter check / Number of live pups at first litter check * 100.
- Precentage live females at first litter check = Number of live female pups at first litter check / Number of live pups at first litter check * 100.
- Viability index (%) = Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering * 100.
- Weaning index (%) = Number of live offspring on Day 21 after littering / Number live offspring on day 4 (after culling) * 100.
- Percentage live males at weaning (%) = Number of live male pups on Day 21 after littering / Number of live pups on Day 21 after littering * 100.
- Percentage live females at weaning (%) = Number of live female pups on Day 21 after littering / Number of live pups on Day 21 after littering * 100.- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
At 3125 ppm, one female had a greasy coat and red staining (i.e. chromodacryorrhoea) on the back during Week 9/10 of treatment, which recovered in early
week 10. Additionally, piloerection was observed for 5 females of the same group (group 4) (Nos. 180, 185, 191, 193 and 199) during week 15/16 of treatment. For most females, piloerection was noted on one or two days only. At the incidence observed and as clinical signs were transient, they were considered to be
unrelated to treatment with the test item.
Other clinical signs noted during the treatment period (i.e. scabs and alopecia) occurred within the range of background findings to be expected for rats of this
age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed,
these were considered to be unrelated to treatment with the test item.
See table 23 in the field below 'Attached backgrowd material'. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No test item-related mortality occurred during the study period.
Two females, one of the control and 1000 ppm groups each (Nos. 125 and 173), were euthanized on Lactation Day 2 or 1, respectively, as these females had a
total litter loss.
See table 22 in the field below 'Attached backgrowd material'. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related differences in body weight were observed at 3125 ppm.
Mean body weight gain of males at 3125 ppm was lower from Day 8 of treatment onwards. This resulted in a lower mean body weight of these males during the pre mating and mating period (not always statistically significant). Mean terminal body weight was 9% lower than control mean (statistically significant).
Mean body weight gain of females at 3125 ppm was lower from Day 22 of treatment onwards up to and including Day 1 of the mating period. On Day 1 of the mating period, mean body weight was 4% lower than control mean. Mean body weight gain was similar between all groups during gestation, but the mean
body weight of these females remained statistically significantly lower throughout gestation. The difference in body weights between high dose and control
group was more or less constant (6-8% lower) and most likely related to the decreased body weights at the end of the pre mating period at 3125 ppm. During lactation, a higher mean body weight gain was observed for these females from Day 7 of lactation onwards, resulting in a slight recovery with regards to
mean body weight. On Day 1 of lactation, mean body weight was 9% lower than control mean, whereas on Day 21 of lactation the difference was reduced
to 5%. Mean terminal body weight of these females was 4% lower than control mean (not statistically significant).
Body weights and body weight gain of animals treated at 300 or 1000 ppm were considered to have been unaffected by treatment with the test item.
See table 24 in the field below 'Attached backgrowd material'. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption: No toxicologically relevant changes in food consumption before or after correction for body weight were noted. Mean absolute food
consumption of males at 3125 ppm was lower during weeks 4-8 of treatment and mean relative food consumption during weeks 4-7 of treatment (statistically significant). As no relevant differences were observed from week 8 of treatment onwards and the mean of means food consumption (absolute and relative) was similar to concurrent control, this temporary and minimal decrease in food consumption was considered not toxicologically relevant.
Mean absolute food consumption of females at 3125 ppm was slightly lower during the pre-mating and gestation periods (not always statistically significant). As relative food consumption was similar to concurrent control mean, this decrease was attributed to the lower body weights observed for these females
rather than a direct effect of the test item. No toxicologically relevant differences were observed in food consumption of these females during the lactation
period.
Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 1000 ppm.
Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment with
the test item since no trend was apparent regarding dose and duration of treatment.
Note: When compared to the pre-mating period, a generally higher food intake (absolute and relative to body weight) was measured in females during the
phases of gestation and lactation, with values for the control group within the normal range. The observed higher food intake is a normal physiological
process. It reflects the increased nutritional need of the dams during the periods of gestation and lactation. Furthermore, from the third week of lactation
onwards also their pups start to consume solid feed.
Test item intake: Mean test article intake over the study period was indicated in the table 26. Lower accuracies were measured for the prepared powder diet. In order to determine the worst case scenario exposure, the table 27 presents the mean of means test item intake corrected for the mean of means accuracy per period. See tables 26 and 27 in the field below 'Any other information on results incl. tables'.
See also table 25 in the field below 'Attached backgrowd material' for details on F0-generation food consumption. - Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Hematology:
The following changes distinguished treated females from control animals:
- Mean concentration of platelets was decreased at 1000 and 3125 ppm (0.92x and 0.89x of control, respectively; not statistically significant). Given the
magnitude or based on a dose-related response, this change was considered to be test item-related.
Hematological parameters of treated males were considered not to have been affected by treatment with the test item.
Mean concentrations of reticulocytes in treated males were lower than concurrent control mean. This was considered to be unrelated to the test item as it
was attributed to a higher control mean due to a relatively high value of Male No. 7.
Any other changes in hematology parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the change,
variation in direction of change, absence of a dose response, relatively high values in single animals and/or absence of biological relevance.
See table 28 in the field below 'Attached backgrowd material'.
Coagulation:
No toxicologically relevant changes were noted in coagulation parameters. The shorter prothrombin time (PT) of males at 3125 ppm was considered not to be of toxicological relevance given the minimal magnitude of the change.
See table 29 in the field below 'Attached backgrowd material'. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The following changes distinguished treated from control animals:
- Mean bile acid concentration was decreased in males at 3125 ppm (0.61x of control; not statistically significant).
- Mean creatinine concentration was decreased in males at 3125 ppm (0.87x of control).
Given the magnitude, these changes were considered to be test item-related.
Mean total bilirubin concentration was decreased in males at 1000 and 3125 ppm (0.84x and 0.83x of control, respectively; not statistically significant at 1000
ppm). Mean inorganic phosphate concentration was increased in females at 3125 ppm (1.11x of control; not statistically significant). Given the magnitude, as
individual values generally remained within concurrent control range and/or in absence of a dose-related trend, these changes were considered not toxicologically relevant.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of
a dose-related trend.
See table 30 in the field below 'Attached backgrowd material'. - Endocrine findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Mean TSH values of treated females were 1.22x, 1.58x and 2.52x of control at 300, 1000 and 3125 ppm, respectively (not statistically significant). The increased mean TSH value at 3125 ppm was partially caused by one female with a relatively high value. Without this female, the group mean would be 1.84x of control.
Other individual values generally remained within the concurrent control range as well as the historical control range (Historical control data of TSH (mU/L) in
female Wistar Han rats (2017-2021); Mean = 0.204, P5-P95 = 0.030-0.626 (n=116)).
Mean T4 values were also increased in treated females (not statistically significant), but this change was considered not to be of toxicological relevance as the
opposite effect (i.e. a decrease) would be expected in case of target organ toxicity and as no clear dose-response was observed.
For males, the mean T4 concentration was lower at 3125 ppm and the mean TSH concentration was higher at all dose levels when compared with concurrent
controls (all not statistically significant). As the decrease in mean T4 was minimal, the increase in mean TSH occurred in the absence of a dose-related response
and as individual values generally remained within concurrent control mean, no relationship with the test item was indicated.
See table 31 in the field below 'Attached backgrowd material'. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Urinalysis parameters of treated rats were considered not to be affected by treatment with the test item.
For 1 and 3 males at 1000 and 3125 ppm, respectively, a small response (1+) was recorded for bilirubin while all control and 300 ppm were negative. At the
incidence and severity observed and as the positive result could not be confirmed, this difference was considered not test item-related.
See table 32 in the field below 'Attached backgrowd material'. - Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings after treatment with 4-tert-butylpyrocatechol were noted in the stomach of the 3125 ppm group males and females and are summarized in the table 35 (See table 35 in the field below 'Any other information on materials and methods incl. tables').
Non-glandular stomach (i.e. forestomach): An increased incidence and/or severity of diffuse hyperkeratosis (up to slight degree) accompanied in a few animals by squamous cell hyperplasia (minimal degree) was present in both sexes at 3125 ppm.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no
test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
See also table 36 in the field below 'Attached backgrowd material'. - Histopathological findings: neoplastic:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Length and regularity of the estrous cycle were considered not to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for control Female No. 106 (with normal litter) and Female No. 187 at 3125 ppm (not pregnant). Given their incidental nature and occurrence in the control group, these findings did not indicate a relation with the test item.
See table 37 in the field below 'Attached backgrowd material'. - Reproductive function: sperm measures:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Mean percentage of motile and progressive sperm were lower in treated males when compared with control. The percentage of motile sperm was 0.79, 0.82 and 0.88x of control and of progressive sperm 0.64, 0.68 and 0.79x of control for the 300, 1000 and 3125 ppm groups, respectively (not statistically significant for
percentage of motile sperm at 3125 ppm). All individual values (except for one male at 300 ppm) remained within the normal range for rats of this age and strain.
In absence of a dose-related response and given the absence of any reproductive/developmental toxicity, these differences were considered not toxicologically relevant.
Sperm concentration and morphology were considered to be unaffected by treatment with the test item.
The mean sperm count in males at 300 and 1000 ppm was increased (1.22 and 1.18x of control, respectively). Due to the direction of change and in the absence of a dose-related response, this difference was considered unrelated to the test item.
See table 38 in the field below 'Attached backgrowd material'. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- - Mating Index: Mating index was considered not to be affected by treatment with the test item for both sexes. Except for one female at 1000 ppm (No. 170),
all females showed evidence of mating.
- Precoital Time: Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 4 days, with
exception of one female (No. 180) at 3125 ppm, for which it took 13 days before mating could be confirmed.
- Number of Implantation Sites: Mean number of implantation sites at 3125 ppm was decreased (0.94x of control; not statistically significant). Based on the
magnitude of change and as individual values remained within concurrent control range, this difference was considered not toxicologically relevant. Number
of implantation sites was considered not to be affected by treatment with the test item up to 1000 ppm.
- Fertility Index: Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 88% for the control, 300 and 1000
ppm groups, and 84% for the 3125 ppm groups for both sexes.
A total of 3 females from the control, 300 and 1000 ppm groups each and 4 females at 3125 ppm were not pregnant. Since these cases of non-pregnancy
showed no clear dose related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment with the test item.
- Histopathological evaluation of reproductive preformance: Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. A total of 3 females from the control, 300 and 1000 ppm groups each and 4 females at 3125 ppm were not pregnant, despite evidence of mating. In addition, mating could not be confirmed for one couple in the 1000 ppm group. Furthermore, one female of the control group and one female at 1000 ppm had
total litter loss at Lactation Day 2 and 1, respectively. There were no microscopic findings in the reproductive organs (and mammary gland in the case of total
litter loss) which could account for this lack of healthy offspring. Since these cases of non-pregnancy and total litter loss showed no clear dose related
incidence across the dose groups and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment with
the test item.
See table 39 in the field below 'Attached backgrowd material'. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effect (general toxicity).
- Remarks on result:
- other:
- Remarks:
- General toxicity: Only non-adverse effects were observed.
- Dose descriptor:
- LOEL
- Effect level:
- ca. 1 000 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- haematology
- clinical biochemistry
- histopathology: non-neoplastic
- Remarks on result:
- other:
- Remarks:
- General toxicity: Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach (local effects) at 3125 ppm (hyperkeratosis and hyperplasia), not considered as adverse.
- Dose descriptor:
- NOEL
- Effect level:
- ca. 300 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- haematology
- clinical biochemistry
- histopathology: non-neoplastic
- Remarks on result:
- other:
- Remarks:
- General toxicity: Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach (local effects) at 3125 ppm (hyperkeratosis and hyperplasia), not considered as adverse.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No reproductive effect.
- Remarks on result:
- other:
- Remarks:
- Reproductive toxicity. No reproduction toxicity was observed up to the highest dose level tested (3125 ppm).
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- F1 - GENERATION - F1-PUPS DURING LACTATION:
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
An abnormal posture of the left or right hindleg was observed for 2 control pups (one of Litter Nos. 107 and 123 each), 1 pup at 300 ppm (Litter No. 147),
1 pup at 1000 ppm (Litter No. 160) and 1 pup at 3125 ppm (Litter No. 196). This clinical sign was observed from PND 7, 20 or 13 onwards. Based on the
absence of a dose-response or and occurrence in the control group, the abnormal posture of one of the hindlegs was considered unrelated to the test item.
Fissures of the left foreleg were observed between PND 7-9 and missing eyes were observed from PND 17 onwards in 2 pups of the control group (both of
Litter No. 103). Additionally, another pup of this litter was sacrificed in extremis on Day 7 to prevent suffering due to the excess of Indian ink (used for
identification on PND 1) observed ventrally. Due to occurrence in the control group only, above mentioned clinical signs were unrelated to the test item.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered unrelated
to the test item.
See tables 40/41 in the field below 'Attached backgrowd material'.
F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
One Cohort 1C female of the control group (No. 540) was weaned while missing both eyes. Based on the body weight gain data, there was no impact on the development of this animal. As such, this clinical sign had no impact on animal welfare or the study results.
See table 47 in the field below 'Attached backgrowd material'.
Note for the table: “Day 1” of treatment is the day that the first animals were weaned. For those animals that were not yet weaned on the day that the first
animals were weaned, a “.” appears in the table. - Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- F1-GENERATION - OFFSPRINGS:
Viability index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by
treatment with the test item.
Viability indices (number of live offspring on PND 4 before culling as a percentage of number of offspring on Day 1 after littering) were 99% for the control,
1000 and 3125 ppm groups, and 98% for the 300 ppm group.
Two pups of the control group (one of Litter Nos. 103 and 121 each), 5 pups at 300 ppm (one of Litter Nos. 128, 138 and 146 each and two of Litter No. 136), 3 pups at 1000 ppm (two of Litter No. 159 and one of Litter No. 171) and 2 pups at 3125 ppm (one of Litter Nos. 193 and 199 each) were found dead or missing on PND 2-4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidencedid not show a dose-related trend and remained within the range considered normal for pups of this age.
In addition, the last pup of Litter No. 125 (control) was missing on PND 2, resulting in a total litter loss for this dam.
See tables 40/41 in the field below 'Attached backgrowd material'.
F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
No mortality occurred during the study period that was related to treatment with the test item.
Animal No. 633 (1000 ppm, Cohort 1A) was sacrificed 5 days post weaning as it appeared to be male upon daily observations while it was assigned to the
females. This male was replaced by Female No. 673 (1000 ppm, Cohort 1C). All data of Animal No. 673 was reported under No. 633 and all data of Animal No. 633 was reported under No. 673 or No. 633-A.
For Animal Nos. 539, 736 and 750, the necropsy date was recorded as being the first date of weaning. These animal numbers were planned in the computer system, but were not used in the study as no animal was available at weaning.
See table 48 in the field below 'Attached backgrowd material'.
