2-propanol and 2-butanol production, distn. residues

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

number of animals was 15/group instead of 20/group

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propan-2-ol

EC Number:

200-661-7

EC Name:

Propan-2-ol

Cas Number:

67-63-0

Molecular formula:

C3H8O

IUPAC Name:

propan-2-ol

Constituent 2

Reference substance name:

2-propanol

IUPAC Name:

2-propanol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): isopropanol (Shell Oil & Co; Deer Park, TX)
- Physical state: colourless liquid
- Analytical purity: 99.95±0.01% (GC: HP 5890A)
- Stability under test conditions: homogenous and stable for at least 49 days
- Storage condition of test material: refrigerated

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc., Denver, PA
- Age at study initiation: 5.5 months
- Weight at study initiation: 2750 to 3800 g
- Fasting period before study: not reported
- Housing: singly in stainless steel cages with mesh flooring (Hoeltge, Inc., Cincinnati, OH)
- Diet (e.g. ad libitum): #5322 Purina Certified Rabbit Chow ad libitum
- Water (e.g. ad libitum): deionized/filtered tap water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.3 to 20.6 °C
- Humidity (%): 42.3 to 60.0%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light


IN-LIFE DATES: From: March 13, 1990 To: April 27, 1990

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionized/distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prior to formulation of study dosing solutions, aliquots of formulations of isopropanol in vehicle, bracketing the range of concentrations to be used in this study, were assayed for homogeneity, stability and dose level verification. Dosing formulations were homogeneous and stable for at least 49 days under refrigerated conditions. Aliquots sufficient for one day of dosing per dose group were stored in containers with septum seal caps, used for one day of dosing and then appropriately disposed of. To prepare dosing formulations, isopropanol was dissolved in deionized/distilled water according to the following formula: Concentration (mg/mL) = Dose level (mg/kg)/Dose volume (2 mL/kg).

The concentration of isopropanol in the dosing solutions varied with dose. Dilutions of isopropanol in deionized/distilled water were formulated independently for each concentration and were prepared in a quantity sufficient for the period of dosing. An aliquot of each formulation of isopropanol and vehicle was analyzed to verify the concentration of the test compound. Dosing formulations with assayed values of 90 - 110% of the nominal concentration were considered to be suitable for use in these studies. Personnel, other than the laboratory supervisor and those involved in analysis of dosage formulations for isopropanol concentration, were not informed of the formulation concentrations until all laboratory work had been completed. For analysis of the dosing formulations, triplicate 0.100 mL samples of the vehicle control solution (deionized/distilled water, CAS No. 7732-18-5) and of the dosing formulations, 80.0, 160.0 and 240.0 mg/mL, were pipetted into separate GC auto sampler vials each containing 0.80 mL of methanol. An 0.10 mL aliquot of the internal standard solution, 0.250 - 0.251 g of t-butanol/mL of water, was added to each vial. The samples were mixed and analyzed by gas chromatography (Hewlett Packard 5890A Gas Chromatograph, Waters 840 data system). Standards encompassing the range of dosing formulation concentrations were prepared and analyzed in the same way. The capillary column was J&W DB-I (30 m x O.32 mm ID), 5 micron film with the injection solvent methanol:water (4:1).


DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: 12.8 - 15.6 °C


VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 0, 60, 120, and 240 mg/mL mixture
- Amount of vehicle (if gavage): 2 mL/kg bw
- Lot/batch no. (if required): not provided
- Purity: not provided

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An aliquot of each formulation of isopropanol and vehicle was analyzed to verify the concentration of the test compound. Dosing formulations with assayed values of 90 - 110% of the nominal concentration were considered to be suitable for use in these studies. For analysis of the dosing formulations, triplicate 0.100 mL samples of the vehicle control solution (deionized/distilled water, CAS No. 7732-18-5) and of the dosing formulations, 80.0, 160.0 and 240.0 mg/mL, were pipetted into separate GC auto sampler vials each containing 0.80 mL of methanol. An 0.10 mL aliquot of the internal standard solution, 0.250 - 0.251 g of t-butanol/mL of water, was added to each vial. The samples were mixed and analyzed by gas chromatography (Hewlett Packard 5890A Gas Chromatograph, Waters 840 data system). Standards encompassing the range of dosing formulation concentrations were prepared and analyzed in the same way. The capillary column was J&W DB-I (30 m x O.32 mm ID), 5-micron film with the injection solvent methanol:water (4:1).

Details on mating procedure:

- Impregnation procedure: Artificial insemination
- Verification of same strain and source of both sexes: No. Test animals consisted of female New Zealand (NZW) rabbits supplied by Hazleton Research Products, Inc., Denver, PA. Male rabbits of the same strain and originally from the same supplier were obtained from the laboratory's (RTI) breeding colony and used to provide semen samples.
- Proof of pregnancy: On gestation day 30, approximately one to one and a half days before expected parturition, all surviving maternal animals were killed and the pregnancy status of the rabbits was confirmed by uterine examination.

Duration of treatment / exposure:

From day 6 to 18 of gestation.

