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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 November 2015 to 20 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state at room temperature: liquid
- Colour: colourless to light yellow
- Lot/batch No.of test material: 15083ACP81
- Expiration date of the lot/batch: not applicable
- Purity: 99.67 %
- Purity test date: 27 March 2015

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 shaking the prepared formulation thoroughly before every dose administration.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl: WI(Han) (Full Barrier)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 148 – 174 g (mean: 160.98 g, ± 20% = 128.78 – 193.17 g) / females: 137 – 154 g (mean: 144.73 g, ± 20% = 115.78 – 173.67 g)
- Fasting period before study: no
- Housing: 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- 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: at least 5 days

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

IN-LIFE DATES: From: 10 December 2015 To: 24 March 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle (corn oil) was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was selected based on the test item’s characteristics, testing guideline and previous studies.
- Concentration in vehicle: 31.25, 62.5 and 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no.: MKBV2080V
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of at least 5 mL were retained from all groups in weeks 1, 5, 9 and in the last week of treatment.
In weeks 1, 5 and in the last week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared low and high dose formulations.
All samples were analysed (HPLC-UV) within 10 days after sampling and until then stored under appropriate conditions based on available stability data.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once a day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of previous studies
- Rationale for animal assignment (if not random): mean body weight of the group-housed animals was used to assign all animals to the experimental groups
- Post-exposure recovery period in satellite groups: none
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day
- Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter.
- Observations: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration, weekly during the treatment period and on the day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (weekly)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period.
- Dose groups that were examined: all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Yes (ketamine, xylazine)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period prior to or as part of the sacrifice of the animals.
- Animals fasted: Yes
- How many animals: all
- Parameters checked: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea
total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals at the end of the treatment period.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once in the last week of exposure.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, thyroid/ parathyroid glands, spleen, brain, pituitary gland, heart) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together.