Note: The date under “Treatment from” in Table 48 is the date that the first animals reached PND 21. Actual treatment of the animals commenced on the date
that the respective animals reached PND 21. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- F1-GENERATION - F1-PUPS - DURING LACTATION:
Body weights of pups were considered not to be affected by treatment with the test item.
The higher mean pup weights on PND 1 (not statistically significant for males) were considered unrelated to the test item as the opposite effect (i.e. an
decrease) would be expected in case of toxicity and as pup body weights were similar to concurrent controls on subsequent intervals.
See table 42 in the field below 'Attached backgrowd material'.
F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
Test item-related differences in body weight were observed at 1000 and 3125 ppm.
Mean body weight gain of males at 1000 and 3125 ppm was lower from Day 15 of treatment onwards, which resulted in a lower mean body weight of these males throughout the treatment period (not always statistically significant). Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower
than control for males at 1000 ppm and 9 and 6% lower than control for males at 3125 ppm, respectively (not statistically significant for Cohort 1B).
Mean body weight gain of females at 3125 ppm was slightly lower from Day 36 of treatment onwards (not statistically significant). No clear differences were observed in mean body weight during the treatment period, but terminal body weight of these females was 5 and 1% lower than control mean for Cohort 1A
and 1B animals, respectively (both not statistically significant). Given the magnitude of change, the observed differences were considered not toxicologically relevant.
Body weights and body weight gain of males treated at 300 and females treated at 300 or 1000 ppm were considered to have been unaffected by treatment with the test item.
Note: “Day 1” of treatment is the day that the first animals were weaned. Body weight of animals that were weaned during the subsequent 7 days were all
entered under that day. Body weight of animals that were weaned in the subsequent week were all entered under “Day 8”. This may have resulted in the
statistically significant changes as described above. Since the majority of animals were weaned within 7 days after commencement of weaning of the first
animals, “Day 8” was used as the reference day for body weight gain calculations.
See table 49 in the field below 'Attached backgrowd material'. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
No toxicologically relevant changes in food consumption before or after correction for body weight were noted.
Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and duration of treatment.
See table 50 in the field below 'Attached backgrowd material'.
Test article intake for F1-animals was indicated in the table 51. Lower accuracies were measured for the prepared powder diets. In order to determine the
worst case scenario exposure, the table 52 presents the mean of means test item intake corrected for the mean of means accuracy per period. See below tables 51 and 52 in the field 'Any other information on materials and methods incl. tables'. - Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- F1 - GENERATION FROM WEANING ONWARDS (Cohort 1A):
Hematology:
Test item-related changes were observed in males and females from 300 ppm onwards.
The mean lymphocyte count (LYMPH) was increased when compared with concurrent control for all treatment groups and both sexes (statistically significant in males at 3125 ppm and in females at 1000 and 3120 ppm), resulting in increased mean total white blood cell (WBC) counts (statistically significant only in females at 1000 and 3125 ppm). Additionally, an increase in mean large unstained cells (LUC) count was observed in all treatment groups and both sexes (statistically significant only in males at 3125 ppm). LUC are normally either larger monocytes or lymphocytes that could not be classified, and given the increase in lymphocytes that was observed in these animals, these increased LUC counts were considered test item-related as well. The fold changes for these parameters when compared with control are presented in table 53, see table 53 in the field 'Any other information on materials an methods incl. tables.
The increased mean basophil concentration for females at 300, 1000 and 3125 ppm was considered to have arisen as a result of slightly low control value
when compared with the historical control range and therefore considered to be of no toxicological significance.
Any other changes in hematology parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the change,
variation in direction of change, absence of a dose response, relatively high values in single animals and/or absence of biological relevance.
See also table 54 in the field below 'Attached backgrowd material'.
Historical Control Data for Wistar Han rats; F1-females (2017-2021): Basophils (109/L) mean = 0.0; P5 – P95 = 0.00 – 0.01 (n=114).
Coagulation:
Coagulation parameters of treated rats were considered not to have been affected by treatment. See table 55 in the field below
'Attached backgrowd material'. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- F1 - OFFSPRINGS (F1-PUPS - DURING LACTATION):
Clinical Biochemistry (T4 levels) on PND 4 F1-Pups:
Serum T4 levels in male and female pups culled at PND 4 were considered not to be affected by treatment with the test item. It should be noted that for
several animals within each group, values of 5.0 ng/mL were reported (i.e. ½ LOQ) as the original value was below LOQ, which is represented by relatively low mean values. If the 5.0 ng/mL values would not be taken into account, mean values would be 12.6, 12.9, 12.9 en 11.9 ng/mL for control, 300, 1000 and 3125 ppm groups, respectively.
See table 43 in the field below 'Attached backgrowd material'.
Note to table: Serum T4 levels of PND 4 pups are reported as females, but these were derived from pooled samples collected from male and female pups.
F1 - GENERATION FROM WEANING ONWARDS (Cohorts 1A and Cohort Surplus)
Clinical chemistry:
The following changes distinguished treated Cohort 1A animals from control animals:
• Mean concentration of total bilirubin was decreased for males at 1000 and 3125 ppm (0.89 and 0.85x of control, respectively; not statistically
significant).
Given the magnitude and based on a dose-related response, this change was considered to be test item-related.
Any other changes in clinical chemistry parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the
change, variation in direction of change, absence of a dose response, relatively high values in single animals and/or absence of biological relevance.
See table 56 in the field below 'Attached backgrowd material'.
Thyroid hormone analyses:
Cohort 1A (PND 89-95):
Serum levels of T4 and TSH in Cohort 1A males and females were considered unaffected by treatment with the test item up to 3125 ppm.
The increased mean TSH concentration in males at 3125 ppm (1.82x of control; not statistically significant) was attributed to the relatively high value in a
single animal (No. 424). As remaining individual values generally remained within concurrent control range, no relationship with the test item was indicated.
The decreased mean TSH concentration in females at 300 and 1000 ppm (0.42 and 0.61x of control, respectively; not statistically significant at 1000 ppm)
was considered unrelated to the test item in absence of a dose-related response.
Cohort Surplus (PND 22-24):
Decreased mean TSH levels were noted for PND 22-24 males and females at 3125 ppm (0.63 and 0.86x of control, respectively; not statistically significant).
Individual values generally remained within the concurrent and historical control range .
Serum T4 levels in male and female pups of Cohort Surplus at PND 22-24 were considered not to be affected by treatment with the test item.
See table 56bis in the field below 'Attached backgrowd material'.
Historical control data for TSH (mU/L) in PND 22-24 pups (2017-2021): Males mean = 0.081, P5 P95 = 0.0245 0.2065 (n=100) / Females mean = 0.076, P5-P95 = 0.0245-0.1765 (n=100). - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- F1 - GENERATION FROM WEANING ONWARS (Cohort 1A):
Urinalysis parameters of treated rats were considered not to be affected by treatment with the test item.
See table 57 in the field below 'Attached backgrowd material'. - Sexual maturation:
- no effects observed
- Description (incidence and severity):
- F1- Generation from weaning onwards (Cohorts 1A, 1B, 1C and Surplus):
Sexual maturation was considered not to be affected by treatment with the test item.
In all treatment groups, females were slightly older and mean body weight was slightly higher than in concurrent control females on the day that vaginal patency was reached (not statistically significant for body weight at 1000 pm). Additionally, females of the treatment groups were slightly older on the day of first estrus
than concurrent control females (not statistically significant). These differences were attributed to relatively low control values and were therefore considered
unrelated to treatment with the test item.
Historical Control Data for Wistar Han rats; F1-females (2017-2021):
- Vaginal opening (VO; PND): mean = 31.4; P5-P95 = 27.00-35.00 (n=722)
- Body weight on day of VO (gram): mean = 95; P5-P95 = 72.5-115.0 (n=720)
- First estrus (PND): mean = 35.3; P5-P95 = 31.00-39.00 (n=239)
See table 58 in the field below 'Attached backgrowd material'. - Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- F1 - GENERATION - F1-PUPS:
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
See table 44 in the field below 'Attached backgrowd material'. - Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- F1 - GENERATION – F1-PUPS:
Treatment up to 3125 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
See table 44 in the field below 'Attached backgrowd material'. - Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- F1 - GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B and Cohort Surplus):
Cohort 1A (PND 89-95)
The differences noted in males for the absolute weights of pituitary gland (at 1000 and 3125 ppm: 0.87 and 0.89x of control, respectively) and thymus (at 1000 and 3125 ppm: 0.84 and 0.86x of control, respectively), and relative weights of brain (at 3125 ppm: 1.08x of control), heart (at 3125 ppm: 1.05x of control),
liver (at 3125ppm: 1.05x of control) and kidney (at 1000 and 3125 ppm: 1.07 and 1.15x of control, respectively) were attributed to the lower terminal body
weights compared to concurrent control (statistically significant in F1 males at 1000 ppm (8% lower) and at 3125 ppm (9% lower)).
In females at 3125 ppm, there were statistically significant organ weight changes in the relative weight of the liver (1.08x of control) and spleen (1.14x control), which were considered to be mainly caused by a lower final body weight (5% lower).
Any other differences in organ weights of males and females including those that reached statistical significance (Males: absolute weights of brain and heart at 1000 ppm, prostate gland at 300 ppm, and relative weight of kidney at 300 ppm) were considered not to be 4 tert-butylpyrocatechol-related due to the lack of a dose-related pattern, lack of a test item-related microscopic correlate and/or general overlap and variability in individual values.
Cohort 1B (≥ PND 90)
There were no test item-related alterations in organ weights up to 3125 ppm.
Cohort Surplus (PND 22-24)
Weights of brain, thymus and spleen (absolute and relative to body weight) of treated PND 22-24 males and females were considered to be similar to those of control animals.
See tables 59 and 59 bis in the field below 'Attached backgrowd material'. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- F1-GENERATION: Macroscopy until Weaning – F1-Pups:
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test
item. For the pup of Litter No. 113 (control) that was found dead on PND 18, the stomach was noted to be grown together with the pancreas at necropsy.
Additionally, for the majority of the pup(s) of Female Nos. 125 (control), 134 (300 ppm), 164, 171, 173 (1000 ppm) that were found dead at first litter check,
beginning autolysis and/or absence of milk in the stomach were noted. For some pups cannibalism was observed. The nature and incidence of these and
other macroscopic findings remained within the range considered normal for pups of this age or occurred in the control group only, and were therefore
considered unrelated to the test item.
See table 45 in the field below 'Attached backgrowd material'.
F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Suplus)
Cohort 1A (PND 89-95)
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B (≥ PND 90)
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1C (males: ≥ PND 35; females: ≥ PND 25)
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort Surplus (PND 22-24)
No macroscopic abnormalities were observed.
See table 60 in the field below 'Attached backgrowd material'. - Histopathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathology – PND 4 F1-PUPS:
There were no test item-related microscopic findings in the stomach of the selected PND 4 pups.
The only recorded finding was a cyst in the stomach of a control male pup.
See table 46 in the field below 'Attached backgrowd material'.
F1-GENERATION FROM WEANING ONWARDS (Cohorts 1A and Suplus):
Cohort 1A (PND 89-95):
Test item-related microscopic findings after treatment with 4-tert-butylpyrocatechol were noted in the stomach of the 3125 ppm males and females and are
summarized in the table 59. See table 59 in the field below 'Any other information on results incl. tables'.
Non-glandular stomach (i.e. forestomach): Hyperkeratosis was present at a high incidence and up to slight degree in both sexes at 3125 ppm. In some
animals, this was accompanied by diffuse squamous cell hyperplasia (up to slight degree) and or focal erosions of the non glandular stomach. The minimal
diffuse hyperkeratosis in a 1000 ppm male and female were considered to be spontaneous findings.
A finding of note was present in a single 3125 ppm male (No. 424) consisting of marked testicular tubular atrophy (mostly Sertoli cell only) and a massive
reduction of the presence of sperm in the lumen of the epididymides, correlating in both organs with the macroscopic finding reduced size. This was
considered to be unrelated to the test item due to the single incidence and absence of test item-related findings in the testes or epididymides and presence of normal progression of the spermatogenic cycle or the expected cell associations and proportions in the various stages of spermatogenesis in all other males of Cohort 1A.
A follicular cell adenoma, a benign tumor, was detected in the thyroid gland (unilateral) of a female at 3125 ppm (No. 705). Since there was no further
evidence of test item-related proliferative lesions in the thyroid gland and this tumor occurred in a single animal, this was regarded as a spontaneous finding.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were no test item-related effects on the ovarian follicle and corpora lutea counts in the F1 3125 ppm group females (Cohort 1A) when compared to control group females. Any variations between group mean counts represented biological variability and were not statistically significant.
See table 61 in the field below 'Any other information on results incl. table'.
Cohort Surplus (PND 22-24):
There were no test item-related findings in the stomach. The few microscopic findings noted (inflammation in a single control male and dilated glands in a few animals) did not distinguish treated animals from control animals and were regarded to be spontaneous in nature.
See table 46 in the field below 'Attached backgrowd material'. - Other effects:
- no effects observed
- Description (incidence and severity):
- ESTROUS CYCLE - F1-GENERATION FROM WEANING ONWARDS (Cohort 1A):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had a regular cycle of 4-5 days between
PND 75 to 88. For one female at 1000 ppm (No. 624) the cycle classification could not be determined and one female at 3125 ppm (No. 710) was acyclic.
Given the incidental nature, these findings did not indicate a relation with the test item.
See table 62 in the field below 'Attached backgrowd material'.
SPLENIC LYMPHOCYTE SUBPOPULATION - F1-GENERATION FROM WEANING ONWARDS (Cohort 1A):
There were no test item-related changed in splenic lymphocyte subpopulations of Cohort 1A animals.
A statistically significantly higher NK-cell subpopulation of splenic lymphocytes was observed for Cohort 1A females at 3125 ppm (1.25x of control). This shift was considered to represent biological variability and therefore not to be related to treatment with the test item, because this shift was slight of nature and
occurred in the absence of either a dose-related trend or apparent changes in lymphoid cellularity of the spleen as revealed by histopathology evaluation.
There were no indications that other splenic lymphocyte subpopulations were affected in Cohort 1A males or females.
See table 63 in the field below 'Attached backgrowd material'.
SPERM ANALYSIS - F1-GENERATION FROM WEANING ONWARDS - Cohort 1A):
There were no test item-related changes in sperm motility, concentration and morphology in males treated up to 3125 ppm.
See table 64 in the field below 'Attached backgrowd material'. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effect (general toxicity)
- Remarks on result:
- other:
- Remarks:
- No adverse effects were observed.
- Dose descriptor:
- LOEL
- Generation:
- F1
- Effect level:
- ca. 300 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- haematology
- clinical biochemistry
- histopathology: non-neoplastic
- Remarks on result:
- other:
- Remarks:
- 1) Body weight: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6 lower than control for males at 3125 ppm, respectively.As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse. 2) Hematology and clinical biochemistry: In F1-generation animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse. 3) Effect on the glandular stomach: at 3125 ppm of TBC diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia (Cohort 1A) were observed. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, these findings was considered as a local, non-adverse test item-related effect.
