4-hydroxybenzophenone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In conclusion, based on the interim results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422, performed under GLP conditions); (no raw data was reviewed and no phase reports were available for dose formulation analysis and histopathology), the following no-observed-adverse-effect level (NOAEL) of 4-HYDROXY-BENZOPHENONE were established:

Parental NOAEL: 100 mg/kg (based on increased liver weights at 300 mg/kg/day)

Reproduction NOAEL: at least 300 mg/kg

Developmental NOAEL: at least 300 mg/kg

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 2019 to 02 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

exposure-related information

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

2000

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Physical Description: White powder
Purity/Composition: 99.72%
Storage Conditions: At room temperature desiccated
Test item handling: No specific handling conditions required
Stability at higher temperatures: Stable
Molecular formula: C13H10O2
Molecular weight: 198.22
Stability in vehicle (Propylene Glycol): Stability for at least 24 hours at room temperature under normal laboratory light conditions, stability and resuspension homogeneity for at least 8 days in the refrigerator and after 30 minutes stirring before sampling, and stability for at least 3 weeks in the freezer (< -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions), Test Facility Study No. 20151996.

Species:

rat

Strain:

Wistar

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.

The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license
AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Females: 6 (nulliparous and non-pregnant)
Target Age at the Initiation of Dosing: 12-14 weeks Females, 10-12 weeks Males
Target Weight at the Initiation of Dosing: 200 to 250 g Females, 250 to 350 g Males

ENVIRONMENTAL CONDITIONS
Temperature: 19 to 21 °C
Humidity: 45 to 61%
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures)
Ventilation: At least 10 air changes per hour with 100% fresh air (no air recirculation)

FOOD
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) provided ad libitum. During motor activity measurements, animals did not have access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Test Facility.

WATER
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals did not have access to water for a maximum of 2 hours. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.

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, height 18 cm).

During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.

In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

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.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.

Test item dosing formulations (w/w) was homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.

Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle will be continuously stirred until and during dosing. Adjustment will be made for specific gravity of the vehicle. No correction will be made for the purity/composition of the test item.

Any residual volumes were discarded.

Details on mating procedure:

Cohabitation/Mating Procedure – F0-Generation
Frequency: Daily, after a minimum of 14 days of treatment. The mating period will consist of a maximum of 14 consecutive days.

Procedure: Animals will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated.

A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible, see section 14) will be used for re-mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis:
Dose formulation samples were collected as below.
Additional samples may be collected and analyzed at the discretion of the Study Director.

Dose Formulation Sample Collection Schedule:
Occasion: Week 1 of treatment
Concentration: All groups(a)
Homogeneity: Groups 2 and 4(a)

(a) The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.

All samples analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility.

Residual samples were discarded after completion of the sample analysis.

Analytical Method:
Analyses described were performed using a validated analytical procedure (Test Facility Study No. 20151996).

Dose Formulation Analyses

Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
No test item was detected in the Group 1 formulation.

Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days/week

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-63 days, i.e. 14 days prior to mating (with the objective 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 before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-54 days.
The first day of dosing was designated as Day 1.
Female nos. 54 and 58 (Group 2), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Details on study schedule:

TERMINAL PROCEDURES – F1-GENERATION
Method of Euthanasia – F1-Generation
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16), except the pups selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 will be anesthetized using isoflurane followed by exsanguination.

Unscheduled Deaths – F1-Generation
Recognizable fetuses of females that die spontaneously or are euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Live fetuses were euthanized by decapitation.
Pups that die or are euthanized before scheduled termination were examined externally and sexed (both externally and internally, if possible). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol if not necropsied on the same day. 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 will be evaluated.

Scheduled Euthanasia – F1-Generation
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally. From two surplus pups per litter, blood will be collected, if possible.
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. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection, and was preserved in 10% buffered formalin.

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Experimental dates: 18 January 2019 to 23 August 2019

A dose-range finding study (DRF) was conducted on three animals at test doses of 500 and 1000 mg/kg/day.

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 10 days.

A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures (Study No. 520990 was used for DCS).

The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg, Test Facility Study No. 20151992).

The justification of the route of administration is identical as for the main study.

