Acetophenone

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Extended One-Generation Reproductive Toxicity Study (OECD TG 443, GLP):

In P generation, Cohort 1A or Cohort 1B animals there were no adverse effects on reproductive parameters at all doses tested. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be: 500 mg/kg/day in both sexes based on absence of adverse effect at this dose level.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
in accordance with ECHA Decision number: CCH-D-2114394437-37-01/F:
Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in Sprague Dawley rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (Developmental neurotoxicity);

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan:SD (CD®)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) carboxymethylcellulose 400-800cPs / 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurs or 14 days have elapsed
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- If mating has not occurred after 2 weeks, the animals will be separated without further opportunity for mating.
- After successful mating each pregnant female was caged (how): alone

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentrations in the administered dose formulations analyzed in each P-premating, P-mating, P-Gestation, P-Lactation periods and 4 times spread over the F1-postweaning period remained within an acceptable range of -5.3% to +2.7% when compared to the nominal values ( ± 15% of the nominal concentrations).
No test item was observed in the control dose formulation.

Duration of treatment / exposure:

P males: at least 18 weeks of treatment (10 weeks before mating, up to 2 weeks during the mating period, until the day before euthanasia (after weaning of the F1 pups))
P females: at least 16 to 18 weeks of treatment (10 weeks before mating, up to 2 weeks during the mating period, during gestation, during lactation until the day before euthanasia, until the day before euthanasia for females with no evidence of mating or no delivery)
Cohort 1A (males and females): from weaning (Day 22 p.p.) until the day before euthanasia (from Day 90 p.p., to 104 p.p. maximum)
Cohort 1B (males and females): from weaning (Day 22 p.p.) until the day before euthanasia (from Day 98 p.p., to 112 p.p. maximum)
Cohort 2A (males and females): from weaning (Day 22 p.p.) until the day before euthanasia (after completion of behavioural testing: after Day 75 p.p. but not exceeding Day 90 p.p.)
Cohort 2B: no direct dosing

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

group 2

Dose / conc.:

225 mg/kg bw/day (nominal)

Remarks:

group 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

20 (P and cohort 1A and 1b animals)
10 (cohort 2A and 2B)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, based on the results of the following previous studies:
> A 90-day day study (see section 7.5.1, Key study 2016) performed in Wistar rats using gavage (in corn oil) at dose levels of 125, 250 and 500 mg/kg/day.
> A pre-natal developmental toxicity study (see section 7.8.2 Key study 2016) performed in Wistar rats using gavage (in corn oil) at dose levels of 125, 300 and 750 mg/kg/day (Gestation Days 5-19).
> An OECD 422 screening study (see section 7.8.1 Key study 2003) performed in Sprague-Dawley rats using gavage (in corn oil) dose levels of 75, 225 and 750 mg/kg/day.
- Fasting period before blood sampling for clinical biochemistry: yes

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day
- Cage side observations checked: morbidity, mortality, general clinical observation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a week

BODY WEIGHT: Yes
- Time schedule for examinations:
> P Males: at least once during the pre-treatment period, on the first day of treatment (Day 1), then at least once a week until euthanasia.
> P Females: at least once during the pre-treatment period, on the first day of treatment (Day 1), then at least once a week until mated (or until euthanasia for females with no evidence of mating), on Days 0, 4, 7, 10, 14, 17 and 20 post-coitum (p.c.) and, on Days 1, 4, 7, 14 and 21 p.p.
> Cohorts 1A, 1B and 2A (males/females): on the first day of treatment (Day 1), then at least once a week until euthanasia.

FOOD CONSUMPTION:
- The quantity of food consumed by each P male was measured at least once a week from the first day of treatment until the start of the mating period and after the mating period until euthanasia.
- The quantity of food consumed by each P female was measured at least once a week from the first day of treatment until the start of the mating period, during gestation for the intervals: Days 0 4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7 14, and 14 21 p.p.
- Cohorts 1A, 1B and 2A: the quantity of food consumed by each animal (males and females from Cohorts 1A, 1B and 2A) will be measured at least once a week from the first day of treatment until euthanasia.

WATER CONSUMPTION:No

Reactivity to manipulation or to different stimuli (Functional Observation Battery):
Cohort 2A animals were observed once between Days 63 and 75 p.p., in the cage, in the hand and in the standard arena. The following parameters will be assessed and graded:
- in the cage: "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of miosis or mydriasis),
- in the standard arena (two-minute recording): grooming, palpebral closure, defecation, and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyper activity), posture, stereotypic behaviour and breathing, ataxia, hypotonia.
In addition, the following parameters, reflexes and responses were recorded:
- pupil reflex,
- auditory startle reflex,
- forelimb grip strength,
- visual stimulus.

MOTOR ACTIVITY:
Cohort 2A animals were subjected to motor activity testing (including habituation) on the same day as the Functional Observation Battery using an automated infra-red sensor equipment recording individual animal activity over a 60 minute period.
The following parameters will be reported:
- horizontal movements,
- vertical movements.

LYMPHOCYTE SUBTYPING (Cohort 1A)
For the investigation of pre- and postnatally induced immunotoxic effects, 10 males and 10 females from Cohort 1A animals were subject to a splenic lymphocyte subpopulation analysis [T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, natural killer (NK) cells and NKT cells].

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning as follows:
- during the last 2 weeks of the premating period,
- during the mating period, until the females are mated or the mating period has ended.

Sperm parameters (parental animals):

Parameters examined in P and Cohort 1A male parental generations:
testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

On Day 22 p.p., 20 pups/group (Cohort 2A) (10 males and 10 females/group; 1 male or 1 female/litter; all litters represented by at least 1 pup; randomly selected) were selected for neurobehavioral testing followed by neurohistopathology assessment as adults.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not included

Auditory startle test:
Cohort 2A animals were subjected to an auditory startle test (including habituation) on Day 24 p.p. (± 1 day) using a computerized shuttle box system to determine the amplitude and time to response.

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, all surviving P animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- P males: after weaning of all F1 pups (the left epidydimis and testis will be sampled at euthanasia, see § Seminology),
- P females: on Days 22 to 25 p.p.,
- P females which do not deliver: on Day 24-26 p.c. (after body weight recording to check for a possible unnoticed delivery; euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation will be used if gestation is suspected),
- P females with no evidence of mating: 24-26 days after the end of the mating period if no delivery occurred (euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation will be used if gestation is suspected),
- P females with total litter loss: as appropriate.
On completion of the treatment period, all surviving F1 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- F1 males (Cohort 1A): on Day 90-104 p.p. (the left epididymis and testis will be sampled at euthanasia, see § Seminology),
- F1 males (Cohort 1B): after euthanasia of Cohort 1A and agreement of the Study Director,
- F1 females (Cohort 1A): on Day 90-104 p.p.,
- F1 females (Cohort 1B): after euthanasia of Cohort 1A and agreement of the Study Director,
- F1 animals (Cohort 2A): between Days 75 and 90 p.p., after completion of behavioural testing,
- F1 animals (Cohort 2B): on Day 22 p.p.

HEMATOLOGY, COAGULATION AND BLOOD BIOCHEMISTRY (P and Cohort 1A animals):
parameters determined: packed cell volume (haematocrit), erythrocyte count, haemoglobin, mean cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, thrombocyte (platelet) count, total leucocyte count, differential white cell count with cell morphology, reticulocyte count (absolute and relative), prothrombin time, fibrinogen, activated partial thromboplastin time.

BLOOD BIOCHEMISTRY (P and Cohort 1A animals):
The following parameters will be determined from each group (10 animals/sex/group):
sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total proteins, albumin, albumin/globulin ratio, bile acids.

THYROID HORMONES (P generation, F1 culled pups and Cohort 1A animals):
Blood samples were collected into tubes containing K3-EDTA as anticoagulant, as follows:
- on Day 4 p.p. from F1 pups to be culled, for potential measurement of thyroid hormone (T4) levels,
- on Day 22 p.p. from F1 pups not selected for cohorts for measurement of thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels,
- at termination from the first 10/group surviving P males and lactating females and the first 10/sex/group surviving Cohort 1A animals for measurement of thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels.

GROSS NECROPSY
A complete macroscopic post-mortem examination will be performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and not selected F1 pups on Day 22 p.p.). This will include examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention will be paid to the reproductive organs.
The numbers of implantation sites will be recorded for females euthanized on Days 22 25 p.p. (P generation).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

NEUROPATHOLOGY ASSESSMENT:
Cohort 2A animals were terminated after behavioural testing (after Day 75 p.p., but not exceeding Day 90 p.p.), with brain weight recorded and full neurohistopathology for purposes of neurotoxicity assessment

Postmortem examinations (offspring):

SACRIFICE
F1 pups were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- pups whose mother dies: as soon as possible,
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- pups not selected at weaning: on Day 22 p.p.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
A microscopic examination was performed on:
• all tissues listed in the Tissue Procedure Table (see report) from animals of the control and high-dose groups from Cohort 1A euthanized at the end of the treatment period,
• all macroscopic lesions of all groups (from P generation and F1 cohorts), including those from found dead and prematurely euthanized animals,

NEUROPATHOLOGY ASSESSMENT:
On Day 22 p.p., 20 pups/group (10 males and 10 females/group; 1 male or 1 female/litter; all litters represented by at least 1 pup; randomly selected) were selected for neurohistopathology (including brain weight) at weaning (Day 22 p.p.). If there are insufficient numbers of animals, preference will be given to assign animals to Cohort 2A.

Statistics:

Body Weight, Food Consumption and Reproductive Data: data were compared by one-way variance analysis and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
PATHDATA software was used to perform the statistical analysis of organ weight data.
Splenic Lymphocyte Immunophenotyping: statistical analysis was performed using the SAS Enterprise Guide software.
Ano-Genital Distance, Locomotor Activity, Number of Nipples and Areolae, Number of Primary Follicles/Corpora Lutea, Time of Preputial Separation/Vaginal Opening, Time to First Estrous after Vaginal Opening/Patency, Seminology, Hematology, Coagulation, Blood Biochemistry, Thyroid Hormones, Post-Implantation Loss, Sex-Ratio, Live Birth, Viability and Lactation Indexes and Auditory Startle Reflex Data: lab's internal sofware (see report).

Reproductive indices:

The following parameters were calculated:
• post-implantation loss = [(Number of implantation sites - Number of live pups) / Number of implantation sites] x 100
• mating index = (Number of mated animals / Number of paired animals) x 100
• fertility index= (Number of pregnant female partners / Number of mated pairs) x 100
• gestation index = (Number of females with live born pups / Number of pregnant females) x 100
• AGD/cube root of body weight ratio (calculated with Excel) = (AGD / ³√Body weight)

Offspring viability indices:

• live birth index = (Number of live pups on Day 1 p.p. / Number of delivered pups) x 100
• viability index on Day 4 p.p. = [Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups] x 100
• lactation index = (Number of surviving pups on Day 21 p.p. / Number of surviving pups on Day 4 p.p. (after culling)] x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 500 mg/kg/day: reflux at dosing (1 animal, Day 45), loud/abdominal breathing (2, Days 45-88), hypoactivity (24, Days 2-131), half-closed eyes (24, Days 5-124), staggering gait (2, Days 21-60), continuous chewing movement (6, Days 5-71) and/or ventral recumbency (1, Day 4) were observed before and/or after treatment as early as 3 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day, findings of hypoactivity (21 animals, Days 34-120) and/or half-closed eyes (14, Days 24-117) were observed after dosing as early as 24 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no adverse clinical signs.
Ptyalism and/or burrowing activity were observed in all treated groups with dose related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia/thinning of hair, chromodacryorrhea/chromorhinorrhea, scabs/wounds, bent tail, soiled
forelimb and/or abnormal growth of teeth) are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item treatment was considered to be unlikely.

Females:
At 500 mg/kg/day, continuous chewing movement (9 animals, Days 5-17), half-closed eyes (12, Days 5-119), hypoactivity (12, Days 3-119), abdomen increased in size (12, Days 10-29) and/or lateral recumbency (2, Day 4) were observed before and/or after treatment as early as 3 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day, continuous chewing movement (1, Day 16), half-closed eyes (18, Days 12-121) and/or hypoactivity (14, Days 34-121) were observed after dosing as early as 12 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no adverse clinical signs.
Ptyalism and burrowing activity were observed in all treated groups with dose related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia, chromodacryorrhea/chromorhinorrhea, hunched posture, loud breathing, pallor of extremities, piloerection, reflux at dosing, scabs/wounds, soiled mouth region, bent tail, thin appearance, vaginal discharge and/or vulva increased in size) are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item-treatment was considered to be unlikely.

Mortality:

mortality observed, treatment-related

Description (incidence):

In males, there were no unscheduled deaths.
In females, there was a dose-related increased incidence of mortality in test item-treated females. A relationship to test item was considered to be likely since no similar findings occurred in controls.
There was a reproduction trouble in 2 females treated at 75 mg/kg/day (more specifically, both females had difficulties in delivery), 4 females treated at 225 mg/kg/day (3 females had a dead litter and 1 female had difficulties in delivery) and in 12 females treated at 500 mg/kg/day (11 females had a dead litter and 1 female had a dead fetus in the uterus and red vagina among other signs). In addition, 1 control female and 1 female treated at 225 mg/kg/day had no evident cause of death.
In pregnant females from 75 mg/kg/day, there were premature deaths because of difficulties to deliver and/or dead litter that were considered to be test item related:
- In the 500 mg/kg/day group: 11 females were prematurely euthanized due to observations of dead litters during the period of Days 1 to 4 p.p., and 1 female was prematurely euthanized on Day 25 p.c. on humane grounds (observations of abnormal color (reddish) vagina, piloerection, hunched posture, pallor of eyes and extremities, hypoactivity, staggering gait, increase in size of the abdomen, scabs, half-closed eyes and ptyalism).
- In the 225 mg/kg/day group: 1 female was prematurely euthanized on Study Day 32 (premating period) on humane grounds (observations of tremors, staggering gait) (No macroscopic finding was observed at necropsy). 1 female was prematurely euthanized on Day 24 p.c. on humane grounds (difficulties in delivery) (At necropsy, in the right uterine horn, one dead fetus and 7 placentas were observed, in the left uterine horn, 9 placentas and 2 scars were present. Vagina was red discolored, and a liquid was present). 3 females were prematurely euthanized due to observations of dead litters during the period of Days 2 to 3 p.p.. Two placentas
were present in the uterus of 1 female ; three placentas and one late resorption were
present in the uterus of another female.
- In the 75 mg/kg/day group: 1 female was prematurely euthanized on Day 24 p.c. on humane grounds (difficulties in delivery) (At necropsy, in the right uterine horn, 1 placenta and 5 scars were observed; in the left uterine horn, one dead fetus, 9 placentas and one scar were present). 1 female was prematurely euthanized on Day 23 p.c. on humane grounds (difficulties in delivery) (At necropsy, in the right uterine horn, 4 dead fetuses, 4 placentas and 2 scars were observed; in the left uterine horn, 2 dead fetuses and 5 placentas were present).
- In the 0 mg/kg/day group: 1 female was prematurely euthanized on Study Day 32 (premating period) on humane grounds (observations of staggering gait and tremors from Study Day 14). No correlating finding were observed at necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

When compared with controls and despite a few statistically significant differences, there were no adverse effects on mean body weight or mean body weight change in P generation males or females.
In males (premating and post-mating periods) and at 500 mg/kg/day when compared with controls, there was a lower mean body weight gain over the Days 1 to 126 period (-9%, p< 0.01). The difference was mainly due to lower change during the first two weeks of the treatment period (Days 1 to 15). A test item treatment relationship cannot be excluded but taking into account the amplitude of the change, this finding was considered to be non-adverse.
During the premating period in females and from 225 mg/kg/day when compared with controls, there was a higher mean body weight gain over the Days 1 to 15 period (up to +30% at 500 mg/kg/day, p< 0.001). The difference was mainly due to higher change during the first two weeks of the treatment period (Days 1 to 15) which returned towards controls values from the Days 8 to 15 period. A test-item treatment relationship cannot be excluded but taking into account the reversibility of the change, this finding was considered to be non-adverse.
During the pregnancy period, when compared with controls and despite a few statistically significant differences, there were no adverse effects on mean body weight or mean body weight change in P generation females. At 500 mg/kg/day and when compared with controls, the initial mean body weight was significantly higher (approx. +7% on Days 0 to 14 p.c.) but weight gain over the period of gestation was comparable with controls. Therefore, the difference was considered to be non-adverse.
During the lactation period, when compared with controls, there were no effects on mean body weight or mean body weight change in P generation females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Premating (P Generation Males and Females) and Post-Mating (P Generation Males) Periods:
When compared with controls and despite a few statistically significant differences, there were no adverse effects on mean food consumption in P generation males or females.
In males and in all treated groups when compared with controls, there was a dose-related decrease in mean food consumption during the first week of the treatment period (down to -17% vs. controls at 500 mg/kg/day on Days 1 to 8, p<0.001). Thereafter, mean food consumption returned towards control values. Therefore these decreases were considered to be test-item treatment related but not adverse.
In females and at 500 mg/kg/day when compared with controls, there was a lower mean food consumption during the first week of the treatment period (-14% vs. control on Days 1 to 8, p<0.001). Thereafter, mean food consumption returned towards controls values. Therefore, this finding was considered to be test-item treatment related but not adverse.

