Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information
The developmental toxicity (oral) was tested in a read-across study with tertiary butanol. The NOAEL based on teratogenicity was determined to be >= 1556 mg/kg bw/day. 
The developmental toxicity (inhalation) was tested in a read-across study with t-butanol. The NOAEL based on teratogenicity was determined to be 5000 ppm.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well described publication and suitable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Pregnant mice were given tertiary butanol by gavage twice daily from day 6 through day 18 of gestation. Examinations were made on day 18.
GLP compliance:
no
Species:
mouse
Strain:
other: CBA/J and C57BL/6J
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME, USA
- Age at study initiation: 25 - 30 weeks
- Diet: standard lab chow; ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug day 0 of pregnancy
Duration of treatment / exposure:
13 days (from day 6 through day 18 of gestation.)
Frequency of treatment:
twice a day (every 12 hour)
Duration of test:
The female mice were sacrificed on day 18 by decapitation.
Remarks:
Doses / Concentrations:
20 mL/kg bw /day of a 10% (v/v) solution
Basis:
other: nominal conc. 20 ml/kg bw of a 10% (v/v) solution every 12 hours
Remarks:
Doses / Concentrations:
21 mmoles / kg bw /day = 1556 mg/kg bw/day
Basis:
other: nominal conc. 10.5 mmoles/ kg bw (=778 mg/ kg bw every 12 hours)
No. of animals per sex per dose:
CBA/J control: 7 females
CBA/J t-butanol: 12 females
C57BL/6J control: 5 females
C57BL/6J t-butanol: 9 females
Control animals:
yes, concurrent vehicle
Ovaries and uterine content:

- Number of implantations: Yes
- Number of resorptions: Yes
- Number of live fetuses: Yes
Fetal examinations:
- External examinations: no data
- Soft tissue examinations: Yes (half per litter)
- Skeletal examinations: Yes (half per litter)
- Head examinations: no data
- Mean fetal weight: Yes (all per litter)
Statistics:
A two way analysis of variance (strain x treatment) was used in comparing the mean numbers of viable fetuses, resorptions and abnormalities per litter between treatment groups. Treatment effects on fetal weight within each strain were analyzed by Student's t test. Chi2-Yates was used to compare the number of litters with resorptions or 100% resorptions between treatment groups.
Details on maternal toxic effects:
Maternal toxic effects:no data
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The effects of t-butanol treatment on fetal resorptions and the weight of the surviving fetuses: Tertiary butanol produced a significant increase in the number of resorptions per litter, F(1,29)=7.361, p.=0.011, and a significant decrease in the number of live fetuses per litter, F(1,29)=10.060, p.=0.004. There were no significant effects of strain on either the number of resorptions per litter, F(1,29)=0.07, p.=0.787, or the
number of live fetuses per litter, F(1,29)=0.322, p.=0.575. Eight of the 21 litters in the t-butanol treated groups had all of the fetuses resorbed vs.
none in the control groups (X2=4.10, p.<0.05). Significantly more of the resorptions in the treated groups (33/75) required ammonium sulfide for
visualization than in the control groups (2/14) (X2=4.66, p.<0.05), suggesting an early effect on fetal viability. No inter-strain differences in fetal
mortality were observed. There was a slight but statistically insignificant decrease in the weight of the surviving fetuses in both strains.
The results of morphological examination of the surviving offspring: Soft tissue examination revealed no teratogenic effects of t-butanol in either strain. There was no evidence that t-butanol produced the dilated ventricles, open eyelids, exencephaly, heart defects, or gastroschisis reported for ethanol in the CBA/J strain (27), even though fetuses were carefully examined for these defects. Skeletal examinations showed defects limited to the skull and sternum. Minor variations (misaligned or underossified sternebrae and underossified supraoccipital bones) occurred more frequently, but were not significantly different between treatments or strains.
Dose descriptor:
NOAEL
Effect level:
>= 1 556 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Dose descriptor:
LOAEL
Effect level:
1 556 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The developmental toxicity was tested in a read-across study with tertiary butanol. The NOAEL based on teratogenicity was determined to be >= 1556 mg/kg bw/day.
Executive summary:

