4-phenylbutan-2-one

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from 2001-03-06 to 2003-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

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:

The Toxicologist 72 (S-1):76-77

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetophenone

EC Number:

202-708-7

EC Name:

Acetophenone

Cas Number:

98-86-2

IUPAC Name:

1-phenylethanone

Constituent 2

Reference substance name:

606-042-00-1

IUPAC Name:

606-042-00-1

Details on test material:

- Name of test material (as cited in study report): acetophenone:
- Physical state: slightly pale yellow liquid
- Analytical purity: 98.80 %
- Lot/batch No.: R012-078
- Expiration date of the lot/batch: none provided

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.
- Age at study initiation: eight weeks
- Weight at study initiation: 246-285 g for males, 162-201 g for females
- Fasting period before study: no
- Housing: individually during acclimation and while on study (except during cohabitation) in suspended stainless steel cages
- Diet (e.g. ad libitum): ad libitum (PMI Certified Rodent Meal® #5002, Purina Mills, Inc)
- Water (e.g. ad libitum): ad libitum (purified by reverse osmosis and ultraviolet light irradiation)
- Acclimation period: 36 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10 to 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:

VEHICLE
- Concentration in vehicle: 0, 37.5, 112.5, 375 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg
- Lot/batch no. (if required): QN0035

Details on mating procedure:

The male rats from the toxicity phase were used to bread the reproduction phase females

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to initiation of the toxicity and reproduction/developmental screening studies, homogeneity and stability analyses were performed on concentrations of the test article in the vehicle which encompassed the low- and high-dose concentrations. Homogeneity analyses were performed on duplicate samples taken from the top, middle and bottom of the two mixtures. Stability of the test article in the vehicle was evaluated on duplicate samples on day 0 and at 3 days and 10 days of room temperature storage. Concentration verification analysis was performed on the vehicle and each test article dosing solution prepared for study weeks 0, 2, 4 and 6.
Results of the homogeneity analyses revealed that the average recoveries of the 37.5 and 375 mg/mL dosing formulations were within 4.9 % of the nominal concentration, indicating that the formulations were homogeneous. Results of the stability analyses revealed that the 37.5 and 375 mg/mL dosing formulations were stable for 10 days following room temperature storage. The average results of the concentration verification analyses were within 5.3 % of the nominal concentrations. No Acetophenone was detected in the vehicle control samples analyzed during the course of the study.

Duration of treatment / exposure:

28 days to male and female rats for the toxicity phase and for a minimum of 14 days through day 3 of lactation to female rats for the reproduction/developmental phase

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 75, 225, 750 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: The high-dose level was expected to produce some toxic effects, but not excessive lethality. The mid-dose level was expected to produce no to minimal observable effects and the low-dose level was expected to produce no observable effects.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily within approximately one-half hour to two hours following dosing.

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 for examinations: individual body weights were recorded on days 0, 3, 7 and 12 prior to mating. When positive evidence of mating was detected, the females were weighed on gestation days 0, 7, 14 and 20. Following parturition, the females were weighed on lactation days 1 and 4. Females without evidence of mating were weighed twice weekly until euthanasia.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
live births, postnatal mortality

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: All surviving animals [F0 females that delivered were euthanized on lactation day 4. Females that failed to deliver were euthanized 25 days after evidence of mating was first detected (post breeding day 25). F0 females with no evidence of mating were euthanized 25 days after completion of the mating period (post breeding period day 25). Females with total litter loss were euthanized and necropsied on the day that no surviving pups remained.]

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surfaces of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. Uterine contents were examined and the number of implantation sites and number of corpora lutea (per ovary) were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for histopathological examination:
Accessory genital organs (uterus and vagina), Adrenals, All gross lesions, Aorta, Brain (including sections of medullalpons, cerebellar cortex and cerebral cortex), Cecum, Colon, Duodenum, Esophagus, Exorbital lachrymal glands, Eyes with optic nerve, Femur (including articular surface) and bone marrow, Heart, Ileum, Jejunum, Kidneys, Liver (three sections collected), Lungs (infused with formalin) with bronchi, Mammary gland, Mandibular lymph node, Mediastinal lymph node, Mesenteric lymph node, Ovaries, Pancreas, Peripheral nerve (sciatic), Pituitary, Rectum, Skeletal muscle (thigh), Skin, Spinal cord (cervical, midthoracic and lumbar), Spleen, Sternum with bone marrow, Stomach (glandularlnonglandular), Submaxillary salivary gland, Thymus, Thyroidlparathyroid, Tongue, Trachea, Urinary bladder.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring: all surviving pups were euthanized on lactation day 4.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: special attention was paid to the organs of the reproductive system. All gross lesions were preserved in 10% neutral buffered formalin for possible future histopathological examination.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including a single incidence of cleft palate, a single incidence of edema, increased incidences of atelectasis, milk not present in the stomach, desquamation and dermal hypoplasia]