During the pregnancy period in females, there were no effects of treatment on mean food consumption.
During the lactation period in females, at 225 and 75 mg/kg/day, when compared with controls, there were no effects on mean food consumption. At 500 mg/kg/day, when compared with controls, low mean food consumption was recorded (down to -32% on Days 14 to 21 p.p., p<0.001). This finding was considered to be test item treatment-related but non-adverse based on the absence of significant effects on mean body weight at this dose level during the lactation period.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no adverse test item treatment-related effects on mean haematology and coagulation parameters, in males and females.
In males and when compared with controls, there were a dose-related increase in reticulocytes (up to +35% in terms of absolute count at 500 mg/kg/day, p< 0.01). However, the differences remained within the range of the Historical Control Data. Therefore, while a test-item relationship cannot be excluded the differences were considered to be non-adverse.
The few other statistically significant differences were observed with no dose level relationship, with minimal magnitude when compared with controls, in one sex only and/or remained within the range of the Historical Control Data. Therefore, any test item-relationship was considered to be unlikely.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no adverse test item treatment-related effects on mean blood biochemistry parameters, in males and females.
In males (at 500 mg/kg/day) and in females (from 225 mg/kg/day), mean concentrations of bile acids were above the upper limit of the Historical Control Data. A test item was not excluded but the amplitude of the change was moderate and considered not to be adverse in the absence of associated pathology findings.
The few other statistically significant differences were observed with no dose level relationship, with minimal magnitude when compared with controls, in one sex only and/or remained within the range of the Historical Control Data. Therefore, any test item-relationship was considered to be unlikely.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid Hormones (P generation males and lactating females):
There were no test-item treatment related effects on mean plasma T4 and TSH concentrations in P-generation males.
There were no test-item treatment related effects on mean plasma T4 and TSH concentrations in P-generation females. At 75 mg/kg/day and when compared with controls, there was an increase in mean TSH plasma concentration (2394 vs. 1054 pg/mL, p<0.05). In the absence of any dose level relationship and associated effects on mean T4 plasma concentration, a test item treatment-related effect was considered to be unlikely.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were noted in the liver (hepatocellular hypertrophy), kidneys (tubular basophilia and accumulation of hyaline droplets), spleen (pigment), forestomach (hyperkeratosis and squamous cell hyperplasia), esophagus (hyperkeratosis) and thyroid gland (follicular cell hypertrophy) in males and females treated at ≥ 75 mg/kg/day. In addition, there was an equivocal slightly increased incidence of vaginal mucification in females treated at 500 mg/kg/day when compared to controls.
- liver: minimal to slight dose-related hepatocellular centrilobular or diffuse hypertrophy was recorded in males and females treated at ≥ 75 mg/kg/day.
- kidneys: minimal to moderate dose-related increased severity and/or incidence of tubular basophilia, and accumulation of hyaline droplets were noted in males treated at ≥ 75 mg/kg/day. The minimally increased incidence and/or severity of hyaline casts and dilatation of pelvis were considered not to be related to the test item administration in view of the low differences with controls.
- spleen: minimal to slight dose-related brown pigment was seen in enlarged macrophages in males and females treated at ≥ 75 mg/kg/day. At the examination of the Perls staining, the pigment was considered to be consistent with hemosiderin (positive blue staining in the macrophages seen with increased severity in the high dose male when compared to the control male.
- forestomach: minimal to slight dose-related hyperkeratosis and sometimes accompanied by squamous cell hyperplasia were seen in the forestomach from males and females treated at ≥ 75 mg/kg/day.
- esophagus: minimal diffuse hyperkeratosis was seen in the esophagus from males and females treated at 500 mg/kg/day. A single male treated at 75 mg/kg/day had also minimal hyperkeratosis in the esophagus. The relationship to test item remained questionable in view of this isolated occurrence at this low dose.
- thyroid gland: minimal follicular cell hypertrophy was noted in males and in one female treated at 500 mg/kg/day.

reproductive organs:
There was a minimal increase in the incidence of vaginal mucification in females treated at 500 mg/kg/day (8/12 = 67%) when compared to controls (9/23 = 39%). The severity was similar in both groups (minimal to moderate). Conversely, there was a decreased number of cycled females, i.e. noted in proestrus/estrus/metestrus/diestrus, in the group treated at 500 mg/kg/day (3/12 = 25%) when compared to controls (14/23 = 61%). There were no test item-related microscopic changes in the uterus, ovaries or oviducts. This minimal increase in vaginal mucification could be related with the difficulties to deliver and/or dead litter in the group treated at 500 mg/kg/day, although these changes were recorded in females not submitted for microscopic examination (prematurely sacrificed).
There were no test item-related changes in the male reproductive organs.
At the examination of males associated with reproductive difficulty (for which the female was
not pregnant or did not mate), the following observations were made: no noteworthy microscopic findings in the male genital system, no adverse changes in the other organs, except 1 male at 225 with a moderate atrophy of prostate and minimal decreased secretory content in seminal vesicles.
At the examination of females with no delivery, the following observations were made:
• 1 female treated at 75 mg/kg/day: no noteworthy microscopic findings in the female genital organs,
• 1 female treated at 75 mg/kg/day: 4 fetuses and 3 scars in the uterus with gestational glands remnants and slight dilatation, and vagina had marked mucification,
• 1 female treated at 225 mg/kg/day: no noteworthy microscopic findings in the female genital organs except slight dilatation of the uterus suggesting normal stage of cycle (pro-estrus/estrus),
• 1 female treated at 500 mg/kg/day: one dead fetus was found in the uterus, while vagina had moderate mucification,
• 1 female treated at 500 mg/kg/day: hemorrhage was seen in placental remnants in uterus but it was also seen in controls, vagina had moderate mucification. There were test item-related lesions in liver and forestomach.
There was a good correlation between the vaginal smears and the histopathological examination of estrus cycle in all examined females.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls. This included the poorly dose-related prostate atrophy seen in 1/24 males treated at 225 mg/kg/day and in 2/24 males treated at 500 mg/kg/day, that was not seen in any other test item-treated males.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects on mean estrous parameters in P generation females.

Enumeration of corpora lutea in HE-stained slides:
At enumeration of corpora lutea in the ovary of high-dose group and control group, there were no statistical differences with a mean number of corpora lutea of 10.42 (± 8.45) and 9.13 (± 3.48) per animal respectively.

Enumeration of the number of primordial follicles on PCNA-stained slides:
There were no statistical differences between the high-dose and control groups, with a mean number of 7.72 (± 6.37) and 6.27 (± 4.76) primordial follicles per animal on PCNA-stained slides respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the test item-treated groups and when compared with controls, there were no effects on sperm motility. Sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate) were unaffected by treatment with the test item at 500 mg/kg/day.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating and Fertility Data:
There were no adverse effects on mating (including the mean number of days taken to mate) or fertility in P generation animals.
At 500 mg/kg/day and when compared with controls or Historical Control Data (HCD), there was a lower female mating index (87.5% vs. 95.8% in controls or 100% in HCD). The difference was considered to be of no biological significance as such comparable fertility indexes were also observed in previous studies (90.0 to 100% in OECD421/422 studies, 2009-2012).

Delivery Data:
When compared with controls, there were no effects on Days 1 and 21 p.p. sex-ratio (percent of males) at any dose levels.
At 500 and 225 mg/kg/day, when compared with controls, there were low mean numbers of live pups on Day 1 p.p. (7.5 and 8.8 vs. 11.9, respectively) and low live birth indices (34.1 and 79.6 % vs. 96.8%, respectively). These decreases were the consequence of high mean percent of post-implantation losses (29.8%, p<0.05 and 24.1% vs. 16.1%, respectively). These findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day and when compared with controls, there were no test item treatment-related effects on delivery data.

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

In P generation, Cohort 1A or Cohort 1B animals there were no adverse effects on reproductive parameters at all doses tested.

Dose descriptor:

NOAEL

Effect level:

< 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Difficulties to deliver

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Lactating pups:
Macroscopic examination of F1 pups on Day 1 p.p. (qualitative external examination, assessment of body temperature and/or activity/reaction to handling): From 225 mg/kg/day onwards and when compared with controls, F1 pups had more findings at qualitative external examination (e.g. malformed/short tail and/or hematoma/desquamation/scab) and assessment of body temperature (e.g. cold to the touch) than controls. These findings were associated with low birth and Day 4 p.p. survival index. Therefore, they were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no evidence of adverse findings at macroscopic examination of F1 pups on Day 1 p.p.
Clinical Signs during lactation: From 225 mg/kg/day onwards, some pups were cold to the touch (Nb pups/Nb litters: 3/3, 7/3 and 24/2 respectively at 0, 225 and 500 mg/kg bw), dehydration (Nb pups/Nb litters: 1/1 and 1/1 respectively at 0 and 500 mg/kg bw), hypoactivity (Nb pups/Nb litters: 1/1 at 500 mg/kg bw) and/or thin appearance (Nb pups/Nb litters: 1/1 at 500 mg/kg bw). These findings were considered to be the consequence of lack of maternal care and, to be test item treatment-related and adverse.
The other observed findings were recorded in controls, observed at low incidences with no dose level relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study.

Cohort 1A (males):
At 500 mg/kg/day, half-closed eyes (10 animals, Days 1-38), hypoactivity (10, Days 1-39), loss of balance (1, Day 3) and/or staggering gait (8, Days 1-9) were observed after treatment as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day, hypoactivity (2 animals, Days 1-2) and/or half-closed eyes (4, Days 2-4) were observed after dosing as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no adverse clinical signs.
Ptyalism was observed in all treated groups with dose related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (abdominal breathing, alopecia/thinning of hair, bent head, burrowing activity, chromodacryorrhea, decreased grasping reflex, difficulty in using limb/forelimb, hunched posture, hyperactivity, loud breathing, nodosities, scabs, piloerection, soiled anus and/or thin appearance) were recorded without any dose level relationship and/or are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item treatment was considered to be unlikely.

Cohort 1A (females):
At 500 mg/kg/day, half-closed eyes (19 animals, Days 1-43), hypoactivity (19, Days 1-43), loss of balance (1, Day 2), loud breathing (5, Days 8-28), piloerection (9, Days 3-7) and/or staggering gait (12, Days 1-9) were observed before and/or after treatment as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day, half-closed eyes (4 animals, Days 2-3), hypoactivity (1, Day 3) and/or loud breathing (3, Days 17-32) were observed before and/or after dosing as early as 2 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no adverse clinical signs.
Ptyalism was observed in all treated groups with dose-related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia and/or thinning of hair, nodosities, scabs and/or shortened/bent tail) were recorded without any dose level relationship and/or are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item treatment was considered to be unlikely.

Cohort 1B (males):
At 225 mg/kg/day, burrowing activity (2 animals, Days 2-49), half-closed eyes (4, Days 2-66), hypoactivity (2, Days 46-56) and/or loud breathing (2, Days 6-7) were observed before and/or after treatment as early as 2 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no adverse clinical signs.
Ptyalism was observed in all treated groups with dose related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia and/or thinning of hair, chromodacryorrhea, scabs/wounds and/or bent tail) were recorded without any dose level relationship and/or are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item-treatment was considered to be unlikely.

Cohort 1B (females):
At 225 mg/kg/day, burrowing activity (2 animals, Days 2-73), half-closed eyes (2, Days 2-77) and/or hypoactivity (3, Days 38-73) were observed after treatment as early as 2 days after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
Ptyalism was observed in all treated groups with dose related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia and/or thinning of hair, nodosities, scabs and/or piloerection) were recorded without any dose level relationship and/or are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item-treatment was considered to be unlikely.

Cohort 2A (males):
At 500 mg/kg/day, half-closed eyes (5 animals, Days 1-24), hypoactivity (5, Days 1-39), piloerection (5, Days 1-6) and/or staggering gait (5, Days 1-7) were observed before and/or after treatment as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day, half-closed eyes (2 animals, Days 1-5) and /or hypoactivity (2, Days 1-2) were observed after treatment as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no test item treatment-related clinical signs.
Ptyalism was observed in all treated groups with dose-related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia, burrowing activity, nodosities, scabs and/or bent tail) were recorded without any dose level relationship and/or are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item-treatment was considered to be unlikely.

