Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effect levels from reproductive toxicity screening assay (OECD Guidelines 422) (Key study: Thorsrud, 2003):
no indication of a possible impairment of male or female fertility in the dose range 75 to 750 mg/kg bw/d
Maternal toxicity: NOAEL 225 mg/kg bw/day, LOAEL 750 mg/kg bw/day


Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2001 to January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study: Study meets criteria of the OECD Guidelines 421 and 422 for reproductive toxicity screening study; study acceptable as screening study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 8 wks plus 36 days acclimation plus a minimum of 14 days for treatment before mating
- Weight at study initiation: females: 162-201 g at receipt
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 36 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
GAVAGE SOLUTIONS
For each test article dose group, a specified amount of acetophenone was weighed into a pre-calibrated beaker. A sufficient quantity of corn oil was added to the beaker to achieve the desired concentration and the mixture was stirred for 30 minutes. Each test article solution was prepared fresh weekly, dispensed into daily aliquots and stored in amber glass containers at ambient conditions.

VEHICLE
- Justification for use and choice of vehicle: low solubility in water
- Concentration in vehicle: 37.5, 112.5, 375 mg/mL
- Amount of vehicle (if gavage): 2 mL
- Lot/batch no.: Lot no. QN0035
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: copulatory plug in the vagina or sperm positive vaginal smear referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged: alone
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration verification performed by gas chromatographic analysis of the vehicle and each test article dosing solution prepared for study weeks 0, 2, 4, and 6
Duration of treatment / exposure:
females: minimum of 14 days before mating, throughout mating and gestation up to lactation day 3
males: minimum of 14 days before mating, throughout mating up to a total treatment period of 28 days
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: about 15 weeks
Remarks:
Doses / Concentrations:
0, 75, 225, 750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females, 10 males
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: high-dose level expected to produce some toxic effects, but no excessive lethality; mid-dose level expected to produce no to minimal observable effects; low-dose level was expected to produce no observable effects
- Rationale for animal assignment: random by computer randomization program
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule for cage side observations: once daily

MORTALITY/GENERAL HEALTH CHECK: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a minimum of weekly until evidence of mating was detected, daily during gestation and lactation and on the day of scheduled euthanasia

BODY WEIGHT: Yes
- Time schedule prior to mating: day 0, 3,7, and 12 of treatment
- Time schedule when positive evidence of mating was detected: weighing on gestation days 0, 7, 14 and 20.
- Time schedule following parturition: days 1 and 4 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined on same days as body weight and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OTHER
- Time parturition was first detected and time parturition was completed
- Signs of difficult or prolonged delivery
- Observation of nesting and nursing behaviour
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations:
testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- viability: daily from day 0 to 4
- number and sex of pups: day 0 and 4
- body weights: day 1 and 4
- presence of gross external anomalies: days 0 and 4

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities with emphasis on developmental anomalies; possible cause of death was not determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 28 days of exposure (These animals were used for the 28 day repeated dose toxicity study, for test results see Section 7.5.1)
- Female animals:
Maternal animals: All surviving animals on lactation day 4.
Females that failed to deliver were euthanized on post-breeding day 25 (25 days after detection of mating).
Females with no evidence of mating were euthanized on post-breeding day 25 (25 days after termination of the mating period).
Females with total litter loss were euthanized on the day that no surviving pups remained.

GROSS NECROPSY
Necropsy examination consisted of the external surfaces of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents.
Investigation of female reproductive tract: vagina and ovaries; uterine contents including number of implantation sites, number of corpora lutea, all abnormalities

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs and tissues preserved for possible future histopathological examination:
Accessory genital organs (epididymides, seminal vescicles and prostate or uterus and vagina), adrenals, all gross lesions, aorta, brain, cecum, colon, duodenum, esophagus, exorbital lachrymal glands, eyes with optic nerve, femur and bone marrow, heart, ileum, jejunum, kidneys, liver, lungs, mammary gland, mandibular lymph node, mediastanal lymph node, mesenteric lymph node, ovaries, pancreas, peripheral nerve, pituitary, rectum, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach (glandular, nonglandular), testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder.
Data from histopathological examinations are available from the investigations during the 28-day repeated dose toxicity study for the males, that had been mated in the reproductive toxicity study (N=5), and for unmated female rats (N=5) (for details see Section 7.5.1). Tissues of the male and female reproductive tract were included in this investigations.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
macroscopic examination for structural abnormalities or other pathological changes with special attention on the organs of the reproductive system

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
- F1 rats from scheduled sacrifice or unscheduled death: All gross lesions were preserved for possible future histopathological examination
Statistics:
ANOVA, Dunnett's test, Chi-square test, Fisher's exact test; minimum significance level p<0.05
Reproductive indices:
Precoital intervall
Gestation length
Number with successful copulation
Number of males paired
Number of females gravid
Copulation index
Fertility index
Offspring viability indices:
Percent live pups
Percent dead pups
Percent litters with live offspring
Sex ratio
Mean live litter size
Pup survival
Mean pup weight
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL FEMALES)
- Mortality: no in any dose group
- Unscheduled euthanasia:
Control group: 1 f on postbreeding day 25 with no evidence of mating
2 f on postbreeding day 25 with evidence of mating but failing to deliver
75 mg/kg: 1 f on postbreeding day 25 with evidence of mating but failing to deliver
750 mg/kg: 1 f on postbreeding day 25 with evidence of mating but failing to deliver
6 f on lactation days 1 to 4 due to total litter loss
- Clinical signs: for details see Table 1
75 mg/kg: single incidence of urine stain in 2 females
225 mg/kg: low incidence of urine stain and predose salivation, of postdose feces small in size; slightly increased incidence of postdose salivation
750 mg/kg: low incidence of decreased activity, skin pale in color, unkempt appearance, rough coat, postdose dark material around nose,
postdose few feces; increased incidences of urine stain, predose and postdose salivation, and postdose wobbly gait
Evaluation: Effects observed at 225 mg/kg are considered to be of no toxicological significance due to low incidence or due to absence of adverse effect as for slight increase of postdose salivation. In contrast, effects observed at 750 mg/kg are considered to be of toxicological significance due to incidence, or type of effect as e.g. wobbly gait indicating central nervous system depression.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL FEMALES) (for details see Table 2)
225 mg/kg: during days 0-3 of the premating period slight increase of body weight loss accompanied by significant decrease of food consumption
750 mg/kg: during days 0-3 of the premating period slight increase of body weight loss accompanied by significant decrease of food consumption
during gestation days 0-7 significant decrease of body weight gain (61% of control group) accompanied by slight decrease
of food consumption (86% of control group); during gestation days 7-14 and 14-20 the weight gain was higher or comparable
to the control group
on gestation day 7 body weight was slightly lower (ca. 6%) than controls
during lactation days 1-4 slight decrease of body weight gain accompanied by slight decrease of food consumption
Evaluation: The slight increase of body weight loss at 225 mg/kg during premating days 0-3 is considered to be of no toxicological significance as it occurred simultaneously to significantly decreased food consumption. There was no significant effect on body weights. The significant decrease of body weight gain during gestation days 0-7 at 750 mg/kg is considered to be of toxicological significance. However, during the rest of the gestation period body weight gain was higher or comparable to the control group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects on testis weight or epididymis weight

