3-(4-tert-butylphenyl)propionaldehyde

Endpoint summary

Description of key information

In a GLP OECD 422 study, Bourgeonal was administered via oral gavage to male and female Crl:CD(SD) Sprague Dawley rats once daily beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until Day 14 of lactation (female rats) at doses of 0.5, 1, or 5 mg/kg/dose. Administration of Bourgeonal did not produce any mortality or clinical signs in the P generation males or females at any dose. There were no Bourgeonal related effects on mating and fertility in the P generation males or females or any effects on estrous cycling and natural delivery parameters in the P generation females. There were no Bourgeonal-related differences in any preweaning developmental parameter evaluated in the F1generation offspring. Furthermore, there were no Bourgeonal-related macroscopic or microscopic findings or alterations in the organ weights in the P generation adults or F1generation pups. Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general toxicity, mating, and fertility for Bourgeonal in P generation males and females was 5 mg/kg/dose. The NOAEL for development of the F1 generation offspring was also 5 mg/kg/dose.

In a GLP non guideline study, rats were orally gavaged over a period of 5 days. Treatment of male rats with Bourgeonal was associated with systemic toxicity at 250 and 100 mg/kg/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Dec 2018 to 16 Dec 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The Crl:CD(SD) strain was used because 1) it is one mammalian species accepted for use in toxicity studies and it has been widely used throughout industry; 2) this species and strain has been demonstrated to be sensitive to reproductive and developmental toxicants; and 3) historical data and experience exist at the Testing Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 29 to 35 days
- Weight at study initiation: 191-230g for males, 283-329g for females
- Housing: Upon arrival, P generation rats were co-housed in solid-bottomed cages (2/sex/cage), except during the cohabitation period and postpartum periods (see Appendix 2, Deviations). During cohabitation, each pair of P generation rats was housed in the male rat’s nesting box (1:1). P generation females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at Gestation Day (GD) 0 and assigned to individual housing in nesting boxes. Each P generation dam and delivered litter were housed in a common nesting box during the postpartum period, except during motor activity and functional observational battery testing. Following cohabitation, P generation males were returned to the previous premating cage mate and were monitored for incompatibility issues post-cohabitation.
Controls were housed on a separate rack from test substance treated rats (see Appendix 2, Deviations).
- Diet (e.g. ad libitum): Rats were given Certified Rodent Diet® #5002 pelleted food (PMI® Nutrition International) available ad libitum from individual feeders throughout the study, except during designated procedures
- Water (e.g. ad libitum): ad libitum from individual bottles attached to the nesting boxes

DETAILS OF FOOD AND WATER QUALITY:
The food was analyzed for environmental contaminants and results of the analysis are on file at the Testing Facility. There were no known contaminants in the food that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES:
Study Initiation Date: 27 Dec 2018
Initiation of Dosing: 15 Jan 2019
Completion of In-life: 12 Mar 2019

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected as requested by the ECHA (European Chemicals Agency).

Vehicle:

corn oil

Details on oral exposure:

The control substance, Corn Oil, NF, was aliquoted once weekly for administration to Group 1, stored at room temperature until use, and dispensed as necessary. The control substance, Corn oil, NF to be used as the vehicle to prepare the test substance dose formulations had an assigned 7 day use period. Therefore, the Corn Oil, NF parent container was used for preparation and or/aliquoted in single use aliquots for up to 7 days from the initial date of opening of the parent container. The single use aliquots were used for the duration of the study, up to the date of expiration.

The test substance, Bourgeonal, was formulated in the control substance and prepared at the appropriate concentrations. The prepared test substance dose formulation was prepared once daily, and maintained at ambient conditions, protected from light throughout the duration of use for dose administration and/or sampling. The test substance dose formulation was fully used within 4 hours of preparation. The dosing formulations were stirred continuously for at least 30 minutes prior to dosing, throughout dose administration and post-dose sampling.

P generation male rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated females, during cohabitation and continuing through the day prior to scheduled euthanasia. P generation males were exposed to the test or control substance for 42 to 45 days.
P generation female rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated males and continuing through Lactation Day (LD) 12 (rats that deliver a litter) or GD 25 (rats that did not deliver a litter).
Doses were adjusted based on the most recently recorded body weight. The gavage needle was wiped clean prior to dose administration for each rat.