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- < 300 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- haematology
- clinical biochemistry
- histopathology: non-neoplastic
- Remarks on result:
- other:
- Remarks:
- 1) Body weight: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6% lower than control for males at 3125 ppm, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse. 2) Hematology and clinical biochemistry: In F1-generation animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse. 3) Effect on the glandular stomach: at 3125 ppm of TBC diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia (Cohort 1A) were observed. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, these findings was considered as a local, non-adverse test item-related effect.
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 3 125 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No developmental effect.
- Remarks on result:
- other:
- Remarks:
- Developmental toxicity: No developmental toxicity was observed up to the highest dose level tested (3125 ppm).
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- - This extended one generation showed that TBC induced mean body weight gain reduction in males and females F0-generation animals in presence of TBC at
3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period and in males F1-generation males at 1000 and 3125 ppm, resulting in a
lower mean body weight throughout the treatment period.
- At 3125 ppm (the higher tested dose), test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item related findings consisted of an increased incidence and severity of diffuse hyperkeratosis with or without squamous cell hyperplasia. Similar effects were observed to the F1-generation animal non-glandular stomach at 3125 ppm. The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia compared to the stomachs of the F0-generation at 3125 ppm.
Based on this study, no general, reproductive or developmental toxicity was observed up to the highest dose tested (3125 ppm). - Executive summary:
The objective of this study was to provide an evaluation of the pre- and postnatal effects of 4-tert-butylpyrocatechol on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of 4-tert-butylpyrocatechol on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. The dose levels in this study were selected to be 0, 300, 1000 and 3125 ppm, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of 4-tert-butylpyrocatechol in rats. The same dietary concentrations were used throughout the study, except for the lactation period. As food intake is considerably higher in lactating females, dietary concentrations were lowered for all treated groups (based on historical control data for relative food consumption) during the lactation period (PND 1-24). Chemical analyses of dietary preparations were conducted at nine occasions during the study to assess accuracy and homogeneity.
For the F0-generation, the following parameters and endpoints were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F0-generation: mating and fertility indices, precoital time, estrous cycle determination, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones) and histopathological examination of the stomach (PND 4 and PND 22-24 pups).
Results: Analysis of diet preparations confirmed that the test item was homogeneously distributed in the diet. The achieved dietary concentrations of 4-tert-butylpyrocatechol were however not always within the acceptance criterion of 80 – 120 %. A thorough check of the diet preparation procedures and analytical methodology revealed no explanation for the lower accuracies. In order to determine the worst case scenario exposure, test item intake was corrected for the mean achieved dietary concentrations per study period.
No mortality occurred during the study period that was considered to be related to treatment with the test item. In the F0-generation, one female of the control and 1000 ppm groups each, were euthanized on Lactation Day 2 or 1, respectively, as these females had a total litter loss. In the F1-generation, one male (1000 ppm, Cohort 1A) was sacrificed 5 days post weaning as it was assigned to the females. No general, reproductive, or developmental toxicity was observed up to the highest dose tested (3125 ppm).
The following test item-related, non-adverse, effects were observed:
Parental results: Mean body weight gain was reduced in males and females at 3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period. For females at 3125 ppm, no relevant differences were noted during the gestation period, whereas a higher mean body weight gain was observed from Day 7 of lactation onwards resulting in a slight recovery in mean body weight. Mean terminal body weight was 9 and 4% lower than concurrent control mean for males and females, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.
In animals at 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected by treatment with the test item, including decreased platelets counts in females, and decreased mean bile acid and creatinine concentrations in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology and as such they were regarded as non-adverse. In addition, mean serum levels oft hyroid stimulating hormone (TSH) were dose-dependently increased for females at all dose levels. As individual values generally remained within the historical control range, the change in serum TSH in the females was considered not to be adverse.
At 3125 ppm, test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item-related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia. Since the recorded severities of these findings in adults remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.
Developmental results: Mean serum levels of TSH were decreased for male and female PND 22-24 pups (Cohort Surplus) at 3125 ppm. As individual values remained within the historical control range, the change in serum TSH was considered not to be adverse.
F1-generation results: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6% lower than control for males at 3125 ppm, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.
In animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected, including increased mean lymphocyte counts and as a result increased mean total white blood cell count and mean large unstained cell count in both sexes, and decreased mean total bilirubin concentration in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse.
Similar to the F0-generation, test item-related findings were present in the non-glandular stomach at 3125 ppm. These test item‑related findings consisted of an increased incidence and severity of diffuse hyperkeratosis(up to slight degree) with or withoutsquamous cell hyperplasia.The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia (incidence of 50% in males and females 60%, up to slight degree) compared to the stomachs of the F0-generation at 3125 ppm (incidence of 20% in males and 8% in females, at minimal degree). Furthermore, there were focal erosions (minimal degree) in the non-glandular stomach in 2/20 F1-rats of both sexes at 3125 ppm. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.
In conclusion, based on the results of this extended one-generation reproductive toxicity study (including Cohorts 1), the following No Observed Adverse Effect Levels (NOAELs) of 4-tert-butylpyrocatechol were established:
General toxicity NOAEL:
F0-generation : at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 172-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
F1-generation: at least 3125 ppm (on average corresponding to a corrected test article intake of 221 and 230 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).Reproduction NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
Developmental NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144 -172 and 176 -194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
The test item (4 -tert-butylpyrocatechol) did not induce reprotoxic effects neither developmental effects up to the highest tested dose (3125 ppm)
Referenceopen allclose all
F0 data
Tables:
Table 1: Test Article Intake
Group No. Nominal dietary inclusion level (ppm) |
Mean over means intake mg test item/kg body weight] (mean range indicated within brackets) |
|
|||
Group 2 |
Group 3 |
Group 4 |
|||
Sex |
Study period |
|
|||
Males |
Pre-mating |
201 (133-223) |
382 (143-464) |
631 (99-875) |
|
|
Post-mating |
185 (147-201) |
359 (306-397) |
726 (586-889) |
|
|
*Mean of means |
193 |
371 |
677 |
|
|
|
|
|
|
|
Females |
Pre-mating |
199 (90-237) |
375 (96-459) |
579 (120-885) |
|
|
Post-mating |
199 (77-234) |
364 (289-405) |
698 (481-946) |
|
|
Lactation |
470 (239-641) |
884 (463-1219) |
470 (239-641) |
|
|
*Mean of means |
273 |
507 |
854 |
|
*Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((14x mean premating) + (13 x mean mating)) / 27
Females Group 2: ((14 x mean premating) + (23 x mean post-coitum) + (14 x mean lactation)) / 51
Females Group 3: ((14 x mean premating) + (24 x mean post-coitum) + (14 x mean lactation)) / 52
Females Group 4: ((14 x mean premating) + (25 x mean post-coitum) + (14 x mean lactation)) / 54
Table 2: Summary test item-related microscopic stomach findings - Scheduled euthanasia animals
|
Males |
Females |
||||||
Dose level (ppm): |
0 |
3125 |
6250 |
12500 |
0 |
3125 |
6250 |
12500 |
|
|
|
|
|
|
|
|
|
STOMACHa |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
Hyperkeratosis - forestomach |
|
|
|
|
|
|
|
|
minimal |
- |
4 |
- |
- |
- |
5 |
4 |
2 |
slight |
- |
6 |
5 |
4 |
- |
2 |
2 |
5 |
moderate |
- |
- |
4 |
6 |
- |
- |
1 |
3 |
|
|
|
|
|
|
|
|
|
Hyperplasia, squamous - forestomach |
|
|
|
|
|
|
|
|
minimal |
- |
5 |
4 |
6 |
- |
2 |
4 |
6 |
slight |
- |
- |
2 |
4 |
- |
- |
- |
3 |
|
|
|
|
|
|
|
|
|
Ulceration - forestomach |
|
|
|
|
|
|
|
|
minimal |
- |
1 |
- |
- |
- |
- |
- |
- |
slight |
- |
- |
1 |
- |
- |
- |
- |
- |
|
|
|
|
|
|
|
|
|
Erosion - glandular: limiting ridge |
|
|
|
|
|
|
|
|
minimal |
- |
1 |
- |
- |
- |
- |
- |
2 |
slight |
- |
- |
- |
- |
- |
- |
- |
2 |
a = Number of tissues examined from each group.
Other tables are attached in the field below 'Attached background material'.
DIET ANALYSES:
Accuracy
In the chromatograms of the Group 1 diets prepared for use in Weeks 3 and 26, a small response was observed at the retention time of the test item. It was considered not to derive from the diet since a similar response was obtained in the analytical blanks. The maximum contribution to the Group 2 samples was 0.71% based on peak area and taking dilution factors into account. In the diets of Group 1 prepared for use in Weeks 1, 6, 11, 14, 15, 16 and 21, no test item was detected.
An overview of the obtained accuracy results for Group 2, 3 and 4 diets is given in the tables below.
Table 19: Mean Accuracy of Diets F0-Generation from Premating up to Lactation
Occasion |
Study Phase |
Group 2 |
Group 3 |
Group 4 |
Week 1 of treatment |
Premating period |
67% |
69% |
78% |
Week 3 of treatment |
61% |
67% |
75% |
|
Week 6 of treatment |
68% |
76% |
82% |
|
Mean of Means Accuracy Premating Period |
65 % |
71% |
78% |
|
Week 11 of treatment |
Mating period |
76% |
80% |
83% |
Table 20: Mean Accuracy of Diets F0-Generation during Lactation
Occasion |
Study Phase |
Group 2a |
Group 3a |
Group 4a |
Week 14 of treatment |
Days 1-3 |
70% |
73% |
77% |
Week 15 of treatment |
Days 4-6 |
69% |
68% |
73% |
Week 16 of treatment |
Day 7 onwards |
63% |
70% |
72% |
a During the lactation period, the following dietary concentrations were used (based on historical control data for relative food consumption):
Lactation |
Group 2 |
Group 3 |
Group 4 |
Days 1-3 |
200 ppm |
666 ppm |
2085 ppm |
Days 4-6 |
150 ppm |
500 ppm |
1563 ppm |
Day 7 onwards |
120 ppm |
400 ppm |
1250 ppm |
Table 21: Mean Accuracy of Diets F1-Generation
Occasion |
Group 2 |
Group 3 |
Group 4 |
Week 21 of treatment |
70% |
73% |
81% |
Week 26 of treatment |
75% |
78% |
81% |
Mean of Means Accuracy Treatment Period |
73% |
76% |
81% |
For the diet samples of Groups 2, 3 and 4, prepared for use in Weeks 1, 3, 6, 11, 14, 15, 16, 21 and 26, the mean accuracy of the diet samples was 61 – 83%. Mean accuracy seemed to increase with an increase in the target concentration of the diet. Since the mean accuracy was not always within the acceptance criterion of 80 – 120 %, several out of specification investigations were performed. During these investigations, the diet preparation procedure as well as the analytical procedure was scrutinized. As part of the investigation for samples from diets prepared for Week 3 of treatment, presence of test item – metal complexes or oxidized test item (Quinones) which may absorb light at a different wavelength was checked in the extracts. The possibility of oxidation was based on information provided by the sponsor and published literature. UV-Vis spectra from 200-900 nm were recorded for one sample of each group. The spectra were recorded after extraction, filtration and further dilution if required. These spectra were compared with spectra for blank extraction solvent and with spectra for two calibration solutions. Since it was not possible to draw a conclusion from the UV-Vis spectra, the samples were subsequently analyzed on the UPLC-UV using the validated method, with UV detection at a general wavelength of 210 nm. The chromatograms of the calibration solutions and diet samples showed one major test item peak at 1.4 min. In the spectra of the diet samples, there were several peaks visible between 1.8 and 4.0 min which were attributed to the diet matrix. No other test item-related peaks such as test item - metal complexes or oxidized test item (Quinones) were visible. However, no detailed examination or identification of these peaks has been carried out, thus it is not possible to assert that these peaks were linked only to the diet matrix.
The conclusion of all out of specification investigations was that no explanation could be found for the low mean accuracy results for the diet samples of Groups 2, 3 and 4.
Homogeneity
The diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation≤ 10%).
F0 - GENERATION RESULTS:
TEST ITEM INTAKE:
Table 26: Test article intake over the study period (F0 - generation).
|
Mean of Means Intake (Mean Range Indicated within Brackets) |
||||||
|
Group No. |
2 |
3 |
4 |
|||
Nominal Dietary Inclusion Level (ppm)(b) |
300 |
1000 |
3125 |
||||
|
|
||||||
Sex |
Study Period |
|
|
|
|||
Males |
Pre-mating |
21 |
(16-30) |
70 |
(55-98) |
221 |
(177-303) |
Post-mating |
15 |
(13-16) |
52 |
(48-56) |
173 |
(159-195) |
|
Mean of Means(a) |
20 |
66 |
211 |
||||
|
|
||||||
Females |
Pre-mating |
24 |
(20-31) |
82 |
(68-103) |
249 |
(210-310) |
Post-coitum Days 0-23 Post-coitum Days 0-24 |
- 22 |
- (12-26) |
74 - |
(41-85) - |
218 - |
(151-255) - |
|
Lactation Days 1-3 |
22 |
(18-26) |
80 |
(65-94) |
251 |
(207-276) |
|
Lactation Days 4-6 |
21 |
(21-21) |
75 |
(72-79) |
241 |
(230-247) |
|
Lactation Days 7-24 |
23 |
(15-31) |
80 |
(43-102) |
254 |
(134-334) |
|
Mean of Means (a) |
23 |
80 |
243 |
||||
- = not applicable.
(a) Mean of means of all periods, weighed for number of measurement intervals per period: Males ((70 * mean premating) + (18 * mean mating)) / 88, Females: ((70 * mean premating) + (23 or 24 mean post-coitum) + (23 * mean lactation)) / 116 or 117.
(b) During the lactation period, dietary concentration of 4 -tert-butylpyrocatechol were lowered to maintain constant test item (based on historical control data for relative food consumption).