Statistics:

Statistical analysis for the reproduction/developmental screening study were performed using the SLI Alpha ReproTox computer system. Data, including body weights, body weight changes, food consumption and mean live litter size were analyzed by ANOVA. If significance was observed with ANOVA, control to treatment group comparisons were performed using Dunnett's test. Count data were analyzed using R x C Chi-Square test followed by Fisher's Exact Test for copulation and fertility indices, pup sex ratios, the number of live and dead pups per group (on lactation day 0) and pup survival (after lactation day 0). All analyses were two-tailed with a minimum significance level of 5 % (p<0.05).

Reproductive indices:

Mating and fertility indices

Offspring viability indices:

F1 viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

a low incidence of urine stain, predose and postdose salivation, a low incidence of postdose feces small in size in the 225 mg/kg/day group. For other detailed symptoms see section "Details on results" (parental animals).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight change of F0 females in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7. For other detailed symptoms see section "Details on results" (parental animals).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean body weight change of F0 females in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7. For other detailed symptoms see section "Details on results" (parental animals).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minimal to mild hyaline droplet nephropathy was observed for males in the 75, 225 and 750 mg/kg/day groups.

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred in the F0 females. The clinical signs included a low incidence of urine stain and predose salivation, a low incidence of postdose feces small in size and postdose salivation in the 225 mg/kg/day group, and a low incidence of decreased activity, skin pale in color, unkempt appearance, rough coat, postdose dark material around nose, postdose few feces, and predose and postdose salivation and postdose wobbly gait in the 750 mglkglday group. In addition, one female in the 750 mg/kg/day group (#1851) appeared to have prolonged parturition. Remarkable clinical signs for females in the 75 mg/kg/day group were limited to a single incidence of urine stain in two females.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weight change of F0 females in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7. F0 females in the 225 and 750 mg/kg/day groups lost slightly more body weight than controls during days 0-3 (prior to mating) and mean body weight change of
F0 females in the 750 mg/kg/day group appeared to be slightly lower than controls during lactation days 1-4.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
no effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
Lower absolute epididymides weight relative to final body weight for males in the 750 mg/kg/day group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects for males. Remarkable gross necropsy findings in F0 females were limited to two females in the 750 mg/kg/day group with total litter loss that had pup tissue mixed with ingesta in the stomach.

HISTOPATHOLOGY (PARENTAL ANIMALS)
A minimal to mild hyaline droplet nephropathy was observed for males in the 75, 225 and 750 mg/kg/day groups.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity and reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: not available

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity and reproductive effects

Effect level:

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

Based on:

other: 4-phenylbutan-2-one (conversion factor 1.23)

Sex:

male/female

Basis for effect level:

other: not available

3

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity and reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: a significantly decreased live birth index (increased rate of stillbirths), as well as decreased pup survival during the lactation phase and a decreased mean pup body weight in the high-dose group.

4

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity and reproductive effects

Effect level:

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

Based on:

other: 4-phenylbutan-2-one (conversion factor 1.23)

Sex:

male/female

Basis for effect level:

other: not available

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Effects in 225 and 750 mg/kg/day groups. For other detailed symptoms see section "Details on results" (offspring).

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

22.9 % in the 750 mg/kg/day group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean F1 pup weights in the 750 mg/kg/day group were statistically lower than controls on lactation days 1 and 4, and outside the range of SLI historical control d

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For detailed symptoms see section "Details on results" (offspring).