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) (for details see Table 3)
Mating and feritlity indices and mean gestation lengths comparable in all groups

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically meaningful differences (for details see Section 7.5.1)

GROSS PATHOLOGY (PARENTAL ANIMALS)
750 mg/kg: 2 females with total litter loss had pup tissue mixed with ingesta in the stomach
No toxicological significant abnormalities were observed in the vagina, uterus, or ovaries.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No toxicological significant abnormalities were observed during a 28-day exposure in the testes, epidymidides, seminal vescicles, and prostrate of mated male rats or in the vagina, uterus, or ovaries of unmated female rats.
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: maternal toxicity indicated from clinical signs and depression of body weight gain during gestation days 0-7
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: reproductive performance
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
VIABILITY (OFFSPRING) (for details see Table 3)
750 mg/kg: statistically significant increase of number of stillborn (percent of stillborn also out of historical control range), of number of F1 pups dying, missing and/or cannibalized during lactation days 1 -4, and of the number of litters with total litter loss up to lactation day 4;
statistically significant decrease of the total number of liveborn, of the number of pups surviving to lactation day 4, of the viability index on lactation day 4, of the mean number of live pups per litter on lactation days 1, 2, 3 and 4; number of mean live pups per liter slightly lower on lactation day 0 and significantly lower on lactation days 1 -4; live birth index out of historical control range

CLINICAL SIGNS/EXTERNAL INVESTIGATION (OFFSPRING) (for details see Table 4)
225 mg/kg: increased incidence of desquamation as single remarkable finding
750 mg/kg: remarkable observations included increased incidences of desquamation, cool to the touch, skin with shiny appearance, skin appearing tight - restricting movement, and a slightly increased incidence of gasping and skin pale in color; 1 female with prolonged parturition
Evaluation: The increase of desquamation at 225 mg/kg which showed no dose relationship was considered to be of no toxicological significance.

BODY WEIGHT (OFFSPRING) (for details see Table 3)
750 mg/kg: significant decrease of the pup weight per litter on lactation day 1 and 4; out of historical control range
GROSS PATHOLOGY (OFFSPRING) (for details see Table 5)
225 mg/kg: the only remarkable finding was observed in pups with scheduled euthanasia on lactation day 4: slightly increased incidence of scabbing, increased incidence of desquamation
750 mg/kg: remarkable effects in pups with scheduled euthanasia on lactation day 4 (SE), in stillborn pups (S), and in died pups (D): single incidence each of cleft palate and edema (S), increased incidences of atelectasis (S), milk not present in the stomach (S, D), of dermal hypoplasia (S, D), of scabbing (D, SE), desquamation (D, SE); in addition, 22 died pups with observed autolysis
Evaluation: The increase of desquamation at 225 mg/kg, which showed no dose relationship, as well as the slight increase of scabbing was considered to be of no toxicological significance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
225 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity up to lactation day 4
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity up to lactation day 4 based on findings from pup viability, pup weight, external observations, and gross necropsy
Reproductive effects observed:
not specified

Table 1: Summary of remarkable clinical observations in F0 females  (frequency of observation/number of animals)

Observation

Dose group (mg/kg bw/d)

Timepoint

Effect

0

75

225

750

General observations

Urine stain

0/0

2/2

8/3

33/6

Unkempt appearance

0/0

0/0

0/0

2/1

Skinpale in color

0/0

0/0

0/0

5/1

Rough coat

0/0

0/0

0/0

15/2

Decreased activity

0/0

0/0

0/0

1/1

Predose

Salivation

0/0

0/0

1/1

13/8

Postdose

Dark material around nose

1/1

0/0

1/1

4/3

Wobbly gait

0/0

0/0

0/0

62/10

Salivation

0/0

0/0

17/7

64/10

Feces small in size

0/0

0/0

2/2

0/0

Few feces

0/0

0/0

0/0

4/4

Other observation

Prolonged parturition

0/0

0/0

0/0

1/1

Table 2: Summary of body weight changes and food consumption in F0 female rats (g)

Time period

Parameter

Dose group (mg/kg bw/d)

0

75

225

750

Prior to mating

Day 0 - 3

Body weight change

-3

-1

-6

-10

Food consumption

16

15

14b

9a

Day 3 – 7

Body weight change

4

3

1

6

Food consumption

18

17

17

17

Day 7 - 12

Body weight change

13

7

10

9

Food consumption

18

16

16

17

Gestation

Day 0 - 7

Body weight change

36

36

33

22b

Food consumption

22

23

22

19

Day 7 - 14

Body weight change

23

30

31

27

Food consumption

20

24

24

22

Day 14 - 20

Body weight change

55

67

62

54

Food consumption

22

23

22

22

Lactation

Day 1 - 4

Body weight change

12

17

14

8

Food consumption

33

35

37

25

Significant effect: a p<0.01, b p<0.05

Table 3: Summary of reproductive indices and F1 pup viability and body weights

Parameter

Dose (mg/kg bw/d)

 

 0

 75

 225

 750

F0 females on study

9

10

10

10

  Mating index (%)

 100

 100

 100

 100

  Fertility index (%)

