Acetophenone

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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);

Test material

Test material information

Constituent 1

Reference substance name:

Acetophenone

EC Number:

202-708-7

EC Name:

Acetophenone

Cas Number:

98-86-2

Molecular formula:

C8H8O

IUPAC Name:

1-phenylethanone

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan:SD (CD®)

Sex:

male/female

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

group 2

Doses / concentrations 3

Dose / conc.:

225 mg/kg bw/day (nominal)

Remarks:

group 3

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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.

2

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

3

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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 (F1)

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.

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

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.