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index on lactation day 4 was 94.3% in the control group, 96.3% in the 75 mg/kg/day group, 94.5% in the 225 mg/kg/day group and 22.9% in the 750 mg/kg/day group.
Two F0 females in the control group (#A867 and #1870), one F0 female in the 75 mg/kg/day group (#1869) and one F0 female in the 750 mg/kg/day (#1842) with evidence of mating failed to deliver and were euthanized on postbreeding day 25. Six F0 females in the 750 mg/kg/day group
(#1841, #1847, #1848, #1857, #1861 and #1876) were euthanized due to total litter loss. These females were euthanized on lactation days 1-4, the day all pups in the litter were found dead. All other F0 females in the control, 75, 225 and 750 mg/kg/day groups survived to scheduled euthanasia on lactation day 4.

CLINICAL SIGNS (OFFSPRING)
Remarkable F1 pup observations during lactation days 0-4 included desquamation in the 225 and 750 mg/kg/day groups and a low incidence of gasping and skin pale in color, cool to the touch, skin with shiny appearance and skin appears tight - restricts movement in the 750 mg/kg/day group.

BODY WEIGHT (OFFSPRING)
Mean F1 pup weights in the 750 mg/kg/day group were statistically lower than controls on lactation days 1 and 4, and outside the range of SLI historical control data.

SEXUAL MATURATION (OFFSPRING)
not examined

ORGAN WEIGHTS (OFFSPRING)
no data

GROSS PATHOLOGY (OFFSPRING)
For F1 pups stillborn included a single incidence of cleft palate, a single incidence of edema, increased incidences of atelectasis, milk not present in the stomach, and dermal hypoplasia in the 750 mg/kg/day group. For F1 pups born alive but found dead during lactation days 0-4 included scabbing, desquamation, dermal hypoplasia and an increased incidence of milk not present in the stomach in the 750 mg/kg/day group. In addition, autolysis was observed for 22 F1 pups found dead in the 750 mg/kg/day group. for F1 pups at scheduled euthanasia on lactation day 4 included scabbing and desquamation in the 225 and 750 mg/kg/day groups.

HISTOPATHOLOGY (OFFSPRING)
no data

OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Key result

Remarks on result:

other: refer to details on results (F1)

Remarks:

refer to details on results (F1)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

As a result of a significantly decreased live birth index (increased rate of stillbirths), as well as decreased pup survival during the lactation phase and a decreased mean pup body weight in the high-dose group, a dosage level of 225 mg/kg/day was considered a NOAEL for reproductive effects. However, the mating and fertility indices and mean gestation length for the test article-treated females appeared unaffected at dosage levels up to 750 mg/kg/day.

Executive summary:

This study, according to OECD guideline 422 was conducted to: serve as a screening study for potential reproductive and developmental effects in male and female rats. This study was not designed to provide a complete assessment of all aspects of reproduction and development; but to offer a limited means of detecting postnatal manifestations of prenatal exposure, as well as effects induced during limited postnatal exposure. The study consisted of a toxicity study and a reproduction/developmental screening study. The test article of 0, 75, 225 and 750 mg/kg/day was administered once daily (seven days a week) by oral gavage in males and females for a minimum of 14 days prior to mating and continuing through lactation day 3. Cage-side observations for overt signs of toxicity were performed once daily within approximately onehalf hour to two hours following dosing. Detailed clinical observations were performed a minimum of weekly until evidence of mating was detected, daily during gestation and lactation and on the day of scheduled euthanasia. Individual body weights were recorded on days 0, 3, 7 and 12 prior to mating, on gestation days 0, 7, 14 and 20 and on lactation days 1 and 4. Individual food consumption was recorded on the same days as body weights (except during cohabitation). After a minimum of 14 days of treatment, females were cohabited with males from the same treatment group in the toxicity study. Following parturition, pup viability, sex determinations, observations and body weights were recorded at specified intervals during lactation days 0 and 4. Surviving females and their pups were euthanized and necropsied on lactation day 4. Selected tissues and organs were collected from the females at necropsy and retained in 10 % neutral buffered formalin for possible future histopathological evaluation.