 77.8

 90

 100

 90

  Mean gestation length (days)

 21.9

 22.0

 22.0

 22.0

Females with liveborn (gestation index in %)

7 (100)

 9 (100)

 10 (100)

 9 (100)

  Number of females with completed delivery (%)

7 (100)

 9 (100)

 10 (100)

 9 (100)

  Females with stillborn pups (%)

 2 (28.6)

4 (44.4)

 3 (30.0)

 7 (77.8)

Litters with liveborn pups

7

9

10

9

Litters with liveborn but with no pups alive on lactation day 4

= total litter loss (%)

0

0

0

6 b (66.7)

Pups delivered: total

                          mean

107

15.3

142

15.8

149

14.9

139

15.4

Number of liveborn

105

136

145

109a

Live birth index

98.1

95.8

97.3

78.4c

Number of stillborn (%)

2 (1.9)

6 (4.2)

4 (2.7)

30a (21.6d)

Number of missing

2

2

4

22

  Pups died or missing and/or cannibalised on lactation day 0 (%)

0

0

0

5 (4.6)

  Pups died or missing and/or cannibalized during lactation days 1 -4

 6 (5.7)

 5 (3.7)

 8 (5.5)

 79 a (72.5)

  Number of pups surviving to lactation day 4

 99

 131

 137

 25 a

  Viability index on lactation day 4 (%)

 94.3

 96.3

 94.5

 22.9 a

Mean number of live pups per litter on lactation day 0

                                                                                   1

                                                                                   2

                                                                                   3

                                                                                    4

15.0

14.6

14.4

14.1

14.1

15.1

14.8

14.7

14.7

14.6

14.5

13.8

13.8

13.7

13.7

12.1

4.1a

3.0a

2.9a

2.8a

Sex ratio - male pups : total pups (%)  on lactation day 0

                                                                                        4

54.3

57.6

44.9

43.5

46.2

45.3

53.2

40.0

Pup weight per litter  on lactation day 1

                                                             4

6.3

8.7

6.5

9.2

6.3

8.4

5.0a,e

5.4a,f

statistically significant change: a p<0.01; b p<0.05

historical control data: c live birth index: range 95.5 -99.1 %; d % stillborn: range 0.9 -4.5%; e mean pup weight on lactation day 1: range 6.5 -7.5 g; f mean pup weight on lactation day 4: range 8.5 -11.1 g

Table 4:  Summary of observations from external examination of F1 pups on lactation days 0 and 4 different from vehicle control
(frequency of observation/ number of dams with the specific observation in their litter)

Observation

Dose group (mg/kg bw/day)

0

75

225

750

Number of dams with litters examined

7

9

10

9

Within normal limits

191/7

254/9

216/10

53/4

Respiration: gasping

0/0

0/0

0/0

4/2

Skin pale in color

0/0

1/1

1/1

4/3

Cool to touch

0/0

9/3

0/0

31/5

Desquamation

0/0

0/0

42/5

15/3

Skin has shiny appearance

0/0

0/0

0/0

43/4

Skin appears tight – restricts movement

0/0

0/0

0/0

27/3

Overall evaluation of external examination

No remarkable effects

Desquamation as
single remarkable effect

All compiled effects considered as remarkable

Table 5:  Summary of gross necropsy observations in stillborn F1 pups, in died F1 pups (on lactation day 0 to 4) and in euthanised F1 pups
(Pup incidence / Litter incidence)

Group of pups

Location

Observation

Dose group (mg/kg bw/d)

0

75

225

750

Stillborn

Litters evaluated

1

4

3

7

Pups evaluated

1

6

4

28

Whole Body

Edema

0

0

0

1 / 1

Oral cavity

Cleft palate

0

0

0

1 / 1

Lung

Atelectasis

1 / 1

6 / 4

3 / 2

15 / 4

Skin

Dermal hypoplasia

0

0

0

12 / 2

Stomach

Milk not present

1 / 1

6 / 4

3 / 2

23 / 5

Overall evaluation

No remarkable findings

No remarkable findings

Compiled effects considered as remarkable

Died pups

Litters evaluated

3

3

2

8

Pups evaluated

4

3

4

62

Skin

Scabbing

1 / 1

0

0

8 / 2

Desquamation

0

0

0

3 / 1

Dermal hypoplasia

0

0

0

6 / 2

Stomach

Milk not present

2 / 2

1 / 1

1 / 1

43 / 7

Gross examination

Autolysis

0

2 / 2

1 / 1

22 / 7

Overall evaluation

No remarkable findings

No remarkable findings

Compiled effects considered as remarkable

Scheduled euthanasia

Litters evaluated

7

9

10

3

Pups evaluated

99

131

137

25

Skin

Scabbing

2 / 2

2 / 1

8 / 4

12 / 2

Desquamation

0

0

43 / 5

14 / 2

Overall evaluation

No remarkable findings

Compiled effects considered as remarkable

Compiled effects considered as remarkable

Conclusions:
During administration of acetophenone via gavage in doses of 0, 75, 225 and 750 mg/kg bw/d to male and female rats, starting at 14 days before mating and continued up to 3 days of the lactation period, maternal toxicity was indicated at 750 mg/kg from observed clinical signs including central nervous system depression, and from reduced weight gain in the gestation period (days 0 to 7). The NOAEL for maternal toxicity was 225 mg/kg bw/d. Reproductive performance was not affected up to the highest dose of 750 mg/kg bw/d. The NOAEL for developmental toxicity is 225 mg/kg bw/d. At 750 mg/kg bw/d developmental toxicity is indicated from decreased live birth index, decreased pup survival and decreased pup weights during lactation days 0 to 4. A toxic effect on the offspring is also obvious from the clinical findings/external observation and the findings from gross necropsy including a single case of cleft palate and a high incidence of atelectasis in stillborn pups.
However, as reproductive effects occurred only at the high dose being associated with maternal toxicity, including reduced weight gain during gestation days 0 -7, there is a high probability that the reproductive effects are a non-specific secondary consequence of a general toxic effect of acetophenone.
Executive summary:

In the course of a combined repeated dose toxicity and reproduction/developmental screening study groups of 10 female Sprague-Dawley rats were exposed to acetophenone at dosages of 0, 75, 225 and 750 mg/kg bw/day by gavage treatment starting at a minimum of 14 days before mating, and continued throughout the mating and gestation period up to lactation day 3. Each rat was mated with a single male from the same dose group. Clinical signs, body weight and food consumption were observed throughout the dosing period. After delivery, females and their offspring remained together until in-life conclusion on lactation day 4. Recorded parameters were reproductive indices as precoital interval, mating indices, fertility indices, and gestation lengths, and, at delivery, mean number of pups and sex ratios. During lactation days 0-4 pup survival and body weight development were recorded. After euthanasia on lactation day 4, a macroscopic examination was performed on the dams including uterine contents, and on the pups to investigate structural abnormalities or other pathological changes.