HISTOPATHOLOGY: Yes: adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland including parathyroid glands, trachea, urinary bladder, uterus with cervix and vagina.
Other examinations:
Immunohistochemistry of α2µ-globulin was performed on kidneys of all 40 male animals of the study.
Statistics:
A statistical assessment of the results of haematology, blood coagulation and clinical biochemistry were performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of body weight, functional observation battery and absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Remarkable clinical signs related to the treatment with the test item were observed in males and females of the HD group. Predominant clinical sign was slightly to moderately reduced spontaneous activity in all males of the HD group and most females of the HD group on most days of treatment. Furthermore, ataxia was observed transiently in some males and one female of the HD group on few days in the first week of treatment. These signs were considered as related to the treatment with the test item based on its known hypnotic characteristics (Acetophenone was used in the past as “Hypnone”).
Salivation and/or moving the bedding, dose dependently observed in the dose groups noted transiently after dose administration, was considered as a slight sign of discomfort attributed to a local reaction to the test item and not as an adverse systemic effect. Likewise, salivation was also noted on several occasions during the weekly detailed clinical observation in the MD and the HD group and in the functional observation battery at the end of the treatment period which was related to the earlier daily dose administration of the animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
All animals survived the treatment period with the exception of one single female of the HD group which was found dead on treatment day 90, the last day of the treatment period. At necropsy a discoloured (dark) lung was seen. Without significant change in body weight or preceded worsened health condition and based on the macroscopic finding, the death is likely related to a technical reason e.g. an accidental gavage error and not considered to be related to an effect of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In both males and females, the mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group.
In males, a tendency towards marginally lower mean body weight (approx. -6 %) was observed in the HD group from treatment day 64 onwards compared to the control group. This was mainly based on the loss of body weight of one individual male due to accidental swallowing a piece of the oral gavage tube on day 62. Differences to control group did not achieve statistical significance. However, mean body weight change was noted to be marginally to moderately lower at several intervals of the treatment period in the male HD group when compared to controls. This reached statistical significance in the first week (26 % below controls, p < 0.01) and the 6th week of treatment (49 % below controls, p < 0.01). Thus, a statistically significantly lower body weight gain was noted over the entire treatment period in the male HD group (15 % below controls, p < 0.05) which was considered as an adverse effect of the test item. Slightly but statistically significantly lower mean body weight change was also noted in the MD group in the first week of treatment (13 % below controls, p < 0.05) which was considered to be related to the treatment with the test item. As there was no statistical significance over the entire treatment period or the remaining study weeks and no effect on mean body weight, this effect was not considered to be adverse.
In females, there was no biologically significant effect of Acetophenone on body weight change in the dose groups when compared to the respective controls. Statistically significantly lower body weight gain in one single week of treatment (6th) in the LD group did not affect the overall body weight change for the entire treatment period or mean body weight and was considered incidental. Marginally but statistically significantly higher mean body weight was noted in the female HD group from day 50 onwards until the end of the treatment period (7 % to 8 % above controls, p < 0.05). As a marginal difference of 5 % to controls was already evident at the initiation of the treatment period and without a significant effect on body weight change, this was not considered as an effect of the test item but incidental in nature.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of toxicological relevance on food consumption during the treatment period of any group.
However, in correlation to the lower mean body weight change in the first week of treatment of the male HD group, transiently slightly lower food consumption was observed (11 % below controls). As no such effect was noticed for the following weeks and total food consumption was comparable to controls, this was not considered adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmoscopic findings in any of the animals of this study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, a dose dependently higher mean percentage of reticulocytes was observed in all male and female dose groups with achieving statistical significance in all groups except for the female LD group when compared to the respective controls. Values in the LD, the MD and the HD group were 33 %, 51 % and 97 % above controls in males and 26 %, 50 % and 94 % above controls in females, respectively. Mean percentage of reticulocytes in the male HD group (2.9 %) was beyond the maximum value of historical control data (2.8 %). This regenerative response in the male and female HD group was correlated to an increase in cell volume. Thus, mean corpuscular volume (MCV) was noted to be marginally, but statistically significantly higher in the male HD group (5 % above controls), the female MD group (4 % above controls) and the female HD group (5 % above controls). As values for reticulocytes of the concurrent control groups were comparably low compared to historical control data, effects on reticulocytes in the male and female LD and MD group were considered as slight. Haematological parameters for the male and female LD and MD group were well within historical control data and not considered to be within a toxicological relevant range and as such not adverse. However, marked changes in the HD group were considered as adverse effects of the treatment with the test item. Furthermore, in correlation to the regenerative response, marginally but statistically significantly lower red blood cell count and haemoglobin were observed in the female HD group (7 % and 5 % below controls, respectively). A tendency towards lower red blood cell count was also observed in the male HD group (5 % below controls) but without reaching statistical significance.
There were no further effects of toxicological relevance on parameters of haematology. Blood coagulation was not affected by Acetophenone.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, at the end of the treatment period, cholesterol was noted to moderately, statistically significantly higher in the HD group when compared to the respective controls (31 % above controls, p < 0.05). The same was seen with dose dependency in females (MD group: 33 % above controls, p < 0.05; HD group: 53 % above controls, p < 0.01). This was considered as test item related effect. However, values were within the range of historical control data.
Slightly higher levels of serum glucose were seen in the male MD and HD group but without clear dose dependency (each 20 % above controls, p < 0.05). Moderately higher levels were also noted for the female HD group (37 % above controls, p < 0.001) when compared to the respective controls. This is considered as an effect of the treatment with the test item, however, as values were very well within historical control data, no adversity was assumed.
There were no further biologically or statistically significant differences between the dose groups and the control group for the other parameters of clinical chemistry for males and females at the end of the treatment period.
Urinalysis findings:
no effects observed
Description (incidence and severity):
All parameters of urinalysis of the dose groups at the end of the treatment period were not considerably different to the corresponding control group and were within the normal range of variation. Slightly deviating values for individual animals throughout all groups were considered as incidental findings without relation to the treatment with the test item.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the functional observation battery at the end of the treatment period, spontaneous activity was noted to be dose dependently decreased in males and females of the MD and the HD group which was related to the treatment with the test item. Furthermore, this was considered to cause the observed dose dependently, slightly lower number of rearing in the dose groups when compared to controls in the last week of the treatment period.
As during the weekly detailed clinical observation, increased salivation was also noted dose dependently in males and females of the MD and the HD group in the functional observation battery at the end of the treatment period.
No relevant effects were observed in any of the remaining parameters of the functional observation battery.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, a toxicologically relevant, dose dependent effect was noted for liver weight in both genders. Slightly to moderately, statistically significantly higher absolute and relative liver weight was recorded in the male MD (absolute weight 18 % above controls, p < 0.01) and HD group (absolute weight 26 % above controls, p < 0.01) and the female MD (absolute weight 20 % above controls, p < 0.01) and HD group (absolute weight 32 % above controls).
In males, the relative but not absolute kidney weight was shown to be dose dependently, marginally but statistically significantly higher in the MD and the HD group compared to the control group (13 %, p < 0.05, and 16 %, p < 0.01, above controls, respectively, when related to body weight). In females, relative kidney weight of the MD but not of the HD group was marginally higher compared to the control group (13 % above controls, p < 0.05). Furthermore, absolute female kidney weight was noted to be marginally but statistically significantly higher in both the MD and the HD group (17 % above controls, p < 0.01, and 18 % above controls, p < 0.01, respectively).
A marginal tendency towards higher absolute and relative thyroid weight was observed in the male MD and HD group (absolute weight 9 % and 13 % above controls, respectively). This did not achieve statistical significance, however, a similar trend was observed in females. In females, mean absolute thyroid weight of the LD, the MD and the HD group was 21 %, 24 % and 30 % higher, respectively, compared to controls but without statistical significance.
Marginally higher absolute adrenals weight was seen in the male HD group (14 % above controls) but did not achieve statistical significance. When related to body weight, adrenal weight was slightly, statistically significantly higher in the male HD group when compared to controls (22 % above controls, p < 0.05). In females, there was no statistically significant difference for adrenals weight. However, a marginal trend towards higher adrenal weight in the MD and the HD group was noted but without dose dependency.
The mean absolute and relative thymus weight was observed to be slightly higher in the female dose groups when compared to controls but did not follow a dose dependent pattern. Statistical significance was only achieved for the LD group (absolute weight 29 % above controls, p < 0.01). Moreover, this was not observed in males.
In females but not in males, absolute and relative spleen weight was noted to be slightly but dose dependently higher when compared to the respective controls (absolute weight: MD group 21 % above controls, p < 0.05 and HD group 29 % above controls, p < 0.01). As spleen weight in females was within the normal range of variation of historical control data and no histopathological effect was observed, this was not considered to be toxicologically relevant.
Mean weight of pituitary gland was noted to be highly variably between the male dose groups without following a dose dependent pattern. No statistical significance was achieved due to high inter-individual variability and no similar effect was seen in females. Thus, this was considered as incidental in nature.
In all dose groups, weight of ovaries was slightly higher when compared to the control group. However, due to high inter-individual variability no statistical significance was achieved. Without histopathological findings this was considered as incidental, possibly related to different stages of estrous cycle in the individual females.
Besides, other organ weights were within the normal range of variation of historical data for this strain. Without histopathological correlate and without dose dependency other single statistically significant differences between the dose groups and the respective controls were not assumed to be biologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Single gross lesions noted at necropsy were within the normal range of background lesions without relation to the treatment with the test item.
One female of the HD group was found dead and partly cannibalized on treatment day 90. Organs were autolytic and a discoloured (dark) lung was observed. Without any preceding signs for worsened health condition, its death was likely related to an accidental gavage error and not related to a systemic effect of the test item.
At the end of the treatment period, few gross pathological changes were recorded. One male of the MD group was noted with discoloured (yellow) head of epididymis which was considered as incidental. Furthermore, the finding of fluid filled uterus was seen in single females of all groups including control and was not related to the treatment with the test item as per microscopic evaluation.
A piece of the oral gavage tube was bitten off and suspected to be swallowed by one animal of the HD group on study day 62. This piece was found at necropsy, stuck between oesophagus and stomach.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
By histopathological examination, a minor, dose dependent hepatocellular hypertrophy was noted in the liver of several animals from all dose groups with males being more affected.
Hyaline inclusions in the kidneys increased in incidence and severity in all male dose groups. The hyaline inclusions corresponded with the presence of α2-microglobulin by immunhistochemistry. The increase of this protein was statistically significant in the male MD and the HD group compared to controls. There were no further changes in the kidneys.
Furthermore, there was a minimal diffuse follicular hypertrophy in the thyroid glands of one female of the MD group and of a few animals of the HD group.
Minimal diffuse cortical hypertrophy in the adrenal glands was observed in two females of the MD group and of a few HD animals.
A minor severity graded atrophy of the thymus was noted in animals at all doses.
All remaining findings recorded were within the range of spontaneous background alterations that may be recorded in Wistar rats at these ages.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no biologically relevant differences in body temperature between the groups.
Details on results:
DOSE FORMULATION ANALYSIS:
Concentration analysis of formulation samples was determined in study weeks 1, 5, 9 and last week of the study for all dose groups The mean recoveries observed in the LD, the MD and the HD group were 104.8 %, 100.6 % and 102.9 % 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 in study weeks 1, 5 and last week of the study for the LD and the HD group. The mean recoveries observed for the LD group were 101.0 %, 103.1 % and 101.0 % of the nominal value and 96.8 %, 100.6 % and 103.4 % of the nominal value for HD group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.7 %, 0.4 % and 0.9 % in LD dose group, 1.1 %, 1.2 % and 2.8 % in HD dose group. All samples were homogenous, as COV was below or equal 10 %.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
haematology

