Acetoacetanilide

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: REPRODUCTIVE/DEVELOPMENTAL TOXICITY SCREEN

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed OECD and GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Only minor deviations exist that do not affect the validity of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetoacetanilide

EC Number:

202-996-4

EC Name:

Acetoacetanilide

Cas Number:

102-01-2

Molecular formula:

C10H11NO2

IUPAC Name:

3-oxo-N-phenylbutanamide

Details on test material:

- Name of test material (as cited in study report): acetoacetanilide

Test animals

Species:

rat

Strain:

other: Crl:CD®BR(SD) VAF/Plus® (abbreviated as CD®)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Inc, Portage, Michigan
- Number of Rats: 75 (male), 75 (female)
- Approx date of birth: 16 Jul 1995 (m), 9 Jul 1995 (f)
- Approx age at arrival: 58 days (m), 65 days (f)
- Weight (g) on day after arrival: 245 - 284 (m), 187 - 228 (f)
- Weight (g) at study assignment: 352 - 374 (m), 238 - 266 (f)
- Room temperature: 70 °F - 78 °F (+/- 2 %)
- Rel. humidity: 40% - 70%
- Diet: Certified Rodent Diet #5002, ad libitum
- Water: Local water processed through a reverse osmosis membrane and chlorinated, ad libitum, from automatic watering system.
- Artificial light: in 12 hr periods, dark period beginning at 1900 EST.
- Identification: Each rat was individually identified with a Monel self-piercing ear tag. F1 generation pups were not individually identified.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 1.0% methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing suspensions were prepared at approximately weekly intervals. The amount of the test substance used in the preparation of the dosing suspensions was not adjusted for the percent active ingredient since the test substance is > 99% pure. The amounts of suspensions that were used for dosage were documented each day.

Details on mating procedure:

- Male and female rats were cohabitated in pairs during the breeding period, which lasted a maximum of 14 days
- Proof of pregnancy: observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ.
- Each pair of rats was returned to individual housing after mating was confirmed.
- Rats that did not mate remained in cohabitation for the entire breeding period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing suspensions were analysed for the concentration of the test substance at Lancaster laboratories, PA. The methods used for these analyses are described in detail in the Analytical Report (Appendix 2 of the full study report).

Duration of treatment / exposure:

Parental generation male and female rats were administered the test substance once daily as a single daily dose. Dosage volumes were based on the most recently recorded body weights. Male rats were given the test substance once daily beginning 14 days before breeding and continued through the day before sheduled sacrifice. Male received a total of 47 to 49 doses. Female rats were given the test substance once daily beginning 14 days before breeding and continued through day 4 postpartum, when they were scrificed. Female rats received a total of 39 to 52 doses.

Frequency of treatment:

Male received a total of 47 to 49 doses. Female rats received a total of 39 to 52 doses.

Details on study schedule:

- Length of Study: approx. 8 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Dosage: 0 mg/kg/day, Conc: 0 mg/l (in vehicle)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Dosage: 3 mg/kg/day, Conc: 0.3 mg/l (in vehicle)
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Dosage: 30 mg/kg/day, Conc: 3 mg/l (in vehicle)
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
Dosage: 100 mg/kg/day, Conc: 10 mg/l (in vehicle)
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of 7-day repeated dose toxicity screen conducted prior to this study.Based on the results of that study a high dosage level of 100 mg/kg/day in this reproductive/developmental toxicity screen was expected to result in a significant amount of systemic toxicity (methemoglobinemia and hemolytic anemia) and was considered to be the highest dosage level that could be tolerated by pregnant animals in the study. The 30 mg/kg/day dosage level was expected to produce an intermediate degree of effect or no effect, and the 3 mg/kg/day dosage level was expected to produce no effect.
- Oral route of exposure and the gavage were selected for use because i) the oral route is one possible route of human exposure and ii) the daily dosage can be accurately administered using the gavage method
- F1 generation pups were not directly given the test substance but may have been exposed to the test substance during maternal gestation (in utero exposure) or via maternal milk during the lactation period

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

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)

- Salivation as only clinical sign
- Body weight was slightly but significantly reduced in the high-dosage group in males between d1 and d8; at the end all weights were comparable
- Organ weights included enlarged spleens for 9 of 10 females in the high-dosage group

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

reproductive and developmental toxicity

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive or developmental effects at any dose level tested

Remarks on result:

other: Generation: P and F1 (migrated information)

2

Dose descriptor:

NOEL

Remarks:

parental systemic toxicity

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic effects included methemoglobinemia at 30 and 100 mg/kg/day dosages and transient reductions in body weight and feed consumption values, indications of hemolytic anemia, and increases in the weight and/or size of the spleen at 100 mg/kg/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

HEMATOLOGY

 

		0 (VEHICLE)	3 MG/KG/DAY	30 MG/KG/DAY	100 MG/KG/DAY
Methhemoglobin in %	male	1.0	1.4	2.3	4.4
	female	0.3	0.6	1.6	3.0
Red blood cells in millions per microliter	male	7.8	7.7	7.4	6.4
	female	5.9	6.0	5.9	4.9

