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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline equivalent study; adequately reported for assessment; GLP status unknown

Data source

Reference Type:
Lack of selective developmental toxicity of three butanol isomers administered by inhalation to rats
Nelson BK, Brightwell WS, Khan A, Burg JR, and Goad PT
Bibliographic source:
Fundamental and Applied Toxicology 12:469-479

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Body weights recorded on gestation days 0-7, 14, and 19; food consumption weekly
GLP compliance:
not specified
Limit test:

Test material

Details on test material:
Reagent grade t-butanol
Source: Curtin Matheson Scientific, Cincinnati, OH
Purity: > 99%

Test animals

Details on test animals and environmental conditions:
- Sprague-Dawley rats specified to be free of Mycoplasma, Sendai virus, and internal and external parasites
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Weight upon receipt: females 176-200g, males over 300g
- Quarantine period: 1-2 weeks
- Housing: Individually housed in 32 X 41 X 18-cm stainless-steel wire-mesh cages equipped with automatic water dispensers except during inhalation exposure and upon a sperm positive smear. Females with sperm (Day 0 of gestation) were placed individually into 30 X 34 X 17-cm polycarbonate cages with autoclavable polyester filter covers. Bedding consisted of cleaned, heat-treated sawdust from a local supplier (Absorb-Dri, from Tasty Foods, Cincinnati, OH)
- Inhalation exposure cage: From gestation days (GD) 1-19, females were placed into 13 X 25 X 18-cm compartments in stainless-steel wire-mesh caging within the exposure chambers for 7 hours a day; then returned to their home cages.
- Diet: Purina or NIH-07 lab chow (Ziegler Bros., Garden, MA) was available ad libitum except during inhalation exposure
- Water: Tap water was available ad libitum except during inhalation exposure

- Temperature (°C): 24 ± 2°C
- Humidity (%): 50 ± 10%.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
other: Air
Details on exposure:
The inhalation exposures were conducted in 0.5-m3 Hinners-type exposure chambers with the vapor generation equipment housed above the exposure chambers in glove boxes which were maintained under negative pressure to prevent any leakage of contaminants. Reagent-grade tertiary butyl alcohol was placed into a flask and a low-flow pump circulated liquid from the reservoir flask into a 10-mL syringe contained within the flask such that the syringe was constantly overflowing. The syringe provided a constant head of chemical for a second pump which injected the specified amount of liquid into a three-way valve which was attached to a Greensmith impinger. Heated compressed air was introduced through the second inlet of the three-way valve and alcohol evaporation was controlled by regulating the preheating of compressed air. In generation of high concentrations, glass beads were also placed at the bottom of the impinger to further increase the heat transfer area between the alcohol and the compressed air. This vapor and air mixture was introduced into the chamber airflow upstream of the orifice plate; the turbulence and pressure drop created by the orifice plate provided uniform mixing downstream of the vapor and air before the mixture entered the chamber. Airflow through the chambers provided approximately one air change per minute.

From Gestation Days 1 to 19, females were transported from the animal quarters to the exposure chambers in their homecage shoe boxes with filter tops in place. Females were placed into the inhalation chamber and controls were placed in similar caging within an adjacent exposure chamber for the same hours as the exposed animals. Exposures were conducted 7 hr/day, 7 days a week. After inhalation exposure was concluded, the animals were removed and returned to their homecages.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration within the chamber was monitored continuously with a Miran 1A general-purpose infrared analyzer connected to a strip chart recorder for continuous recording of the concentration throughout the day. On an hourly basis, the chamber concentration, temperature, and humidity were recorded and at the end of each day, the mean, range, and time-weighted average concentrations were calculated. At the conclusion of the study, these daily values were averaged for an overall study mean for each concentration. The analyzer was also interfaced with a computer which recorded 5-min means of chemical concentration. These means were averaged to give hourly and daily means, which were used to calculate a study mean for each concentration.