Table 27: Corrected test article intake over the study period (F0 -generation).
|
Mean of Means Intake[mg Test Item/kg Body Weight] Corrected for Mean of Mean Accuracy of Prepared Diets |
|||
|
Group No. |
2 |
3 |
4 |
Nominal Dietary Inclusion Level (ppm) |
300 |
1000 |
3125 |
|
|
|
|||
Sex |
Study Period |
|
|
|
Males |
Pre-mating |
14 |
50 |
172 |
Post-mating |
11 |
42 |
144 |
|
|
|
|||
Females |
Pre-mating |
16 |
58 |
194 |
Post-coitum Days 0-23
|
- 17 |
59 - |
181 - |
|
Lactation Days 1-3 |
16 |
59 |
193 |
|
Lactation Days 4-6 |
14 |
51 |
176 |
|
Lactation Days 7-24 |
14 |
56 |
183 |
|
HISTOPATHOLOGY:
Table 35: Summary of test item-related microscopic findings (F0 - generation).
|
Males |
Females |
||||||
Dose level (ppm): |
0 |
300 |
1000 |
3125 |
0 |
300 |
1000 |
3125 |
|
|
|
|
|
|
|
|
|
NON-GLANDULAR STOMACHa |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
Squamous cell hyperplasia |
|
|
|
|
|
|
|
|
Minimal |
- |
- |
- |
5 |
- |
- |
- |
2 |
Hyperkeratosis, diffuse |
|
|
|
|
|
|
|
|
Minimal |
- |
1 |
1 |
22 |
1 |
3 |
2 |
21 |
Slight |
- |
- |
- |
3 |
- |
- |
- |
- |
a = Number of tissues examined from each group.
F1- GENERATION:
- F1 - GENERATION FROM WEANING ONWARDS (Cohorts 1A, 1B, 1C and Surplus):
TEST ITEM INTAKE:
Table 51: Mean test article intake (F1 - generation)
|
Mean of Means Intake (Mean Range Indicated within Brackets) |
||||||
|
Group No. |
2 |
3 |
4 |
|||
Nominal Dietary Inclusion Level (ppm) |
300 |
1000 |
3125 |
||||
|
|
||||||
Sex |
Study period |
|
|
|
|||
Males |
Treatment |
26 |
(15-42) |
85 |
(48 - 139) |
273 |
(175-439) |
|
|
||||||
Females |
Treatment |
27 |
(20-41) |
89 |
(65-134) |
284 |
(217-436) |
Table 52: Corrected mean test article intake (F1 generation):
|
Mean over Means Intake [mg Test Item/kg Body Weight] Corrected for Mean of Mean Accuracy of Prepared Diets |
|||
|
Group No. |
2 |
3 |
4 |
Nominal Dietary Inclusion Level (ppm) |
300 |
1000 |
3125 |
|
|
|
|||
Sex |
Study period |
|
|
|
Males |
Treatment |
19 |
65 |
221 |
|
|
|||
Females |
Treatment |
20 |
68 |
230 |
F1 -GENERATION - COHORT 1A
Table 53: Summary Test Item-Related Hematology Changes (fold change to Control) – F1-Cohort 1A
|
Males |
Females |
||||
Dose level (ppm): |
300 |
1000 |
3125 |
300 |
1000 |
3125 |
No. of animals analyzed |
10 |
10 |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
WBC (109/L) |
1.19 |
1.20 |
1.34 |
1.15 |
1.39* |
1.39* |
LYMPH (109/L) |
1.26 |
1.25 |
1.49** |
1.17 |
1.41* |
1.47** |
LUC (109/L) |
1.11 |
1.41 |
2.14# |
1.46 |
1.62 |
1.92 |
#Kruskal-Wallis & Dunn: = p ≤ 0.05
* / ** Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01
Table 61. Summary test-item-related microscopic findings - F1 -Cohort 1A.
|
Males |
Females |
||||||
Dose level (ppm): |
0 |
300 |
1000 |
3125 |
0 |
300 |
1000 |
3125 |
|
|
|
|
|
|
|
|
|
NON-GLANDULAR STOMACHa |
20 |
20 |
20 |
20 |
20 |
20 |
20 |
20 |
Erosion, |
|
|
|
|
|
|
|
|
Minimal |
- |
- |
- |
2 |
- |
- |
- |
2 |
Squamous cell hyperplasia |
|
|
|
|
|
|
|
|
Minimal |
- |
- |
- |
7 |
- |
- |
- |
8 |
Slight |
- |
- |
- |
3 |
- |
- |
- |
4 |
Hyperkeratosis, diffuse |
|
|
|
|
|
|
|
|
Minimal |
- |
- |
1 |
13 |
- |
- |
1 |
11 |
Slight |
- |
- |
- |
5 |
- |
- |
- |
8 |
a = Number of tissues examined from each group. |
||||||||
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 144 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- This study followed the test guideline OECD 443 (basic design). Klimish cotation 1.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
An extended one-generation study has been conducted on rats (testing proposal) based on some reproductive toxicity alerts in 3-month repeated dose toxicity studies in rats and mice, where sperm and vaginal cytology observations were performed (ref. 1) (see also section 7.8.1, study endpoint records 'Tox.repro rat V1 2002NTP' and 'Tox.repro. mouse V1 2002NTP'). Significant reductions in male reproductive organ weights and sperm count or motility were noted in male rats at the highest dose level of 12500 ppm after 14 weeks of administration. In female rats, significantly fewer estrous cycles than controls were seen at all dose levels assessed (from 3125 ppm) and the estrous cycle duration increased with concentration, the females spending more time in diestrus than controls. In female mice also, significantly longer estrous cycles were seen at the highest dose level of 12500 ppm.
In order to perform the extended one-generation study, a Dose Range finding study by gavage, similar to the OECD 421 test guideline (see section 7.8.1, the endpoint study record 'DRF_Repro screening test rats (gavage)_Charles River 2020a') was first conducted. In this Dose Range finding study, rats were exposed by gavage to 0, 50, 100 and 200 mg/kg/day of TBC. During this study, it was discovered that TBC was too corrosive to test high enough dose levels when given by gavage. The Dose Range Finding study by gavage was cancelled mid-study. For animal welfare reasons and in order to test higher dose levels, a palatability study (see section 7.8.1, the endpoint study record '14 day palatability study rats (diet)_Charles River V1 2020b'), a reproduction/developmental toxicity screening study (see section 7.8.1, the endpoint study record 'DRF_Repro screening test rats (diet)_Charles River V1 2020b') and the extended one-generation study (see section 7.8.1, the endpoint study record 'Extended one generation study rats (diet)_Charles River 2021') were performed via dietary administration of the registered substance. The dose levels in the extended one-generation study are 0, 300, 100 and 3125 ppm and were selected based on the results of the preliminary reproductive toxicity study (Range finding study - reproduction/developmental toxicity screening test) with dietary of TBC in rats. Based on the extended one-generation study performed by diet, no reproductive or developmental toxicity was observed in rats up to the highest dose tested (3125 ppm). It can be therefore concluded that 4-tert-butylpyrocatechol does not induce effect on reproduction and on development up to the dose level of 3125 ppm. The effects noted in the 3-month repeated toxicity studies (reductions in male reproductive organ weights and sperm count or motility, fewer estrous cycles in females compared to the control group and increase of estrous cycle duration) were not observed in the extended one-generation study or were not considered as adverse or with toxicological relevance. In the F0-generation animals (extended one-generation study), there were no statistically changes in absolute organ weights of the males. Statistically significant higher mean organ weights relative to body weights compared to the current control group values were observed at 3125 ppm in males in testes (1.12x of control) and epididymes (1.10x of control). However, these organ weight changes were without microscopic correlate and were in line with lower body weight (statistically significant in 3125 ppm F0-males, 9% lower compared to concurrent controls). Mean percentage of motile and progressive sperm were lower in treated males when compared with control. The percentage of motile sperm was 0.79, 0.82 and 0.88x of control and of progressive sperm 0.64, 0.68 and 0.79x of control for the 300, 1000 and 3125 ppm groups, respectively (not statistically significant for percentage of motile sperm at 3125 ppm). All individual values (except for one male at 300 ppm) remained within the normal range for rats of this age and strain. In absence of a dose-related response and given the absence of any reproductive/developmental toxicity, these differences were considered not toxicologically relevant. Sperm concentration and morphology were considered to be unaffected by treatment with the test item. The mean sperm count in males at 300 and 1000 ppm was increased (1.22 and 1.18x of control, respectively). Due to the direction of change and in the absence of a dose-related response, this difference was considered unrelated to the test item. In F0-generation females, length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days. In F1-generation animals (cohorts 1A and 1B), there were no difference between treated groups and control group in absolute and relative weights of reproductive organs. In cohort 1A, there were no test item-related changes in sperm motility, concentration and morphology in males treated up to 3125 ppm. In F1-generation females (cohort 1A), length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had a regular cycle of 4-5 days between PND 75 to 88.
1.Dunnick J.K. NTP Technical Report on the Toxicity Studies of 'p-tert-Butylcatechol' (CAS No. 98-29-3) Administered in Feed to F344/N Rats and B6C3F1 Mice. Toxicity Report Series Number 70 (2002).
Dose level selection for the extended one-generation study:
The dose levels in the extended one-generation study were selected based on the results of the preliminary reproductive toxicity study (see section 7.8.1, the endpoint study record ‘DRF_Repro screening test rats (diet)_Charles River V1 2020a) and in an attempt to produce graded responses to the test item. In the preliminary study, Wistar Han rats received 0, 3125, 6250 and 12500 ppm of TBC by dietary administration for at least 28 days. No mortalities were observed during the treatment period. The following major findings were noted:
- At 12500 ppm, females showed piloerection and some showed a hunched posture during the second and third week of treatment. Body weight loss up to 9% was observed in both males and females and the first week, body weight (gain) and food consumption of both sexes remained distinctively lower when compared to concurrent controls throughout the treatment period. At necropsy, the majority of the animals had an irregular surface of the stomach. Most females delivered a healthy litter, however pup body weights were distinctively lower. During the last body weight measurement (PND 13), mean combined pup body weights were 40% lower than concurrent controls.
- At 6250 ppm, no clinical signs were noted. Body weight (gain) and food consumption of both sexes were lower than concurrent control throughout the treatment period, however to a lesser extent than observed for animals treated at 12500 ppm. At necropsy, the majority of the animals had an irregular surface of the stomach. Most females delivered a healthy litter, however pup body weights were distinctively lower. During the last body weight measurement (PND 13), mean combined pup body weights were 25% lower than concurrent controls.
- At 3125 ppm, no clinical signs were noted. Food consumption of both sexes was slightly lower during the first week of administration, but recovered to similar values as concurrent controls during subsequent intervals. Mean body weight gain of males was lower than concurrent controls during the treatment period. For females, body weight gain was similar to concurrent controls during the premating and post-coitum periods but slightly lower during lactation. An irregular surface of the stomach was observed in 2 males only at necropsy. All females delivered a healthy litter, however pup body weights were distinctively lower. During the last body weight measurement (PND 13), mean combined pup body weights were 15% lower than concurrent controls.
In the extended one-generation study, the total duration of exposure was longer than in the preliminary reproductive toxicity study; there was be a 10-week premating period as opposed to the 2-week premating period. It was therefore expected that the effects on body weight and food consumption may become more severe after a longer treatment period. In addition, during extended one-generation study, F1-animals were weaned at PND 21 and treated afterwards. Given the effects observed in the preliminary study in the F0-animals and the pup body weights, dose levels of 6250 and 12500 ppm were not considered appropriate for the extended one-generation study. Considering this information, a dose level 3125 ppm was selected as high dose. Low and mid dose levels were selected to be 300 and 1000 ppm, respectively.
3125 ppm was selected as highest dose level, based on the clear toxicity that was observed from 6250 ppm onwards during a preliminary reproductive toxicity study . Due to the longer treatment period in the current study, dose levels higher than 3125 ppm were considered not appropriate.
Possible degradation of 4-tert-butylpyrocatechol during the extended one-generation study:
Chemical analyses of dietary preparations were conducted at nine occasions during the extended one-generation study to assess accuracy and homogeneity.
Lower accuracies were measured for the prepared powder diets in the extended one-generation study. For the diet samples of Groups 2, 3 and 4, prepared for use in Weeks 1, 3, 6, 11, 14, 15, 16, 21 and 26, the mean accuracy of the diet samples was 61 – 83% (for more detail, see the endpoint study record for the extended one-generation study). Mean accuracy seemed to increase with an increase in the target concentration of the diet. The achieved dietary concentration of 4 -tert butylpyrocatechol were therefore not always within the acceptance criterion of 80% - 120%. Lower accuracies were not observed in the preliminary study, where analysis of the accuracy revealed acceptable levels within the acceptance criterion of 80% to 120%. This difference in accuracies can be related to the fact that higher concentration of TBC have been tested in the preliminary study. Since in the extended one-generation study the mean accuracy was not always within the acceptance criterion of 80 – 120 %, several out of specification investigations have been performed by the laboratory in which the extended one-generation study was conducted. During these investigations the diet preparation procedure as well as the analytical procedure was scrutinized. The details on these investigations are summaried in the endpoint study record for the extended one-generation study. The conclusion of all out of specification investigations was that no explanation could be found for the low mean accuracy results for the diet samples of Groups 2, 3 and 4.
4-tert-butylpyrocatechol in contact with air oxidizes to Quinones (Menter and Willis, 1980). See the publication attached below in the field 'Attached background material'. In this publication it is specified that 4-tert-butylpyrocatechol oxidizes to Quinones in contact with air and that this oxidation leads to different Quinones depending on the pH. At pH 7.4, 4-tert-butylpyrocatechol is 6-hydroxylated to 2,4,5-trihydroxy-t-butylbenzene and then further oxidized to the corresponding p-quinone, 2-hydroxy-5-(t-butyl)-1,4-benzoquinone. The oxidized product at pH 9 may be the o-quinone of TBC, 4-(t-butyl)-1,2-benzoquinone.
During diet preparation with the test item, the test item (TBC), may be in contact with air and can oxidized. The decrease of the test item measured in the diet preparation (compared to the mesures done in the preliminary study) can be explained by the lower concentrations of TBC administered in the extended one-generation study. Indeed, the result of TBC oxidation (% of initial TBC oxidized) may be more important in proportion if initial concentration of TBC is low.
Calculation of the test item intake in the extended one-generation study:
Mean test article intake over the study period (for the extended one-generation study) was calculated. Lower accuracies were measured for the prepared powder diets. In order to determine the worst case scenario exposure, the mean of means test item intake corrected for the means accuracy per period was calculated. The corrected test article intake are the following:
- Corrected test article intake for F0-Generation:
300 ppm of TBC: corresponds to 11 to 14 mg/kg/day (males) / 14 to 16 mg/kg/day (females) (anticipated dose level = 30 mg/kg/day)
1000 ppm of TBC: corresponds to 42 to 50 mg/kg/day (males) / 51 to 59 mg/kg/day (females) (anticipated dose level = 100 mg/kg/day)
3125 ppm of TBC: corresponds to 144 to 172 mg/kg/day (males) / 181 to 194 mg/kg/day (females) (anticipated dose level = 300 mg/kg/day)
- Corrected test article intake for F1-Generation
300 ppm of TBC: corresponds to 26 mg/kg/day (males) / 27 mg/kg/day (females) (anticipated dose level = 30 mg/kg/day)
1000 ppm of TBC: corresponds to 85 mg/kg/day (males) / 89 mg/kg/day (females) (anticipated dose level = 100 mg/kg/day)
3125 ppm of TBC: corresponds to 273 mg/kg/day (males) / 284 mg/kg/day (females) (anticipated dose level = 300 mg/kg/day)
The corrected test article intakes are lower than the anticipated dose levels. In the extended one-generation study, the animals received less test item than targeted. No adverse toxicity were observed in F0-generation animals. However, non-adverse test item-related effects were observed in F0 and F1-generation animals. In parents (F0-generation), mean body weight gain was reduced in males and females at 3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period. Mean terminal body weight was 9 and 4% lower than concurrent control mean for males and females, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse. In animals at 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected by treatment with the test item, including decreased platelets counts in females, and decreased mean bile acid and creatinine concentrations in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. At 3125 ppm, test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item-related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia. Since the recorded severities of these findings in adults remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.