In females of the parent (F0 generation), clinical signs observed at 225 mg/kg are considered to be of no toxicological significance. At 750 mg/kg, significant maternal toxicity appeared as more severe clinical signs including e.g. decreased activity, skin pale in color, and postdose wobbly gait, and as significantly reduced body weight gain during gestation days 0-7 (61% of the control group; food consumption only slightly reduced). One female showed prolonged parturition. Remarkable gross necropsy findings of F0 females were limited to two females in the 750 mg/kg-group with total litter loss that had pup tissue mixed with ingesta in the stomach.

Mating indices, fertility indices, mean gestation lengths, mean number of pups delivered, and sex ratios were comparable to the control in all dose groups.

Reproductive toxicity was indicated at 750 mg/kg by a decreased live birth index (number of liveborn pups/total number of pups delivered) out of the range of the historical control, by statistically significant reductions of the number of mean live pups per litter on lactation days 1-4, the total number of pups surviving to lactation day 4, and a statistically significant increase of the number of stillborn pups/total number of pups delivered and of the number of F1 pups dying, missing and/or cannibalized during lactation days 1-4.

Pups of the 750 mg/kg-group, showed the following changes during lactation days 1 -4. Weight was significantly decreased (outside range of historical control data) and toxicity was indicated by external observations as e.g. gasping, skin pale in color, cool to the touch, skin with shiny appearance, and skin appears tight - restricts movement . At gross necropsy, stillborn pups showed a single incidence each of cleft palate and edema, increased incidences of atelectasis, milk not present in the stomach, and dermal hypoplasia. Pups born alive but found dead during lactation showed scabbing, desquamation, dermal hypoplasia, increased incidence of milk not present in the stomach, and in addition, 22 pups with observed autolysis. Findings in pups with scheduled euthanasia on lactation day 4 were scabbing and desquamation. There were no significant toxicological findings in pups of the 75 or 225 mg/kg-dose groups.

Based on these findings, maternal toxicity is indicated at 750 mg/kg from observed clinical signs including central nervous system depression, and from reduced weight gain in the gestation period (days 0 to 7). The NOAEL for maternal toxicity is 225 mg/kg bw/d.

Reproductive performance is not affected up to the highest dose of 750 mg/kg.

The NOAEL for developmental toxicity is 225 mg/kg. At 750 mg/kg, developmental toxicity is indicated from decreased live birth index, from decreased pup survival and decreased pup weights during lactation days 0 to 4. A toxic effect on the offspring is also obvious from the clinical findings/external observations and the findings from gross necropsy including a single case of cleft palate and a high incidence of atelectasis in stillborn pups. However, as these reproductive effects occurred only at the high dose being associated with maternal toxicity, including reduced weight gain during gestation days 0 -7, there is a high probability that the reproductive effects are a non-specific secondary consequence of a general toxic effect of acetophenone.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study: Study meets criteria of the OECD Guidelines 421 and 422 for reproductive toxicity screening study; study acceptable as screening study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a combined repeated dose toxicity and reproduction/developmental screening assay according to OECD Guidelines 422 (Key study, Thorsrud, 2003), there was no indication of a possible impairment of male fertility from histopathological examination and organ weights of testes, epididymides, and prostrate. Also, the macroscopic and histological examination of the female reproductive tract including corpora lutea and implantation sites showed no adverse effects. Parameters describing reproductive performance, including precoital interval, mating index, fertility index, and gestation index were not affected in rats up to the high test dose of 750 mg/kg bw/d after daily gavage treatment starting at 14 days before mating and continued throughout the 14-day mating period up to lactation day 3 (males treated for 28 days in total).

The combination of a pre-mating dosing period of 14 days and subsequent mating/fertility with an overall dosing period of 28 days, followed by detailed histopathology of the male gonads, is considered sufficient to enable detection of the majority of effects on male fertility and spermatogenesis. The dosing period for females is also sufficient to enable detection of effects on estrous cycle, on conception and pregnancy.

The high dose of 750 mg/kg bw/d was accompanied by signs of systemic and central nervous system toxicity both in male and female rats.

Effects on developmental toxicity

Description of key information

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

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

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

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 17-SEP-2019 to 04-JUN-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
31.5.2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, 01400 Châtillon sur Chalaronne, France
- Age at study initiation: 17 to 19 weeks old (at mating)
- Weight at study initiation: 2819 g to 4170 g (at mating)
- Housing: 2 air-conditioned rooms in a barrier protected unit. Females were individually housed in plastic cages meeting European Directive 2010/63/EU.
requirements
- Diet: approximately 200 g of food per day (Pelleted complete rabbit diet (diet Reference No. 3409) sterilised by irradiation and analysed for chemical and bacterial contaminants)
- Water: Softened and filtered (0.2 μm) mains drinking water ad libitum
- Acclimation period: 6 days between animal arrival and the start of treatment

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

IN-LIFE DATES: From: 16 Sep 2019 To: 25 Oct 2019
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) carboxymethylcellulose 400-800cPs/0.5% (w/v) Tween® 80 in purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared weekly as an emulsion (prepared and stirred using the same method as for a suspension (magnetic stirring for at least 15 minutes) in the control item at
concentrations of 20, 56.7 and 166.7 mg/mL and divided into daily aliquots.