Samples of the bulk chemical were analyzed by gas chromatography for purity. Charcoal tube samples were also collected from the chamber atmosphere for independent verification of chamber concentrations. Sampling times varied from 10-30 min in duration, and samples were collected at the rate of 5-10 per week. The samples were independently analyzed by NIOSH analytical methods. NIOSH Division of Physical Sciences and Engineering, Arthur A. Little, and Southern Research Institute provided the purity analyses of the bulk chemical and analyzed the charcoal tube samples.
Details on mating procedure:
For mating, virgin females weighing 200-300 g were placed individually with breeder males. Each morning, the litter paper under each male's cage was examined for sperm plugs; if no plugs were detected, vaginal smears were taken. Females with sperm (Day 0 of gestation) were transferred into individual polycarbonate cages.
Duration of treatment / exposure:
7 hours/day
Frequency of treatment:
Duration of test:
Gestation days 1-19, inclusive.
No. of animals per sex per dose:
15-20 dams/group
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of concentrations for initial pilot exposures to evaluate toxicity of a series of butanol isomers, including tertiary butyl alcohol, was based on a previous publication on the toxicity of propanols (Nelson et al., 1988). Five to six nonpregnant females were exposed for 7 hr to 9000 or 10,000 ppm of each butanol isomer. Depending on the subjective toxicity observed in these animals, a similar number of other animals were exposed to lower concentrations in the pilot phase. For the teratology study, the high concentration was selected to be maternally toxic, but not lethal, and two lower concentrations were included. Fifteen to twenty sperm-positive females were randomly assigned to each exposure group.

Reference cited: Nelson et al., 1988. Teratogenicity of n-propanol and isopropanol administered at high inhalation concentrations to rats. Food Chem. Toxicol. 26: 247-254.


Maternal examinations:
Body weights were recorded on gestation days 0-7, 14, and 20.

Food consumption was measured on gestation days 0, 7, 14, and 20

Water consumption was recorded on gestation days 0, 7, 14, and 20
Ovaries and uterine content:
On gestation day 20, pregnant females were individually weighted and euthanised by CO2 asphyxiation. The entire uterus (with ovaries attached) was removed, and corpora lutea, resorptions (classified as early, middle, or late), and live fetuses were counted.
Fetal examinations:
Fetuses were serially removed, examined for external malformations, blotted of excess fluids, weighed, and external sex was determined.

One-half of the fetuses were randomly selected and placed into 80% ethanol, eviscerated, macerated in 1.5% KOH, stained in alizarin red S, and examined for skeletal malformations and variations. The other half were placed in Bouin's solution and examined for visceral malformations and variations using a razor blade cross-sectioning technique (Wilson 1965).

Reference cited: Wilson JG, 1965. Methods for administering agents and detecting malformations in experimental animals. In: Teratology: Principles and Techniques (J.G. Wilson and J. Warkany, Eds), pp. 262-277. Univ. of Chicago Press, Chicago.
For maternal data, multivariate analysis (with baseline as covariate) was used for weight comparisons across groups. Group differences in food and water intake were analyzed by multivariate analysis of variance. Gestation body weights utilized analysis of variance with initial weight as a covariate (ANCOVA) for comparing treatment groups with their respective controls. These were followed by multivariate analyses (MANCOVA) to compare the entire gestational weight gain. A Kruskal-Wallis test was used for group comparisons of corpora lutea per animal. For the fetal data, analysis of variance was used to compare fetal weights across both groups and sex. The Kruskal-Wallis test was used for group comparisons of variables including: litter size, percentage alive/litter, percentage normal/litter, and percentage females/litter. The Fisher's exact test was used to compare numbers of litters with one or more of the following variables of interest: skeletal malformations, skeletal variations, visceral malformations, visceral variations, external malformations, and nonnormal fetuses. The results of the tests were adjusted for multiple comparisons, when appropriate, using the Bonferroni technique. A probability of p ≤ 0.05 was required for significance.
No data provided in publication
Historical control data:
No data provided in publication

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All exposure concentrations of TBA produced an unsteady gait at the end of the 7-hour exposure. All animals responded to a tap on the cage, but locomotor activity was impaired. No mortality was produced in the study.

Exposure to 5000 ppm TBA produced statistically significant lower body weights when compared with controls.

The 5000 ppm group consumed significantly less food than the controls but the decrease was statistically significant only in the first 2 weeks.

Water intake increased as pregnancy progressed and was generally higher, though not statistically significant, in treatment groups than in controls.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
2 000 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
2 000 ppm (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetotoxicity was generally increased with increasing concentrations of tertiary butyl alcohol. There was a statistically significant decrease in fetal weights for both sexes at 2000 ppm (9%-M, 10%-F); 3500 ppm (12%-M, 13%-F); and 5000 ppm (32%-M, 31%-F).