Despite the fact that the doses of TBC actually administered in rats in the extended one-generation study is lower than targeted and that only minimal general toxic effects were observed in F0-generation animals, it can still be concluded based on the extended one-generation study that the registered substance (TBC) is not reprotoxic up to 3125 ppm, the highest dose tested. For F0-generation animals this dose corresponds on a average to a corrected test article intake of 144 -172 for males and of 176 -194 mg/kg/day for females. For F1-generation animals, this dose corresponds to a corrected test article of 221 mg/kg/day for males and of 230 mg/kg/day for females.
In conclusion, based on the results of the extended one-generation reproductive toxicity study (including Cohorts 1), the following No Observed Adverse Effect Levels (NOAELs) of 4-tert-butylpyrocatechol were established:
General toxicity NOAEL:
- F0-generation at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm)
- F1-generation at least 3125 ppm (on average corresponding to a corrected test article intake of 221 and 230 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
Reproduction NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
Developmental NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
The dose of 3125 ppm was selected as highest dose level, based on the clear toxicity that was observed from 6250 ppm onwards during a preliminary reproductive toxicity study. Due to the longer treatment period in extended one-generation study, dose levels higher than 3125 ppm were considered not appropriate.
For information, the DNELs for systemic effects and long term exposure used for the risk assessment of the registered substance have been calculated from a LOAEL of 70 mg/kg/day identified based on the 14 -week rat dietary study. This LOAEL is lower than the reproductive/developmental NOAEL (144 mg/kg/day - 3125 ppm) identified in the extended one-generation study.
Effects on developmental toxicity
Description of key information
Two OECD 414 test guideline studies have been performed, one on rats (2013) and the other one on rabbits (2017). These studies have been selected as key studies.
The OECD 414 test guideline study performed on rats (CIT, 2013 - reliability 1) showed at 300 mg/kg/day (highest tested dose) non-statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations in a context of maternal toxicity. No malformation was noted and the increasing in fetal variations was considered to be of minor toxicological significance.
On the OECD 414 test guideline study performed on rabbits (CharlesRiver, 2017 - reliability 1), time-mated New Zealand rabbits were treated from day 6 to day 28 post-coitum by daily gavage at dose levels of 30, 30 and 100 mg/kg/day. No adverse developmental toxicity related to the substance was observed at all dose groups.
Therefore based on these two prenatal developmental studies TBC does not induce teratogenic effect.
In addition, an extended one-generation study perfomed in 2021 in diet (CharlesRiver, 2021 - reliability 1) (selected as supporting study for the development) showed no effect on the development in rats up to the highest dose tested (3125 ppm). This extented one-generation provided an evaluation of the pre- and postnatal effects of 4-tert-butylpyrocatechol on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was done.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 15 May 2013 to
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study, OECD 414 compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certificate n°2012/96 dated 10 January 2013
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- - Number: 96 female rats were received at CiToxLAB France between 22 and 31 May 2013.
- Strain and sanitary status: Sprague-Dawley, Rj Han: SD (IOPS Han).
- Breeder: Janvier, le Genest-Saint-Isle, France.
- Age/Weight: at the beginning of the treatment period, the females were 10-11 weeks old and had a mean body weight of 250 g (range: 202 g to 309 g). The females were sexually mature and primigravid.
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm², 48 cm x 26.5 cm x 21 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was selected in order to not jeopardize gestation.
Each cage contained an object for the environmental enrichment of the animals (rat hut).
The cages were placed in numerical order on the racks.
- Food and water: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 4898480 and 6318584 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation: the animals were acclimated to the conditions of the study for a period of 5 days before the beginning of the treatment period (arrival of the females on day 1 post-coitum (p.c.)).
- Allocation to study: during the acclimation period, the animals were allocated to the groups, according to a stratification procedure based on body weight recorded on day 2 p.c., to ensure comparatively similar mean body weights of the groups.
- Identification: each animal was individually identified by an ear tattoo (unique CiToxLAB France identity number).
ENVIRONMENTAL CONDITIONS
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: about 12 cycles/hour of filtered, non-recycled air.
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- DOSE FORMULATION PREPARATION
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, mixed with the required quantity of vehicle and kept under continuous stirring for at least 30 minutes.
The test item dose formulations were prepared for up to 7 days (stability results up to 11 days, CiToxLAB France/Study No. 39960 AHS), stored at room temperature and protected from light.
Dose formulations were delivered at room temperature and protected from light (in brown flasks).
VEHICLE
The vehicle was corn oil, batch No. MKBH4894V. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Concentrations of the test item in the dose formulations have been quantified by a validated analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 39959 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report. - Details on mating procedure:
- - Mating: the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).
- Duration of treatment / exposure:
- The dose formulations were administered daily from day 6 to day 20 p.c., inclusive.
- Frequency of treatment:
- Daily
- Duration of test:
- One month
- No. of animals per sex per dose:
- 24 mated females per group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2 week dose-range finding study by oral route (gavage) in rats (CiToxLAB France/Study No. 39961 RSR) where a total of 20 time-mated female Sprague-Dawley rats were allocated to 4 groups and received the test item at 50, 150 or 400 mg/kg/day, or the vehicle (corn oil) once daily from day 6 until day 20 post-coitum (p.c.).
In this study, one pregnant dam given 400 mg/kg/day was prematurely sacrificed on day 12 p.c. for ethical reasons (emaciated appearance, hypoactivity, loud breathing, abdominal breathing, dyspnea, ptyalism, eyes half-closed and 8% of body weight loss between days 6 and 9). At 150 mg/kg/day, all females had ptyalism and one had loud breathing. At 400 mg/kg/day all females had ptyalism and loud breathing. Furthermore, when compared with controls, there were marked decreases in mean body weight ( 17% vs. controls on day 21 p.c.) and mean body changes (-38% for the period of days 6 to 21 p.c.) associated with reduced mean food consumption at the same dose level (up to -61% for the period of days 6 to 9 p.c.). At hysterectomy, there were no obvious effects on mean reproductive parameters but a significant decrease in mean fetal body weight at 400 mg/kg/day (-12.5% vs. controls). There were no findings at external examination of the fetuses.
Therefore, 400 mg/kg/day was considered to be a dose-level associated with excessive maternal toxicity and 300 mg/kg/day was selected as the high dose-level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 100 mg/kg/day). - Maternal examinations:
- MORBIDITY AND MORTALITY:
Each animal was checked for mortality and morbidity at least once a day before and after the treatment period and at least twice a day during the treatment period, including weekends.
CLINICAL SIGNS:
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.
BODY WEIGHT:
The body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..
FOOD CONSUMPTION:
The quantity of food consumed by each female was recorded for the following intervals:
- days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..
POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21 p.c.
- Macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- Other organs examined: liver, vagina, colon, jejunum and thymus
Any macroscopic lesions observed were sampled and kept preserved in 10% buffered formalin The corresponding tissues of five control animals were sampled and preserved. - Ovaries and uterine content:
- The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.
The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with discernible digits.
A gross evaluation of placentas was also undertaken. - Fetal examinations:
- Fetal examination was conducted for all litters where the female had at least one live fetus.
BOBY WEIGHT OF FETUSES:
The body weight of each live fetus was recorded.
SEX oF FETUSES:
The sex of each fetus (excluding any autolyzed fetuses) was determined at the time of hysterectomy.
The sex of fetuses was determined by visual assessment of anogenital distance and was confirmed by examination of sexual organs at detailed dissection of the soft tissues or at evisceration. Autolyzed fetuses were not sexed.
EXTERNAL EXAMINATION:
Each fetus (excluding any autolyzed fetus) was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
SOFT TISSUE EXAMINATION:
As soon as possible after sacrifice, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed with Harrison's fluid for examination of the structures of the head. A fetus was submitted to skeletal examination instead of visceral examination due to its fetal external malformation.
SKELETAL EXAMINATION:
The remaining live fetuses per litter were eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs. - Statistics:
- Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test. - Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
PREGNANCY STATUS: At termination on day 21 p.c., there were 24, 24, 24 and 19 females with live fetuses in the groups treated at 0, 30, 100 and 300 mg/kg bw/day, respectively.
MORTALITY: There were no unscheduled deaths in control, 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day, five females (all pregnant) were found dead:
- two females were found dead on day 16 or 14 p.c., respectively. Loud breathing and ptyalism were noted from days 9 to 15 p.c. or 8 to 13 p.c. before death, respectively. At necropsy, both females had a transverse vaginal septum with a colored content (brownish thick),
- one female was found dead on day 18 p.c.. Loud breathing, ptyalism and abdominal breathing were noted from day 7 p.c. until death. At necropsy, this female had a dilated colon,
- one female was found dead on day 8 p.c.. No clinical signs were noted before death and there were no findings at necropsy,
- one female was found dead on day 7 p.c.. Loud breathing, ptyalism and hypoactivity were noted on day 7 p.c. before death. At necropsy, this female had reddish colored focus in thymus and reddish color liquid in intestines (jejunum).
All these deaths were considered to be associated to the test item treatment due to the incidence of mortality which was limited to the high dose.
CLINICAL SIGNS: At 300 mg/kg bw/day, hypoactivity, loud breathing and ptyalism were noted in most females.
Ptyalism noted at 100 and 300 mg/kg bw/day was considered to be of minor toxicological significance.
BODY WEIGHT: When compared with controls, there were no effects on mean body and mean body weight change at 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day and from day 6 to day 9 p.c. there was a body weight loss (-16 g vs. +11 g in controls, p<0.001). Thereafter, mean body weight gain returned towards control values but mean body weight remained lower than controls. When compared with controls, all differences were statistically significant (up to -12.9%, p<0.001).
FOOD CONSUMPTION: When compared with controls, there were no effects on mean food consumption at 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day, there was a marked decrease in mean food consumption on initiation of the treatment period (-57.1% vs. controls on the period of days 6-9 p.c.). Thereafter, mean food consumption remained lower than control (-17.2% vs. controls on days 18-21 p.c., p<0.001).
MACROSCOPIC post-mortem EXAMINATION: There were no test item treatment-related findings at macroscopic post-mortem examination.
In the 100 mg/kg bw/day group, one female had a liver with colored area. This finding was isolated and considered to be fortuitous.
NET BODY WEIGHT CHANGE: When compared with controls, there were no effects on mean gravid uterus weight, mean carcass weight and net body weight change at 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day and when compared with controls, females had lower mean gravid uterus weight (-13%), mean carcass weight (-13%) and mean net body weight gain from day 6 p.c. (-70%).
HYSTERECTOMY DATA (Table 1): When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg/day.
At 300 mg/kg bw/day and while not statistically significant, there were increased mean number of dead fetuses (higher than the upper limit of Historical Control Data) and a tendency towards dose-related increased mean post-implantation loss (within the range of Historical Control Data). - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Maternal toxicity
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical signs
- mortality
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHT AND SEX RATIO: When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg bw/day.
At 300 mg/kg bw/day, there was a decrease in mean fetal body weight, while treatment-related this slight finding (-7% vs. controls) was considered to be of minor toxicological significance.
There were no test item treatment-related effects on sex ratio.
EXTERNAL EXAMINATION (Tables 2 and 3): There were no external variations in the 30 and 100 mg/kg/day groups.
At 300 mg/kg bw/day and when compared with Historical Control Data, there was an increased litter incidence of fetuses with autolysis. However a similar finding was recorded in the contemporaneous control group and a test item treatment-related effect cannot be ascertained.
When compared with controls, there were no test item treatment-related findings in the number of fetuses with external malformations.
SOFT TISSUE EXAMINATION (Table 4): There were no malformations in control and test item-treated groups at soft tissue examination.
When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg bw/day.
At 300 mg/kg bw/day and when compared with controls, there was a statistically significant increase in the number of fetuses variations. This increase was considered to be associated to the treatment to the test item but of minor toxicological significance.
CARTILAGE AND SKELETAL EXAMINATION (Table 5): At cartilage examination and when compared with controls, there were a few statistical significant differences but none were of toxicological relevance.
There were no relevant malformations in control and test item-treated groups at skeletal examination.
When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg bw/day. In the absence of any dose-relationship, the statistically significant increase in unossified hindpaws at 100 mg/kg bw/day was considered fortuitous.
At 300 mg/kg bw/day and when compared with controls or Historical Control Data, there was a statistically significant increase in the number of fetuses with ossification delays (thoracic vertebra(e) with dumbbell ossification centrum, incomplete ossification of the 1st to 4th sternebrae, metacarpals with incomplete ossification and/or unossified 1st metatarsal). This increase was considered to be associated to the treatment to the test item but of minor toxicological significance. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- fetal/pup body weight changes
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- no
- Conclusions:
- The test item, 4-Tert Butylpyrocatechol (batch No. C 492 L 2262), was administered by gavage, once daily, from days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosage of 30, 100 or 300 mg/kg bw/day.
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on clinical signs, deaths, decreased body weight and decreased food consumption, and decreased mean carcass weight at 300 mg/kg bw/day,
- the NOAEL for embryo-fetal development was considered to be 100 mg/kg bw/day, based on a non statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations at 300 mg/kg/day (the increasing in fetal variations was considered to be of minor toxicological significance).
4-Tert Butylpyrocatechol did not elicit any teratogenic potential. - Executive summary:
The objective of this prenatal development toxicity study (2013) was to evaluate the potential toxic effects of the test item, 4-Tert Butylpyrocatechol, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.), inclusive).
Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, 4‑Tert Butylpyrocatechol (batch No. C2262), once daily from day 6 to day 20 p.c., by gavage at dosages of 30, 100 or 300 mg/kg bw/day. An additional group of 24 time-mated females received the vehicle, corn oil, under the same experimental conditions and acted as the control group.
A dose volume of 5 mL/kg/day was used.
The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.
On the basis of the results obtained in this study:
. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on clinical signs, deaths, decreased body weight and decreased food consumption, and decreased mean carcass weight at 300 mg/kg bw/day,
. the NOAEL for embryo-fetal development was considered to be 100 mg/kg bw/day, based on a non‑statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations at 300 mg/kg/day (the increasing in fetal variations was considered to be of minor toxicological significance).