F1 generation pups were not directly exposed to the test or control substance, but may have been possibly exposed during maternal gestation (in utero exposure), via maternal milk during the lactation period, or from exposure to maternal urine/feces.

The doses were selected from a 14-day Dose-Range Finding (DRF) study (CRL 20153551). Based on this study the maximum concentration dose was determined to be 5 mg/kg/dose. Aditional details of dose selection rational could be found in section 7.8.1 (OECD 422).

Animals received the test material or vehicle control formulations orally at a volume-dosage of 15 ml/kg/dose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate (1 mL, aliquot weights to be measured to at least 0.001 g) sets of top, middle, and bottom test substance samples for the sampling time points were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 20% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Duration of treatment / exposure:

Bourgeonal, or the control substance, corn oil, were administered orally by gavage through mating and continuing for at least 28 days (P generation male rats) or through parturition until Day 14 of lactation (P generation female rats) at 0 (Control), 0.5, 1, and 5 mg/kg/dose.

Frequency of treatment:

Once daily beginning before cohabitation.

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

P generation male rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated females, during cohabitation and continuing through the day prior to scheduled euthanasia. P generation males were exposed to the test or control substance for 42 to 45 days. P generation female rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated males and continuing through Lactation Day (LD) 12 (rats that deliver a litter) or GD 25 (rats that did not deliver a litter).

F1 generation pups were not directly exposed to the test or control substance, but may have been possibly exposed during maternal gestation (in utero exposure), via maternal milk during the lactation period, or from exposure to maternal urine/feces.
Doses were adjusted based on the most recently recorded body weight. The gavage needle was wiped clean prior to dose administration for each rat.


The in-life procedures, observations, and measurements listed below were performed for all P generation rats:
The rats were assessed for viability at least twice daily during the study.

General Appearance:
The P generation rats were observed for general appearance at least once during the acclimation period, at least once weekly during the predose estrous evaluation (females), daily during the exposure period, and on the day of scheduled euthanasia.

Detailed Clinical observations:
The P generation rats were observed for detailed clinical observations once prior to initiation of exposure (baseline), and once weekly thereafter. During the weeks of Functional Observational Battery (FOB) testing, detailed clinical observations were not
conducted on the 5 rats/sex/group.

Postdose Observations:
Postdose observations were recorded between 1 and 2 hours following each daily dose administration.

Body Weights:
P generation male rats had body weights recorded on the day after arrival at the Testing Facility, on the first day of exposure, at least once weekly thereafter, and on the day of scheduled euthanasia.
P generation female rats had body weights recorded on the day after arrival at the Testing Facility, on the first day of exposure, at least once weekly thereafter, and on GD 0, 4, 7, 11, 14, 17, 20, and 25 (for P generation females with no confirmed mating date), and on LD 0, 3, 6, 9, and 12. A terminal weight was also recorded.

Food Consumption:
In P generation males, food consumption was recorded at least once weekly during the exposure period and once during the week of euthanasia (food left value). In P generation females, food consumption was recorded at least once weekly during the exposure period, on GD 0, 4, 7, 11, 14, 17, 20, and 25 (for P generation females with no confirmed mating date) and on LD 0, 3, 6, 9, and 12.

Estrous Cycle Evaluations:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Samples were collected from P generation females beginning 13 days prior to treatment, the first 14 days of treatment and during cohabitation, and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Also, on the day of scheduled euthanasia (LD 13), an examination of vaginal cytology was performed prior to necropsy examination to determine the stage of estrous cycle.

Cohabitation:
P generation rats were assigned to cohabitation (i.e., pairing), one male per one female (within each dose group). The cohabitation period consisted of a maximum of 14 days. P generation females that did not mate with a P generation male within the first 7 days of cohabitation were assigned an alternate P generation male (same dose group) that successfully mated with a P generation female from the same dose group and remained in cohabitation for a maximum of 7 additional days. P generation females observed with spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be GD 0 and assigned to individual housing. P generation female 1420 (Group 1, 0 mg/kg/dose) did not mate after completion of the 14-day cohabitation period and was considered to be GD 0 on the last day of cohabitation. This female was assigned to individual housing (solid bottom cage) and euthanized at the discretion of the Study Director.