Justification of Route and Dose Levels:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of 4-HYDROXY-BENZOPHENONE in rats (Test Facility Study No. 20151995), and in an attempt to produce graded responses to the test item.

The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Parental animals: Observations and examinations:

Mortality/Moribundity Checks – F0-Generation:
Frequency: At least twice daily throughout the study.
Procedure: Animals were observed for general health/mortality and moribundity. Animals were not removed from the cages during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F0-Generation:
Frequency: During treatment, animals were observed at least once daily, up to the day prior to necropsy.
These clinical observations were conducted on the peak period of anticipated effects after treatment (to be amended based on the results of the dose range finder).
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 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 in summary tables.

Arena Observations – F0-Generation:
Frequency: Once before the first administration of the test item and 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 Weights – F0-Generation:
Frequency: Males and females will be weighed on the first day of treatment (prior to dosing), 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.
Procedure: Animals were individually weighed.

Food Consumption – F0-Generation:
Frequency: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females will be measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
Procedure: Food consumption was quantitatively measured..

Water Consumption – F0-Generation:
Frequency: Regular basis throughout the study.
Procedure: Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

Functional Tests – F0-Generation:
Frequency: Once during the treatment period. 5 males were tested once during Week 4 of treatment and 5 females were tested once during the last week of lactation (i.e. PND 6-13). These tests were performed after clinical observations (including arena observation).
Procedure: The following tests were performed:
-hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
-fore- and hind-limb grip strength were recorded as the mean of three measurements , using a grip strength meter.
-locomotor activity (recording period: 1 hour under normal laboratory light conditions) were tested using the Kinder Scientific Motor Monitor System. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
Clinical Pathology:
Sample Collection:
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead and females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Additional blood samples were obtained (e.g. due to clotting of non-serum samples) if necessary in both the animal facility and in the necropsy room if permissible sampling frequency and blood volume were not exceeded. After collection, samples were transferred to the appropriate laboratory for processing.

F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling, but water was still available. F0-females were not fasted overnight.

Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.

On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and was pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. If the target volume of 0.5 mL couldn't be reached by pooling from two pups, blood from a third surplus pup of the same litter would be added, if available.

On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). If the target volume of 1.0 mL/pup couldn't be reached, a separate blood sample was collected from another pup of the same litter and sex (if possible). Any incomplete blood samples were discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure.

Samples were collected according to the table ("Samples for Clinical Pathology Evaluation") below:

Hematology:
Target Volume: 0.5 mL.
Anticoagulant: K3-EDTA (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria).

A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. If additional examination of blood smears was deemed necessary, the smears were subsequently evaluated.

Hematology parameters:
White blood cells (WBC)
Red Blood Cell Distribution Width (RDW)
Neutrophils (absolute)
Haemoglobin
Lymphocytes (absolute)
Haematocrit
Monocytes (absolute)
Mean corpuscular volume (MCV)
Eosinophils (absolute)
Mean corpuscular haemoglobin (MCH)
Basophils (absolute)
Mean corpuscular haemoglobin concentration (MCHC)
Red blood cells
Platelets
Reticulocytes (absolute)
Large unstained cells (absolute) (LUC)



Coagulation:
Target Volume: 0.45 mL.
Anticoagulant: Citrate (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria).

Coagulation Parameters:
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)

Clinical Chemistry:
Target Volume: 0.5 mL [For bile acid measurement: 1.0 mL (same sample as for thyroid hormone measurement)]
Anticoagulant: Li-Heparin (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Not applicable for serum tubes.
Processing: To serum (bile acids) or to plasma

Clinical Chemistry Parameters:
Alanine aminotransferase (ALAT):
Creatinine
Aspartate aminotransferase (ASAT):
Glucose
Alkaline Phosphatase (ALP):
Cholesterol
Total protein:
Sodium
Albumin:
Potassium
Total Bilirubin:
Chloride
Bile Acids
Calcium
Urea:
Inorganic Phosphate (Inorg. Phos)

Thyroid Hormone:
Target Volume: F0-animals: 1.0 mL (same sample as for bile acid measurement).
PND 4 pups: 0.5 mL in total (pooled).
PND 14-16 pups: 1.0 mL per pup.

Anticoagulant: Not applicable for serum. (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria).