Cohort 2A (females):
At 500 mg/kg/day, burrowing activity (1 animal, Days 8-9), half-closed eyes (4, Days 1-54), hypoactivity (4, Days 1-32), loud/abdominal breathing (before / after treatment) (2, Days 9-25), piloerection (4, Days 1-16) and/or staggering gait (3, Days 2-4) were observed before and/or after treatment as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day, burrowing activity (1 animal, Day 3), half-closed eyes (2, Days 2-4), hypoactivity (2, Days 1-4) and/or loud/abdominal breathing (2, Days 11-29) were observed before and/or after treatment as early as 1 day after the start of repeated dosing. Taken together, these findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day, there were no test item treatment-related clinical signs.
Ptyalism and or reflux at dosing were observed in all treated groups with dose-related increased incidence and onset. These findings are commonly observed after a gavage procedure and were considered to be test item treatment-related but not adverse. Other findings (alopecia and/or thinning of hair, chromodacryorrhea, scabs and/or bent tail) were recorded without any dose level relationship and/or are commonly observed in this species and strain maintained in the experimental conditions of this study. Therefore, any relationship to the test item-treatment was considered to be unlikely.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Lactating pups (mortality during lactation):
On Days 1-4 p.p. from 225 mg/kg/day, when compared with controls, there was a higher percentage of pups found dead, missing and/or cannibalized (25.9 and 79.1 %, p<0.001 vs. 3.9 % in controls at 225 and 500 mg/kg/day, respectively). At necropsy of found dead pups, absence of milk and/or cannibalism were noted. Absence of milk in the stomach is likely to represent nursing difficulties or absence of maternal care. These findings were considered to be test item treatment-related and adverse.
At 75 mg/kg/day and despite a slight increase in the number of found dead pups when compared with controls, there were no statistically significant differences in the number of died, missing and/or cannibalized pups in P generation offspring.
Viability and lactation indexes: from 225 mg/kg/day, when compared with controls or Historical Control Data, there was a low Day 4 p.p. viability index (77.7 and 38.1% vs. 96.3% in controls at 225 and 500 mg/kg/day, respectively). This finding was considered to be test item treatment-related and adverse. After culling on Day 4 p.p., there were no effects on lactation index. At 75 mg/kg/day and when compared with controls, there were no effects on the viability or lactation indices.

Cohort 1A:
In the 75 mg/kg/day group: 1 male was found dead on Day 1 (at necropsy, fracture and red discoloration of the cranium were observed and, in the brain, red discoloration was present), and 1 female was prematurely euthanized on Day 34 on humane grounds (a necropsy, a scab on neck was observed and dessicated and exophthalmia of right eye were observed).
In the 500 mg/kg/day group: 1 female was prematurely euthanized on Day 1 on humane grounds (Hypoactivity, Lateral recumbency, Staggering gait, Half-closed eyes). No macroscopic finding was observed at necropsy.

Cohort 1B:
There were no unscheduled deaths in Cohort 1B males and females (dose levels of 75 and 225 mg/kg/day).

Cohort 2A:
In the 225 mg/kg/day group: 1 male was found dead after treatment on Day 20 (no clinical signs, no correlating findings were observed, at necropsy).
In the 500 mg/kg/day group: 1 female was found dead after treatment on Day 24 (Hypoactivity and ptyalism were observed before death). No correlating findings were observed, at necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lactating pups:
At 500 mg/kg/day, when compared with controls, mean body weight was low in males and females from Day 1 p.p. (-20% vs. controls in both sexes, p<0.001). Mean body weight and mean body weight gain remained lower than controls during the lactation period.
At 225 mg/kg/day, when compared with controls, mean body weight was low in males and females from Day 1 p.p. (-11 and 9% vs. controls in males and females respectively, p<0.001), with a return to control values thereafter. Therefore, while a test item-relationship could not be excluded, this finding was of minimal magnitude and considered to be non-adverse.
At 75 mg/kg/day, there were no test item treatment-related effects.

Cohort 1A (males):
At 500 mg/kg/day, when compared with controls, mean body weight and mean body weight gain were lower (down to -18%, p<0.01 vs. controls and +40 g, p<0.01 vs. +50 g on Day 8, respectively) with a tendency towards a return to control values thereafter (-8% for mean body weight on Day 64). Considering the magnitude of the differences, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day and when compared with controls, there were a few statistically significant differences (less than 10% for mean body weight) which were considered to be test item treatment-related and non-adverse effects based on the magnitude of the change. Mean body weight change was not affected.
At 75 mg/kg/day, there were no effects on mean body weight or mean body weight change.

Cohort 1A (females):
There were no adverse effects on mean body weight or mean body weight change.
In females and from 225 mg/kg/day when compared with controls, there was a higher mean body weight gain over the Days 1 to 64 period (up to +14% at 500 mg/kg/day, p< 0.01). The difference was mainly due to higher change during the first two weeks of the treatment period (Days 1 to 15) with a return towards controls values on the period of Days 50 to 57. A test-item treatment relationship cannot be excluded but taking into account the reversibility of the change, this finding was considered to be non-adverse.

Cohort 1B:
There were no effects on mean body weight or mean body weight change in Cohort 1B animals.
The statistically significant differences in terms of mean body weight change in the 225 mg/kg/day females (on periods of Days 8 to 15 and Days 36 to 43) were isolated and recorded with minimal magnitude when compared to controls. Therefore, they were considered to be fortuitous.

Cohort 2A (males):
At 500 mg/kg/day, and when compared with controls, mean body weight and mean body weight gain were lower (down to -18% vs. controls and +38 vs. +51 g on Day 8, respectively). Taking into account the magnitudes of the differences, these findings were considered to be test item treatment-related and adverse.
At 225 mg/kg/day and when compared with controls, there were a few statistically significant differences (less than 10% for mean body weight) which were considered to be test item treatment-related and non-adverse effects based on the magnitude of the change.
At 75 mg/kg/day, there were no effects on mean body weight or mean body weight change.

Cohort 2A (females):
There were no adverse effects on mean body weight or mean body weight change.
In females and from 225 mg/kg/day when compared with controls, there was a higher mean body weight gain over the Days 1 to 50 period (up to +13% at 500 mg/kg/day, p< 0.01). The difference was mainly due to higher change during the first month of the treatment period (Days 1 to 29) with a tendency towards a return to controls values from the period of Days 29 to 32. A test-item treatment relationship cannot be excluded but taking into account the reversibility of the change, this finding was considered to be non-adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Cohort 1A:
At 500 mg/kg/day and when compared with controls, mean food consumption was low on Days 8 to 15 (-24%, p<0.05 for males and -11%, p<0.05 for females). Thereafter, mean food consumption returned towards control values. Taking into account the magnitude and duration of the change, this finding was considered to be test item treatment-related but non adverse.
At 225 and 75 mg/kg/day, there were no effects on mean food consumption.

Cohort 1B:
There were no effects on mean food consumption in Cohort 1B animals.

Cohort 2A:
There were no effects on mean food consumption in Cohort 2A animals.

Haematological findings:

no effects observed

Description (incidence and severity):

Cohort 1A:
There were no adverse, test item treatment-related effects on mean haematology and coagulation parameters.
The few statistically significant differences were observed with no dose level relationship, with minimal magnitude when compared with controls, in one sex only and/or remained within the range of the Historical Control Data. Therefore, any test item-relationship was considered to be unlikely.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no adverse test item treatment-related effects on mean blood biochemistry parameters.
In males and females there were dose related increased mean concentrations of bile acids (above the upper limit of the Historical Control Data for males). A test-item effect was not excluded but the amplitudes of the changes were considered not to be adverse in the absence of associated adverse findings at pathology examination.
The few statistically significant differences were observed with no dose level relationship, with minimal magnitude when compared with controls, in one sex only and/or remained within the range of the Historical Control Data. Therefore, any test item-relationship was considered to be unlikely.

Sexual maturation:

no effects observed

Description (incidence and severity):

In Cohort 1A males, at 500 mg/kg/day and when compared with controls, there was a delay in the mean age of balanopreputial separation in the absence of significant change in mean body weight on the day of occurrence. A test item treatment-relationship cannot be excluded but taking into account the magnitude of the change (+5% vs. controls, p<0.01), this finding was considered to be non-adverse. At 225 and 75 mg/kg/day, there were no effects on the mean age at which balanopreputial separation occurred.
In Cohort 1A females, there were no significant effects on the mean age at vaginal opening. At 500 mg/kg/day and when compared with controls, there was a delay in the mean age at vaginal opening (+9% vs. controls, not statistically significant) and an increased mean body weigh on the day of occurrence (+5% vs. controls, not statistically significant). Taking into account the small/minimal amplitudes of the changes the differences were considered to be fortuitous and a test item treatment-relationship excluded.
In Cohort 1A females, there were also no effects on the mean time of the first estrous after vaginal opening, and no effects on mean estrous cycle parameters.
At enumeration of corpora lutea in the ovary of high-dose and control groups, there were no differences, with a mean number of corpora lutea of 24.89 (± 7.50) and 23.50 (± 6.42) per animal respectively, without statistical significance.
At enumeration of primordial follicles in the ovary of high-dose and control groups, there were no differences, with a mean number of primordial follicles of 6.52 (± 4.44) and 4.94 (± 3.15) per animal respectively, without statistical significance.

Cohort 1B:
There were no effects on the mean age of balanopreputial separation or on mean body weight on
the day of occurrence in Cohort 1B males.
In Cohort 1B females, there were no effects on the mean age of vaginal opening or on mean body weight on the day of occurrence. At 225 mg/kg/day and when compared with controls, there was a delay in the mean age at vaginal opening (+8% vs. controls, not statistically significant) and an increased mean body weigh on the day of occurrence (+11% vs. controls, not statistically significant). Taking into account the minimal amplitudes of the changes these differences were considered to be fortuitous and a test item treatment-relationship excluded.

Cohort 2A:
In males at 500 mg/kg/day and when compared with controls, there was a delay in the mean age of balanopreputial separation with no impact on mean body weight on the day of occurrence. A test item treatment-relationship cannot be excluded based on the magnitude of the change (+13% vs. controls, p<0.05). However, the difference was considered to be non-adverse based on the absence of adverse effects in Cohort 1A and 1B males. At 225 and 75 mg/kg/day, there were no effects on the mean age at which balanopreputial separation occurred.
In Cohort 2A females, there were no effects on the mean age at vaginal opening. At 500 mg/kg/day and when compared with controls, there was a delay in the mean age at vaginal opening (+7% vs. controls, not statistically significant) and an increased mean body weigh on the day of occurrence (+3% vs. controls, not statistically significant). Taking into account the minimal amplitudes of the changes these differences were considered to be fortuitous and a test item treatment-relationship excluded.

Sperm Analysis (F1 Generation, Cohort 1A):
In the test item-treated groups and when compared with controls, there were no effects on sperm motility. Sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate) were unaffected by treatment with the test item at 500 mg/kg/day.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no test item treatment-related effects on Day 1 p.p. mean anogenital distance (AGD) and normalized AGD, both in males and females.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no nipples or areolae detected in male pups examined on Day 12 p.p..

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
Increased absolute and relative-to-body liver weights were recorded in females treated at ≥ 75 mg/kg/day and in males treated at ≥ 225 mg/kg/day (up to +38%; p<0.01 or 0.05). This correlated with hepatocellular hypertrophy at microscopic examination.
Increased absolute and/or relative-to-body kidney weights were recorded in males and females treated at ≥ 75 mg/kg/day (up to +14%; p<0.01 or 0.05). Although poorly dose-related and with microscopic correlates in males only (accumulation of hyaline droplets), the relationship to test item administration was considered to be likely, as seen in males of the parental generation.
Increased absolute and relative-to-body spleen weights were recorded in males and females treated at ≥ 225 mg/kg/day (up to +35%; p<0.01 or 0.05). This correlated with pigment deposition at microscopic examination at 500 mg/kg/day.
Increased absolute and/or relative-to-body ovary weights were recorded in females treated at ≥ 75 mg/kg/day (up to +25%; p<0.01 or 0.05). In spite the absence of microscopic correlates, the relationship to test item administration could not be ruled out.
The other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 1B:
Increased absolute and/or relative-to-body liver weights were recorded in females treated at ≥ 75 mg/kg/day and in males treated at 225 mg/kg/day (up to +22%; p<0.01 or 0.05).
Increased absolute and/or relative-to-body kidney weights were recorded in males treated at ≥ 75 mg/kg/day and in females treated at 225 mg/kg/day (up to +15%; p<0.01 or 0.05).
The other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 2A:
Increased absolute and/or relative-to-body liver weights were recorded in females treated at ≥ 75 mg/kg/day and in males treated at ≥ 225 mg/kg/day (up to +33%; p<0.01 or 0.05).
Increased absolute and/or relative-to-body kidney weights were recorded in females treated at ≥ 75 mg/kg/day (up to +23%; p<0.01 or 0.05).
The increase in kidney weights noted in males treated at 500 mg/kg/day in the relative-to-body weight was considered to be due to the decrease in body weight (-13%).
The other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 2B:
There were no test item-related differences.
The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
This included the higher absolute and relative-to-body thyroid gland weights in males treated at 225 mg/kg/day (respectively + 26 and +33%) that did not reach statistical significance.

Non-selected pups:
There were not test item-related organ weight differences. The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pups euthanized after weaning:
There were no test item treatment-related findings in any groups. The few findings observed were recorded in both controls, observed at low incidences with no dose level relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study.

Cohort 1A:
There were no test item-related findings.
The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the yellow mass seen in the liver from one high-dose female that correlated generally with lobar necrosis which is a common background finding.

Cohort 1B:
There were no test item-related findings.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the yellow mass in the deferent duct from 1/20 males treated at 225 mg/kg/day and the black discoloration in the stomach from 1/20 females treated at 225 mg/kg/day which had no microscopic correlates.

Cohort 2A:
There were no test item-related findings.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the deformed liver from 1/4 females treated at 500 mg/kg/day that correlated with microscopic malformation (strangulation of one lobe).

Cohort 2B:
There were no test item-related findings.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Non-selected pups:
There were no gross changes recorded.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
Test item-related changes were noted in the liver (hepatocellular hypertrophy), kidneys (tubular basophilia and accumulation of hyaline droplets), spleen (pigment), forestomach (hyperkeratosis and squamous cell hyperplasia), esophagus (hyperkeratosis) and thyroid gland (follicular cell hypertrophy) in males and females treated at ≥ 75 mg/kg/day.
- liver: minimal to slight dose-related hepatocellular centrilobular hypertrophy was observed in males and females treated at ≥ 75 mg/kg/day.
- kidneys: minimal to moderate dose-related increased severity and/or incidence of tubular basophilia, and accumulation of hyaline droplets were noted in males treated at ≥ 75 mg/kg/day. The minimally increased incidence and/or severity of tubular dilatation were considered not to be related to the test item administration in view of the low differences with controls.
- spleen: minimal dose-related brown pigment was seen in enlarged macrophages located in red pulp from males and females treated at 500 mg/kg/day.
- forestomach: minimal to slight dose-related hyperkeratosis and minimal squamous cell hyperplasia were seen in the forestomach from males and females treated at ≥ 75 mg/kg/day.
- esophagus: minimal diffuse hyperkeratosis was seen in the esophagus from males and females treated at 500 mg/kg/day.
- thyroid gland: minimal follicular cell hypertrophy was noted in males treated at 500 mg/kg/day. The minimal adrenal gland hypertrophy seen in 1/20 females treated at 225 mg/kg/day (correlated with gross enlargement) and in 2/19 females treated at 500 mg/kg/day was considered to be probably unrelated to the test item administration in view of the very low incidence of this finding. This was confirmed by the examination of the adrenal glands from all females of cohort 1A: no other females were affected.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included the mammary gland adenocarcinoma seen in one female treated at 75 mg/kg/day that can be encountered spontaneously (see Kuzutani et al., 2012), and the lobar necrosis seen in the liver from one male treated at 225 mg/kg/day and in one female treated at 500 mg/kg/day.
Finally, the slight necrosis in the brain from one control male was considered to be an incidental lesion.
There were test item-related changes neither in the male or female reproductive organs.
There was a good correlation between the vaginal smears and the histopathological examination of estrus cycle in all examined females.