Natural Delivery Observations:
P generation females were evaluated for adverse clinical signs, the duration of gestation (GD 0 to the time the first pup was observed), litter size (defined by all pups delivered), and pup viability at birth.

Maternal Observations:
Maternal observations were recorded daily during the postpartum period.

Functional Observational Battery (FOB):
A functional observational battery (FOB) evaluation was conducted on one occasion for 5 rats/sex/group, during the exposure period, when possible. Due to early deaths, the evaluations were conducted on 4 female rats in Groups 1, 2, and 3 to ensure
sufficient data for clinical pathology analysis. The FOB 3,4,5,6 was conducted by an observer who was unaware of the group assignment of the rat. The observer examined the rat in its home cage, while handling the animal, and/or in an open field to assess parameters including, but not limited to the following: lacrimation, salivation, palpebral closure, prominence of the eye, pupillary reaction to light, piloerection, respiration, and urination and defecation (autonomic functions); sensorimotor responses to visual, acoustic, tactile and painful stimuli (reactivity and sensitivity); reactions to handling and behavior in the open field (excitability); gait pattern in the open field, severity of gait abnormalities, air righting reaction, visual placing response, and landing foot splay (gait and sensorimotor coordination); forelimb and hindlimb grip strength; and abnormal clinical signs including but not limited to convulsions, tremors and other unusual behavior, hypotonia or hypertonia, emaciation, dehydration, unkempt appearance and deposits around the eyes, nose, or mouth. Body temperature was measured at the completion of the FOB.

Motor Activity Evaluation
Motor activity was conducted on one occasion during the exposure period using the same five rats/sex/group that were selected for FOB evaluation, when possible.
The rats were placed in an individual enclosure held within a Smartframe containing 7 x 15 photobeams utilizing infra-red pyroelectric detectors. Movement was detected in 2 dimensions anywhere in the enclosure and was differentiated into fine movement and ambulation. Each animal was monitored for one session of 60 minutes. For the purpose of data tabulation, activity data files were reduced to Excel® format into successive periods of 10 minutes each at the completion of testing. Fine movements and ambulation were analyzed in these six 10-minute periods and compared across the dose groups.

In-life Procedures, Observations, and Measurements - F1 Generation
Preweaning
The in-life procedures, observations, and measurements listed below were performed for all F1 generation litters, with the litter as the unit of measure.

Viability Checks
Litters were observed for dead pups at least twice daily and the pups in each litter were counted once daily during the preweaning period.

Clinical Observations
Litters were observed at least once daily.

Body Weights
F1 generation pups had body weights recorded on Day 0 (birth), 3, 6, 9, and 12 postpartum.

Anogenital Distance
On Day 0 (birth) anogenital distance was recorded for all F1 generation pups using a calibrated stereomicroscope, micrometer, and ruler. The anogenital distance was measured from the cranial edge of the anus, which comes to a point, to the base of the
genital tubercle.

Nipple Retention
On Day 12 postpartum (LD 12) nipple presence was evaluated and the number present was recorded for all F1 generation male pups.

Laboratory Evaluations
Clinical Pathology:
Blood samples were collected under isoflurane/oxygen anesthesia from the inferior vena cava from 5 rats/sex/group.
Hematology Parameters:Red blood cell count; Hemoglobin concentration; Hematocrit; Mean corpuscular volume; Red blood cell distribution width; Mean corpuscular hemoglobin concentration; Mean corpuscular hemoglobin; Reticulocyte count (absolute); Platelet count; White blood cell count; Neutrophil count (absolute); Lymphocyte count (absolute); Monocyte count (absolute); Eosinophil count (absolute); Basophil count (absolute); Large unstained cells.
Coagulation Parameters: Activated partial thromboplastin time; Fibrinogen; Prothrombin time
Clinical Chemistry Parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubina, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride.
Thyroid Sample Collection: P Generation On the day of scheduled euthanasia, blood samples were collected from all P generation male and female rats. F1 Generation Pups: On Day 3 postpartum, blood samples were collected from 2 culled pups/litter/group. On Day 12 postpartum, blood samples were collected from 1 pup/sex/litter/group.
Thyroid Sample Analysis: Serum samples were analyzed for Thyroxine (T4) levels using a validated analytical procedure.