Thyroid Hormone Parameters:
Thyroxine (T4): Thyroid-Stimulating Hormone (TSH; only if required)



After clotting and centrifugation, serum was used as listed below.
F0-Males: Serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of total T4, and the remaining volume of serum for possible future measurement of TSH.

F0-Females: The serum was used for possible future measurement of total T4 and/or thyroid-stimulating hormone TSH.

PND 4 Pups: The pooled serum was used for possible future measurement of total T4.

PND 14-16 Pups: Serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of total T4, and the remaining volume of serum for possible future measurement of TSH.

Serum samples retained for possible future analysis was maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples would be stable for 6 months. Any remaining sample was discarded

Estrous Cycle Evaluations – F0-Generation:
Frequency: Daily vaginal lavage were performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage would continue for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or had died spontaneously.
Procedure: Estrous cycles will be evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Cohabitation/Mating Procedure – F0-Generation:
Frequency: Daily, after a minimum of 14 days of treatment. The mating period ran for a maximum of 14 consecutive days.
Procedure: 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 occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. In case less than 9 females per group showed evidence of mating, each non-mated female was re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible) was used for re-mating.

General Reproduction Data – F0-Generation
Frequency: Daily from the mating period onwards.
Procedure: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used when necessary to aid in confirmation of pregnancy. The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that are littering will be left undisturbed.

Cage debris of pregnant females was examined for evidence of premature delivery. Signs of difficult or prolonged parturition were also recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, was also recorded, if applicable.

Oestrous cyclicity (parental animals):

Frequency: Daily vaginal lavage were performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage would continue for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or had died spontaneously.
Procedure: Estrous cycles will be evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Litter observations:

IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS – F1-GENERATION:
The in-life procedures, observations, and measurements listed below will be performed for the pups.

Mortality/Moribundity Checks – F1-Generation
Frequency: The number of live and dead pups were determined on PND 1 and daily thereafter.
Procedure: Pups were observed for general health/mortality and moribundity. If possible, defects or cause of death were evaluated. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F1-Generation
Frequency: At least once daily.
Procedure: Detailed clinical observations were made for all pups. Only days on which clinical signs are present between the first and last litter check were given in the respective report tables.

Body Weights – F1-Generation
Frequency: On PND 1, 4, 7, and 13.
Procedure: Live pups were individually weighed.

Sex – F1-Generation
Frequency: On PND 1 and 4.
Procedure: Sex was externally determined for all pups.

Anogenital Distance – F1-Generation
Frequency: On PND 1.
Procedure: Anogenital distance (AGD) was measured for all live pups. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention – F1-Generation
Frequency: On PND 13.
Procedure: All males in each litter was examined for the number of areola/nipples. ]

Postmortem examinations (parental animals):

See "Any other information on materials and methods incl. tables" below

Postmortem examinations (offspring):

See "Any other information on materials and methods incl. tables" below

Statistics:

See "Any other information on materials and methods incl. tables" below

Reproductive indices:

See "Any other information on materials and methods incl. tables" below

Offspring viability indices:

See "Any other information on materials and methods incl. tables" below

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Test item-related clinical observations were observed starting at treatment with 100 mg/kg/day.

Rales were noted in some animals (males and females) of the 100 and 300 mg/kg dose group on multiple consecutive days throughout the treatment period. Hunched posture and piloerection were incidentally noted in females at 100 mg/kg during week 2 of treatment. Salivation seen after dosing among animals of the 100 and 300 mg/kg dose group during most days of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Incidental findings that were noted included alopecia. These findings 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. At the incidence observed, these were not considered to be signs of toxicological relevance.

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post coitum and lactation phase.

Mortality:

no mortality observed

Description (incidence):

(Appendix 1 and Appendix 2)
No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology
(Appendix 1 and Appendix 2)
No toxicologically relevant changes were noted in haematological parameters. Haematocrit values in treated females at 300 mg/kg achieving a level of statistical significance when compared to controls were considered not toxicologically relevant due to the minimal magnitude of the change (0.95x of control) and since values remained within the normal range(1).

(1)Historical control data in (non-fasted) female Wister Han rats (period 2015-2019):
Haematocrit (L/L): Mean = 0.435; P5-P95 = 0.401 - 0.469 (n = 206).