Cohort 1B:
There were no test item-related findings.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

Cohort 2A:
There were no test item-related findings.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

NEUROHISTOPATHOLOGY (Cohort 2A):
Brain weights:
There were no test item-related brain weight differences.
The few observed differences were minimal and/or not dose related.
The lower absolute brain weight in males treated at 500 mg/kg/day (-6.40%; p<0.05) when compared to controls was considered to be unrelated to the test item administration. This difference of low magnitude was related in part to the decreased body weight (-13.46%; p<0.01).
Qualitative microscopic examination:
The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brainstem and cerebellum. There were no test item-related findings in these areas.
There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.
Morphometric measurements of regions of interest in cerebral cortex (level 3), hippocampus (level 4) and cerebellum (level 7):
A minimum of 6 (up to 10) brains/group/sex considered as appropriate were obtained for hippocampus and cerebellum measurements in control males and females, and for the cortex measurements for the control females.
For the hippocampus and cerebellum of males treated at 500 mg/kg/day, there were 5 brains submitted. For the hippocampus of females treated at 500 mg/kg/day, there were 4 brains submitted. The comparisons were considered to be acceptable since the number of samples were close to the target and since the standard deviation was less than 10 %. It is noteworthy that the total number of animals per group was 5 in males and females treated at 500 mg/kg/day (and not 10 as in the other groups for which the recommendation was established). It was considered not to have compromised the statistical comparison.
For the cortex of controls males and males treated at 500 mg/kg/day, there were respectively 1 or 2 brain(s) submitted. For the cortex and cerebellum of females treated at 500 mg/kg/day, there were 1, 0 or 3 brain(s) submitted per group. These numbers for evaluations were not satisfactory and thus excluded from the scope of this study. Of note, no differences were noted for these measurements between high-dose and control groups, although in insufficient numbers.
There were no differences between controls and high-dose groups for the measurements performed except the lower hippocampus measurements in males treated at 500 mg/kg/day when compared to controls (-19%, -15% and -12% respectively; p<0.01). These differences may be due to the decreased body weight, as noted in the brain weight section. In addition, similar difference was not seen in 2A females, or in the males and females from cohort 2B, that had adequate number of samples submitted. Thus the relationship to test item of the difference seen at hippocampus level was considered to be unlikely (see discussion).

Cohort 2B:
There were no test item-related findings.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

NEUROHISTOPATHOLOGY (Cohort 2B):
Brain weights:
There were no test item-related brain weight differences.
The few observed differences were minimal and/or not dose related.
Qualitative microscopic examination:
The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brainstem and cerebellum. There were no test item-related findings in these areas.
There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.
Morphometric measurements of regions of interest in cerebral cortex (level 3), hippocampus
(level 4) and cerebellum (level 7):
A minimum of 6 (up to 10) brains/group/sex considered as appropriate were obtained for cortex and hippocampus in all groups.
For the cerebellum of controls and of males and females treated at 225 mg/kg/day, there were 5 brains submitted. However, the comparisons were considered to be acceptable since the number of samples were close to the target (6) and since the standard deviation was less than 10 %. It was considered not to have compromised the statistical comparison.
There were no differences between controls and animals treated at 225 mg/kg/day for the measurements performed.

Non-selected pups:
No examination was performed.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormones (Non-selected F1 male and female offspring, Day 22 p.p.):
In males, from 225 mg/kg/ day and when compared with controls or Historical Control Data, there was a dose-related decrease in mean TSH plasma concentrations associated with increased mean T4 plasma concentrations. A test item treatment-related effect cannot be excluded and was considered not to be adverse in the absence of adverse findings at microscopic examination the thyroids. At 75 mg/kg/day and when compared with controls, there were no adverse effects on mean T4 or TSH plasma concentrations.
In females at 225 mg/kg/ day and when compared with controls or Historical Control Data, there was a decrease in mean TSH plasma concentrations associated with increased mean T4 plasma concentrations. A test-item treatment related effect cannot be excluded and was considered not to be adverse in the absence of adverse findings at microscopic examination the thyroids. At 75 mg/kg/day and when compared with controls, there were no effects on mean T4 or TSH plasma concentrations.

Thyroid Hormones (Cohort 1A animals):
There were no test item treatment-related effects on mean TSH and T4 plasma concentrations in Cohort 1A males and females. Significantly higher T4 concentrations in males at 75 mg/kg/day; and significantly lower TSH concentrations in males at 75 and 225 mg/kg/day are not considered to be related to treatment, in the absence of a dose-response relationship. Mean T4 and TSH concentrations in males at 500 mg/kg/day were not significantly different to controls. There were no statistically significant difference in T4 or TSH concentrations in females in any of the treated groups.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Auditory Startle Test (Cohort 2A):
In males: At 500 g/kg/day and when compared with controls, there were low magnitude of responses (down to 45% vs. controls on Trial 41 to 50, p<0.01). Taking into account the magnitude of the difference, this finding was considered to be adverse. These decreases were recorded in males
only, but a test item treatment-related effect cannot be excluded. At 225 and 75 mg/kg/day, there were no test item treatment-related adverse effects on mean latency times and amagnitude of the responses.
In females: There were no effects.

Reactivity to Manipulation or to Different Stimuli (Functional Observation Battery) (Cohort 2A):
Cohort 2A animals were tested once between Days 67 to 71 p.p..
There was no abnormal reactivity to manipulation or to different stimuli.
Detailed clinical examination:
• "touch escape" or "ease of removal from the cage" (reactivity to handing) were normal,
• fur appearances were normal when compared with controls,
• there were no excessive grooming, defecation, urination in treated groups when compared with the control group,
• there were no salivation, no lacrimation, no piloerection and no palpebral closure,
• all pups had a normal pupil size (myosis) at examination and there were no exophthalmos,
• all pups had a normal gait, posture, behavior and breathing,
• there were no tremors, no twitches, no clonic/tonic convulsions, no ataxia, no hypoactivity, no hyperactivity, no hypotonia and no stereotypies.
Reactivity to stimuli:
• visual stimulus response and pupillary reflex were normal in all groups,
• auditory startle reflex was normal in all groups,
• forelimb grip strength was normal in all groups.

Motor Activity (Cohort 2A):
In test item-treated groups and when compared with controls, there were no effects on mean number of horizontal movements and rearings.

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

Lymphocyte Subtyping (Cohort 1A):
Immunophenotyping analyses by flow cytometry on dissociated spleen from Sprague-Dawley rats (Cohort 1A) were successfully performed using a standard panel in order to assess the impact of the test item (acetophenone) administration on the different lymphocyte subpopulations.
In Cohort 1A males, there were decreased absolute counts of splenocytes, B cells, T cells, helper T cells and cytotoxic T cells in all test item-treated groups and decreased absolute NKT cells in mid- and high-dose level groups. In terms of relative counts, there were a decrease in B cells and an increase in NK cells in the mid-dose level group.
In Cohort 1A females, there were no significant variations in any cell subpopulation either in terms of relative (%) and absolute (cells/mg of spleen) counts. Therefore, the test item did not induce any immunotoxic effects on females from Cohort 1A animals.

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2B)

Effect level:

225 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

< 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

brain

Treatment related:

yes

Relevant for humans:

not specified

Reproductive effects observed:

no

Executive summary:

In an Extended One-Generation Reproductive Toxicity Study (OECD TG 443, GLP), acetophenone was administered to sexually-mature male and female rats (Parental (P) generation) by oral gavage, at test item dose levels of 0, 75, 225 or 500 mg/kg/day, daily, starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were also treated daily by oral gavage (from Day 22 post-partum, p.p.), at the same test item dose levels of 0, 75, 225 or 500 mg/kg/day. Pups were assigned to Cohorts for:
• reproductive/developmental toxicity testing (Cohorts 1A and 1B, without extension to mate the Cohort 1B animals to produce an F2 generation),
• developmental neurotoxicity testing (Cohorts 2A and 2B).

P generation
Mortality: in males, there were no unscheduled deaths. In pregnant females from 75 mg/kg/day, there were premature deaths because of difficulties to deliver and/or dead litter that were considered to be test item related.
Clinical signs: in both sexes from 225 mg/kg/day, hypoactivity and/or half-closed eyes were observed after dosing. At 500 mg/kg/day, these observations were associated with loud/abdominal breathing, staggering gait, continuous chewing movement and/or ventral recumbency. Taken together, these findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day, there were no adverse clinical signs.
Mean body weight, mean body weight change and mean food consumption: there were no adverse effects during the premating, mating, gestation or lactation periods.
Estrous cycle, mating and fertility: there were no effects on estrous cycle, mating (including the mean number of days taken to mate) or fertility.
Delivery data: when compared with controls, there were no effects on the Days 1 and 21 p.p. sex-ratio (percent of males) at any dose level. At 500 and 225 mg/kg/day, there were low mean numbers of live pups on Day 1 p.p. (7.5, p<0.01 and 8.8, p<0.01 vs. 11.9, respectively) and low live birth indexes (34.1 and 79.6 % vs. 96.8%, respectively). These decreases were the consequence of high mean percent of post-implantation losses (29.8, p<0.05 and 24.1% vs. 16.1% in controls, respectively) in these dose groups. These findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day and when compared with controls, there were no test item treatment-related effects on delivery data.

P generation offspring (pre-weaning F1 pups):
On Days 1-4 p.p. from 225 mg/kg/day, when compared with controls, there was a higher percentage of pups found dead, missing and/or cannibalized (25.9 %, p<0.001 and 79.1 %, p<0.001 vs. 3.9 % in controls at 225 and 500 mg/kg/day, respectively). At necropsy of pups found dead, absence of milk and/or cannibalism were noted. Absence of milk in the stomach is likely to represent nursing difficulties or absence of maternal care. These findings were considered to be test item treatment-related and adverse. These findings were associated with a low Day 4 p.p. viability index (77.7 and 38.1% vs. 96.3% in controls at 225 and 500 mg/kg/day, respectively). These findings were considered to be test item treatment-related and adverse. After culling on Day 4 p.p., there were no effects on
lactation index.
At 75 mg/kg/day and when compared with controls, there were no effects on viability and lactation indexes.

Cohorts 1A and 1B
Mortality: there were no test-item related unscheduled deaths.
Clinical signs: in both sexes from 225 mg/kg/day, half-closed eyes, hypoactivity, loss of balance and/or staggering gait were observed. Taken together, these findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day, there were no adverse clinical signs.
Body weight, body weight change: in Cohort 1A males at 500 mg/kg/day, mean body weight and mean body weight gain were lower (down to -18% vs. controls and +40 vs. +50 g on Day 8, respectively) with a tendency towards a return to control values thereafter (-8% for mean body weight on Day 64). Considering the magnitude of the differences, these findings were considered to be test item treatment-related and adverse. At 225 and 75 mg/kg/day, there were no adverse effects.
Food consumption: there were no adverse effects.
Sexual development: there were no adverse effects.
Estrous cycles: there were no effects on the mean time of the first estrous after vaginal opening, or on mean estrous cycle parameters in Cohort 1A females.

Cohorts 2A
Mortality: there were no test item-related unscheduled deaths.
Clinical signs: in both sexes from 225 mg/kg/day, half-closed eyes, hypoactivity, piloerection and/or staggering gait were observed. Taken together, these findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day, there were no test item treatment-related clinical signs.
Body weight, body weight change: in males at 500 mg/kg/day, mean body weight and mean body weight gain were lower (down to -18% vs. controls and +38 vs. +51 g on Day 8, respectively). Considering the magnitude of the differences, these findings were considered to be test item treatment-related and adverse. At 225 and 75 mg/kg/day there were no effects on mean body weight or mean body weight change. In females there were no effects at any dose-levels.
Food consumption: there were no effects.
Sexual development: there were no adverse effects.
Neurobehavioral testing: there was no abnormal reactivity to manipulation or to different stimuli at the Functional Observatory Battery and no effects of treatment on motor activity testing.
However, in males at 500 g/kg/day, there were low magnitudes of response in the auditory startle test (down to 45% vs. controls on Trial 41 to 50, p<0.01). Considering the magnitude of the difference, this finding was considered to be adverse. At 225 and 75 mg/kg/day, there were no test item treatment-related adverse effects on mean latency times and magnitude of the responses. In females, there were no effects at any dose-levels.

Laboratory investigations
Hematology, coagulation, blood biochemistry and urinalysis: there were no adverse effects on P generation or Cohort 1A animals.
Thyroid hormones: from 225 mg/kg/ day, in non-selected F1 pups (euthanized on Day 22 p.p.) and when compared with controls or Historical Control Data, there was a dose-related decrease in mean TSH plasma concentrations associated with increased mean T4 plasma concentrations. A test-item treatment related effect cannot be excluded. However, the differences were considered to be non-adverse based on the absence of adverse findings at microscopic examination of the thyroids. There were no test-item treatment related effects in P-generation and Cohort 1A animals.
Sperm analysis (P generation and Cohort 1A males): there were no effects in P generation or Cohort 1A males.
Lymphocyte subtyping (Cohort 1A):
In Cohort 1A males, there were decreased absolute counts of splenocytes, B cells, T cells, helper T cells and cytotoxic T cells in all test-item treated groups and decreased absolute NKT cells count in mid- and high-dose groups. In terms of relative counts, there were a decrease in B cells and an increase in NK cells in the mid-dose group.
In Cohort 1A females, there were no significant variations in any cell subpopulation either in terms of relative (%) and absolute (cells/mg of spleen) counts. Therefore, the test item did not induce any immunotoxic effects on females from Cohort 1A animals.

Pathology
P generation: there was a dose-related increased incidence of mortality in test item-treated females at ≥ 75 mg/kg/day due to reproductive troubles. This was associated with equivocal minimal increased incidence of vaginal mucification in females from terminal sacrifice treated at 500 mg/kg/day.
Increased liver, kidney and thyroid gland weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross test item-related findings. Test item-related non-adverse changes were noted in the liver (hepatocellular hypertrophy), kidneys (tubular basophilia and accumulation of hyaline droplets; males only), spleen (pigment), forestomach (hyperkeratosis and squamous cell hyperplasia), esophagus (hyperkeratosis) and thyroid gland (follicular cell hypertrophy) at ≥ 75 mg/kg/day.
At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the high-dose and the control groups.
Cohort 1A: there was no test item-related mortality. Increased liver, kidney, spleen and ovary weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross test item-related findings. Test item-related non-adverse changes were noted in the liver (hepatocellular hypertrophy), kidneys (tubular basophilia and accumulation of hyaline droplets; males only), spleen (pigment), forestomach (hyperkeratosis and squamous cell hyperplasia), esophagus (hyperkeratosis) and thyroid gland (follicular cell hypertrophy; males only) at ≥ 75 mg/kg/day.
At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the high-dose and the control groups.
Cohort 1B: there was no test item-related mortality. Increased liver and kidney weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross or microscopic test item-related findings.
Cohort 2A: there was no test item-related mortality. Increased liver and kidney weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross or microscopic test item-related findings.
At neurohistopathology, there were lower hippocampus measurements in males treated at 500 mg/kg/day when compared to controls. The relationship to test item administration was considered to be "possible".
Cohort 2B: there was no test item-related mortality. There were no test item-related organ weight differences, gross or microscopic findings.
At neurohistopathology, there were no test item-related changes.
Non selected pups: There was no mortality. There were no test item-related organ weight differences or gross findings.