Ovarian and Uterine Examinations
The reproductive tract was dissected from the abdominal cavity. The number of implantation sites was recorded.

Necropsy
All P generation males and females were subjected to a complete necropsy examination.

Organ Weights
The organs were weighed at necropsy for all P generation rats at scheduled euthanasia. Paired organs were weighed together, unless otherwise indicated.

Tissue Collection and Preservation
Representative samples of the tissues were collected from all rats and preserved in 10% neutral buffered formalin.

Histology
Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology
Histopathological evaluation was performed by a board-certified veterinary pathologist. The following tissues were evaluated:
• Thyroid and parathyroid were evaluated from all P generation males and females in the control and high dose groups.
• Gross lesions were evaluated in all P generation males and females in all groups.
• Tissues identified were evaluated from all P generation males and females in the control and high dose groups.
• Special attention was paid to the stages of spermatogenesis in the testes, epididymides, and interstitial testicular cell structures.
• Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated, processed, and reported.
Thyroid and parathyroid were evaluated from one F1 generation pup/sex/litter in the control and high dose groups

STATISTICAL ANALYSIS
Descriptive statistics including number, mean, percentages and/or standard deviation were reported as appropriate.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the study


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:once weekly

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / isoflurane/oxygen anesthesia
- Animals fasted: Yes
- How many animals:5/sex/group
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: :5/sex/group
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:5/sex/group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Functional Observation Battery, Motor Activity.

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Descriptive statistics including number, mean, percentages and/or standard deviation were reported as appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no Bourgeonal-related clinical observations in the P generation males at any dose.
All clinical observations were considered to be unrelated to Bourgeonal because: 1) the observations were not dose dependent; 2) the observations were limited to the control group; and/or 3) the observation was transient and did not persist.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of 1, 1, and 1 P generation females in the 0, 0.5, and 1 mg/kg/dose groups, respectively, did not survive to scheduled euthanasia. Of these unscheduled necropsies, one P generation female was euthanized due to no surviving pups and the remaining two P generation females were found dead prior to or after dose administration. These unscheduled necropsies were considered unrelated to Bourgeonal because: 1) the death was not dose-dependent; 2) the death was related to a gavage error; and/or 3) it was a protocol specific timepoint (i.e., euthanize after the last pup is found dead or euthanized).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no Bourgeonal-related effects on mean body weight gains or mean body weights in the P generation males at any dose.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no Bourgeonal-related effects on hematology parameters in P generation males or females at any dose. All differences in hematology parameters were considered unrelated to Bourgeonal because: 1) the observations were not dose dependent; 2) the differences were of small magnitude; and/or 3) the differences were inconsistent in direction.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no Bourgeonal-related effects on clinical chemistry parameters in the P generation males and females at any dose.
All differences in clinical chemistry parameters, including those that reached statistical significance, were considered unrelated to Bourgeonal because: 1) the observations were not dose dependent; 2) the differences were of small magnitude; and/or 3) the differences were inconsistent in direction.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no Bourgeonal-related effects on functional observational battery assessments in the P generation females at any dose.
There were no Bourgeonal-related effects on either ambulation or fine movement during motor activity testing in the P generation females at any dose.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related organ weight changes were noted. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of Bourgeonal.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of Sprague-Dawley rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Bourgeonal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No microscopic changes in the P generation animals were attributed to the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

highest dose

Effect level:

5 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

mortality

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

In conclusion, Bourgeonal was administered via oral gavage to male and female Crl:CD(SD) Sprague Dawley rats once daily beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until Day 14 of lactation (female rats) at doses of 0.5, 1, or 5 mg/kg/dose. Administration of Bourgeonal did not produce any mortality or clinical signs in the P generation males or females at any dose. There were no Bourgeonal related effects on mating and fertility in the P generation males or females or any effects on estrous cycling and natural delivery parameters in the P generation females. There were no Bourgeonal-related differences in any preweaning developmental parameter evaluated in the F1generation offspring. Furthermore, there were no Bourgeonal-related macroscopic or microscopic findings or alterations in the organ weights in the P generation adults or F1generation pups. Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general toxicity, mating, and fertility for Bourgeonal in P generation males and females was 5 mg/kg/dose. The NOAEL for development of the F1 generation offspring was also 5 mg/kg/dose.