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
nervous system
Organ:
not specified
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In this study, the NOAEL is considered to be 250 mg/kg body weight/day, based on reduced body weight gain, reduced spontaneous activity and increased % reticulocytes.
Executive summary:

On the basis of a 90-Day Repeated Dose Oral Toxicity Study (OECD 408, GLP) in Wistar rats with acetophenone at dose levels of 125, 250 and 500 mg/kg body weight/day (in corn oil, by gavage) the following conclusions can be made:

No adverse effects of acetophenone were found at the dose level of 125 mg/kg body weight/day. At a dose level of 500 mg/kg body weight/day statistically significantly lower mean body weight gain was noted in the male HD group (15 % below controls when related to the entire treatment period). No toxicologically relevant effect for body weight was noted for females.

Clinical signs related to the known hypnotic effect of acetophenone were observed mainly in the male and female groups treated with 500 mg/kg body weight/day such as reduced spontaneous activity. Slight effects were also observed transiently in some animals of the MD group during the weekly detailed clinical observation.

At the end of the treatment period, adverse effects on parameters of haematology were noted in the male and female groups at 500 mg/kg body weight/day. A dose dependently higher mean percentage of reticulocytes was observed in all male and female dose groups with achieving statistical significance in all groups except for the female low dose group when compared to the respective controls. Markedly higher values for the male group at 500 mg/kg body weight/day (97 % above controls) and the female group at 500 mg/kg body weight/day (94 % above controls) was considered as adverse effect of the test item. This regenerative response in the male and female groups at 500 mg/kg body weight/day was correlated to an increase in cell volume. Thus, mean corpuscular volume (MCV) was noted to be marginally, but statistically significantly higher in the male group at 500 mg/kg body weight/day (5 % above controls), the female group at 250 mg/kg body weight/day (4 % above controls) and the female group at 500 mg/kg body weight/day (5 % above controls). In correlation to the regenerative response, marginally but statistically significantly lower red blood cell count and haemoglobin were observed in the female group at 500 mg/kg body weight/day (7 % and 5 % below controls, respectively). Slight changes for parameters of haematology in the male and female groups at 125 and 250 mg/kg body weight/day were well within historical control data and not considered to be in a toxicological relevant range.

Acetophenone caused in the liver a minor hepatocellular hypertrophy in animals of all test item treated groups which was correlated to a significantly higher liver weight in the male and female groups at 250 and 500 mg/kg body weight/day when compared to the respective controls. Without accompanying lesions, this was considered as an adaptive metabolic effect. Secondary to the liver lesions, a minimal diffuse follicular hypertrophy in one female at 250 and few animals at 500 mg/kg body weight/day was observed which is known not to be of relevance for human. Furthermore, stress related lesions (minimal diffuse cortical hypertrophy in the adrenal glands at 250 and 500 mg/kg body weight/day and minor atrophy of the thymus in all dose groups) were not considered as adverse effect. Hyaline inclusions in male kidneys from animals of all test item treated groups corresponded with the presence of α2-microglobulin which was statistically significantly higher in the male groups dosed with 250 and 500 mg/kg body weight/day compared to controls. This is a typical issue in male rats and was considered as a non-adverse metabolic change as there were no further lesions in the kidneys.

Thus, the NOAEL in this study is considered to be 250 mg/kg body weight/day. Based on the histopathological findings the NOEL of Acetophenone could not be established.