Conclusion
The test item, Acetophenone, was administered daily by oral gavage, at dose levels of 0, 75, 225 or 500 mg/kg/day, to sexually-mature male and female rats (Parental (P) generation) starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to Cohorts of animals for the assessment of reproductive/developmental toxicity or developmental neurotoxicity.
Systemic toxicity evaluation:
The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) were considered to be:
• 75 mg/kg/day in males based on adverse clinical signs from 225 mg/kg onwards,
• lower than 75 mg/kg/day in females based on difficulties to deliver from this dose level.
Reproductive/developmental toxicity testing:
In P generation, Cohort 1A or Cohort 1B animals there were no adverse effects on reproductive parameters at all doses tested. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be:
• 500 mg/kg/day in both sexes based on absence of adverse effect at this dose level.
Developmental neurotoxicity testing:
• in Cohort 2A: lower hippocampus measurements possibly related to the test item were observed from 75 mg/kg/day,
• in Cohort 2B: no neurotoxicity could be evidenced at 225 mg/kg/day for the measurements performed.
Therefore, the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be lower than 75 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study: Study meets criteria of the OECD Guideline 443 and was done according to GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Two reprotoxicity studies are available with acetphenone: an Extended One-Generation Reproductive Toxicity Study (OECD TG 443, GLP) considered as the key data, and a combined repeated dose toxicity and reproduction/developmental screening assay according to OECD Guidelines 422 (supporting study).

In the Extended One-Generation Reproductive Toxicity Study (OECD TG 443, GLP), acetophenone was administered to sexually-mature male and female rats (Parental (P) generation) by oral gavage, at test item dose levels of 0, 75, 225 or 500 mg/kg/day, daily, starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were also treated daily by oral gavage (from Day 22 post-partum, p.p.), at the same test item dose levels of 0, 75, 225 or 500 mg/kg/day. Pups were assigned to Cohorts for:
• reproductive/developmental toxicity testing (Cohorts 1A and 1B, without extension to mate the Cohort 1B animals to produce an F2 generation),
• developmental neurotoxicity testing (Cohorts 2A and 2B).

P generation
Mortality: in males, there were no unscheduled deaths. In pregnant females from 75 mg/kg/day, there were premature deaths because of difficulties to deliver and/or dead litter that were considered to be test item related.
Clinical signs: in both sexes from 225 mg/kg/day, hypoactivity and/or half-closed eyes were observed after dosing. At 500 mg/kg/day, these observations were associated with loud/abdominal breathing, staggering gait, continuous chewing movement and/or ventral recumbency. Taken together, these findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day, there were no adverse clinical signs.
Mean body weight, mean body weight change and mean food consumption: there were no adverse effects during the premating, mating, gestation or lactation periods.
Estrous cycle, mating and fertility: there were no effects on estrous cycle, mating (including the mean number of days taken to mate) or fertility.
Delivery data: when compared with controls, there were no effects on the Days 1 and 21 p.p. sex-ratio (percent of males) at any dose level. At 500 and 225 mg/kg/day, there were low mean numbers of live pups on Day 1 p.p. (7.5, p<0.01 and 8.8, p<0.01 vs. 11.9, respectively) and low live birth indexes (34.1 and 79.6 % vs. 96.8%, respectively). These decreases were the consequence of high mean percent of post-implantation losses (29.8, p<0.05 and 24.1% vs. 16.1% in controls, respectively) in these dose groups. These findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day and when compared with controls, there were no test item treatment-related effects on delivery data.

P generation offspring (pre-weaning F1 pups):
On Days 1-4 p.p. from 225 mg/kg/day, when compared with controls, there was a higher percentage of pups found dead, missing and/or cannibalized (25.9 %, p<0.001 and 79.1 %, p<0.001 vs. 3.9 % in controls at 225 and 500 mg/kg/day, respectively). At necropsy of pups found dead, absence of milk and/or cannibalism were noted. Absence of milk in the stomach is likely to represent nursing difficulties or absence of maternal care. These findings were considered to be test item treatment-related and adverse. These findings were associated with a low Day 4 p.p. viability index (77.7 and 38.1% vs. 96.3% in controls at 225 and 500 mg/kg/day, respectively). These findings were considered to be test item treatment-related and adverse. After culling on Day 4 p.p., there were no effects on lactation index.
At 75 mg/kg/day and when compared with controls, there were no effects on viability and lactation indexes.

Cohorts 1A and 1B
Mortality: there were no test-item related unscheduled deaths.
Clinical signs: in both sexes from 225 mg/kg/day, half-closed eyes, hypoactivity, loss of balance and/or staggering gait were observed. Taken together, these findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day, there were no adverse clinical signs.
Body weight, body weight change: in Cohort 1A males at 500 mg/kg/day, mean body weight and mean body weight gain were lower (down to -18% vs. controls and +40 vs. +50 g on Day 8, respectively) with a tendency towards a return to control values thereafter (-8% for mean body weight on Day 64). Considering the magnitude of the differences, these findings were considered to be test item treatment-related and adverse. At 225 and 75 mg/kg/day, there were no adverse effects.
Food consumption: there were no adverse effects.
Sexual development: there were no adverse effects.
Estrous cycles: there were no effects on the mean time of the first estrous after vaginal opening, or on mean estrous cycle parameters in Cohort 1A females.

Cohorts 2A
Mortality: there were no test item-related unscheduled deaths.
Clinical signs: in both sexes from 225 mg/kg/day, half-closed eyes, hypoactivity, piloerection and/or staggering gait were observed. Taken together, these findings were considered to be test item treatment-related and adverse. At 75 mg/kg/day, there were no test item treatment-related clinical signs.
Body weight, body weight change: in males at 500 mg/kg/day, mean body weight and mean body weight gain were lower (down to -18% vs. controls and +38 vs. +51 g on Day 8, respectively). Considering the magnitude of the differences, these findings were considered to be test item treatment-related and adverse. At 225 and 75 mg/kg/day there were no effects on mean body weight or mean body weight change. In females there were no effects at any dose-levels.
Food consumption: there were no effects.
Sexual development: there were no adverse effects.
Neurobehavioral testing: there was no abnormal reactivity to manipulation or to different stimuli at the Functional Observatory Battery and no effects of treatment on motor activity testing.
However, in males at 500 g/kg/day, there were low magnitudes of response in the auditory startle test (down to 45% vs. controls on Trial 41 to 50, p<0.01). Considering the magnitude of the difference, this finding was considered to be adverse. At 225 and 75 mg/kg/day, there were no test item treatment-related adverse effects on mean latency times and magnitude of the responses. In females, there were no effects at any dose-levels.

Laboratory investigations
Hematology, coagulation, blood biochemistry and urinalysis: there were no adverse effects on P generation or Cohort 1A animals.
Thyroid hormones: from 225 mg/kg/ day, in non-selected F1 pups (euthanized on Day 22 p.p.) and when compared with controls or Historical Control Data, there was a dose-related decrease in mean TSH plasma concentrations associated with increased mean T4 plasma concentrations. A test-item treatment related effect cannot be excluded. However, the differences were considered to be non-adverse based on the absence of adverse findings at microscopic examination of the thyroids. There were no test-item treatment related effects in P-generation and Cohort 1A animals.
Sperm analysis (P generation and Cohort 1A males): there were no effects in P generation or Cohort 1A males.
Lymphocyte subtyping (Cohort 1A):
In Cohort 1A males, there were decreased absolute counts of splenocytes, B cells, T cells, helper T cells and cytotoxic T cells in all test-item treated groups and decreased absolute NKT cells count in mid- and high-dose groups. In terms of relative counts, there were a decrease in B cells and an increase in NK cells in the mid-dose group.
In Cohort 1A females, there were no significant variations in any cell subpopulation either in terms of relative (%) and absolute (cells/mg of spleen) counts. Therefore, the test item did not induce any immunotoxic effects on females from Cohort 1A animals.

Pathology
P generation: there was a dose-related increased incidence of mortality in test item-treated females at ≥ 75 mg/kg/day due to reproductive troubles. This was associated with equivocal minimal increased incidence of vaginal mucification in females from terminal sacrifice treated at 500 mg/kg/day.
Increased liver, kidney and thyroid gland weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross test item-related findings. Test item-related non-adverse changes were noted in the liver (hepatocellular hypertrophy), kidneys (tubular basophilia and accumulation of hyaline droplets; males only), spleen (pigment), forestomach (hyperkeratosis and squamous cell hyperplasia), esophagus (hyperkeratosis) and thyroid gland (follicular cell hypertrophy) at ≥ 75 mg/kg/day.
At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the high-dose and the control groups.
Cohort 1A: there was no test item-related mortality. Increased liver, kidney, spleen and ovary weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross test item-related findings. Test item-related non-adverse changes were noted in the liver (hepatocellular hypertrophy), kidneys (tubular basophilia and accumulation of hyaline droplets; males only), spleen (pigment), forestomach (hyperkeratosis and squamous cell hyperplasia), esophagus (hyperkeratosis) and thyroid gland (follicular cell hypertrophy; males only) at ≥ 75 mg/kg/day.
At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the high-dose and the control groups.
Cohort 1B: there was no test item-related mortality. Increased liver and kidney weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross or microscopic test item-related findings.
Cohort 2A: there was no test item-related mortality. Increased liver and kidney weights were recorded in males and/or females treated at ≥ 75 mg/kg/day. There were no gross or microscopic test item-related findings.
At neurohistopathology, there were lower hippocampus measurements in males treated at 500 mg/kg/day when compared to controls. The relationship to test item administration was considered to be "possible".
Cohort 2B: there was no test item-related mortality. There were no test item-related organ weight differences, gross or microscopic findings.
At neurohistopathology, there were no test item-related changes.
Non selected pups: There was no mortality. There were no test item-related organ weight differences or gross findings.

Conclusion
The test item, Acetophenone, was administered daily by oral gavage, at dose levels of 0, 75, 225 or 500 mg/kg/day, to sexually-mature male and female rats (Parental (P) generation) starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to Cohorts of animals for the assessment of reproductive/developmental toxicity or developmental neurotoxicity.
Systemic toxicity evaluation:
The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) were considered to be:
• 75 mg/kg/day in males based on adverse clinical signs from 225 mg/kg onwards,
• lower than 75 mg/kg/day in females based on difficulties to deliver from this dose level.
Reproductive/developmental toxicity testing:
In P generation, Cohort 1A or Cohort 1B animals there were no adverse effects on reproductive parameters at all doses tested. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be:
• 500 mg/kg/day in both sexes based on absence of adverse effect at this dose level.
Developmental neurotoxicity testing:
• in Cohort 2A: lower hippocampus measurements possibly related to the test item were observed from 75 mg/kg/day,
• in Cohort 2B: no neurotoxicity could be evidenced at 225 mg/kg/day for the measurements performed.
Therefore, the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be lower than 75 mg/kg/day.

In a combined repeated dose toxicity and reproduction/developmental screening assay according to OECD Guidelines 422 (Supporting study, GLP, 2003), there was no indication of a possible impairment of male fertility from histopathological examination and organ weights of testes, epididymides, and prostrate. Also, the macroscopic and histological examination of the female reproductive tract including corpora lutea and implantation sites showed no adverse effects. Parameters describing reproductive performance, including precoital interval, mating index, fertility index, and gestation index were not affected in rats up to the high test dose of 750 mg/kg bw/d after daily gavage treatment starting at 14 days before mating and continued throughout the 14-day mating period up to lactation day 3 (males treated for 28 days in total).
The combination of a pre-mating dosing period of 14 days and subsequent mating/fertility with an overall dosing period of 28 days, followed by detailed histopathology of the male gonads, is considered sufficient to enable detection of the majority of effects on male fertility and spermatogenesis. The dosing period for females is also sufficient to enable detection of effects on estrous cycle, on conception and pregnancy.
The high dose of 750 mg/kg bw/d was accompanied by signs of systemic and central nervous system toxicity both in male and female rats.

Effects on developmental toxicity

Description of key information

Prenatal Developmental Toxicity in rats (OECD TG 414, GLP):

NOAEL for maternal toxicity and foetal toxicity is considered to be 125 mg/kg body weight/day.

Prenatal Developmental Toxicity in rabbits (OECD TG 414, GLP):

NOAEL for maternal toxicity and foetal toxicity is considered to be 170 mg/kg body weight/day.

Extended One-Generation Reproductive Toxicity Study in rat (OECD TG 443, GLP):

- NOAEL for maternal toxicity (P-generation) is considered to be lower than 75 mg/kg/day based on difficulties to deliver.

- NOAEL for developmental toxicity in Cohort 1A and 1B is considered to be 500 mg/kg body weight/day (no adverse effect).

- NOAEL for developmental neurotoxtoxicity in Cohort 2A is considered to be lower than 75 mg/kg body weight/day (due to lower hippocampus measurements possibly related to the test item).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 17-SEP-2019 to 04-JUN-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

31.5.2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, 01400 Châtillon sur Chalaronne, France
- Age at study initiation: 17 to 19 weeks old (at mating)
- Weight at study initiation: 2819 g to 4170 g (at mating)
- Housing: 2 air-conditioned rooms in a barrier protected unit. Females were individually housed in plastic cages meeting European Directive 2010/63/EU.
requirements
- Diet: approximately 200 g of food per day (Pelleted complete rabbit diet (diet Reference No. 3409) sterilised by irradiation and analysed for chemical and bacterial contaminants)
- Water: Softened and filtered (0.2 μm) mains drinking water ad libitum
- Acclimation period: 6 days between animal arrival and the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C
- Humidity: > 35%
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours light (artificial)/12 hours dark

IN-LIFE DATES: From: 16 Sep 2019 To: 25 Oct 2019

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) carboxymethylcellulose 400-800cPs/0.5% (w/v) Tween® 80 in purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared weekly as an emulsion (prepared and stirred using the same method as for a suspension (magnetic stirring for at least 15 minutes) in the control item at
concentrations of 20, 56.7 and 166.7 mg/mL and divided into daily aliquots.