Coagulation
(Appendix 1 and Appendix 2)
The following statistically significant changes distinguished treated from control animals at the end of the treatment period (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
• At 300 mg/kg, the activated partial thromboplastin time (APTT) was increased in females (1.18x of control). Values remained within the range of the historical controls(2).

(2)Historical control data (non-fasted) female Wistar Han rats (period 2017 - 2019):
APTT (s): Mean = 17.5; P5-P95 = 13.1 - 22.2 (n = 203).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals at the end of the treatment period (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):

• Increased alanine aminotransferase (ALAT) values in males at 300 mg/kg (1.60 x of control). Values were close to the upper limit of the historical control range(1).
• Decreased creatinine values in females at 30, 100 and 300 mg/kg (0.91, 0.91 and 0.86x of control, respectively). Values at 300 mg/kg were below the lower limit of the historical control range(2).
• Increased cholesterol values in females at 300 mg/kg (1.43x of control). Noteworthy, a trend towards the opposite effect was observed in males at 300 mg/kg (0.84x of control), although not reaching statistical significance. All values remained within normal ranges(3).

Thyroid hormone analyses:
Serum T4 analyses in F0-males are ongoing.

(1)Historical control data (fasted) male Wistar Han rats (period 2017 - 2019):
ALAT (U/L): Mean = 50.9; P5-P95 = 32.7 – 75.1 (n = 270).
(2)Historical control data (non-fasted) female Wistar Han rats (period 2017 - 2019):
Creatinine (umol/L): Mean = 39.7; P5-P95 = 35.4 - 44.3 (n = 210).
(3)Historical control data Wistar Han rats (period 2017 - 2019):
(fasted) Males: Cholesterol (mmol/L): Mean = 1.95; P5-P95 = 1.41 - 2.59 (n = 270).
(non-fasted) Females: Cholesterol (mmol/L): Mean = 2.05; P5-P95 = 1.48 - 2.69 (n = 210).
(4) Historical control data (fasted) male Wistar Han rats (period 2017 – 2019):
Total T4 (ug/dL): Mean = 4.51; P5-P95 = 2.85 – 6.37 (n=557)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

(Appendix 5)
There were no test item-related microscopic observations.
The apparent dose-dependent increased incidence and severity of extramedullary hematopoiesis in the spleen of treated females is most likely due to an abnormal low incidence (i.e. absence) of this finding in the control group and therefore not considered to be test item-related.
All other histologic changes were considered to be incidental findings and 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.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Food Consumption
(Appendix 1 and Appendix 2)
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.

In males and females of the 300 mg/kg group, relative food consumption was slightly decreased during the first two weeks of treatment. Normal values for food consumption and relative food consumption were noted during the rest of the treatment period. As the decrease in food consumption during the first two weeks of treatment was transient and values remained within normal range, this finding was considered not toxicologically relevant.

At 300 mg/kg, relative food consumption was statistically significantly increased in females between Day 17-20 post coitum. Given the direction of the effect and since values returned to control values in the next days, this finding was considered not toxicologically relevant. Please note that food consumption in males during the mating phase was not yet corrected for the days spend with the female (i.e. not in the male home cage).

Functional Tests
(Appendix 1 and Appendix 2)

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

In males, grip strength of the fore leg was statistically significantly decreased at 300 mg/kg (0.65x of control). A similar trend was observed for hind leg grip strength at 300 mg/kg (0.86x of control), although values did not reach statistical significance. All values remained within the range of our historical control data1. In females, grip strength was considered similar between control and high dose animals.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

(Appendix 2)
Length and regularity of the estrous cycle were considered not to have been affected by
treatment. All females had regular cycles of four days.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating Index:
(Appendix 1 and Appendix 2)
Mating index was considered not to be affected by treatment. Except for female no. 51 (Group 2), all females showed evidence of mating.
The mating indices were 100, 90, 100 and 100% for the control, 30, 100 and 300 mg/kg groups, respectively.

Precoital Time:
(Appendix 1 and Appendix 2)
Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within four days.

Number of Implantation Sites:
(Appendix 1 and Appendix 2)
Number of implantation sites was considered not to be affected by treatment. One female at 30 mg/kg (No. 56) and one female at 100 mg/kg (No. 63), which did not deliver a viable litter, had a single implantation site only. As this occurred in absence of a dose response relationship, this was considered to be unrelated to treatment.