Frequency of treatment:

Daily

Duration of test:

from the start of the test (i.e., animals in cages) to necropsy

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

A total of 15 females per dose were treated; 2 females in the 120 mg/kg bw/day dose group were not pregnant at sacrifice.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a range-finding study in pregnant rabbits performed at RTI (RTI STUDY CODE: RbgO-ISPT). The highest dose level was chosen to induce overt maternal toxicity, but not to cause a weight loss greater than 20% when compared to concurrent controls, nor to cause greater than 10% maternal mortality. The low dose was selected to be a maternal/developmental no-observable-adverse-effect level (NOAEL). The mid-dose was halfway between the high and low doses.
- Rationale for animal assignment (if not random): Animals were assigned to treatment groups by a stratified randomization method designed to provide uniform mean body weights across dose groups at the initiation of the study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily on gestation days 0 - 5 (prior to dosing) and 19 - 30 (after dosing period); twice daily at dosing and 1 - 2 hours after dosing, throughout the dosing period (gestation days 6 through 18).
- Cage side observations checked in table No. 3 were included.


DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 15, 18, 24, and 30.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: thoracic and abdominal cavities, and organs; liver and uterus weights obtained.

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
- Other: dead and live fetuses

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:

Parametric statistical procedures were applied to selected measures. Appropriate General Linear Models (GLM) procedures (SAS Institute Inc., 1985a, 1985b) for the proposed Analyses of Variance (ANOVA) were employed. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967) and Bartlett’s test for homogeneity of variance (alpha level = 0.001) was performed on all data to be analyzed by ANOVA (Winer, 1962). GLM analysis was used to determine the significance of the dose-response relationships (Test for Linear Trend), and to determine whether significant dose effects had occurred for selected measures (ANOVA). When a significant (p < 0.05) main effect for dose occurred, Williams’ Multiple Comparison Test (Williams, 1971; 1972) and/or Dunnett’s Multiple Comparison Test (Dunnett, 1955; 1964) was used to compare each exposed group to the vehicle control group for that measure. A one-tailed test (i.e., Williams’ Test and/or Dunnett’s Test) was used for all pairwise comparisons except that a two-tailed test was used for maternal body and organ weight parameters, maternal food consumption, fetal body weight, and percent males per litter. Nominal scale measures were analyzed by Chi-Square Test for Independence for differences among treatment groups, and by a test for linear trend on proportions (Snedecor and Cochran, 1967). When Chi-Square revealed significant (p < 0.05) differences among groups, then a one-tailed Fisher’s Exact Probability Test was used for pair wise comparisons between each treated group and the vehicle control group.

Indices:

Dams: pregnancy; corpora lutea; implantation sites per litter; percent preimplantation loss; live fetuses per litter; total and percent: resorptions per litter, litters with resorptions, late fetal deaths per litter, litters with late fetal deaths, nonlive implants per litter, litters with nonlive implants, adversely affected implants per litter; male and female fetuses per litter; average fetal body weight per litter; average male fetal body weight per litter; and average female fetal body weight per litter.

Historical control data:

Large historical databases for reproductive performance and prevalence of spontaneous malformations in 200 control rabbit litters are available from studies conducted at RTI, as well as from historical data on 1,142 New Zealand White rabbit control litters summarized across teratology studies from 11 pharmaceutical companies or biological research organizations by the Middle Atlantic Reproduction and Teratology Association (Woo and Hoar, 1982).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects: Yes

Details on maternal toxic effects:
Profound maternal toxicity at 480 mg/kg/day, expressed as 26.7% mortality, reduced body weight gain during the treatment period and for the gestational period corrected for the contribution of the gravid uterus, reduced food consumption during the treatment period and relatively severe clinical signs of toxicity. At 240 and 120 mg/kg/day, the only findings were transient, relatively mild and nonspecific clinical signs of toxicity.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: No effects

Details on embryotoxic / teratogenic effects:
There was no demonstrable developmental toxicity at a dose resulting in significant maternal toxicity (480 mg/kg/day) or at doses with only relatively mild and transient clinical signs of toxicity (240 and 120 mg/kg/day).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

The apparent non-significant reduction in fetal body weight per litter observed at 480 mg/kg/day may be viewed in the context of the RTI historical control data base of 200 NZW rabbit litters. The mean historical control fetal body weight per litter is 45.60 ±0.56 g with a range of 26.26 to 67.19 g and an average litter size of 7.20 ±0.19. The lower and upper 95% confidence intervals for the historical control weights are 44.48 and 46.71 g, respectively. For historical control males, the mean fetal weight per litter is 45.90 ±0.60 g; for historical control females, it is 44.36 ±0.58 g. The values in the current study at 480 mg/kg/day are 46.48 ±3.31 g for all fetuses, 46.04 ±2.94 g for males and 42.79 ±3.05 g for females, with an average litter size of 8.2 ±1.0. All of the current study values are within ±95% confidence interval of the RTI historical control values except for the weight of females, which also was the only parameter with a statistically significant dose-related downward trend.

The no-observable-adverse-effect level (NOAEL) for maternal toxicity in rabbits in the present study is 240 mg isopropanol/kg bw/day. The NOAEL for developmental toxicity in rabbits is 480 mg isopropanol/kg bw/day.

Applicant's summary and conclusion

Conclusions:

Upon oral exposure of rabbit to isopropanol there was no statistically significant indication of developmental toxicity and no evidence of teratogenicity at any dose tested. The no-observable-adverse-effect level (NOAEL) for maternal toxicity in rabbits was therefore 240 mg isopropanol/kg bw/day and the NOAEL for developmental toxicity in rabbits was 480 mg isopropanol/kg bw/day, based on general toxicity effects.