VEHICLE
Carboxymethylcellulose sodium 400-800 cps from Sigma Aldrich. Batch No.: SLBK6619V. Expiry Date: 18 Sep 2021.
Tween® 80 from Merck. Batch No.: K51034361. Expiry Date: 31 Dec 2020.
Water for injection from Laboratoire Aguettant. Batch Nos.: 3013728 and 3013806. Expiry Dates: 28 Feb 2021 and 31 Mar 2021.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected from the four testing groups for analysis on Gestational Day 6 and on Gestational Day 28.
Analyses for concentration, homogeneity and stability in the vehicle were performed by Standard HPLC-UV.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

from GD6 to GD28

Frequency of treatment:

once daily

Duration of test:

28 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

1. Control

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

2. Low dose

Dose / conc.:

170 mg/kg bw/day (nominal)

Remarks:

3. Intermediate dose

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

4. High dose

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor on the basis of the results of a preliminary study of prenatal developmental toxicity where pregnant female New Zealand White rabbits were treated at 80, 250 or 700 mg/kg/day from GD6 to GD28. In dams at 700 g/kg/day, one was found dead on GD12 and all females lost weight from GD6 to GD9. Mean food consumption was lower than in the control group during the majority of the treatment period and especially from GD6 to GD9. In fetuses, a lower mean fetal weight was noted when compared with controls. At 250 mg/kg/day, body weight loss and strongly reduced food consumption were noted for a few females from GD6 to GD9. There were no toxicologically significant effects at 80 mg/kg/day. Therefore, 500 mg/kg/day was selected as the high dose level for the current study. The low dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e., 60 and 170 mg/kg/day).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a full clinical examination was performed on each weighing day (GD0, GD6, GD9, GD12, GD15, GD18, GD21, GD24, GD27 and GD29)

BODY WEIGHT: Yes
- Time schedule for examinations: GD0, GD6, GD9, GD12, GD15, GD18, GD21, GD24, GD27 and GD29

FOOD CONSUMPTION: Yes
Food consumption of each animal was measured daily from the day of arrival to GD29.
The mean (g/animal/day) was calculated for the periods GD0 to GD6, GD6 to GD9, GD9 to GD12, GD12 to GD15, GD15 to GD18, GD18 to GD21, GD21 to GD24, GD24 to GD27 and GD27 to GD29. The period GD6 to GD29 has also been calculated and reported.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
Moribund females were euthanized by sodium pentobarbitone injection and exsanguination. They were submitted to a full macroscopic examination to determine their pregnancy status, number of corpora lutea and numbers and types of uterine implantations. Any fetuses from these females were examined externally where possible and discarded.
Dead animals were also necropsied in order to establish, where possible, the cause of death and submitted to the examinations described above. When necessary, animals were refrigerated before necropsy to minimize autolysis.
All surviving females were euthanized on GD29 by intravenous injection of sodium pentobarbitone followed by exsanguination. They were submitted to a full macroscopic examination and any abnormalities observed were recorded. Abnormal organs or tissues
were sampled and preserved. For each female euthanized on GD29, the ovaries and uterus were removed and examined including examination of the placentae.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset.
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

Parametric/Non-Parametric (Body Weight, Body Weight Gains, Food Consumption, Gravid Uterine Weight and Corrected Maternal Body Weights, Litter Observations (Litter Means)):
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric (Ovarian and Uterine Examinations, Litter % of Fetuses with Gross/External/Visceral/Skeletal Abnormalities):
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

Incidence (Parental Indices and Mortality):
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

Pre-Implantation Loss (in %)
Post-Implantation Loss (in %)
Sex Ratio (% males)
Litter % of Fetuses with Abnormalities

Historical control data:

Values compiled from untreated and control New Zealand White rabbits in previous studies

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased activity and/or abnormal gait, sometimes associated with low carriage, were noted for all females given 500 mg/kg/day, 1 hour after dosing, from GD6 to GD27. Animals recovered within the day. In addition, Female Nos. 74 and 76 given 500 mg/kg/day, had labored and shallow breathing,
lying on the side and subdued behavior on GD19-20 and GD9, respectively, 1-2 hours after dosing, but recovered within the day. These clinical signs were considered to be test item-related.
Few faeces were noted in all groups, including the controls, at the same incidence (20 to 22 females per group), but with a dose-related increased frequency of days affected at 170 and 500 mg/kg/day groups, which was consistent with the reduced food consumption. Therefore, the increased frequency of this finding was considered to be test item-related.
Red discharge, observed on GD29 for Female No. 31 given 60 mg/kg/day and for Female No. 53 given 170 mg/kg/day, was associated with the end of gestation. Red discharge, observed on GD15 for Female No. 83 given 170 mg/kg/day, without viable fetuses, might be associated with the numerous early resorptions. In addition, red discharge was observed on GD8 (predose) for Female No. 49 given 170 mg/kg/day, without any clear explanation.
On GD25, red discharge was observed during treatment and red traces were observed on the gavage cannula during intubation for Female No. 68 given 500 mg/kg/day. Therefore, the gavage was stopped and the animal was treated approximately 2 hours later when it had recovered. In addition, red discharge was observed during treatment for Female No. 65 given 170 mg/kg/day. These clinical signs were considered to be associated to the administration procedure.
Other clinical signs, such as thin fur or skin lesions, were observed in all groups including control and/or are commonly seen in this species. Therefore, they were considered as incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

2 females aborted in the 500 mg/kg/day dosed group. This represented 9% of the animals, more than commonly seen in the species (2% in the historical control data). Moreover, a third female given 500 mg/kg/day was found dead, with red discharge in the cage. Even if the cause of death was not clearly determined at necropy, the possibility of an association with the test item cannot be excluded.
In addition, 6 females (2, 1 and 3 in the control, 60 mg/kg/day and 170 mg/kg/day dosed groups, respectively) were found dead or sacrificed moribund due to, or probably due to, gavage error. These premature decedents were not test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 60 and 170 mg/kg/day, there were no major changes in body weight gain when compared with controls, only transient low body weight gain or body weight loss between GD18 and GD21 at 60 mg/kg/day and between GD15 and GD21 at 170 mg/kg/day, for a few females.
At 500 mg/kg/day, there was a body weight loss between GD6 and GD9 for all females (mean loss: -88 g ; range: -8 g to -231 g). Then, animals subsequently gained weight from GD9, as for the control group, with occasional low body weight gain or isolated body weight loss. This change had no impact on the terminal mean body weight on GD29, which was comparable in all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg/day, there were no major changes in food consumption when compared with control and pretest values.
At 170 mg/kg/day, there was lower mean food consumption from GD6 to GD21 when compared with the controls (-16% to -29%). One female had negligible food consumption from the pretest period up to GD21, which was thus considered not to be test item-related.
At 500 mg/kg/day, there was a more pronounced reduction in mean food consumption from GD6 to GD24 when compared with the controls (-15% to -51% ; -28% on average) with a particularly low food consumption for three females.

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no changes in mean gravid uterus weight in treated groups when compared with controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no remarkable macroscopic findings at 60 and 170 mg/kg/day.
There was a dark fluid accumulation in the uterine horn for one female given 500 mg/kg/day. This isolated finding was considered as incidental.
Other findings such as cysts in the oviduct or skin scabs, were noted in control or treated groups and are commonly seen in the species. Therefore, they were considered as incidental.

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

2 females aborted in the 500 mg/kg/day dosed group. This represented 9% of the animals, more than commonly seen in the species (2% in the historical control data).

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The pre-implantation data (number of corpora lutea, implantation sites and pre-implantation loss) were comparable in all groups.

There was no test item-related effect on embryo-foetal survival at 60 and 170 mg/kg/day.
At 500 mg/kg/day, the mean number of late resorptions (and thus also the total number of resorptions) was slightly higher than in the control group (0.7 vs. 0.5 for the late resorptions) and than the historical control range (0.1 to 0.6 for main embryofetal studies). This was mainly due to Female Nos. 67 and 77 (3 late resorptions) and Female No. 83 with no viable fetuses (all early resorptions). Therefore, the mean percentage post-implantation loss was slightly higher when compared with the control group (11.13% vs. 9.20%), and was close to the higher values of the historical control data (2.4% to 11.4% for main embryo-fetal studies). The mean number of live fetuses per litter remained comparable with the control group.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

There were 17, 20, 17 and 18 pregnant females at terminal caesarean in the control, 60, 170 and 500 mg/kg/day groups, respectively, all of which had viable fetuses, except Female No. 83 given 500 mg/kg/day with no viable fetuses.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 500 mg/kg/day induced a slightly higher number of late resorptions.

Dead fetuses:

no effects observed

Changes in number of pregnant:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

170 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

number of abortions

Abnormalities:

effects observed, treatment-related

Localisation:

other: low food consumption and body weight loss leading to 2 abortions and 1 premature death, and clinical signs such as decreased activity, abnormal gait and red discharge.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Consistent with the maternal toxicity, there was a slight dose-related lower mean fetal weight in the 170 and 500 mg/kg/day groups, when compared with the controls (-7.0% and -9.5%, respectively). At 500 mg/kg/day, the difference was statistically significant (p < 0.05) and the mean value was lower than the historical control range (37.44 g vs. 38.4-43.2 g for main embryo-fetal studies). This finding was more pronounced for female fetuses than for the males (difference compared with controls: -12.9% for females vs. -9.1% for males, at 500 mg/kg/day). This change was not associated with any ossification delay.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no changes in mean gravid uterus weight in treated groups when compared with the controls.
The mean number of live fetuses per litter is comparable with the control group.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no test item-related external malformations in any group.
The abnormalities noted in treated groups, such as hyperflexion of forepaw or short tail, are part of the historical control data and were incidental.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were 2 (2) fetuses (litters) with branched rib and/or multiple vertebral malformations in the 60 mg/kg/day group compared with none in the control group. There was 1 (1) fetus (litter) with absent caudal vertebra in the 60 mg/kg/day group, compared with none in the control group. There was also 1 (1) fetus (litter) with fused nasals in the 170 mg/kg/day group, compared with none in the control group.
These malformations were observed only at the low doses and/or are part of the background of changes noted for this strain of rabbit and were therefore considered as incidental.
Less severe skeletal anomalies were noted with sporadic incidence and/or are a part of the background data for this strain of rabbit and were considered as incidental.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were 2 (2) and 2 (2) fetuses (litters) with retroesophagial subclavian artery in the 170 and 500 mg/kg/day groups, respectively, compared with none in the control group.
However, this malformation is part of the background of changes noted for this strain of rabbit (7 cases between 2016 and 2018, with individual study frequency of up to approximately 1%, as noted in the current study) and these cases were therefore considered incidental in view of the lack of any dose-related increase in the incidence.
Other malformations were noted in the control and/or treated groups at the same incidence and thus were also considered as incidental.
Soft tissue anomalies were noted with sporadic incidence and/or are a part of the background data for this strain of rabbit and were considered as incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

170 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

170 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Summary of malformations - Individual descriptions:

Dose Level (mg/kg/day)	Female No.	Fetus No.	Malformation(s) including external, visceral and skeletal examinations.
0	6	1	Aortic arch, interrupted (terminating with left carotid artery) Heart, muscular ventricular septal defect Small lung Left subclavian artery originmalpositioned Sternebra, 1 or more, fused, severe (1st to 5th)
	8	8	Right kidneymalpositioned(caudally, centrally)
	17	5	Dilatation of aortic arch Narrowed ductus arteriosus Pulmonary trunk atresia
60	25	8	Rib, L5, branched
	36	11	Vertebra, general, multiple malformations (first lumbar centrum small and misaligned; first right lumbar arch absent; first left lumbar arch fused with 12th left thoracic arch; 12th left rib branched; scoliosis)
	38	2	Short tail and caudal vertebra absent (9th to 14th)
170	47	1	Both nasals fused
	50	6	Hyperflexion of left forepaw Subclavian artery, right, retroesophageal (arising between left subclavian artery and ductus arteriosus)
	52	5	Subclavian artery, right, retroesophageal (arising between left subclavian artery and ductus arteriosus)
	62	1	Aortic arch, interrupted (aortic arch terminating with left subclavian artery)
500	68	7	Subclavian artery, right, retroesophageal (arising from the descending aorta)
	71	5	Heart, muscular ventricular septal defect
	78	5	Right kidneymalpositioned(caudally, centrally)
	88	8	Subclavian artery, right, retroesophageal (arising from the descending aorta)

 

See also tables attached:

- Summary of Clinical Observations: Gestation

- Summary of Body Weights: Gestation

- Summary of Body Weight Gains: Gestation

- Summary of Food Consumption: Gestation

- Summary of Macroscopic Pathology: Gestation

- Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation

- Summary of Ovarian and Uterine Examinations and Litter Observations

- Summary of Fetal Abnormalities by Finding

Conclusions:

In this prenatal developmental toxicity study in rabbit with acetophenone:
The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 170 mg/kg/day.
The No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 170 mg/kg/day.
Despite the maternal toxicity with associated embryo-fetal effects, there was no evidence of a teratogenic potential of Acetophenone, or delays in skeletal ossification.

Executive summary:

In this prenatal developmental toxicity study in rabbit (OECD TG 414, GLP), acetophenone was administered by daily oral gavage at dose levels of 60, 170 and 500 mg/kg/day to groups of 22 mated female New Zealand White rabbits from Days 6 to 28 of gestation (GD6 to GD28). A control group of 22 rabbits received the vehicle (0.5% (w/v) carboxymethylcellulose 400-800cPs/0.5% (w/v) Tween® 80 in purified water) at the same dose volume (3 mL/kg/day).

Clinical condition, body weights and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on GD29 and litter parameters were recorded. At necropsy, the females were examined macroscopically and the gravid uterus was weighed. All live fetuses were weighed. Each fetus was examined for external defects and all live fetuses were killed by oral administration of sodium pentobarbitone. All live fetuses were examined viscerally and sexed at the time of caesarean section. The heads of approximately half of the fetuses were fixed for internal examination by serial sectioning. The eviscerated carcasses of all fetuses were processed for skeletal examination.

Results

At 500 mg/kg/day, 2 females aborted and a third one was found dead, with red discharge in the cage. These premature decedents were considered to be associated with test item-related maternal toxicity. In addition, 6 females from the control, 60 or 170 mg/kg/day dosed groups were found dead or sacrificed moribund, due to, or probably due to, gavage error.

Decreased activity and abnormal gait, sometimes associated with low carriage, labored breathing, lying on the side or subdued behavior, were noted after dosing in all females given 500 mg/kg/day, throughout the study. In addition, a higher frequency of few faeces was noted at 170 and 500 mg/kg/day, when compared with controls.

At 500 mg/kg/day, there was body weight loss between GD6 and GD9, without any impact on the body weight on GD29. At 60 and 170 mg/kg/day, there were no major changes in body weight gain when compared with control.

At 170 and 500 mg/kg/day, there was lower mean food consumption during the study when compared with controls, which was more pronounced at the high dose. At 60 mg/kg/day, there were no remarkable changes in food consumption.

At necropsy, there were no test item-related macroscopic findings in treated groups.

There were no changes in mean gravid uterus weight in treated groups when compared with the controls.

There were 17, 20, 17 and 18 pregnant females at terminal caesarean in the control, 60, 170 and 500 mg/kg/day groups, respectively, all of which had viable fetuses, except one female given 500 mg/kg/day with no viable fetuses.

The pre-implantation data were comparable in all groups.

There was no test item-related effect on embryo-fetal survival at 60 and 170 mg/kg/day. At 500 mg/kg/day, the mean number of late resorptions (and thus total resorptions) was slightly higher than the control group and slightly higher than the historical control data.

Consistent with the maternal toxicity, there were slight dose-related lower mean foetal weights in the 170 and 500 mg/kg/day groups, when compared with controls. At 500 mg/kg/day, this change was statistically significant and the mean value was lower than the historical control data. This finding was more pronounced for female fetuses than for the males and was not associated with any ossification delay.

There were no test item-related fetal external, visceral or skeletal abnormalities.

Conclusion

Daily oral (gavage) administration of Acetophenone at doses of 60, 170 and 500 mg/kg/day in the pregnant female New Zealand White rabbit from implantation through to the day before caesarean section (Gestation Days 6 to 28) was associated with marked maternal toxicity in the high dose group including low food consumption and body weight loss leading to 2 abortions and 1 premature death, and clinical signs such as decreased activity, abnormal gait and red discharge. Treatment at 170 mg/kg/day induced slightly lower food consumption, considered as non-adverse, due to the low incidence and severity.