Fertility Index
(Appendix 1 and Appendix 2)
Fertility index was considered not to be affected by treatment. The fertility indices were 80,
89, 100 and 100% for the control, 30, 100 and 300 mg/kg groups, respectively. A total of two females of the control group (female nos. 47 and 49) and one female at 30 mg/kg (female no. 59) were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment.

Gestation Index and Duration
(Appendix 1 and Appendix 2)
Gestation index and duration of gestation were considered not to be affected by treatment.
Except for one female at 30 mg/kg (female no. 59) and one female at 100 mg/kg (female no. 63), all pregnant females had live offspring. The gestation indices were 100, 88, 90 and 100% for the control, 30, 100 and 300 mg/kg groups, respectively.
The failed pregnancies of female No.59 (Group 2) and No. 63 (Group 3), without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

Wistar Han rats were treated with 4-HYDROXY-BENZOPHENONE by daily oral gavage at dose levels of 30, 100 and 300 mg/kg. The rats of the control group received the vehicle, Propylene glycol, alone. 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 14-16 days of lactation (for 51-63 days). Females that failed to deliver pups were treated for 40-54 days.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results:
No mortality occurred during the study period.

Test item-related clinical observations were observed starting at treatment with 100 mg/kg/day. Rales were noted in some animals (males and females) of the 100 and 300 mg/kg dose group on multiple consecutive days throughout the treatment period. Further evaluation of the adversity of this clinical sign is pending histopathological evaluation.

Moreover, hunched posture and piloerection were incidentally noted in females at 100 mg/kg during week 2 of treatment. Given the low incidence and transient nature of these clinical signs of toxicity, these findings were considered not toxicologically relevant. Salivation seen after dosing among animals of the 100 and 300 mg/kg dose group during most days of the treatment period was considered not toxicologically relevant. This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. In males, grip strength of the fore leg was statistically significantly decreased at 300 mg/kg (0.65x of control). A similar trend was observed for hind leg grip strength at 300 mg/kg (0.86x of control), although values did not reach statistical significance. As all values remained within normal ranges, this was considered not toxicologically relevant, pending histopathological examination of related tissues. In females, grip strength was considered similar between control and high dose animals.

The following statistically significant changes in coagulation parameters distinguished treated from control animals at the end of the treatment period: At 300 mg/kg, the activated partial thromboplastin time (APTT) was increased in females (1.18x of control). Values remained within normal limits. Further evaluation of coagulation parameters is pending histopathological evaluation.

Clinical laboratory investigations showed treatment-related changes, mainly at 300 mg/kg: Increased alanine aminotransferase (ALAT) values in males, decreased creatinine values in females (also at 30 and 100 mg/kg) and increased cholesterol values in females. Noteworthy, a trend towards the opposite effect on cholesterol levels was observed in males, although not reaching statistical significance. Except for creatinine levels in females at 300 mg/kg, all values remained within normal ranges. Further evaluation of the adversity of these clinical biochemistry parameters is pending histopathological evaluation.

Test item-related higher kidney weights (relative to body weights) were noted in the 300 mg/kg group in males. Given the magnitude of the effect (<10%), this increase was considered not adverse, pending histopathological evaluation of the kidneys.

In females of the 300 mg/kg group, test item-related higher liver weights were noted. Given the magnitude of the effect, this test-item related increased liver weights were considered adverse, pending histopathological evaluation of the liver. There were no other test item-related organ weight changes.

No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. remaining functional observations, motor activity measurements, body weight, food consumption, haematological parameters, and macroscopic examination).

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

(Appendix 2)
No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Post-Implantation Survival Index:
(Appendix 1 and Appendix 2)
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 85, 93, 88 and 92% for the control, 30, 100 and 300 mg/kg groups, respectively.

Litter Size:
(Appendix 1 and Appendix 2)
Litter size was considered not affected by treatment. Live litter sizes were 9.3; 11.4; 9.7 and 10.3 living fetuses/litter for the control, 30, 100 and 300 mg/kg groups, respectively.