VEHICLE
Carboxymethylcellulose sodium 400-800 cps from Sigma Aldrich. Batch No.: SLBK6619V. Expiry Date: 18 Sep 2021.
Tween® 80 from Merck. Batch No.: K51034361. Expiry Date: 31 Dec 2020.
Water for injection from Laboratoire Aguettant. Batch Nos.: 3013728 and 3013806. Expiry Dates: 28 Feb 2021 and 31 Mar 2021.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected from the four testing groups for analysis on Gestational Day 6 and on Gestational Day 28.
Analyses for concentration, homogeneity and stability in the vehicle were performed by Standard HPLC-UV.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
from GD6 to GD28
Frequency of treatment:
once daily
Duration of test:
28 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
1. Control
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
2. Low dose
Dose / conc.:
170 mg/kg bw/day (nominal)
Remarks:
3. Intermediate dose
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
4. High dose
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in agreement with the Sponsor on the basis of the results of a preliminary study of prenatal developmental toxicity where pregnant female New Zealand White rabbits were treated at 80, 250 or 700 mg/kg/day from GD6 to GD28. In dams at 700 g/kg/day, one was found dead on GD12 and all females lost weight from GD6 to GD9. Mean food consumption was lower than in the control group during the majority of the treatment period and especially from GD6 to GD9. In fetuses, a lower mean fetal weight was noted when compared with controls. At 250 mg/kg/day, body weight loss and strongly reduced food consumption were noted for a few females from GD6 to GD9. There were no toxicologically significant effects at 80 mg/kg/day. Therefore, 500 mg/kg/day was selected as the high dose level for the current study. The low dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e., 60 and 170 mg/kg/day).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

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

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

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
Moribund females were euthanized by sodium pentobarbitone injection and exsanguination. They were submitted to a full macroscopic examination to determine their pregnancy status, number of corpora lutea and numbers and types of uterine implantations. Any fetuses from these females were examined externally where possible and discarded.
Dead animals were also necropsied in order to establish, where possible, the cause of death and submitted to the examinations described above. When necessary, animals were refrigerated before necropsy to minimize autolysis.
All surviving females were euthanized on GD29 by intravenous injection of sodium pentobarbitone followed by exsanguination. They were submitted to a full macroscopic examination and any abnormalities observed were recorded. Abnormal organs or tissues
were sampled and preserved. For each female euthanized on GD29, the ovaries and uterus were removed and examined including examination of the placentae.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset.
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

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

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

Incidence (Parental Indices and Mortality):
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Indices:
Pre-Implantation Loss (in %)
Post-Implantation Loss (in %)
Sex Ratio (% males)
Litter % of Fetuses with Abnormalities
Historical control data:
Values compiled from untreated and control New Zealand White rabbits in previous studies
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased activity and/or abnormal gait, sometimes associated with low carriage, were noted for all females given 500 mg/kg/day, 1 hour after dosing, from GD6 to GD27. Animals recovered within the day. In addition, Female Nos. 74 and 76 given 500 mg/kg/day, had labored and shallow breathing,
lying on the side and subdued behavior on GD19-20 and GD9, respectively, 1-2 hours after dosing, but recovered within the day. These clinical signs were considered to be test item-related.
Few faeces were noted in all groups, including the controls, at the same incidence (20 to 22 females per group), but with a dose-related increased frequency of days affected at 170 and 500 mg/kg/day groups, which was consistent with the reduced food consumption. Therefore, the increased frequency of this finding was considered to be test item-related.
Red discharge, observed on GD29 for Female No. 31 given 60 mg/kg/day and for Female No. 53 given 170 mg/kg/day, was associated with the end of gestation. Red discharge, observed on GD15 for Female No. 83 given 170 mg/kg/day, without viable fetuses, might be associated with the numerous early resorptions. In addition, red discharge was observed on GD8 (predose) for Female No. 49 given 170 mg/kg/day, without any clear explanation.
On GD25, red discharge was observed during treatment and red traces were observed on the gavage cannula during intubation for Female No. 68 given 500 mg/kg/day. Therefore, the gavage was stopped and the animal was treated approximately 2 hours later when it had recovered. In addition, red discharge was observed during treatment for Female No. 65 given 170 mg/kg/day. These clinical signs were considered to be associated to the administration procedure.
Other clinical signs, such as thin fur or skin lesions, were observed in all groups including control and/or are commonly seen in this species. Therefore, they were considered as incidental.
Mortality:
mortality observed, treatment-related
Description (incidence):
2 females aborted in the 500 mg/kg/day dosed group. This represented 9% of the animals, more than commonly seen in the species (2% in the historical control data). Moreover, a third female given 500 mg/kg/day was found dead, with red discharge in the cage. Even if the cause of death was not clearly determined at necropy, the possibility of an association with the test item cannot be excluded.
In addition, 6 females (2, 1 and 3 in the control, 60 mg/kg/day and 170 mg/kg/day dosed groups, respectively) were found dead or sacrificed moribund due to, or probably due to, gavage error. These premature decedents were not test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 60 and 170 mg/kg/day, there were no major changes in body weight gain when compared with controls, only transient low body weight gain or body weight loss between GD18 and GD21 at 60 mg/kg/day and between GD15 and GD21 at 170 mg/kg/day, for a few females.
At 500 mg/kg/day, there was a body weight loss between GD6 and GD9 for all females (mean loss: -88 g ; range: -8 g to -231 g). Then, animals subsequently gained weight from GD9, as for the control group, with occasional low body weight gain or isolated body weight loss. This change had no impact on the terminal mean body weight on GD29, which was comparable in all groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg/day, there were no major changes in food consumption when compared with control and pretest values.
At 170 mg/kg/day, there was lower mean food consumption from GD6 to GD21 when compared with the controls (-16% to -29%). One female had negligible food consumption from the pretest period up to GD21, which was thus considered not to be test item-related.
At 500 mg/kg/day, there was a more pronounced reduction in mean food consumption from GD6 to GD24 when compared with the controls (-15% to -51% ; -28% on average) with a particularly low food consumption for three females.
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in mean gravid uterus weight in treated groups when compared with controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no remarkable macroscopic findings at 60 and 170 mg/kg/day.
There was a dark fluid accumulation in the uterine horn for one female given 500 mg/kg/day. This isolated finding was considered as incidental.
Other findings such as cysts in the oviduct or skin scabs, were noted in control or treated groups and are commonly seen in the species. Therefore, they were considered as incidental.
Histopathological findings: non-neoplastic:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
2 females aborted in the 500 mg/kg/day dosed group. This represented 9% of the animals, more than commonly seen in the species (2% in the historical control data).
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The pre-implantation data (number of corpora lutea, implantation sites and pre-implantation loss) were comparable in all groups.