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 170 mg/kg/day.

Consistent with the severity of maternal toxicity, treatment at 500 mg/kg/day induced a slightly higher number of late resorptions and lower mean fetal weight. Treatment at 60 and 170 mg/kg/day was not associated with any adverse embryo-fetal effects.

The No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 170 mg/kg/day.

Despite the maternal toxicity with associated embryo-fetal effects, there was no evidence of a teratogenic potential of Acetophenone, or delays in skeletal ossification.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02-SEP-2015 to 14-JUL-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: based on available stability data, test item formulation was prepared at least once every ten days and stored at room temperature.
- Solubility and stability of the test substance in the solvent/vehicle: Homogeneity of the test item in the vehicle was maintained by vortexing the prepared formulation thoroughly before every dose administration.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: ca. 11-12 weeks old (females), ca. 21-22 weeks old (males batch 1) and ca. 12-13 weeks old (males, batch 2)
- Weight at study initiation: males (batch 1): 343 - 470 g; females (batch 1): 189 - 223 g; males (batch 2): 327 - 359 g; females (batch 2): 205 - 239 g
- Fasting period before study: no
- Housing: individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male)
- Diet: free access to Altromin 1324 maintenance diet for rats and mice
- Water: free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 55 ± 10%
- Air changes: 10 x / hour
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: From: 17 September 2015 To: 22 April 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared with corn oil. The test item was weighed and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes. The formulations were prepared at least once every ten days based on available stability data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): selected based on the test item’s characteristics and testing guideline
- Concentration in vehicle: 31.25, 75 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: MKBP7039V and MKBV2080V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of concentration of the test item in the vehicle were taken in the first and last week of the study for all doses. Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation, in the first and last week of the study.
All samples were analysed by HPLC/UV within 10 days after sampling and until then stored under appropriate conditions based on available stability data.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 (male to female)
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: /

Duration of treatment / exposure:

from gestation day (GD) 5 to gestation day (GD) 19

Frequency of treatment:

once daily

Duration of test:

from mating to gestation day (GD) 20

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

35 females in the control group and the high dose group, and 25 females in the low dose and the medium dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a dose range finding study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays where observations were made once daily.
- Any clinical observations (including spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and Foetusil size) were recorded if observed. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded if observed.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: the sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Yes
- Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- On gestation day 20, sperm positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally.
- At the time of termination or death during the study, dams were examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings is preserved in 10% neutral buffered formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (fetuses used for soft tissue examination)

Statistics:

A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- The predominant clinical sign was slightly to severely reduced spontaneous activity in most females of the high dose group (750 mg/kg bw/day) on several treatment days short (mid-morning), as well as late post-application (afternoon). In several cases, this was also associated with half eyelid closure and prone position. Additionally, high dose group specific clinical signs observed on several occasions were short and late post-application ataxia as well as piloerection. Fully closed eyes and apathy were also seen in single females of the high doe group on few days of treatment. All these signs were considered as related to the treatment with the test item (Acetophenone was used in the past as “Hypnone”). Transiently reduced spontaneous activity short post-application was observed in threesingle females of the medium dose group (300 mg/kg bw/day) on one or two days of treatment.

- Salivation shortly after application was noted transiently in all females of the high dose group, few of the mild dose group and one of the low dose group (125 mg/kg bw/day). Salivation was also rarely observed late post-application in few females of the high dose group.

- Another slight test item related clinical sign was moving the bedding, which was with single exceptions, predominantly observed immediately after dose application in almost all females of the high dose and the medium dose group and some of the low dose group on most days throughout the treatment period. Moving the bedding and salivation in close timely relation to dose application was considered as a slight sign of discomfort attributed to a local reaction to the test item and not as an adverse systemic effect.

- Low incidences of alopecia on various body parts, crust on the skin and nasal discharge in single females irrespective of the group were considered incidental.

- None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One single female of the high dose group (750 mg/kg bw/day) was found dead in the afternoon of gestation day 19. According to the macroscopic finding (dark discoloured lung at necropsy) and the absence of body weight loss or preceded worsened health condition, this death was considered to be likely related to a gavage error.
One female of the control group was found dead after 2 days of treatment. At necropsy, the pericardial sac was found to be filled with blood. This death was considered spontaneous and incidental as no preceded sign of morbidity was observed.
All the remaining animals survived until the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- The mean body weight increased in all groups before initiation of the treatment and further increased with the progress of the study comparably in the control and the low dose group.
- A treatment related slightly to moderately lower mean terminal body weight was observed in the medium dose group (92 % of controls, p < 0.001) and the high dose group (88 % of controls, p < 0.001) when compared to the control group.
- A moderately and dose dependently lower body weight gain was observed over the entire study period (GD 0-20) in the medium group (77 % of controls, p < 0.001) and the high dose group (63 % of controls, p < 0.001). This resulted in a slightly to moderately, statistically significantly reduced mean body weight in the medium dose and the high dose group from Gestation Day (GD) 8 to GD 20, with the greatest effect at the end of the treatment period (medium dose group: 92 % of controls, p < 0.001; high dose group: 88 % of controls, p < 0.001). Effects on body weight and body weight gain in the medium and the high dose groups were considered to be test item related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Mean food consumption of the low dose group was unaffected by the treatment with the test item. Differences to the control group were slight and not statistically significant.
- In correlation to the body weight and body weight gain, food consumption in the medium dose and the high dose groups was noted to be moderately and statistically significantly lower compared to the control group during the entire treatment period (except for the interval of GD 11-14 in the MD group). Over the entire study period (GD 0-20), a dose dependently moderately lower food consumption was observed in the medium dose group (87 % of controls, p < 0.001) and the high dose group (77 % of controls, p < 0.001). Effects on food consumption in the medium dose and the high dose group were considered to be test item related.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- The uterus weight at necropsy was dose dependently, moderately lower in the medum dose group (86 % of controls, p < 0.05) and the high dose group (75 % of controls, p < 0.001). Furthermore, slightly but statistically significantly lower mean adjusted maternal weight (maternal weight minus gravid uterus weight) was also noted in the medium dose group (93 % of controls, p < 0.001) and the high dose group (90 % of controls, p < 0.001). This was considered as a treatment related effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- At necropsy of the female of the high dose group which was found dead on gestation day 19, the lung was noted to be discoloured (dark). Without preceded morbidity, its death was likely related to a gavage error.
- At necropsy of the control female which was found dead on gestation day 7, the pericardial sac was noted to be blood filled and could be accounted as reason for its spontaneous death.
- In one single female of the control group, a fluid filled uterus was noted, which was considered to be incidental in nature reflecting cyclic changes.

No further gross pathological changes were observed during the macroscopic examination of the females of any group.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

External examination: low incidences of haematoma on various body parts were noted in single females without dose dependency and were considered to be spontaneous in nature without relation to the treatment with the test item.

Number of abortions:

no effects observed

Description (incidence and severity):

- No test item related effects of toxicological relevance were noted for number of live foetuses.
- Marginally but not statistically significantly lower mean number of foetuses (9.81) was observed in the high dose group when compared to the control group (11.22). However, without statistical significance, and as treatment in this study was not initiated before actual nidation and values were within the normal range of variation, this was considered as incidental in nature.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- No test item related effects of toxicological relevance were noted for number of corpora lutea, implantation sites,and percent pre- and post-implantation loss.
- Marginally but not statistically significantly lower mean number of implantation sites (10.88) was observed in the high dose group when compared to the control group (11.70). However, without statistical significance, and as treatment in this study was not initiated before actual nidation and values were within the normal range of variation, this was considered as incidental in nature.
- Marginally but not statistically significantly higher mean number of post-implantation loss (10.70 %) in the high dose group compared to the control group (3.74 %) was predominantly related to 100 % post-implantation loss in one single female of the high dose group. Without statistical significance, and as mean values were within the range of normal variation, this was considered incidental.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

- No test item related effects of toxicological relevance were noted for early and late resorptions.
- Marginally but not statistically significantly higher mean number of early resorptions loss (1.06 %) in the high dose group compared to the control group (0.4 %) was predominantly related to 100 % post-implantation loss in one single female of the high dose group. Without statistical significance, and as mean values were within the range of normal variation, this was considered incidental.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were observed in any of the treated groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Successful mating resulted in 24/25 pregnancies in the low dose group, 24/25 in the medium dose group and 33/35 in the high dose group compared to 33/35 pregnancies in the control group.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

organ weights and organ / body weight ratios

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Dose dependent, test item related effects were noted for foetal weight data in the medium dose and the high dose group. Mean foetus weight (calculated on litter basis) of the medium dose group (3.44 g), was slightly but statistically significantly lower (94 % of controls, p < 0.01) when compared to the control group (3.67 g). Moderately lower mean foetus weight was observed in the high dose group (3.11 g, 85 % of controls, p < 0.001). Similarly, the male and female foetus weight was slightly lower in the medium dose group and moderately lower in the high dose group with achieving statistical significance.
- Mean foetal weight of the low dose group was unaffected by the test item administration.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see higher.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- No test item related effects of toxicological relevance were noted for male and female foetuses sex ratio.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

- Dose dependent, test item related effects were noted for litter weight data in the medium dose and the high dose group. Changes in foetal weights resulted in slight effects on litter weight data of the medium dose group with slightly but not statistically significantly lower total litter weight (86 % of controls) and male litter weight (94 % of controls), and statistically significantly lower female litter weight (79 % of controls, p < 0.05). Moderate effects on litter weight data were noted for the high dose group with moderately lower total litter weight (77 % of controls, p < 0.001), female litter weight (72 % of controls, p < 0.01) and slightly but not statistically significantly lower male litter weight (82 % of controls).
- Mean litter weight of the low dose group was unaffected by the test item administration.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

- There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group.
- By external examination, agnathia was observed in one single foetus of the high dose group and in one single foetus of the control group. Considering the low incidence, this was considered incidental. No skeletal correspondence for this finding was observed at skeletal examination of the control foetus, whereas the mandible was shown to be short in the foetus of the high dose group (micrognathia).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings in all groups including the control group:
- Bilateral pelvic girdle caudal shift was noted in moderately more litters of the high dose group (7 litters) with statistical significance (p < 0.05) when compared to the control group (2 litters) (litter incidences: 0.00 % in the low dose, 14.29 % in the medium dose, 30.43 % in the high dose group (p < 0.05) compared to 6.67 % in controls; foetal incidences: 0.00 % in the low dose, 3.48 % in the medium dose, 10.92 % in the high dose group compared to 1.13 % in controls). Values in the high dose group were slightly above historical control data with a maximum historical litter incidence of 30.00 % and a maximum foetal incidence of 6.20 %. As this finding is considered as possible indicator of a treatment effect, a relation to the treatment cannot be excluded.
- Furthermore, as it can typically be seen, the increased number of pre-pelvic vertebrae linked to the observed malposition of the pelvic girdle, was associated with bilateral full supernumerary thoracolumbal ribs in several of the foetuses. Accordingly, 14th full bilateral rib was observed at a moderately but not statistically significantly higher litter incidence in the high dose group (43.48 %) when compared to the control group (23.33 %). Though not statistically significant, litter incidence (43.48 %) and foetal incidence (14.29 %) in the high dose group was observed to be considerably higher when compared to historical control data with a maximum historical litter incidence of 31.58 % and a maximum foetal incidence of 11.65 %.

There were few other statistically significant findings which, however, were not considered as toxicologically relevant:
- Slightly variable status of ossification was seen for the skull bones between the dose groups and the control group with a statistically significantly lower litter incidence of incomplete ossification of interparietal bone in the high dose group when compared to the control group (21.74 % compared to 53.33 % in controls, p < 0.05). As values were within the normal range of variation and historical control data and there was no indication of an overall trend towards accelerated or delayed ossification in the dose groups compared to the control group, this single statistically significant finding was considered incidental and not related to the treatment with the test item.
- Extra ossification sites at the 4th sacral vertebra arches are a common finding based on slight variations in status of ossification. This finding was noted with statistical significance at a moderately lower litter incidence in the high dose group (p < 0.05) when compared to the control group (bilateral, left-sided and right-sided). As values were within the normal range of variation and historical control data, it was considered incidental.
- Moderately and statistically significantly lower litter incidence for wavy ribs was noted in the high dose group (17.39 %, p < 0.05) when compared to controls (50.00 %). Wavy ribs are a common finding in rodent studies and are classified as variation as they are likely to be postnatally reversible. With a lower incidence in the high dose group, this was considered as incidental in nature.
- One foetus of the high dose group was observed with several malformations such as fused, misshapen and short mandible, fused tympanic annulus and misshapen sphenoid. As these were single findings in one single foetus, this was not considered to be related to the treatment with the test item.
- Fused zygomatic arch (bilateral, left-sided, right-sided) was seen at low incidences without dose dependency throughout all groups including control and thus, was considered as incidental.
- Structural anomalies in sternebra such as split and misshapen were seen in single foetuses including controls without a dose dependent trend. Likewise, in vertebra few structural anomalies such as misshapen, split and absent arches were observed in single foetuses including control and were considered as spontaneous and not related to the treatment with the test item.

There was no statistical significance and no indication of a test item related trend in the type and/or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.

Visceral malformations:

no effects observed

Description (incidence and severity):

VISCERAL EXAMINATION
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including the control group:

- Litter incidences were statistically insignificant except for ureter dilated (bilateral) which was observed at lower frequency in the medium dose group in comparison to the control group without dose dependency (20.83 % in the low dose, 4.17 % in the medium dose (p < 0.05) and 30 % in the high dose group compared to 34.38 % in controls. This was considered incidental.
- Discoloured (spotted red) urinary bladder wall was noted without dose dependency in two single foetuses of the high dose group and in one foetus of the low dose group. Discoloured (spotted red) thymus was observed in one single foetus of the high dose group. No statistical significance was achieved for these findings. Discolouration of organs was considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies. As only single foetuses were affected these findings were considered incidental.
- Remaining visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

CRANIOFACIAL EXAMINATION

- Examination revealed few findings (retinal fold, dilated third ventricle, dilated lateral ventricle, subcutaneous edema) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls. Statistical analysis of the data revealed no significant effect. These findings were considered to be spontaneous in nature and not related to the treatment with the test item.
- The finding of a small pituitary gland was seen without statistical significance in single foetuses (4 of the high dose group, 0 of the medium dose group, 1 of the low dose group and 1 of the control group) with a litter incidence of 10.00 % in the high dose group, 0.00 % in the medium dose group and 4.17 % in the low dose group compared to 3.13 % in the control group. Without indication of a test item related trend, this was considered incidental.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Developmental effects observed:

not specified

Results of the dose formulation analysis:

Formulation analysis for concentration verification and homogeneity was performed on collected samples in the first and last week of the study. Concentration analysis of formulation samples was determined for all groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 105.6%, 102.5% and 103.4% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. Homogeneity of formulation samples was determined for the LD and HD group. The mean recoveries observed for the LD group were 105.4% and 103.9% of the nominal value. For the HD group mean recovery was between 100.5% and 104.5% of the nominal value. The coefficients of variation (COV) of the different sampling locations (top, middle, bottom) were 1.1% and 0.9% in the LD dose group and between 0.1% and 1.9% in the HD dose group. All samples were homogenous, as the COV were below or equal to 10%.