Live Birth Index:
(Appendix 1 and Appendix 2)
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.
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. The live birth indices were 100, 100, 95 and 100 % for the control, 30, 100 and 300 mg/kg groups, respectively. Five pups at 100 mg/kg were found dead at first litter check. 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.

Viability Index:
(Appendix 1 and Appendix 2)
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.

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. Viability indices were 100, 100, 98 and 98% for the control, 30, 100 and 300 mg/kg groups, respectively. Two pups at 100 mg/kg and two pups at 300 mg/kg were found missing on PND 2 or 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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Body weights of pups were considered not to be affected by treatment.

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Analysis of serum T4 levels in male and female PND 14-16 pups are ongoing.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

9.4.12. Areola/Nipple Retention
(Appendix 1 and Appendix 2)
Treatment up to 300 mg/kg had no effect on areola/nipple retention examined male pups nipples were observed at PND 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

(Appendix 2)
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex Ratio:
(Appendix 1 and Appendix 2)
Sex ratio was considered not to be affected by treatment.

Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (300 mg/kg).
No treatment-related toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle and histopathological examination of reproductive organs).

Developmental results
No developmental toxicity was observed up to the highest dose level tested (300 mg/kg).
No treatment-related changes toxicologically significant changes were noted in any of the 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, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

other: sex ratio, anogenital distance and areola/nipple retention

Reproductive effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Conclusions:

In conclusion, based on the interim results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422, performed under GLP conditions); (no raw data was reviewed and no phase reports were available for dose formulation analysis and histopathology), the following no-observed-adverse-effect level (NOAEL) of 4-HYDROXY-BENZOPHENONE were established:

Parental NOAEL: at least 300 mg/kg
Reproduction NOAEL: at least 300 mg/kg
Developmental NOAEL: at least 300 mg/kg

Executive summary:

The objectives of this study were to determine the potential toxic effects of 4-Hydroxy-benzophenone when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 0, 30, 100 and 300 mg/kg/day, based on the results of the dose range finder (Test Facility Study No. 20151995). 10 male and 10 female animals were tested for each dose level. The dose volume was 5 mL for each dose level.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 and TSH (F0-males and females), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, 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 and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously.

Parental results:

Test item related effects were noted in the following parameters and end point evaluation: clinical signs, functional observations, clinical pathology, measurement of thyroid hormone T4 (F0-males) and organ weights.

Test item-related clinical observations were observed starting at treatment with 100 mg/kg. Rales were noted in some animals (males and females) of the 100 and 300 mg/kg dose group on multiple consecutive days throughout the treatment period. This sign was considered non-adverse in absence of effects on body weight and food consumption and supporting histopathological findings of respiratory distress.

In males, grip strength of the fore legs was statistically significant decreased at 300 mg/kg (0.65x of control). A similar trend was observed for hind leg grip strength at 300 mg/kg (0.86x of control), although values did not reach statistical significance. As all values remained within normal ranges and there were no correlating histopathological findings in muscle or skeletal tissue, this decrease was considered not toxicologically relevant. In females, grip strength was considered similar between control and high dose animals.

Clinical laboratory investigations showed the following treatment-related changes at 300 mg/kg: decreased creatinine values in females (also at 30 and 100 mg/kg). At 300 mg/kg values were outside the normal ranges. Based on the minimal magnitude of the change and absence of corroborative findings this was considered non-adverse.

Decreased serum T4 levels in F0-males was observed 100 and 300 mg/kg. Values at 300 mg/kg were below the normal limits.

In females of the 300 mg/kg group, test item-related higher liver weights were noted. Based on the absence of macroscopic and microscopic liver findings or changes in blood liver parameters, this increased weight was considered to be non-adverse.

Reproductive and developmental results:

No reproduction or developmental toxicity was observed up to the highest dose level tested (300 mg/kg).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for 4-Hydroxy-benzophenone were established:

Parental NOAEL: at least 300 mg/kg (Note: In this study, a marked reduction of total T4 was observed in mid and high dose groups (in males) which was considered to be compound-related. However, under the conditions of this screening study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental NOAEL.

Reproduction NOAEL: at least 300 mg/kg

Developmental NOAEL: at least 300 mg/kg.

Note: Higher doses could not be tested as they were not tolerated (see dose range finder study).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available