Executive summary:

The objectives of this study were to detect potential effects that may result from daily exposure of Bourgeonal to Crl:CD(SD) male and female rats beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until Day 14 of lactation (female rats). The study design was as follows:

Text Table1
Experimental Design

Group No.	Test Material	Dose Level (mg/kg/dose)	Concentration (mg/mL)	Dose Volume (mL/kg/dose)	No. of Rats
					Males	Females
1	Control	0	0	15	10	10
2	Bourgeonal	0.5	0.033	15	10	10
3	Bourgeonal	1	0.067	15	10	10
4	Bourgeonal	5	0.33	15	10	10

A total of 80 naïve Crl:CD(SD) Sprague Dawley P generation rats (10 rats/sex) were randomly assigned to four dose groups (Groups 1 through 4), 10 rats/sex/group. Formulations of the test substance, Bourgeonal, or the control substance, corn oil, were administered orally by gavage once daily beginning before cohabitation, through mating and continuing for at least 28 days (P generation male rats;actual: 42 to 45 doses) or through parturition until Day 14 of lactation (P generation female rats; actual: 38 to 56 doses)at 0 (Control), 0.5, 1, and 5 mg/kg/dose.

The following parameters and end points were evaluated in the P generation rats assigned in this study: viability, clinical observations, detailed clinical signs, body weights and body weight changes, food consumption, neurobehavioral evaluations (functional observation battery and motor activity), mating and fertility assessments, organ weights, and macroscopic and microscopic evaluations. P generation females were further evaluated for estrous cyclicity, natural delivery parameters, and maternal observations.

The following parameters and end points were evaluated in the F1 generation pups: viability, clinical signs, pup body weights and internal sex, preweaning parameters (anogenital distance and nipple retention), and macroscopic and microscopic examinations. Blood samples were collected from all P generation males and females for clinical pathology assessments (hematology, coagulation, clinical chemistry) and from P generation males and F1 generation pups for thyroid hormone assessments.

P Generation Males

All P generation males survived to scheduled euthanasia, and there were no Bourgeonal‑related clinical signs in the P generation males at any dose. Mean body weights, mean body weight gains, and mean food consumption values were similar across all groups in the P generation males. There were no Bourgeonal-related effects on any neurobehavioral parameter (functional observation battery or motor activity) at any dose.

There were no Bourgeonal-related effects on any mating and fertility parameter in the P generation males at any dose. There were no Bourgeonal-related macroscopic or microscopic observations or alterations in organ weights at any dose. In addition, there were no Bourgeonal-related effects on hematology, clinical chemistry, or coagulation parameters in the P generation males at any dose.

There were no Bourgeonal-related changes in serum T4 concentrations in the P generation males at any dose. In the P generation males, mean serum T4 concentrations were 104%, 87%, and 90% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on DS 43/44. 

P Generation Females

There was no Bourgeonal-related mortality in the P generation females at any dose. A total of 1, 1, and 1 P generation females in the 0, 0.5, and 1 mg/kg/dose groups, respectively, did not survive to scheduled euthanasia. Of these unscheduled necropsies, one P generation female was euthanized due to no surviving pups and the remaining two P generation females were found dead prior to or after dose administration. These unscheduled necropsies were considered unrelated to Bourgeonal because: 1) the death was not dose-dependent; 2) the death was related to a gavage error; and/or 3) it was a protocol specific timepoint (i.e., euthanize after the last pup is found dead or euthanized). All remaining P generation females survived to scheduled euthanasia.

There were no Bourgeonal-related clinical observations or effects on mean body weights, mean body weight gains, and mean food consumption values during the premating, gestation, or lactation periods in the P generation females at any dose. There were no Bourgeonal-related effects on any neurobehavioral parameter (functional observation battery or motor activity) at any dose.