Conclusions:

Under the experimental conditions of this study, no effects of the test item on females and foetuses were found at the dose level of 125 mg/kg bw/day. Based on these results, the NOAEL for both maternal toxicity and foetal toxicity of the test item is considered to be 125 mg/kg bw/day.
Developmental NOAEL is based on a slight body weight reduction at 300 mg/kg (92-93% of control) and skeletal findings at 750 mg/kg (low concern, at the limit of historical control level and with no other adverse effects).
Maternal NOAEL is 125 mg/kg only based on a slight effect on body weight (92-93% of control, with some peak on shorter periods). The only other toxicological effect is the “hypnotic” effect at 750 mg/kg.

Executive summary:

This GLP-compliant study was performed to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure to the test item via oral administration (gavage) according to the method described in OECD Guidelines for Testing of Chemicals, Section 4, No. 414, “Prenatal developmental toxicity study” adopted 22 January, 2001.

Material and methods

The test material - diluted in corn oil (vehicle) - was administered by gavage to pregnant female Wistar rats at doses of 125, 300 and 750 mg/kg bw/day (25 females in the low and the medium dose groups and 35 females in the high dose group). A concurrent control group was treated in the same conditions with the vehicle alone (35 females). Females were treated daily from Gestation Day 5 (GD5) up to and including GD19 (sperm positive smears = day 0 of pregnancy, GD0). Test item formulations were prepared at least once every ten days based on available stability data. Animals were monitored, and the parameters examined during the study included body weight, food consumption, mortality and signs of toxicity. Animals that died during the study were examined macroscopically. All surviving female animals were sacrificed on the respective gestation day 20. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine and ovarian examination were evaluated and included weight, number of implantations, resorptions (early and late), and live and dead foetuses. Uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for early embryonic deaths. Foetuses were identified using strings with numbered plates, sexed and weighed. All foetuses were observed for external abnormalities, half of the fetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities.

Results

At dose levels of 300 and 750 mg/kg bw/day, treatment related clinical signs (known hypnotic effect of Acetophenone) and moderately, statistically significantly reduced body weight and food consumption were noted. Furthermore, dose dependently, statistically significantly lower uterus weight and adjusted maternal weight were noted at the same dose levels. At dose levels of 300 and 750 mg/kg bw/day, mean foetus weight and litter weight were dose dependently slightly-to-moderately and statistically significantly lower when compared to control. Furthermore, at 750 mg/kg bw/day, skeletal examination showed a moderately, statistically significantly higher incidence of bilateral pelvic girdle caudal shift when compared to concurrent controls. This change of position of pelvic girdle relative to the number of pre-pelvic vertebrae was associated with a moderately higher litter incidence of supernumerary bilateral full 14th thoracolumbal rib but without achieving statistical significance. Both findings were noted at higher incidences at 750 mg/kg bw/day compared to maximum litter and foetal incidence of historical data. No effects on remaining prenatal data, foetal external, visceral and craniofacial parameters were observed at 300 and 750 mg/kg bw/day. No effects of the test item were found on females and foetuses at the dose level of 125 mg/kg bw/day.

Conclusion

Under the experimental conditions of this study, no effects of the test item on females and foetuses were found at the dose level of 125 mg/kg bw/day. Based on these results, the NOAEL for both maternal toxicity and foetal toxicity of the test item is considered to be 125 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good quality as a guideline and GLP study is 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

Additional information

Two Prenatal Developmental Toxicity according to OECD TG 414 (GLP, Klimisch reliability 1) are available with acetophenone in two different species: rat and rabbit. Additionally, an Extended One-Generation Reproductive Toxicity Study (OECD TG 443, GLP, Klimisch reliability 1) in rat is reported in section 7.8.1 Toxicity to reproduction.

Prenatal Developmental Toxicity in rats (OECD TG 414, GLP, 2016):

A developmental toxicity study with acetophenone in rats was carried out in 2016 and flagged as key study. The following doses, formulated in corn oil and administered by gavage on gestation days 5 to 19, were evaluated: 0, 125, 300 and 750 mg/kg bw/d.

At dose levels of 300 and 750 mg/kg body weight/day test item related clinical signs (known hypnotic effect of Acetophenone) and moderately, statistically significantly reduced body weight and food consumption were noted. Furthermore, dose dependently, statistically significantly lower uterus weight and adjusted maternal weight was noted at the same dose levels. At dose levels of 300 and 750 mg/kg body weight/day mean foetus weight and litter weight was dose dependently slightly to moderately and statistically significantly lower when compared to the control group. Furthermore, at 750 mg/kg body weight/day at skeletal examination a moderately, statistically significantly higher incidence of bilateral pelvic girdle caudal shift was revealed when compared to concurrent controls. This change of position of pelvic girdle relative to the number of pre-pelvic vertebrae was associated with a moderately higher litter incidence of supernumerary bilateral full 14th thoracolumbal rib but without achieving statistical significance. Both findings were noted at higher incidences at 750 mg/kg body weight/day compared to maximum litter and foetal incidence of historical data. No effects on remaining prenatal data, foetal external, visceral and craniofacial parameters were observed at 300 and 750 mg/kg body weight/day.

No effects of Acetophenone on females and foetuses were found at a dose level of 125 mg/kg body weight/day. The NOAEL for both maternal toxicity and foetal toxicity of Acetophenone in this study is considered to be 125 mg/kg body weight/day.

Prenatal Developmental Toxicity in rabbits (OECD TG 414, GLP, 2020):

In this prenatal developmental toxicity study in rabbit (OECD TG 414, GLP), acetophenone was administered by daily oral gavage at dose levels of 60, 170 and 500 mg/kg/day to groups of 22 mated female New Zealand White rabbits from Days 6 to 28 of gestation (GD6 to GD28). A control group of 22 rabbits received the vehicle (0.5% (w/v) carboxymethylcellulose 400-800cPs/0.5% (w/v) Tween® 80 in purified water) at the same dose volume (3 mL/kg/day).

Clinical condition, body weights and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on GD29 and litter parameters were recorded. At necropsy, the females were examined macroscopically and the gravid uterus was weighed. All live fetuses were weighed. Each fetus was examined for external defects and all live fetuses were killed by oral administration of sodium pentobarbitone. All live fetuses were examined viscerally and sexed at the time of caesarean section. The heads of approximately half of the fetuses were fixed for internal examination by serial sectioning. The eviscerated carcasses of all fetuses were processed for skeletal examination.

Results

At 500 mg/kg/day, 2 females aborted and a third one was found dead, with red discharge in the cage. These premature decedents were considered to be associated with test item-related maternal toxicity. In addition, 6 females from the control, 60 or 170 mg/kg/day dosed groups were found dead or sacrificed moribund, due to, or probably due to, gavage error.

Decreased activity and abnormal gait, sometimes associated with low carriage, labored breathing, lying on the side or subdued behavior, were noted after dosing in all females given 500 mg/kg/day, throughout the study. In addition, a higher frequency of few faeces was noted at 170 and 500 mg/kg/day, when compared with controls.

At 500 mg/kg/day, there was body weight loss between GD6 and GD9, without any impact on the body weight on GD29. At 60 and 170 mg/kg/day, there were no major changes in body weight gain when compared with control.

At 170 and 500 mg/kg/day, there was lower mean food consumption during the study when compared with controls, which was more pronounced at the high dose. At 60 mg/kg/day, there were no remarkable changes in food consumption.

At necropsy, there were no test item-related macroscopic findings in treated groups.

There were no changes in mean gravid uterus weight in treated groups when compared with the controls.

There were 17, 20, 17 and 18 pregnant females at terminal caesarean in the control, 60, 170 and 500 mg/kg/day groups, respectively, all of which had viable fetuses, except one female given 500 mg/kg/day with no viable fetuses.

The pre-implantation data were comparable in all groups.

There was no test item-related effect on embryo-fetal survival at 60 and 170 mg/kg/day. At 500 mg/kg/day, the mean number of late resorptions (and thus total resorptions) was slightly higher than the control group and slightly higher than the historical control data.

Consistent with the maternal toxicity, there were slight dose-related lower mean foetal weights in the 170 and 500 mg/kg/day groups, when compared with controls. At 500 mg/kg/day, this change was statistically significant and the mean value was lower than the historical control data. This finding was more pronounced for female fetuses than for the males and was not associated with any ossification delay.

There were no test item-related fetal external, visceral or skeletal abnormalities.

Conclusion
Daily oral (gavage) administration of Acetophenone at doses of 60, 170 and 500 mg/kg/day in the pregnant female New Zealand White rabbit from implantation through to the day before caesarean section (Gestation Days 6 to 28) was associated with marked maternal toxicity in the high dose group including low food consumption and body weight loss leading to 2 abortions and 1 premature death, and clinical signs such as decreased activity, abnormal gait and red discharge. Treatment at 170 mg/kg/day induced slightly lower food consumption, considered as non-adverse, due to the low incidence and severity.
The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 170 mg/kg/day.
Consistent with the severity of maternal toxicity, treatment at 500 mg/kg/day induced a slightly higher number of late resorptions and lower mean fetal weight. Treatment at 60 and 170 mg/kg/day was not associated with any adverse embryo-fetal effects.
The No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 170 mg/kg/day.
Despite the maternal toxicity with associated embryo-fetal effects, there was no evidence of a teratogenic potential of Acetophenone, or delays in skeletal ossification.

In an Extended One-Generation Reproductive Toxicity Study (OECD TG 443, GLP), acetophenone was administered to sexually-mature male and female rats (Parental (P) generation) by oral gavage, at test item dose levels of 0, 75, 225 or 500 mg/kg/day, daily, starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were also treated daily by oral gavage (from Day 22 post-partum, p.p.), at the same test item dose levels of 0, 75, 225 or 500 mg/kg/day. Pups were assigned to Cohorts for:
• reproductive/developmental toxicity testing (Cohorts 1A and 1B, without extension to mate the Cohort 1B animals to produce an F2 generation),
• developmental neurotoxicity testing (Cohorts 2A and 2B).

In P generation, Cohort 1A or Cohort 1B animals there were no adverse effects on reproductive parameters at all doses tested. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be: 500 mg/kg/day in both sexes.

At neurohistopathology of cohort 2B (PND 22), there were no test item-related changes.
At neurohistopathology of cohort 2A (PND 76-78), there were no differences between controls and high-dose groups for the measurements performed except the lower hippocampus measurements in males treated at 500 mg/kg/day when compared to controls. Therefore, the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be lower than 75 mg/kg/day.
Based on the integrative weight-of-evidence approach of Garman et al (2016), possible evidence of developmental neurotoxicity in hippocampus was concluded because of the following reasons:
• in males from cohort 2A treated at 500 mg/kg/day, there was a decreased body weight of -13%, similarly to the decreased absolute brain weights of -6%; although these decreases were of low magnitudes, a relationship between low body weight and smaller brain is likely;
• there was an adverse maternal toxicity (difficulties to deliver and/or dead litter), that may suggest a reproduction trouble and/or maternal care issues that could have triggered the decreased food consumption, decreased body weight and decreased hippocampal measurements in the progeny. The brain development in the progeny can be affected by undernutrition (as described by Barnes et al, 1973);
• there were statistically significant differences in linear hippocampal measurements in males treated at ≥ 75 mg/kg/day that appeared to be inconsistent: similar difference was not seen in 2A females, or in the males and females from cohort 2B,
• there was an absence of test item treatment-related gross abnormalities (brain size reduction readily visible as a gross lesion) or microscopic alteration in the central nervous system, either in parents, 1A, 2A or 2B F1 cohorts, either in males or females. No microscopic degenerative abnormalities as dysplasia, hypoplasia, hypo/-dysmyelination or ectopia, or neuronal necrosis, myelinic edema or vacuolation were present;
• there was evidence of test item treatment-related nervous behavioral effects (adverse lower magnitude of responses in auditory startle test in 2A males treated at 500 mg/kg/day when compared to controls). At in-life examination, adverse hypoactivity and half-closed eyes were noted at ≥ 225 mg/kg/day. Historically, Acetophenone was used as an anesthetic agent, to induce analgesia and also as a hypnotic agent. It induces sedation and unsteadiness in laboratory animals given high oral dose. Nervous system effects (salivation, decreased activity, weakness, nerve damage) and decreased body weight were observed in animals exposed to moderate oral doses over time. The main roles of hippocampus are learning, memory and spatial coordination. The relationship between the lower linear hippocampal measurements and these clinical signs is thus questionable. Ptyalism was also observed from 75 mg/kg/day onwards and with dose-related increased incidences. This finding is commonly seen.
Even if direct effects on the nervous system cannot be completely ruled out, decreases in morphometric measurements and brain weight of juveniles from neuropathology cohort in conjunction with decreased juvenile body weights and/or maternal care issues can be evidenced: the brain alterations are related to a more general effect on growth resulting from maternally toxic doses rather than a specific neurotoxic effect (Miller et al, 1984; Rice et al; 2000). Consistent patterns of biologically relevant changes have to take into consideration both quantitative and qualitative assessments. In this study, no microscopic abnormalities were present.
In conclusion, there were lower hippocampus measurements in males treated at ≥ 75 mg/kg/day mg/kg/day when compared to controls. The relationship to test item was considered to be "possible" according to the decision tree mentioned in the reference article of Garman et al (2016) in view of the test item-related decreased body weight, in relationship also with minor decreased brain weight at 500 mg/kg/day and the nervous behavioral effects.
(see also Effect on fertility part for other information on this study)

Justification for classification or non-classification

Concerning the reproductive toxicity, no effects were observed in an OECD 443 study in rats (oral gavage), in an OECD 422 in rats (oral gavage) and in an OECD 408 in rats (oral gavage).

Regarding the developmental effects observed in rats in the OECD 414 study, no classification is required for the following reasons:
- The findings are of low level of concern (permanent, but most of the time not adverse),
- No other foetal effects (except on body weight) or malformations were observed, and no mortality was observed. At 300 mg/kg only a slight body weight reduction is observed (93-94% of control, in parallel of a lower maternal body weight and lower food consumption), but no abnormalities.
- The skeletal findings are observed at maternal toxic dose (750 mg/kg),
- Even if a dose-effect is observed (statistically significant at 750 mg/kg), the % of litter with bilateral pelvic girdle caudal shift is only slightly above the maximum incidence when compared to historical control (30.43% vs 30.00%).

In a second study in rabbits, no evidence of a teratogenic potential of Acetophenone, or delays in skeletal ossification was observed.

In the OECD 443 study in rat, no developmental toxicity or neurotoxicity was observed in Cohorts 1A, 1B or 2B. In Corhort 1A, there does appear to be an effect on hippocampus thickness in males across all dose groups. However, there are a number of factors suggesting that the effects are not directly linked to the exposure to acetophenone, but secondary to body weight effects:
- Bodyweight effects were seen in males at 225 and 500 mg/kg bw/d (pups were smaller);
- There were no effects in females (on body weight or hippocampus measurements);
- Values for males do not always show a dose-response relationship;
- The historical control data are very variable, and show that the concurrent control values are very high.

Thus according to CLP (Reg. EC 1271/2008) no classification of acetophenone for reproductive toxicity results is required.