4-Tert Butylpyrocatechol did not elicit any teratogenic potential.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 15 June 2017 to 22 september 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- > Animal Identification: each animal was identified by tattoo in the ear.
> Environmental Acclimation: the animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
> Selection, assignment, replacement, and disposition of Animals: one day after receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the mean per subgroup. Females which were mated on the same day were classified in the same subgroup.
> Housing: following randomization, females were housed individually in cages with perforated floors equipped with water bottles.
> Environmental conditions: target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air were maintained in the animal rooms.
> Food: pelleted diet for rabbits was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay and wooden sticks were provided during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
> Water: municipal tap water was freely available to each animal via water bottles/containers. Periodic analysis of the water was performed. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.
> Animal enrichment: animals were provided with shelters.
> Veterinary care: veterinary care was available throughout the course of the study; however, no examinations or treatments were required. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS AND STORAGE:
> Preparation: The dosing formulations were prepared daily and dosed within 5 hours after adding the vehicle to the test item.
> No adjustment was made for specific gravity of the vehicle and test item.
> No correction was made for the purity/composition of the test item.
> Storage: Test item dosing formulations were kept at room temperature until dosing.
> The dosing formulations were stirred continuously during dose administration. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dose formulation samples were collected for analysis once on 7 August 2017 as following:
> Concentration: all groups
> Homogeneity: groups 2 and 4
All samples were stored on dry ice immediately after sampling. All samples to be analyzed were shipped on dry ice to the analytical laboratory. Upon receipt at the analytical laboratory, the samples were stored in the freezer ≤ -70 °C until analysis.
Analyses were performed by using a validated analytical procedure. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions as those used in the present study - Details on mating procedure:
- > The females were purchaed timed pregnant and were arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating)
- Duration of treatment / exposure:
- The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum
- Frequency of treatment:
- Daily from Day 6 to Day 28 post-coitum
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group 1 (control)
Dose volume = 2 mL/kg
Dose concentration = 0 mg/mL - Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- Group 2
Dose volume = 2 mL/Kg
Dose concentration = 5 mg/mL - Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Group 3
Dose volume = 2 mL/Kg
Dose concentration = 15 mg/mL - Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Group 4
Dose volume = 2 mL/kg
Dose concentration = 50 mg/mL - No. of animals per sex per dose:
- 22 females per dose
- Control animals:
- yes
- yes, concurrent vehicle
- Details on study design:
- > Dose selection rationale: The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of the dose range finder
- Maternal examinations:
- > CAGE SIDE OBSERVATIONS: Yes
Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
> DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy. During the dosing period, these observations were performed at least after dosing.
> BODY WEIGHT: Yes
Animals were individually weighed on Days 2, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.
> FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.
> WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 post-coïtum or within 24 hours for females with abortion or early delivery
- All animals (including animals found dead or sacrificed before planned necropsy and females with abortion or early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed. No tissues, except for the uterus, were weighed. - Ovaries and uterine content:
- Each ovary and uterine content of all animals was dissected and examined as quickly as possible to determine:
- Number of corpora lutea
- Gravid uterus weight
- Number and distribution of live and dead fetuses
- Number and distribution of embryo-fetal deaths - Fetal examinations:
- > Live fetuses were euthanized.
Fetuses of animals found dead or sacrificed before planned necropsy were externally examined in detail and euthanized (if necessary).
> External examinations:
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight (not for fetuses of animals found dead or sacrificed before planned necropsy) was determined.
For late resorptions and recognizable fetuses of females found dead or were euthanized in extremis, a gross external examination was performed.
> Visceral examinations:
All fetuses were sexed and examined for visceral anomalies. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.
> Skeletal examinations:
The skeletal examination was done on all fetuses from all groups.
> Head examinations:
The heads were removed from approximately one-half of the fetuses for soft-tissue examination of all groups.
The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. - Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Parametric test: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric test: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss. - Historical control data:
- The laboratory has historical data on the background incidence of fetal malformations and developmental variations in the species used (same strain and source).
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- > For clinical signs noted for unscheduled deaths at 100 mg/kg, see the section "Mortality"
> For animals surviving until scheduled necropsy:
- Reduced feces production noted for animals of all groups, including controls, with a trend for increased incidence and severity with increasing dose (moderate degree reduction at dose levels up to 30 mg/kg, and up to severe degree reduction at 100 mg/kg). This finding is correlated to the reduced food consumption and of a transient nature, therefore it is considered to be non-adverse.
- Test item-related clinical signs were noted at dose levels of 30 and 100 mg/kg: grey discolouration of the mouth (one 30 mg/kg female and six 100 mg/kg females) and a lean appearance.
- For the control group laboured respiration was noted on two consecutive days for one animal (no. 4).
- All other clinical signs noted during the treatment period were considered to be unrelated to treatment. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Treatment related unscheduled deaths were noted at 100 mg/kg.
> One female at 100 mg/kg was found dead in the morning of Day 11 post-coitum before dosing (severe reduction of feces production and food consumption, 14% body weight loss, dark red contents of the stomach noted at necropsy).
> Three females at 100 mg/kg were euthanized in extremis on Day 15 post-coitum: For the three females: severe reduction of feces production and food consumption, 8-19% body weight loss / For one female: perforation in the forestomach, and grown liver and stomach, and distended caecum with gas (peforation possibly caused by a complication during the dosing procedure, which may also have caused the liver to grow together with the stomach). This complication may have added to the deteriorating state of the female.
> One female at 100 mg/kg was euthanized in extremis on Day 20 post-coitum: excessive salivation, labored respiration, rales, piloerection, pale and lean appearance, severe reduction of feces production and food consumption and body weight loss, red fluid on the manure tray, emaciation and many dark red foci in the stomach.
No mortality occurred in the 10 mg/kg and 30 mg/kg groups. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- > Body weight loss was noted for almost all females at 30 mg/kg and all females at 100 mg/kg on Day 9 post-coitum, when compared to start of treatment on Day 6 post-coitum (mean body weight loss was 4% and 8%, respectively). Body weights increased from Day 12 post-coitum onwards, but body weight gain remained statistically significantly reduced, when compared to controls, up to Day 24 post-coitum for these females at both dose levels.
> In addition, body weight gain corrected for gravid uterus was dose-dependently lower for the females at 30 and 100 mg/kg, when compared to controls (but without reaching statistical significance): mean corrected body weight gain was -64.3 gram (-1.6%) in controls, -69.9 gram (-1.8 %) at 10 mg/kg, -145.4 gram (-4.0%) at 30 mg/kg and -169.2 gram (-4.7%) at 100 mg/kg. These changes were only minimal and remained within the normal range .
> Body weights, body weight gain and body weight gain corrected for gravid uterus of animals treated at 10 mg/kg remained in the same range as controls over the study period. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- > Food consumption before or after correction for body weight was statistically significantly lower over Days 6-9 post-coitum for females at 30 mg/kg and over Days 6-12 post-coitum for females at 100 mg/kg.
> Average relative food consumption values were 38% and 7% of controls, for 30 and 100 mg/kg respectively on Days 6-9 post-coitum.
> No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females at 10 mg/kg. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- > Macroscopic findings noted for unscheduled deaths are described in the section "Mortality".
> In the animals surviving until scheduled necropsy, macroscopic observations at necropsy did not reveal any alterations considered toxicologically relevant.
- Black discoloration of the skin at the lip region was noted for one female at 30 mg/kg (correlating with the grey discoloration of the mouth that was noted during in-life).
- Incidental findings were noted among control and treated animals: reddish foci located on the lungs (two control females), a wound in the skin of the foreleg (one female at 10 mg/kg), grey-white foci on the liver (one female at 100 mg/kg). Since these are occasionally seen among rabbits used and at the incidences observed in absence of a dose-relationship, they were considered to be of no toxicological significance. - Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): One control female delivered her offspring early on Day 26 post-coitum. No clinical signs were noted for this animal prior to early delivery, and food intake and body weight were normal. At necropsy, reddish foci were noted in the lungs. No abnormalities were noted for the fetuses.
As this occurred in a single control female only, this early delivery was not related to treatment with the test item. - Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment- elated effect observed.
The numbers of pregnant females in the control and test groups were similar and in the range of normal biological variation.
In total, 8 females across the dose groups were not pregnant, based on no visually observed implantation sites at necropsy: one control female, three females at 10 mg/kg, two females at 30 mg/kg and two females at 100 mg/kg. Based on the absence of a dose-relationship and as treatment started on Day 6 post-coitum, this was considered to be unrelated to treatment. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- mortality
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- Fetal body weights were considered to be unaffected by treatment.
Mean combined (male and female) fetal body weights were 39.0, 38.9, 37.4 and 38.0 grams for the control, 10, 30 and 100 mg/kg groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed - Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was unaffected by treatment up to 100 mg/kg.
Mean sex ratios (males:females) were 49.7:50.3, 46.6:53.4, 52.1:47.9, 46.3:53.7 for the control, 10, 30 and 100 mg/kg groups, respectively. - Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on litter size of any group.
Mean litter sizes were 8.6, 9.1, 9.4 and 9.0 viable fetuses/litter for the control, 10, 30 and 100 mg/kg groups, respectively. - Changes in postnatal survival:
- not examined
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- External examinations were conducted for all fetuses available for fetal morphological examination.
The number of fetuses (litters) examined for this endpoint were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively.
There were no treatment related effects on external morphology following treatment up to 100 mg/kg/day. All the below malformations occurred singly in different dose groups, no dose response relationship was noted and no malformations occurred in the high dose group. These were therefore considered chance findings and were not related to treatment.
In total five externally malformed fetuses were observed: three in Group 2 (10 mg/kg) and one in Group 1 (control) and Group 3 (30 mg/kg).
> GROUP 3
The fetus at 30 mg/kg had flexure of both tarsals without underlying skeletal abnormality.
> GROUP 2:
One fetus at 10 mg/kg also had flexure of both tarsals without underlying skeletal abnormality. In addition this fetus had carpal flexures whereby the radius appeared to be missing. Besides, this fetus had several other external malformations, namely acrania and thoracoschisis and was viscerally totally malformed.
One other malformed Group 2 fetus had an omphalocele.
The last other malformed fetus at 10 mg/kg had gastroschisis, and also had a diaphragmatic hernia and small eye viscerally and sternoschisis skeletally.
> GROUP 1 (control):
The malformed control fetus had a distended abdomen that was confirmed viscerally by ascites and that appeared to have a cardiac cause (missing atrioventricular valve and large atrium). Moreover, both testes of this fetus were malpositioned. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Skeletal examinations were conducted for all fetuses available for fetal morphological examination.
The number of fetuses (litters) examined for this endpoint were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively.
There was a statistically significant increase of the litter incidence of fetuses with 13th full ribs and caudal shift of pelvic girdle in the high dose group. These respective variations were observed at an incidence of 37.8%, 44.0%, 55.6%, 76.7% and 14.5%, 14.6%, 23.9%, 38.9% per litter in Groups 1, 2, 3, and 4, respectively.
The incidences in Group 3 were above the historical control mean values (48.9% and 16.9 % per litter for 13th full ribs and caudal shift of pelvic girdle, respectively).
For Group 4 the incidences were higher than the historical control maximum values (71.4% and 38.5% per litter for 13th full ribs and caudal shift of pelvic girdle, respectively).
Although, both these skeletal variations were considered to be treatment related at 30 and 100 mg/kg, these were considered to be non-adverse since these are commonly occurring variations, and these variations are considered to have no significant biological effect on animal health or body conformity and represent slight deviations from normal).
For Group 4, the variations noted in the 13th full ribs and caudal shift of pelvic girdle resulted in a statistically significant increase in the percentage of litters with a skeletal variation and the total percent per litter with variations.
Other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Skeletal malformations in fetuses of which the dam received test item were costal cartilage anomaly (one Group 2 fetus ), rib anomaly (one Group 3 fetus), vertebral anomaly with or without associated rib anomaly (three Group 2 fetuses and two Group 4 fetuses), vertebral centra anomaly (one Group 4 fetus), sternoschisis (one Group 2 fetus) and sternal anomaly (one Group 3 fetus).
Because these malformations occurred singly, in concurrent controls (costal cartilage anomaly and vertebral anomaly with associated rib anomaly) and/or were noted previously in historical controls and no clear dose response relationship was noted, they were not considered to be treatment related. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- External examinations were conducted for all fetuses available for fetal morphological examination.
The number of fetuses (litters) examined for this endpoint were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively.
There were no treatment related effects on visceral morphology following treatment up to 100 mg/kg/day.
Visceral malformations occurred in 4 (4), 2 (2), 5 (5) and 4 (2) fetuses (litters) in Groups 1, 2, 3, and 4, respectively.
Three out of the four affected Group 4 fetuses had a fistula and two of these fetuses had distended intestine. Historical control data include one case of a fistula in one litter and no cases of distended intestine. However, as the above two malformations occurred in one litter only, it was considered that there was a hereditary origin and that they were not toxicologically relevant.
The other malformation at the high dose was tetralogy of Fallot which was also observed in Group 3. Because this is not a rare malformation among historical controls and fell within historical control data, it was considered to be a chance finding.
In Group 3 two fetuses in two litters had intestine-diverticulum, since this malformation was observed in the absence of a dose response relationship, it was considered to be a chance finding.
The remaining visceral malformations that were observed in this study, occurred singly, and/or were noted previously in historical controls and therefore do not indicate a relationship to treatment.
All the variations noted, were not considered treatment related as they occurred infrequently, in control fetuses only, and/or occurred at frequencies that were within the range of available historical control data. - Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- SOFT TISSUE CEPHALIC EXAMINATION
The numbers of fetuses (litters) available for morphological examination were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively. Soft tissue cephalic examination was done for approximately half of these fetuses for all groups. Since this effect have been observed in the control group and was not dose related, this effect was not considered to be related to the treatment.
No treatment- related effect observed. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: No treatment related effect observed.
- Key result
- Abnormalities:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No adverse developmental toxicity related to the treatment was observed at all dose groups.
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Based on the results in this prenatal developmental toxicity study, no maternal toxicity was observed in the 10 and 30 mg/kg groups, and no adverse developmental toxicity was observed at all dose levels. The malformations noted in this study were considered to be not related to the substance.
The No Observed Adverse Effect Level (NOAEL) for 4-Tert-Butylpyrocatechol were established as:
Maternal NOAEL: 30 mg/kg/day
Developmental NOAEL: at least 100 mg/kg/day - Executive summary:
The design of this study is based on the guideline OECD 414, Prenatal Developmental Toxicity Study. The objectives of this study were to determine the potential of 4-Tert-Butylpyrocatechol to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum. The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of the dose range finder.