There were no Bourgeonal-related effects on estrous cycling, mating or fertility, or any natural delivery or litter parameter in the P generation females at any dose. There were no Bourgeonal‑related macroscopic or microscopic observations or alterations in organ weights at any dose.

In P generation females, there were no Bourgeonal-related effects on any hematology, coagulation, or clinical pathology parameter at any dose.

F1 Generation Pups

In the F1 generation pups, there were no Bourgeonal-related clinical observations or effects on anogenital distance, nipple retention (males), or mean pup body weights at any dose. In addition, there were no Bourgeonal-related macroscopic or microscopic observations in the F1 generation pups at any dose.

There were no Bourgeonal-related changes in serum T4 concentrations in the F1 generation males or females at any dose. In the F1 generation male pups, mean serum T4 concentrations were 99%, 82%, and 78% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on Day 13postpartum. Inthe F1 generation female pups, mean serum T4 concentrations were 83%, 74%, and 74% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on Day 13 postpartum. There were no Bourgeonal-related microscopic changes in the thyroid or parathyroid glands of the single F1 generation pup/sex/litter that was microscopically examined from 5 mg/kg/dose group. The differences observed in mean serum T4 concentrations in the F1 generation females did not reflect any other evidence at the tissue level, therefore, were considered unrelated to administration of Bourgeonal.

In conclusion, Bourgeonal was administered via oral gavage to male and female Crl:CD(SD) Sprague Dawley rats once daily beginning before cohabitation, through mating and continuing for at least 28 days (P generationmale rats;actual: actual: 42 to 45 doses)[TD1] or through parturition until Day 14 of lactation (P generation femalerats;actual:38 to 56 doses) at doses of 0.5, 1, or 5 mg/kg/dose. Administration of Bourgeonal did not produce any mortality or clinical signs in the P generation males or females at any dose. There were no Bourgeonal-related effects on mating and fertility in the P generation males or females or any effects on estrous cycling and natural delivery parameters in the P generation females. There were no Bourgeonal-related differences in any preweaning developmental parameter evaluated in the F1 generation offspring. Furthermore, there were no Bourgeonal-related macroscopic or microscopic findings or alterations in the organ weights in the P generation adults or F1 generation pups. Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general toxicity, mating, and fertility for Bourgeonal in P generation males and females was 5 mg/kg/dose. The NOAEL for development of the F1 generation offspring was also 5 mg/kg/dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP and OECD guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated Dose Oral:

In a GLP OECD 422 study, Bourgeonal was administered via oral gavage to male and female Crl:CD(SD) Sprague Dawley rats once daily beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until Day 14 of lactation (female rats) at doses of 0.5, 1, or 5 mg/kg/dose. Administration of Bourgeonal did not produce any mortality or clinical signs in the P generation males or females at any dose. There were no Bourgeonal related effects on mating and fertility in the P generation males or females or any effects on estrous cycling and natural delivery parameters in the P generation females. There were no Bourgeonal-related differences in any preweaning developmental parameter evaluated in the F1generation offspring. Furthermore, there were no Bourgeonal-related macroscopic or microscopic findings or alterations in the organ weights in the P generation adults or F1generation pups. Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general toxicity, mating, and fertility for Bourgeonal in P generation males and females was 5 mg/kg/dose. The NOAEL for development of the F1 generation offspring was also 5 mg/kg/dose.

In a GLP non guideline study rats were orally gavaged over a period of 5 days. Treatment of male rats with Bourgeonal was associated with systemic toxicity at 250 and 100 mg/kg/day.

Repeated Dose Inhalation:

No Dermal repeated dose toxicity study was available on the substance.

Repeated Dose Dermal:

No Dermal repeated dose toxicity study was available on the substance.

Justification for classification or non-classification

Although no test item related systemic toxicity was observed in the OECD 422 GLP study performed on rat up to 5mg/kg/day, some test item related systemic toxicity effects were obeserved from 100mg/kg/day and above in the 5 -day study in rats (GLP, non guideline).

Based on this data described above, the substance has shown some systemic toxicity potential at higher concentrations and should therefore be classified as STOT Rep. Exp. 2 (Stomach, liver) according to the (EC) No 1272/2008 Regulation (CLP).