There was no test item-related effect on embryo-foetal survival at 60 and 170 mg/kg/day.
At 500 mg/kg/day, the mean number of late resorptions (and thus also the total number of resorptions) was slightly higher than in the control group (0.7 vs. 0.5 for the late resorptions) and than the historical control range (0.1 to 0.6 for main embryofetal studies). This was mainly due to Female Nos. 67 and 77 (3 late resorptions) and Female No. 83 with no viable fetuses (all early resorptions). Therefore, the mean percentage post-implantation loss was slightly higher when compared with the control group (11.13% vs. 9.20%), and was close to the higher values of the historical control data (2.4% to 11.4% for main embryo-fetal studies). The mean number of live fetuses per litter remained comparable with the control group.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
There were 17, 20, 17 and 18 pregnant females at terminal caesarean in the control, 60, 170 and 500 mg/kg/day groups, respectively, all of which had viable fetuses, except Female No. 83 given 500 mg/kg/day with no viable fetuses.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 500 mg/kg/day induced a slightly higher number of late resorptions.
Dead fetuses:
no effects observed
Changes in number of pregnant:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
170 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
number of abortions
Abnormalities:
effects observed, treatment-related
Localisation:
other: low food consumption and body weight loss leading to 2 abortions and 1 premature death, and clinical signs such as decreased activity, abnormal gait and red discharge.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Consistent with the maternal toxicity, there was a slight dose-related lower mean fetal weight in the 170 and 500 mg/kg/day groups, when compared with the controls (-7.0% and -9.5%, respectively). At 500 mg/kg/day, the difference was statistically significant (p < 0.05) and the mean value was lower than the historical control range (37.44 g vs. 38.4-43.2 g for main embryo-fetal studies). This finding was more pronounced for female fetuses than for the males (difference compared with controls: -12.9% for females vs. -9.1% for males, at 500 mg/kg/day). This change was not associated with any ossification delay.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes in mean gravid uterus weight in treated groups when compared with the controls.
The mean number of live fetuses per litter is comparable with the control group.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no test item-related external malformations in any group.
The abnormalities noted in treated groups, such as hyperflexion of forepaw or short tail, are part of the historical control data and were incidental.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were 2 (2) fetuses (litters) with branched rib and/or multiple vertebral malformations in the 60 mg/kg/day group compared with none in the control group. There was 1 (1) fetus (litter) with absent caudal vertebra in the 60 mg/kg/day group, compared with none in the control group. There was also 1 (1) fetus (litter) with fused nasals in the 170 mg/kg/day group, compared with none in the control group.
These malformations were observed only at the low doses and/or are part of the background of changes noted for this strain of rabbit and were therefore considered as incidental.
Less severe skeletal anomalies were noted with sporadic incidence and/or are a part of the background data for this strain of rabbit and were considered as incidental.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were 2 (2) and 2 (2) fetuses (litters) with retroesophagial subclavian artery in the 170 and 500 mg/kg/day groups, respectively, compared with none in the control group.
However, this malformation is part of the background of changes noted for this strain of rabbit (7 cases between 2016 and 2018, with individual study frequency of up to approximately 1%, as noted in the current study) and these cases were therefore considered incidental in view of the lack of any dose-related increase in the incidence.
Other malformations were noted in the control and/or treated groups at the same incidence and thus were also considered as incidental.
Soft tissue anomalies were noted with sporadic incidence and/or are a part of the background data for this strain of rabbit and were considered as incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
170 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
170 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Summary of malformations - Individual descriptions:

Dose Level

(mg/kg/day)

Female

No.

Fetus

No.

Malformation(s) including external, visceral and skeletal examinations.

0

6

1

Aortic arch, interrupted

(terminating with left carotid artery)

Heart, muscular ventricular septal defect

Small lung

Left subclavian artery originmalpositioned

Sternebra, 1 or more, fused, severe (1st to 5th)

8

8

Right kidneymalpositioned(caudally, centrally)

17

5

Dilatation of aortic arch

Narrowed ductus arteriosus

Pulmonary trunk atresia

60

25

8

Rib, L5, branched

36

11

Vertebra, general, multiple malformations (first lumbar centrum small and misaligned; first right lumbar arch absent; first left lumbar arch fused with 12th left thoracic arch; 12th left rib branched; scoliosis)

38

2

Short tail and caudal vertebra absent (9th to 14th)

170

47

1

Both nasals fused

50

6

Hyperflexion of left forepaw

Subclavian artery, right, retroesophageal (arising between left subclavian artery and ductus arteriosus)

52

5

Subclavian artery, right, retroesophageal (arising between left subclavian artery and ductus arteriosus)

62

1

Aortic arch, interrupted (aortic arch terminating with left subclavian artery)

500

68

7

Subclavian artery, right, retroesophageal (arising from the descending aorta)

71

5

Heart, muscular ventricular septal defect

78

5

Right kidneymalpositioned(caudally, centrally)

88

8

Subclavian artery, right, retroesophageal (arising from the descending aorta)

 

See also tables attached:

- Summary of Clinical Observations: Gestation

- Summary of Body Weights: Gestation

- Summary of Body Weight Gains: Gestation

- Summary of Food Consumption: Gestation

- Summary of Macroscopic Pathology: Gestation

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

- Summary of Ovarian and Uterine Examinations and Litter Observations

- Summary of Fetal Abnormalities by Finding

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

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

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

Results

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

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

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

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

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

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

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

The pre-implantation data were comparable in all groups.

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

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

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

Conclusion

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

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

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

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

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

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02-SEP-2015 to 14-JUL-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: based on available stability data, test item formulation was prepared at least once every ten days and stored at room temperature.
- Solubility and stability of the test substance in the solvent/vehicle: Homogeneity of the test item in the vehicle was maintained by vortexing the prepared formulation thoroughly before every dose administration.