At 100 mg/kg, four females were euthanizedin extremis, due to their deteriorating physical condition, and one female was found dead on Day 11 post-coitum. All five females showed severely reduced food consumption, with consequent moderate to severe body weight losses. One female showed excessive salivation, rales and labored respiration . At necropsy, emaciation and many dark red foci in the stomach were observed for this animal. For the female found dead, dark red contents of the stomach were noted at necropsy. No other signs or macroscopic findings supporting the deteriorating condition of these animals were observed. Based on the severely reduced food consumption and body weight losses, all these decedents were considered to be related to treatment with the test item. Food consumption before or after correction for body weight was statistically significantly lower over Days 6-9 post-coitum for females at 30 mg/kg and over Days 6-12 post-coitum for females at 100 mg/kg. Average relative food consumption values were 38% and 7% of controls, for 30 and 100 mg/kg respectively on Days 6-9 post-coitum. Body weight loss related to the reduced food consumption was noted for almost all females at 30 mg/kg and all females at 100 mg/kg on Day 9 post-coitum, when compared to start of treatment on Day 6 post-coitum (mean body weight loss was 4% and 8%, respectively). Body weights increased from Day 12 post-coitum onwards, but body weight gain remained statistically significantly reduced, when compared to controls, up to Day 24 post-coitum for females at both dose levels.
As towards the end of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse. No clear test item related, adverse macroscopic findings were noted at necropsy.
No maternal toxicity was observed in the 10 and 30 mg/kg groups. Based on these findings a NOAEL of 30 mg/kg/day was identified for maternal toxicity
No adverse developmental toxicity was observed at all dose levels. The treatment related increased incidence of fetuses with13thfull ribs and a caudal shift of pelvic girdle at 30 and 100 mg/kg were considered non adverse since these are commonly occurring variations, and these variations are considered to have no significant biological effect on animal health or body conformity and represent slight deviations from normal.
No adverse treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. number of pregnant females, corpora lutea and implantation sites, or pre- or post-implantation loss,litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations) were noted up to 100 mg/kg. Based on this study the NOAEL for developmental effects is at least 100 mg/kg/day.
In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Level (NOAEL) for 4-Tert-Butylpyrocatechol were established as:
Maternal NOAEL: 30 mg/kg.
Developmental NOAEL: at least 100 mg/kg
- Endpoint:
- developmental toxicity
- Remarks:
- Extended one-generation study (developmental results).
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 22 April 2020 to 01 July 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD guideline 443
- Version / remarks:
- June, 2018.
- Deviations:
- yes
- Remarks:
- The achieved dietary concentrations of 4-tert-butylpyrocatechol were not always within the acceptance criterion of 80 – 120 %.
- GLP compliance:
- yes
- Specific details on test material used for the study:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Details on mating procedure:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Duration of treatment / exposure:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Frequency of treatment:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Duration of test:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Dose / conc.:
- 0 ppm (nominal)
- Dose / conc.:
- 300 ppm (nominal)
- Remarks:
- Anticipated dose level: 30 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 11-14 mg/kg/day for males and 14-17 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 19 mg/kg/day for males and 20 mg/kg/day for females. - Dose / conc.:
- 1 000 ppm (nominal)
- Remarks:
- Anticipated dose level: 100 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 42-50 mg/kg/day for males and 51-59 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 65 mg/kg/day for males and 68 mg/kg/day for females. - Dose / conc.:
- 3 125 ppm (nominal)
- Remarks:
- Anticipated dose level: 300 mg/kg/day.
For F0-animals: on average corresponding to a corrected test article of 144-172 mg/kg/day for males and 176-194 mg/kg/day for females.
For F1-animals: on average corresponding to a corrected test article of 221mg/kg/day for males and 230 mg/kg/day for females. - No. of animals per sex per dose:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Control animals:
- yes, plain diet
- Details on study design:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Twice daily.
DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least once daily, beginning during the first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy.
BODY WEIGHT: Yes.
- Time schedule for examinations: On the first day of treatment (prior to administration), and weekly thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
POST-MORTEM EXAMINATIONS: Yes.
Other: Parturition and maternal care.
For more information, see data detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River 2021 described in the section 7.8.1 Toxicity to reproduction. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: No.
- Number of implantations: Yes.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.
- Blood sampling:
- On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter.
If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. When the target volume of 0.5 mL was not reached by pooling two pups, a third surplus pup of the same litter was added.
Thyroid hormones were evaluated (T4 and TSH). - Fetal examinations:
- EXAMINATION OF PUPS:
- External examinations: Yes:
Culled pups (PND 4): Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
- Soft tissue examinations: No.
- Skeletal examinations: No.
- Head examinations: No.
- Anogenital distance of all live rodent pups: yes. - Statistics:
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Indices:
- Calculation of :
- Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) * 100.
- Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) * 100.
- Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) * 100.
- Percentage live males at first litter check (%): Number of live male pups at first litter check/Number of live pups at first litter check) * 100.
- Percentage live females at first litter check (%): Number of live female pups at first litter check/Number of live pups at first litter check) * 100.
- Viability index (%): (Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering) *100.
- Weaning index (%): (Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling)) * 100.
- Percentage live males at weaning (%): (Number of live male pups on day 21 after littering/Number of live pups on day 21 after littering) * 100.
- Percentage live females at weaning (%): (Number of live female pups on day 21 after littering/Number of live pups on day 21 after littering) * 100.
The following parameters were examined:
Duration of gestation (number of days between confirmation of mating and the beginning of parturition), litter size, total number of offspring born, number of
live offspring on Day 1 after littering, number of live offspring on day 4 (before culling), number of live offspring on day 4 (after culling), number of live
offspring on day 21 after littering. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Endocrine findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- See information detailed in the endpoint study record 'Extended one generation study rats (diet)_Charles River V1 2021 described in the section 7.8.1 Toxicity to reproduction.
- Other effects:
- no effects observed
- Description (incidence and severity):
- Parturition/Maternal care:
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. - Number of abortions:
- no effects observed
- Description (incidence and severity):
- Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Post-implantation loss:
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 93, 95 and 94% for the
control, 300, 1000 and 3125 ppm groups, respectively.
See table 1 and 2 in the field below 'Attached background material'. - Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- Litter size was considered not affected by treatment with the test item.
Live litter sizes were 10.6, 11.4, 11.1 and 10.3 living pups/litter for the control, 300, 1000 and 3125 ppm groups, respectively.
See table 2 in the field below 'Attached background material'. - Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Duration of gestation was considered not to be affected by treatment with the test item.
See table 2 in the field below 'Attached background material'. - Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- Gestation index was considered not to be affected by treatment with the test item. Except for one female at 1000 ppm (No. 173), that had a total litter loss, all pregnant females had live offspring on Day 1 of lactation. The gestation indices were 100% for the control, 300 and 3125 ppm groups and 95% for the 1000 ppm group.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 3 125 ppm (nominal)
- Based on:
- test mat.
- Remarks on result:
- other:
- Remarks:
- No adverse maternal toxicity.
- Dose descriptor:
- LOEL
- Effect level:
- ca. 1 000 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- haematology
- histopathology: non-neoplastic
- Remarks on result:
- other:
- Remarks:
- Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach at 3125 ppm (hyperkeratosis and hyperplasia). Not considered as adverse.
- Dose descriptor:
- NOEL
- Effect level:
- ca. 300 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- haematology
- histopathology: non-neoplastic
- Remarks on result:
- other:
- Remarks:
- Reduction of body weight and body weight gain in males and females at 3125 ppm (not considered to be adverse since these effects on body weight were minimals (less than 10%). Few hematology or clinical biochemistry parameters were affected by treatment with the test item at 1000 and 3125 ppm. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology. Findings in forestomach at 3125 ppm (hyperkeratosis and hyperplasia). Not considered as adverse.
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- Body weights of pups during lactation:
Body weights of pups were considered not to be affected by treatment with the test item.
The higher mean pup weights on PND 1 (not statistically significant for males) were considered unrelated to the test item as the opposite effect (i.e. an decrease) would be expected in case of toxicity and as pup body weights were similar to concurrent controls on subsequent intervals.
See table 3 in the field below 'Attached background material'. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- - Litter size:
Litter size was considered not affected by treatment with the test item.
Live litter sizes were 10.6, 11.4, 11.1 and 10.3 living pups/litter for the control, 300, 1000 and 3125 ppm groups, respectively.
See table 2 in the field below 'Attached background material'.
- Live Birth Index:
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the
test item.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the
test item. The live birth indices were 97, 100, 94 and 100% for the control, 300, 1000 and 3125 ppm groups, respectively.
The lower live birth index at 1000 ppm was mostly attributed to the total litter loss of Female No. 173 (11 dead pups in total). Additionally, 6 pups of the control group (Litter No. 125), 1 pup at 300 ppm (Litter No. 134) and 3 pups at 1000 ppm (Litter No. 164) were found dead at first litter check. No toxicological
relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered
normal for pups of this age.
See table 2 in the field below 'Attached background material'. - Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- Sex ratio was considered not to be affected by treatment with the test item.
For 2 pups from Litter No. 173 (1000 ppm), sex could not be determined due to cannibalism and were therefore assigned at random as males.
See table 2 in the field below 'Attached background material'. - Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- - Viability Index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by
treatment with the test item.
Viability indices (number of live offspring on PND 4 before culling as a percentage of total number of offspring on day 1 after littering) were 99% for the control,
1000 and 3125 ppm groups, and 98% for the 300 ppm group.
Two pups of the control group (one of Litter Nos. 103 and 121 each), 5 pups at 300 ppm (one of Litter Nos. 128, 138 and 146 each and two of Litter No. 136), 3 pups at 1000 ppm (two of Litter No. 159 and one of Litter No. 171) and 2 pups at 3125 ppm (one of Litter Nos. 193 and 199 each) were found dead or missing on
PND 2-4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not
show a dose-related trend and remained within the range considered normal for pups of this age.
In addition, the last pup of Litter No. 125 (control) was missing on PND 2, resulting in a total litter loss for this dam.
See table 2 in the field below 'Attached background material'. - Anogenital distance of all rodent fetuses:
- no effects observed
- Description (incidence and severity):
- Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
See table 4 in the field below 'Attached background material'. - Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- - The number of live pups offspring on day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by
treatment with the test item (see field above for details 'Changes in litter size and weights').
- Weaning index: The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be
affected by treatment with the test item. The weaning indices were 99% for the control and 3125 ppm groups, and 100% for the 300 and 1000 groups.
Two pups of the control group (one of Litter Nos. 103 and 113 each) and 1 pup at 3125 ppm (Litter No. 177), were sacrificed in extremis or
found dead on PND 7, 18 or 17, respectively. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a
dose-related trend and remained within the range considered normal for pups of this age.
See table 2 in the field below 'Attached background material'. - External malformations:
- no effects observed
- Description (incidence and severity):
- - Clinical signs during lactation (F1-Pups): No clinical signs occurred among pups that were considered to be related to treatment with the test item.
- Macroscopy until weaning (F1-Pups): No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be
related to treatment with the test item. For the pup of Litter No. 113 (control) that was found dead on PND 18, the stomach was noted to be grown together with
the pancreas at necropsy. Additionally, for the majority of the pup(s) of Female Nos. 125 (control), 134 (300 ppm), 164, 171, 173 (1000 ppm) that were
found dead at first litter check, beginning autolysis and/or absence of milk in the stomach were noted. For some pups cannibalism was observed. The nature and incidence of these and other macroscopic findings remained within the range considered normal for pups of this age or occurred in the control group only, and were therefore considered unrelated to the test item.
- Histopathology PND 4 F1-Pups: There were no test item-related microscopic findings in the stomach of the selected PND 4 pups. The only recorded finding was a cyst in the stomach of a control male pup.
See table 5 in the field below 'Attached background material'. - Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- Thyroid hormones: Serum T4 levels in male and female pups culled at PND 4 were considered not to be affected by treatment with the test item. It should be noted that for several animals within each group, values of 5.0 ng/mL were reported (i.e. ½ LOQ) as the original value was below LOQ, which is represented by
relatively low mean values. If the 5.0 ng/mL values would not be taken into account, mean values would be 12.6, 12.9, 12.9 en 11.9 ng/mL for control, 300, 1000 and 3125 ppm groups, respectively.
Note to table: Serum T4 levels of PND 4 pups are reported as females, but these were derived from pooled samples collected from male and female pups.
See table 6 in the field below 'Attached background material'. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 3 125 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other:
- Remarks:
- No effect on fetuses and on pups.
- Conclusions:
- - This extended one generation showed that TBC induced mean body weight gain reduction in males and females F0-generation animals in presence of TBC at
3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period and in males F1-generation males at 1000 and 3125 ppm, resulting in a
lower mean body weight throughout the treatment period.
- At 3125 ppm (the higher tested dose), test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item related findings consisted of an increased incidence and severity of diffuse hyperkeratosis with or without squamous cell hyperplasia. Similar effects were observed to the F1-generation animal non-glandular stomach at 3125 ppm. The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia compared to the stomachs of the F0-generation at 3125 ppm.
Based on this study, no general, reproductive or developmental toxicity was observed up to the highest dose tested (3125 ppm). - Executive summary:
The objective of this study was to provide an evaluation of the pre- and postnatal effects of 4-tert-butylpyrocatechol on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of 4-tert-butylpyrocatechol on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. The dose levels in this study were selected to be 0, 300, 1000 and 3125 ppm, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of 4-tert-butylpyrocatechol in rats. The same dietary concentrations were used throughout the study, except for the lactation period. As food intake is considerably higher in lactating females, dietary concentrations were lowered for all treated groups (based on historical control data for relative food consumption) during the lactation period (PND 1-24). Chemical analyses of dietary preparations were conducted at nine occasions during the study to assess accuracy and homogeneity.
For the F0-generation, the following parameters and endpoints were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F0-generation: mating and fertility indices, precoital time, estrous cycle determination, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones) and histopathological examination of the stomach (PND 4 and PND 22-24 pups).
Results: Analysis of diet preparations confirmed thatthe test itemwas homogeneously distributed in the diet. The achieved dietary concentrations of 4-tert-butylpyrocatechol were however not always within the acceptance criterion of 80 – 120 %. A thorough check of the diet preparation procedures and analytical methodology revealed no explanation for the lower accuracies. In order to determine the worst case scenario exposure, test item intake was corrected for the mean achieved dietary concentrations per study period.
No mortality occurred during the study period that was considered to be related to treatment with the test item. In the F0-generation, one female of the control and 1000 ppm groups each, were euthanized on Lactation Day 2 or 1, respectively, as these females had a total litter loss. In the F1-generation, one male (1000 ppm, Cohort 1A) was sacrificed 5 days post weaning as it was assigned to the females.
No general, reproductive, or developmental toxicity was observed up to the highest dose tested (3125 ppm).
The following test item-related, non-adverse, effects were observed:
Parental results: Mean body weight gain was reduced in males and females at 3125 ppm, resulting in a lower mean body weight during the pre-mating and mating period. For females at 3125 ppm, no relevant differences were noted during the gestation period, whereas a higher mean body weight gain was observed from Day 7 of lactation onwards resulting in a slight recovery in mean body weight. Mean terminal body weight was 9 and 4% lower than concurrent control mean for males and females, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.