 

Applicant's summary and conclusion

Conclusions:

Based on the data collected in this study, repeated oral administration of acetoacetanilide to rats did not produce reproductive or developmental toxicity at dosage levels that produced systemic toxicity in the parental rats. Systemic effects included methemoglobinemia at 30 and 100 mg/kg/day dosages and transient reductions in body weight and feed consumption values, indications of hemolytic anemia, and increases in the weight and/or size of the spleen at 100 mg/kg/day. The no-observable-effect level (NOEL) for reproductive and developmental toxicity in this study was at least 100 mg/kg/day. The no effect level for systemic toxicity was 3 mg/kg/day.

Executive summary:

Male and female rats (10/sex/group) were orally administered suspensions of acetoacetanilide at dosage levels of 0, 3, 30 and 100 mg/kg/day. The suspensions of acetoacetanilide were prepared in 1% aqueous methylcellulose and were administered at a dose volume of 10 mL/kg body weight based on the most recent body weight. Male rats were treated daily for a minimum of six weeks comprised of a two-week prebreed period, a breeding period of two weeks maximum, and a two to four week postbreed period. Female rats were treated daily during a two-week prebreed period and throughout gestation and lactation until sacrifice on day 5 postpartum. Animals in the control group received 1% methylcellulose according to the same dosing regimen.

The rats were observed for viability at least twice each day. The rats also received a detailed clinical examination before and approximately one hour after each dose. Body weight and feed consumption were measured weekly throughout the study for male rats and weekly until gestation for female rats and then on gestation days (GD) 0, 7, 14 and 20 and on days 1 (birth) and 5 postpartum. Reproductive and developmental end points evaluated in the study included mating performance, gestation length, fertility and gestation indices, number of implantation sites, number and sex of offspring, litter size, and pup viability indices.

Parental animals were sacrificed after the postbreed period for males and on day 5 postpartum for females. Just prior to sacrifice, blood samples were collected from each rat and evaluated for methemoglobin, counts of erythrocytes, leucocytes and platelets, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. All male and female rats were subjected to a detailed necropsy examination. The spleen and, in male rats, the epididymides and testes were weighed, and these tissues were retained along with the pituitary and the prostate and seminal vesicles in male rats and the ovaries, uterus, vagina in female rats. Histopathological evaluation of the epididymides, testes and ovaries were conducted for rats in the control and high dosage groups. Female rats that did not deliver a litter or that did not have a confirmed mating date were sacrificed on GD 25 of presumed gestation, necropsied, and examined for pregnancy.

The pups were examined for physical abnormalities once each day during the 5-day postpartum period and pup body weight and observations of nursing behavior were recorded on days 1 and 5 postpartum. Pups found dead were necropsied and, evaluated to determine if they were alive at birth. All remaining pups were sacrificed on postpartum day 5 and necropsied.

No deaths occurred during the study. Treatment-related clinical signs of toxicity in the parental animals were limited to excess salivation for five of ten male rats in the 100 mg/kg/day dosage group. Body weight gain and feed consumption for male rats in the 100 mg/kg/day dosage group were reduced during days 1 through 8 of the prebreed period. In female rats, prebreed body weight and body weight gain were unaffected by treatment with the test substance. However, body weight gain during gestation was decreased for female rats in the 100 mg/kg/day dosage group as compared with the control group. There were no other differences in body weight, body weight gain, or feed consumption for parental males or females that were attributed to treatment with the test substance.

Treatment-related hematology changes were observed for male and female rats in the 30 and 100 mg/kg/day dosage groups. These included methemoglobinemia for animals in the 30 and 100 mg/kg/day dosage groups, and increases in the total leucocyte cell counts and apparent hemolytic anemia for animals in the 100 mg/kg/day dosage group. Evidence for hemolytic anemia in the high dosage group male and female rats included reductions in the erythrocyte cell count, hemoglobin and hematocrit and increases in the mean corpuscular volume and mean corpuscular hemoglobin content as well as increased spleen size and/or weight. There were no other changes in the hematology endpoints evaluated in this study that could be clearly attributed to treatment with the test substance.

There were no treatment-related necropsy findings for parental animals or F1 generation pups, and there were no treatment-related microscopic alterations observed in the testes and epididymides of the parental male rats or in the ovaries of the parental female rats in the high-dosage group. Spleen weights were increased for male and female parental animals in the 100 mg/kg/day dosage group. There were no other treatment-related organ weight changes observed.

Mating and fertility for male and female parental rats were unaffected by treatment with the test substance. In addition, there were no treatment-related effects on the duration of gestation and the number of implantation sites per dam, the number of stillborn pups or the gestation and viability indices. Average litter size, liveborn litter size, surviving pups per litter, percent male pups per litter, average pup weight, and the incidences of clinical and necropsy observations in the pups were also comparable among the four dosage groups.