No external malformations were observed. There was no statistically significant increase in the number of skeletal or visceral malformations or visceral variations. There was a statistically significant exposure-related increase in the number of skeletal variations at the mid- and high-exposure levels. Variations seen were typical of fetotoxicity, particularly reduced ossification.

Effect levels (fetuses)

Dose descriptor:
Effect level:
5 000 ppm (nominal)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Throughout the exposures, concentrations of tertiary butyl alcohol were easily generated and were uniform. The mean concentrations ± SD from the daily infrared readings of tertiary butyl alcohol were 5030 ± 100 ppm (23 exposure days), 3510 ± 30 ppm (22 exposure days), and 2200 ± 20 ppm (42 exposure days); charcoal tube results were typically only slightly lower than the infrared means (means were respectively 4500, 3500, and 2010 ppm).  No contaminants were detected in the charcoal tubes collected from the control chambers. When rounded to the appropriate number of significant digits, the means from the infrared analyzer were equivalent to the target concentrations.  Target concentration values were used for discussion throughout this document.


Fetal Observations after Maternal Exposure to Tertiary Butyl Alcohol
Tertiary Butyl Alcohol (ppm)
0 2000 3500 5000
Number of pregnant/number bred 15/16 18/20 15/15 13/15
Corpora lutea/litter 16 ± 2 16 ± 2 16 ± 2 16 ± 2
Resorptions/litter 1.1 ± 1.2 1.2 ± 1.1 0.9 ± 1.0 1.1 ± 0.9
Live fetuses/litter 13 ± 2 13 ± 4 15 ± 2 14 ± 2
Fetal weight (g)
    Female 3.2 ± 0.23 2.9 ± 0.20* 2.8 ± 0.20* 2.2 ± 0.34*
    Male 3.4 ± 0.21 3.1 ± 0.19* 3.0 ± 0.24* 2.3 ± 0.34*
    %Females/litter 56 ± 16 53 ± 13 50 ± 12 46 ± 16
Skeletal Observations, litters (fetuses)
    Number examined 15 (96) 17 (104) 14 (103) 12 (83)
    Number malformations 0 0 2 (2) 2 (4)
    Number variations 10 (18) 14 (35) 14 (53)* 12 (76)*
    % Fetuses normal 100 100 98 ± 1 95 ± 4
Visceral Observations, litters (fetuses)
    Number examined 15 (100) 17 (116) 14 (102) 12 (83)
    Number malformations 1 (1) 1 (1) 2 (4) 1 (1)
    Number variations 6 (6) 4 (4) 6 (6) 12 (27)
    % Fetuses normal 99 ± 1 99 ± 1 96 ± 3 99 ± 1
* p 0.05 statistically significant when compared with control 

Applicant's summary and conclusion

In a teratology study in which groups of pregnant Sprague-Dawley rats were exposed to 2000, 3500 and 5000 ppm tertiary butyl alcohol by whole body inhalation for 7 hr/day on gestation days 1-19, central nervous system effects were observed in the dams at all exposure concentrations. There was no evidence of teratogenicity, even at maternally toxic doses. There was an exposure-related increase in the incidence of fetotoxicity, i.e., reduced weights in both sexes and increases in skeletal variations, although these were also associated with maternal toxicity. The no-observed-adverse-exposure-concentration (LOAEC) for maternal toxicity and developmental toxicity was 2000 ppm under conditions of the study.

Executive summary:

In a well-conducted teratology study in which groups of pregnant Sprague-Dawley rats were exposed to 2000, 3500 or 5000 ppm tertiary butyl alcohol for 7 hr/day on GD 1-19, there was no evidence of teratogenicity, even at maternally toxic doses. All exposure concentrations were maternally toxic and produced CNS effects such as impaired locomotor activity in the dams. Fetotoxicity was observed at maternally toxic exposure concentrations but was limited to decreases in fetal weights for both sexes at all concentrations and a statistically significant exposure-related increase in the number of skeletal variations, e.g., reduced ossification at the mid- and high-exposure levels. Based on this information, tertiary butyl alcohol is not selectively toxic to the developing fetus and is not classified for “Developmental or Reproductive Toxicity” according to the GHS guidelines.