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

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

IN-LIFE DATES: From: 17 September 2015 To: 22 April 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared with corn oil. The test item was weighed and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes. The formulations were prepared at least once every ten days based on available stability data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): selected based on the test item’s characteristics and testing guideline
- Concentration in vehicle: 31.25, 75 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: MKBP7039V and MKBV2080V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of concentration of the test item in the vehicle were taken in the first and last week of the study for all doses. Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation, in the first and last week of the study.
All samples were analysed by HPLC/UV within 10 days after sampling and until then stored under appropriate conditions based on available stability data.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 (male to female)
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: /
Duration of treatment / exposure:
from gestation day (GD) 5 to gestation day (GD) 19
Frequency of treatment:
once daily
Duration of test:
from mating to gestation day (GD) 20
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
35 females in the control group and the high dose group, and 25 females in the low dose and the medium dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a dose range finding study
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays where observations were made once daily.
- Any clinical observations (including spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and Foetusil size) were recorded if observed. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded if observed.

DETAILED CLINICAL OBSERVATIONS: No

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

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- On gestation day 20, sperm positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally.
- At the time of termination or death during the study, dams were examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings is preserved in 10% neutral buffered formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (fetuses used for soft tissue examination)
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Historical control data:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- The predominant clinical sign was slightly to severely reduced spontaneous activity in most females of the high dose group (750 mg/kg bw/day) on several treatment days short (mid-morning), as well as late post-application (afternoon). In several cases, this was also associated with half eyelid closure and prone position. Additionally, high dose group specific clinical signs observed on several occasions were short and late post-application ataxia as well as piloerection. Fully closed eyes and apathy were also seen in single females of the high doe group on few days of treatment. All these signs were considered as related to the treatment with the test item (Acetophenone was used in the past as “Hypnone”). Transiently reduced spontaneous activity short post-application was observed in threesingle females of the medium dose group (300 mg/kg bw/day) on one or two days of treatment.

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

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

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

- None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Description (incidence and severity):
Not applicable.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One single female of the high dose group (750 mg/kg bw/day) was found dead in the afternoon of gestation day 19. According to the macroscopic finding (dark discoloured lung at necropsy) and the absence of body weight loss or preceded worsened health condition, this death was considered to be likely related to a gavage error.
One female of the control group was found dead after 2 days of treatment. At necropsy, the pericardial sac was found to be filled with blood. This death was considered spontaneous and incidental as no preceded sign of morbidity was observed.
All the remaining animals survived until the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- The mean body weight increased in all groups before initiation of the treatment and further increased with the progress of the study comparably in the control and the low dose group.
- A treatment related slightly to moderately lower mean terminal body weight was observed in the medium dose group (92 % of controls, p < 0.001) and the high dose group (88 % of controls, p < 0.001) when compared to the control group.
- A moderately and dose dependently lower body weight gain was observed over the entire study period (GD 0-20) in the medium group (77 % of controls, p < 0.001) and the high dose group (63 % of controls, p < 0.001). This resulted in a slightly to moderately, statistically significantly reduced mean body weight in the medium dose and the high dose group from Gestation Day (GD) 8 to GD 20, with the greatest effect at the end of the treatment period (medium dose group: 92 % of controls, p < 0.001; high dose group: 88 % of controls, p < 0.001). Effects on body weight and body weight gain in the medium and the high dose groups were considered to be test item related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Mean food consumption of the low dose group was unaffected by the treatment with the test item. Differences to the control group were slight and not statistically significant.
- In correlation to the body weight and body weight gain, food consumption in the medium dose and the high dose groups was noted to be moderately and statistically significantly lower compared to the control group during the entire treatment period (except for the interval of GD 11-14 in the MD group). Over the entire study period (GD 0-20), a dose dependently moderately lower food consumption was observed in the medium dose group (87 % of controls, p < 0.001) and the high dose group (77 % of controls, p < 0.001). Effects on food consumption in the medium dose and the high dose group were considered to be test item related.
Food efficiency:
not examined
Description (incidence and severity):
Not applicable.
Description (incidence and severity):
Not applicable.
Ophthalmological findings:
not examined
Description (incidence and severity):
Not applicable.
Haematological findings:
not examined
Description (incidence and severity):
Not applicable.
Clinical biochemistry findings:
not examined
Description (incidence and severity):
Not applicable.
Urinalysis findings:
not examined
Description (incidence and severity):
Not applicable.
Behaviour (functional findings):
not examined
Description (incidence and severity):
Not applicable.
Immunological findings:
not examined
Description (incidence and severity):
Not applicable.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- The uterus weight at necropsy was dose dependently, moderately lower in the medum dose group (86 % of controls, p < 0.05) and the high dose group (75 % of controls, p < 0.001). Furthermore, slightly but statistically significantly lower mean adjusted maternal weight (maternal weight minus gravid uterus weight) was also noted in the medium dose group (93 % of controls, p < 0.001) and the high dose group (90 % of controls, p < 0.001). This was considered as a treatment related effect.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- At necropsy of the female of the high dose group which was found dead on gestation day 19, the lung was noted to be discoloured (dark). Without preceded morbidity, its death was likely related to a gavage error.
- At necropsy of the control female which was found dead on gestation day 7, the pericardial sac was noted to be blood filled and could be accounted as reason for its spontaneous death.
- In one single female of the control group, a fluid filled uterus was noted, which was considered to be incidental in nature reflecting cyclic changes.