No mortality occurred for the F0 females in the reproduction/developmental screening study. Clinical signs were observed for F0 females in the 225 and 750 mg/kg/day groups. The clinical signs included urine stain, predose salivation, postdose feces small in size and postdose salivation in the 225 mg/kg/day group, and decreased activity, skin pale in color, unkempt appearance, rough coat, postdose dark material around nose, postdose few feces, predose and postdose salivation and postdose wobbly gait in the 750 mg/kg/day group. In addition, one female in the 750 mg/kg/day group appeared to have prolonged parturition. Remarkable clinical signs for females in the 75 mg/kg/day group were limited to a single incidence of urine stain in two females. Mean body weight change of F0 females in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7. There were no statistically significant differences in mean body weights; however, mean body weight of F0 females in the 750 mg/kg/day group was slightly (approximately 6 %) lower than controls on gestation day 7. No other toxicologically meaningful differences in mean body weights or body weight change were noted during the study. Mean food consumption of F0 females in the 225 and 750 mg/kg/day groups was statistically lower than controls during days 0-3. In addition, mean food consumption appeared to be slightly, but not statistically lower than controls for F0 females in the 750 mg/kg/day group during gestation days 0-7 and lactation days 1-4. No other toxicologically meaningful differences in mean food consumption were noted between the control, 75, 225 and 750 mg/kg/day groups. The F0 female mating and fertility indices and mean gestation lengths were comparable between the control group, 75, 225 and 750 mg/kg/day groups. The mean number of F1 pups delivered was comparable in the control, 75, 225 and 750 mg/kg/day groups. However, the live birth index (number of liveborn pups of total number of pups delivered) was statistically lower in the 750 mg/kg/day group compared to controls, and the number of stillborn pups in the 750 mg/kg/day group was statistically higher than controls. The number of F1 pups dying, missing and/or cannibalized during lactation days 1-4 and the number of litters with total litter loss in the 750 mg/kg/day group were statistically higher than controls and the number of surviving pups on lactation day 4 in the 750 mg/kg/day group was statistically lower than controls. The mean live pups/litter in the 750 mg/kg/day group was slightly, but not statistically lower than controls on lactation day 0 and statistically lower than controls on lactation days 1-4. Mean F1 pup weights in the 750 mg/kg/day group were statistically lower than controls on lactation days 1 and 4. F1 pup viability and mean F1 pup weights in the 75 and 225 mg/kg/day groups was comparable to controls during lactation days 0-4. The F1 pup sex ratio was comparable between the control, 75, 225 and 750 mg/kg/day groups on lactation days 0 and 4. Remarkable F1 pup observations included desquamation in the 225 and 750 mg/kg/day groups and a low incidence of gasping and skin pale in color, cool to the touch, skin with

shiny appearance and skin appears tight - restricts movement in the 750 mg/kg/day group. No remarkable F1 pup observations were noted in the 75 mg/kg/day group. Remarkable gross necropsy observations for F1 pups stillborn included a single incidence each of cleft palate and edema, increased incidences of atelectasis and milk not present in the stomach, and dermal hypoplasia in the 750 mg/kg/day group. Remarkable gross necropsy findings for F1 pups born alive but found dead during lactation days 0-4 included

scabbing, desquamation, dermal hypoplasia and an increased incidence of milk not present in the stomach in the 750 mg/kg/day group. In addition, autolysis was observed for 22 F1 pups found dead in the 750 mg/kg/day. No remarkable gross necropsy findings were noted for F1 pups stillborn or found dead in the 75 and 225 mg/kg/day groups. Remarkable gross necropsy findings for F1 pups at scheduled euthanasia (lactation day 4) included scabbing and desquamation in the 225 and 750 mg/kg/day groups. No remarkable gross necropsy findings were noted for surviving F1 pups in the 75 mg/kg/day group. Remarkable gross necropsy findings in F0 females were limited to two females in the 750 mg/kg/day group with total litter loss that had pup tissue mixed with ingesta in the stomach. No other remarkable findings were noted at necropsy for F0 females in the control, 75, 225 or 750 mg/kg/day groups.

A no-observed-adverse-effect level (NOAEL) for the systemic toxicity of 75 mg/kg bw/day was proposed in the original study basing on urine stain, predose and postdose salivation in the 225 and 750 mg/kg bw/day groups. In addition, the highest dose group animals showed postdose wobbly gait, postdose urine stain, in male rats statistically lower mean forelimb grip strength and mean motor activity. From regulatory point of view, the effects at 225 mg/kg bw/day were interpreted as not adverse and therefore the systemic toxicity NOAEL was considered at 225 mg/kg bw/day.

As a result of a significantly decreased live birth index (increased rate of stillbirths), as well as decreased pup survival during the lactation phase and a decreased mean pup body weight in the high-dose group, a dosage level of 225 mg/kg/day was considered a NOAEL for reproductive effects. However, the mating and fertility indices and mean gestation length for the test article-treated females appeared unaffected at dosage levels up to 750 mg/kg/day.