In animals at 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected by treatment with the test item, including decreased platelets counts in females, and decreased mean bile acid and creatinine concentrations in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with (adverse) anatomic pathology and as such they were regarded as non-adverse. In addition, mean serum levels oft hyroid stimulating hormone (TSH) were dose-dependently increased for females at all dose levels. As individual values generally remained within the historical control range, the change in serum TSH in the females was considered not to be adverse.
At 3125 ppm, test item-related findings were present in the non-glandular stomach (forestomach) in adult rats. These test item-related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or without squamous cell hyperplasia.Since the recorded severities of these findings in adults remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.
Developmental results: Mean serum levels of TSH were decreased for male and female PND 22-24 pups (Cohort Surplus) at 3125 ppm. As individual values remained within the historical control range, the change in serum TSH was considered not to be adverse.
F1-generation results: Mean body weight gain was reduced in males at 1000 and 3125 ppm, resulting in a lower mean body weight throughout the treatment period. Mean terminal body weights of Cohort 1A and 1B animals were 8 and 1% lower than control for males at 1000 ppm, and 9 and 6% lower than control for males at 3125 ppm, respectively. As these effects on body weight were minimal (<10%) and occurred in the absence of any toxicologically relevant changes in food consumption, they were considered not to be adverse.
In animals at 300, 1000 and 3125 ppm, few hematology or clinical biochemistry parameters were affected, including increased mean lymphocyte counts and as a result increased mean total white blood cell count and mean large unstained cell count in both sexes, and decreased mean total bilirubin concentration in males. None of these changes, either alone or as a combination, are considered to be detrimental to organ system function or vitality of the animals. Moreover, these findings were not associated with adverse anatomic pathology and as such they were regarded as non-adverse.
Similar to the F0-generation, test item-related findings were present in the non-glandular stomach at 3125 ppm. These test item‑related findings consisted of an increased incidence and severity of diffuse hyperkeratosis (up to slight degree) with or withoutsquamous cell hyperplasia.The glandular stomach of F1-rats of Cohort 1A showed a higher incidence and severity of squamous cell hyperplasia (incidence of 50% in males and females 60%, up to slight degree) compared to the stomachs of the F0-generation at 3125 ppm (incidence of 20% in males and 8% in females, at minimal degree). Furthermore, there were focal erosions (minimal degree) in the non-glandular stomach in 2/20 F1-rats of both sexes at 3125 ppm. Since the recorded severities of these findings remained low (up to slight) and since there was no evidence for inflammation or involvement of the deeper layers of the stomach, the combination of these findings was considered as a local, non-adverse test item-related effect.
In conclusion, based on the results of this extended one-generation reproductive toxicity study (including Cohorts 1), the following No Observed Adverse Effect Levels (NOAELs) of 4-tert-butylpyrocatechol were established:
General toxicity NOAEL:
F0-generation: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 172-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose(3125 ppm).
F1-generation: at least 3125 ppm (on average corresponding to a corrected test article intake of 221 and 230 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).Reproduction NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
Developmental NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144 -172 and 176 -194 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
The test item (4 -tert-butylpyrocatechol) did not induce reprotoxic effects neither developmental effects up to the highest tested dose (3125 ppm)
Referenceopen allclose all
Table 1: Hysterectomy data
Dose-level (mg/kg bw/day) |
0 |
30 |
100 |
300 |
HCD |
Number of females with live fetuses |
24 |
24 |
24 |
19 |
150 |
Mean number ofcorpora luteaper animal |
15.3 |
14.8 |
14.8 |
14.7 |
14.0 - 15.5 |
Mean number of implantations per animal |
14.0 |
13.7 |
13.4 |
13.6 |
12.8 - 14.0 |
Mean pre-implantation loss (%) |
8.0 |
6.2 |
9.0 |
7.4 |
7.2 - 13.9 |
Mean number of fetuses per animal |
13.8 |
13.3 |
12.8 |
12.8 |
12.0 - 13.2 |
Dead fetuses (%) |
0.3 |
0 |
0 |
0.8 |
0.0 - 0.38 |
Mean number of implantation scars |
0 |
0 |
0 |
0 |
0.3 - 1.0 |
Mean number of early resorptions |
0.2 |
0.4 |
0.5 |
0.7 |
/ |
Mean number of late resorptions |
0 |
0 |
0 |
0 |
/ |
Mean post-implantation loss (%) |
1.5 |
3.0 |
3.8 |
6.2 |
2.0 - 8.7 |
HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012).
/: not available in HCD.
Table 2: Litter (L) and Fetal (F) incidences of external variations
Dose-level (mg/kg bw/day) |
0 |
30 |
100 |
300 |
HCD |
Dams with live fetuses, n |
24 |
24 |
24 |
19 |
143 |
Fetuses examined, n |
332 |
319 |
308 |
244 |
1824 |
- Autolysis,L(F) % |
4.2 (0.3) |
0 (0.0) |
0 (0.0) |
10.5 (0.8) |
8.3 (2.8) |
Litters affected, n (%) |
1 (4.2) |
0 (0.0) |
0 (0.0) |
2 (10.5) |
3 (2.1) |
Fetus affected, n (%) |
1 (0.3) |
0 (0.0) |
0 (0.0) |
2 (0.8) |
16 (0.9) |
HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (143 out of 150 litters had their fetuses submitted to external examination)).
Table 3: Litter (L) and Fetal (F) incidences of external malformations
Dose-level (mg/kg bw/day) |
0 |
30 |
100 |
300 |
HCD |
Dams with live fetuses, n |
24 |
24 |
24 |
19 |
143 |
Fetuses examined, n |
332 |
319 |
308 |
244 |
1824 |
- Agnathia,L(F) % |
0 (0.0) |
4.2 (0.3) |
0 (0.0) |
0 (0.0) |
- |
Litters affected, n (%) |
0 (0.0) |
1 (4.2) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
Fetus affected, n (%) |
0 (0.0) |
1 (0.3) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (143 out of 150 litters had their fetuses submitted to external examination)).
-: not present in HCD.
Table 4: Litter (L) and Fetal (F) incidences of soft tissues variations
Dose-level (mg/kg bw/day) |
0 |
30 |
100 |
300 |
HCD |
Dams with live fetuses, n |
24 |
24 |
24 |
19 |
140 |
Live fetuses, n |
159 |
153 |
149 |
115 |
861 |
- Cranial cavity, hematoma, L(F) % |
0 (0.0) |
4.2 (0.7) |
4.2 (0.7) |
5.3 (0.9) |
- |
- Liver, colored nodule, L(F) % |
0 (0.0) |
4.2 (0.7) |
0 (0.0) |
0 (0.0) |
- |
- Short innominate artery, L(F) % |
0 (0.0) |
4.2 (1.3) |
4.2 (0.7) |
5.3 (0.9) |
8.3 (2.0) |
- Absent innominate artery, L(F) % |
0 (0.0) |
0 (0.0) |
0 (0.0) |
5.3 (2.6) |
- |
Litters affected, n (%) |
0 (0.0) |
3 (12.5) |
2 (8.3) |
3 (15.8) |
17 (12.1) |
Fetus affected, n (%) |
0 (0.0) |
4 (2.6) |
2 (1.3) |
5* (4.3) |
24 (2.8) |
HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (140 out of 150 litters had their fetuses submitted to soft tissues examination)).
-: not present in HCD.
*: p<0.05.
Table 5: Litter (L) and Fetal (F) incidences of skeletal variations
Dose-level (mg/kg bw/day) |
0 |
30 |
100 |
300 |
HCD |
Dams with live fetuses, n |
24 |
24 |
24 |
19 |
141 |
Fetuses examined, n |
172 |
165 |
159 |
127 |
930 |
- Interparietal, incomplete ossification, L(F) % |
4.2 (0.6) |
20.8 (3.0) |
12.5 (3.1) |
31.6* (5.5*) |
40.0 (12.9) |
- Thoracic vertebra(e), |
4.2 (0.6) |
16.7 (2.4) |
8.3 (1.3) |
31.6* (6.3**) |
21.7 (4.8) |
- Caudal vertebra(e), unossified arch, L(F) % |
4.2 (0.6) |
0 (0.0) |
0 (0.0) |
10.5 (4.7*) |
8.3 (2.5) |
- Sternebra(e), incomplete ossification |
4.2 (0.6) |
16.7 (3.0) |
8.3 (1.3) |
15.8 (7.1**) |
10 (2.4) |
- Sternebra, incomplete ossification of the 6th, L(F) % |
4.2 (0.6) |
4.2 (0.6) |
0 (0.0) |
21.1 (5.5*) |
25.0 (7.4) |
- Metacarpal(s), incomplete ossification, L(F) % |
0 (0.0) |
4.2 (1.2) |
0 (0.0) |
10.5 (7.1#) |
12.5 (4.3) |
- Metatarsal(s), 1st unossified, L(F) % |
33.3 (9.3) |
45.8 (11.5) |
29.2 (6.9) |
57.9 (21.3**) |
37.5 (12.3) |
- Hindpaw, unossified distal phalanx, L(F) % |
29.2 (7.6) |
41.7 (12.7) |
54.2 (18.2**) |
31.6 (7.9) |
37.5 (20.2) |
Litters affected, n (%) |
24 (100.0) |
24 (100.0) |
23 (95.8) |
19 (100.0) |
122 (86.5) |
Fetus affected, n (%) |
113 (65.7) |
102 (61.8) |
95 (59.7) |
80 (63.0) |
377 (40.5) |
Statistical significance: *: p<0.05, **: p<0.01, #: p<0.001.
HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (141 out of 150 litters had their fetuses submitted to skeletal examination)).
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- Two reliability 1 studies according to the OECD 414 guideline are available (one on rats and the other one on rabbits) and selected as key studies.
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Two OECD 414 test guideline studies with reliability 1 have been performed. One on rats (2013) and the other one on rabbits (2017). These prenatal developmental toxicity studies are selected as key studies. In addition, an extended one-generation study has been conducted in 2021 and selected as supporting study for developmental effects.
The objective of the prenatal development toxicity study in rats (2013) was to evaluate the potential toxic effects of the test item, 4-Tert Butylpyrocatechol, on the pregnant female rats and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.), inclusive). Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, 4‑Tert Butylpyrocatechol, once daily from day 6 to day 20p.c., by gavage at dosages of 30, 100 or 300 mg/kg bw/day. An additional group of 24 time-mated females received the vehicle, corn oil, under the same experimental conditions and acted as the control group.
The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.
On the basis of the results obtained in this study:
- The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on clinical signs, deaths, decreased body weight and decreased food consumption, and decreased mean carcass weight at 300 mg/kg bw/day,
- The NOAEL for embryo-fetal development was considered to be 100 mg/kg bw/day, based on a non‑statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations at 300 mg/kg/day (the increasing in fetal variations was considered to be of minor toxicological significance).
Based on the test conditions, 4-Tert Butylpyrocatechol did not elicit any teratogenic potential in rats.
The potential of 4-Tert-Butylpyrocatechol to induce developmental toxicity after maternal exposure during the critical period of organogenesis was also investigate in an OECD 414 study in rabbit. Time-mated female New Zealand White rabbits were exposed orally by gavage to 4-Tert-Butylpyrocatechol from day 6 to 28 post-coitum, inclusive. The dose levels selected were 0, 10, 30, 100 mg/kg/day. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of fetuses (live and dead), early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.
At 100 mg/kg, four females were euthanized in extremis, due to their deteriorating physical condition (three on Day 15 post-coitum and one on Day 20 post-coitum), and one female was found dead on Day 11 post-coitum. All five females showed severely reduced food consumption, with consequent moderate to severe body weight losses (8-19%) prior to early sacrifice or death.
Reduced feces production was noted for animals of all groups, including controls, with a trend for increased incidence and severity with increasing dose. Feces production was up to moderate degree decreased at dose levels up to 30 mg/kg, and up to severe degree reduced at the highest dose level of 100 mg/kg. Food consumption before or after correction for body weight was statistically significantly lower over Days 6-9 post-coitum for females at 30 mg/kg and over Days 6-12 post-coitum for females at 100 mg/kg. Average relative food consumption values were 38% and 7% of controls, for 30 and 100 mg/kg respectively on Days 6-9 post-coitum. Body weight loss related to the reduced food consumption was noted for almost all females at 30 mg/kg and all females at 100 mg/kg on Day 9 post-coitum, when compared to start of treatment on Day 6 post-coitum (mean body weight loss was 4% and 8%, respectively). Body weights increased from Day 12 post-coitum onwards, but body weight gain remained statistically significantly reduced, when compared to controls, up to Day 24 post-coitum for females at both dose levels. As towards the end of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse.
No clear test item related, adverse macroscopic findings were noted at necropsy.
No maternal toxicity was observed in the 10 and 30 mg/kg groups.
No adverse developmental toxicity was observed at all dose levels.
In conclusion, based on the results of this prenatal developmental toxicity study in rabbits the following No Observed Adverse Effect Level (NOAEL) for 4-Tert-Butylpyrocatechol were established as:
- Maternal NOAEL: 30 mg/kg.
- Developmental NOAEL: at least 100 mg/kg
Based on the test conditions, 4-Tert Butylpyrocatechol did not elicit any teratogenic potential in rabbits.
In the extended one-generation study (basic design) in rats by diet following the OECD 443 test guideline including 10 weeks pre-mating, Wistar Han rats were exposed to 0, 300, 100 and 3125 pppm of TBC. For the F0-generation, the following parameters and endpoints were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and endpoints were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F0-generation: mating and fertility indices, precoital time, estrous cycle determination, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones) and histopathological examination of the stomach (PND 4 and PND 22-24 pups).
No mortality occurred during the study period that was considered to be related to treatment with the test item. No developmental toxicity was observed up to the highest dose tested (3125 ppm).
In conclusion, based on the results of the extended one-generation reproductive toxicity study, the following No Observed Adverse Effect Levels (NOAELs) of 4-tert-butylpyrocatechol were established:
General toxicity NOAEL: F0-generation at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and
females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm). F1-generation at least 3125 ppm (on average corresponding to a corrected test article intake of 221 and 230 mg/kg/day for males and females, respectively) since no adverse effects were observed up to the highest tested dose (3125 ppm).
Developmental NOAEL: at least 3125 ppm (on average corresponding to a corrected test article intake of 144-172 and 176-194 mg/kg/day for males and females, respectively) since
no adverse effects were observed up to the highest tested dose (3125 ppm).
Based on this study, 4 -tert butylpyrocatechol have no impact on rat development.
Justification for classification or non-classification
Based on the extended one-generation study performed by diet in rats in 2021 (OECD 443) and based on the two OECD 414 test guideline studies, one performed on rats (2013) and the second conducted on rabbits (2017), TBC is not considered as reprotoxic neither teratogen. Therefore, no classification is required for these endpoints according to EU and UN GHS criteria.
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