No further gross pathological changes were observed during the macroscopic examination of the females of any group.
Neuropathological findings:
not examined
Description (incidence and severity):
Not applicable.
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Not applicable.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Not applicable.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
External examination: low incidences of haematoma on various body parts were noted in single females without dose dependency and were considered to be spontaneous in nature without relation to the treatment with the test item.
Number of abortions:
no effects observed
Description (incidence and severity):
- No test item related effects of toxicological relevance were noted for number of live foetuses.
- Marginally but not statistically significantly lower mean number of foetuses (9.81) was observed in the high dose group when compared to the control group (11.22). However, without statistical significance, and as treatment in this study was not initiated before actual nidation and values were within the normal range of variation, this was considered as incidental in nature.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- No test item related effects of toxicological relevance were noted for number of corpora lutea, implantation sites,and percent pre- and post-implantation loss.
- Marginally but not statistically significantly lower mean number of implantation sites (10.88) was observed in the high dose group when compared to the control group (11.70). However, without statistical significance, and as treatment in this study was not initiated before actual nidation and values were within the normal range of variation, this was considered as incidental in nature.
- Marginally but not statistically significantly higher mean number of post-implantation loss (10.70 %) in the high dose group compared to the control group (3.74 %) was predominantly related to 100 % post-implantation loss in one single female of the high dose group. Without statistical significance, and as mean values were within the range of normal variation, this was considered incidental.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
- No test item related effects of toxicological relevance were noted for early and late resorptions.
- Marginally but not statistically significantly higher mean number of early resorptions loss (1.06 %) in the high dose group compared to the control group (0.4 %) was predominantly related to 100 % post-implantation loss in one single female of the high dose group. Without statistical significance, and as mean values were within the range of normal variation, this was considered incidental.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed in any of the treated groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 24/25 pregnancies in the low dose group, 24/25 in the medium dose group and 33/35 in the high dose group compared to 33/35 pregnancies in the control group.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
organ weights and organ / body weight ratios
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Dose dependent, test item related effects were noted for foetal weight data in the medium dose and the high dose group. Mean foetus weight (calculated on litter basis) of the medium dose group (3.44 g), was slightly but statistically significantly lower (94 % of controls, p < 0.01) when compared to the control group (3.67 g). Moderately lower mean foetus weight was observed in the high dose group (3.11 g, 85 % of controls, p < 0.001). Similarly, the male and female foetus weight was slightly lower in the medium dose group and moderately lower in the high dose group with achieving statistical significance.
- Mean foetal weight of the low dose group was unaffected by the test item administration.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see higher.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- No test item related effects of toxicological relevance were noted for male and female foetuses sex ratio.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
- Dose dependent, test item related effects were noted for litter weight data in the medium dose and the high dose group. Changes in foetal weights resulted in slight effects on litter weight data of the medium dose group with slightly but not statistically significantly lower total litter weight (86 % of controls) and male litter weight (94 % of controls), and statistically significantly lower female litter weight (79 % of controls, p < 0.05). Moderate effects on litter weight data were noted for the high dose group with moderately lower total litter weight (77 % of controls, p < 0.001), female litter weight (72 % of controls, p < 0.01) and slightly but not statistically significantly lower male litter weight (82 % of controls).
- Mean litter weight of the low dose group was unaffected by the test item administration.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
- There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group.
- By external examination, agnathia was observed in one single foetus of the high dose group and in one single foetus of the control group. Considering the low incidence, this was considered incidental. No skeletal correspondence for this finding was observed at skeletal examination of the control foetus, whereas the mandible was shown to be short in the foetus of the high dose group (micrognathia).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings in all groups including the control group:
- Bilateral pelvic girdle caudal shift was noted in moderately more litters of the high dose group (7 litters) with statistical significance (p < 0.05) when compared to the control group (2 litters) (litter incidences: 0.00 % in the low dose, 14.29 % in the medium dose, 30.43 % in the high dose group (p < 0.05) compared to 6.67 % in controls; foetal incidences: 0.00 % in the low dose, 3.48 % in the medium dose, 10.92 % in the high dose group compared to 1.13 % in controls). Values in the high dose group were slightly above historical control data with a maximum historical litter incidence of 30.00 % and a maximum foetal incidence of 6.20 %. As this finding is considered as possible indicator of a treatment effect, a relation to the treatment cannot be excluded.
- Furthermore, as it can typically be seen, the increased number of pre-pelvic vertebrae linked to the observed malposition of the pelvic girdle, was associated with bilateral full supernumerary thoracolumbal ribs in several of the foetuses. Accordingly, 14th full bilateral rib was observed at a moderately but not statistically significantly higher litter incidence in the high dose group (43.48 %) when compared to the control group (23.33 %). Though not statistically significant, litter incidence (43.48 %) and foetal incidence (14.29 %) in the high dose group was observed to be considerably higher when compared to historical control data with a maximum historical litter incidence of 31.58 % and a maximum foetal incidence of 11.65 %.

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

There was no statistical significance and no indication of a test item related trend in the type and/or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.
Visceral malformations:
no effects observed
Description (incidence and severity):
VISCERAL EXAMINATION
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including the control group:

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

CRANIOFACIAL EXAMINATION

- Examination revealed few findings (retinal fold, dilated third ventricle, dilated lateral ventricle, subcutaneous edema) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls. Statistical analysis of the data revealed no significant effect. These findings were considered to be spontaneous in nature and not related to the treatment with the test item.
- The finding of a small pituitary gland was seen without statistical significance in single foetuses (4 of the high dose group, 0 of the medium dose group, 1 of the low dose group and 1 of the control group) with a litter incidence of 10.00 % in the high dose group, 0.00 % in the medium dose group and 4.17 % in the low dose group compared to 3.13 % in the control group. Without indication of a test item related trend, this was considered incidental.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Developmental effects observed:
not specified

Results of the dose formulation analysis:

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

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

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

Material and methods

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

Results

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

Conclusion

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good quality as a guideline and GLP study is available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Two Prenatal Developmental Toxicity according to OECD TG 414 (GLP, Klimisch reliability 1) are available with acetophenone in two different species: rat and rabbit.

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

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

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

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

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

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

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

Results

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

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

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

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

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

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

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

The pre-implantation data were comparable in all groups.

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

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

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

Conclusion

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

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

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

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

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

Justification for classification or non-classification

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

Regarding the developmental effects observed in rats, no classification is required for the following reasons:

- The findings are of low level of concern (permanent, but most of the time not adverse),

- No other foetal effects (except on body weight) or malformations were observed, and no mortality was observed. At 300 mg/kg only a slight body weight reduction is observed (93-94% of control, in parallel of a lower maternal body weight and lower food consumption), but no abnormalities.

- The skeletal findings are observed at maternal toxic dose (750 mg/kg),

- Even if a dose-effect is observed (statistically significant at 750 mg/kg), the % of litter with bilateral pelvic girdle caudal shift is only slightly above the maximum incidence when compared to historical control (30.43% vs 30.00%).

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

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

Additional information