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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Reference Type:
publication
Title:
A combined repeated dose toxicity study and reproduction/developmental screening study in Sprague-Dawley rats with acetophenone (OECD guideline No. 422)
Author:
Kapp RW, Thorsrud BA, Moffatt WJ, Lawton L
Year:
2003
Bibliographic source:
Toxicologist 72: 76-77

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetophenone
EC Number:
202-708-7
EC Name:
Acetophenone
Cas Number:
98-86-2
Molecular formula:
C8H8O
IUPAC Name:
1-phenylethan-1-one
Details on test material:
- Name of test material: Acetophenone
- Physical state: slight pale yellow liquid
- Analytical purity: 98.80 %
- Impurities (identity and concentrations): no data available
- Lot/batch No.: Lot No. R012-078
- Expiration date of the lot/batch: none provided
- Stability under test conditions: no data available
- Storage condition of test material: ambient conditions

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

open allclose all
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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.