The developmental toxicity was tested in a read-across study with tertiary butanol. Pregnant mice were given tertiary butanol by gavage twice daily from day 6 through day 18 of gestation. Examinations were made on day 18. The females were sacrificed on day 18 by decapitation. The following concentrations were used: 20 mL/kg bw/day of a 10 % (v/v) solution equivalent to 1556 mg/kg bw/day (Basis=nominal conc. 10.5 mmoles/kg bw (=778 mg/kg bw every 12 hours). Two different strains were used to determine the toxicity: CBA/J (7 females control and 12 females test item) and C57BL/6J (5 females control and 9 females test item). The ovaries and uterine content was examined by determining the number of implantations, the number of resoprtions and number of live fetuses. Also fetal examinations were performed by determining soft tissue examinations, skeletal examinations and the mean fetal weight. As a result the NOAEL was determined to be >= 1556 mg/kg bw/day based on teratogenicity. The LOAEL was determined to be 1556 mg/kg bw/day based on embryotoxicity. No maternal toxicity was observed during the study. Tertiary butanol produced a significant increase in the number of resorptions per litter, F(1,29)=7.361, p.=0.011, and a significant decrease in the number of live fetuses per litter, F(1,29)=10.060, p.=0.004. There were no significant effects of strain on either the number of resorptions per litter, F(1,29)=0.07, p.=0.787, or the number of live fetuses per litter, F(1,29)=0.322, p.=0.575. Eight of the 21 litters in the t-butanol treated groups had all of the fetuses resorbed vs.none in the control groups (X2=4.10, p.<0.05). Significantly more of the resorptions in the treated groups (33/75) required ammonium sulfide for visualization than in the control groups (2/14) (X2=4.66, p.<0.05), suggesting an early effect on fetal viability. No inter-strain differences in fetal mortality were observed. There was a slight but statistically insignificant decrease in the weight of the surviving fetuses in both strains. The results of morphological examination of the surviving offspring: Soft tissue examination revealed no teratogenic effects of t-butanol in either strain. There was no evidence that t-butanol produced the dilated ventricles, open eyelids, exencephaly, heart defects, or gastroschisis reported for ethanol in the CBA/J strain (27), even though fetuses were carefully examined for these defects. Skeletal examinations showed defects limited to the skull and sternum. Minor variations (misaligned or underossified sternebrae and underossified supraoccipital bones) occurred more frequently, but were not significantly different between treatments or strains.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well described publication and acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The publication presents the results of teratology assessment of industrial alcohols including t-butanol administered by inhalation to rats. For that, approx. 15 rats were exposed to 0, 2000, 3500 or 5000 ppm of t-butanol for 7h/day on gestation days 1-19. Dams were sacrificed on gestation day 20, and fetuses were individually weighted and examined.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Weight at study initiation: female: 200-300 g; male: > 300 g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26 °C
- Humidity (%): 40-60 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The inhalation exposures were conducted in 0.5 m3 Hinners-type exposure chambers. The vapor generation equipment was housed above the exposure chambers in glove boxes which were maintained under negative pressure. Reagent-grade t-butanol was placed into a fIask. A low-flow pump (RP Model lab pump; Fluid Metering Inc., Oyster Bay, NY, USA) circulated liquid from the reservoir f0lask into a 10-ml syringe contained within the flask such that the syringe was constantly overflowing. Thus, the syringe provided a constant head of chemical for a second pump (controlled by a micrometer adjustment) 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 thc second inlet ofthe three-way valve. Alcohol evaporation was controlled by regulating the preheating ofcompressed air. The impinger provided increased contact time between the air and the liquid to ensure total evaporation. In generation of high concentrations, glass beads were also placed at the bottom ofthe 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 down-stream ofthe vapor and air before the mixture entered the chamber. Airflow through the chambers provided approximately one air change per minute.

EXPOSURE
Females with sperm (Day 0 of gestation) were placed individually into 30 X 34 x 17 cm polycarbonate cages having autoclavable polyester filter covers. Bedding consisted of cleaned, heat-treated sawdust. Weekly food and water intake, along with maternal weights, were measured on Gestation Days 0, 7, l4, and 20. Females were also weighed each morning for the first week of exposure. 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 13 X 25 X I8-cm
compartments in stainless-steel wire-mesh caging within thc exposure chambers. 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, and the animals were left in tlle chambers for degassing tor approximately 1/2 hr after vapor generation was terminated. They were then removed and returned in their homecages to the animal quarters where the water bottles were replaced.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration in the chamber was monitored continuously with a Miran 1A general-purpose infrared analyzer (Wilks/Foxboro Analytical, South Norwalk, CT). The Miran 1A was connected to a strip chart recorder to record continously the concentration throughout the day. Hourly, the chamber concentration, temperature, and humidity were recorded on a daily observation sheet. At the end of each day, the mean, range, and time-weighted average concentrations were calculated.
Purity of the bulk chemical was analyzed by gas chromatography.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
Duration of treatment / exposure:
gestation days 1-19
Frequency of treatment:
daily, 7 hours per day
Remarks:
Doses / Concentrations:
0, 2000, 3500, 5000 ppm estimated as 200, 350, and 500 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Pilot study was performed with up to 10.000 ppm
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: mortality, posture and behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: on the first week, daily afterwards weekly, GD 7, 14, 20

POST-MORTEM EXAMINATIONS: No data
- Sacrifice on gestation day 20
- Organs examined: No data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea
- Number of resorptions
- Number of live fetuses
Fetal examinations:
Examinations:
- external malformations
- fetus weight
- external determination of sex
- skeletal malformations: One-half of the fetuses were randomly selected, placed into 80% ethanol, and subsequently eviscerated, macerated
in 1.5% KOH, stained in alizarin red S, and examined for skeletal malformations and variations.
- visceral malformations: The other half were placed in Bouin's solution and subsequently examined far visceral malformations and variations using a razor blade cross-sectioning technique (Wilson, 1965).
Statistics:
Multivariate analysis was performed for maternal weight comparison and differences in food and water intake. The Kruskal-Wallis test was used for comparison of corpora lutea per animal. Analysis of variance was used to compare fetal weight. Group comparisons of the variables such as Iitter size, percentage alive/litter, percentage normal/litter, and percentage females/litter were made using the Kruskal-Wallis test. For the variables including skeletal malformations, skeletal variations, visceral malformation, visceral variations, external malformations, and nonnormal fetuses, the number of litters with one or more of the variables of interest was compared using Fisher's exact test. The results of the tests were adjusted for multiple comparisons, when appropriate, using Bonferroni. p<= 0.05 was required for statistical significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Exposure to 5000 ppm induced narcosis of all animals, an unsteady gait, reduced body weight gain (significant at 5000 ppm), reduced food consumption, and impaired locomotor activity.
Exposure to 3500 and 2000 ppm also resulted in an unsteady state and impaired locomotor acitity.
Dose descriptor:
LOAEC
Effect level:
2 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Body weight and body weight gain were reduced in all treatment groups.
Dose descriptor:
LOAEC
Effect level:
2 000 ppm
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

No external malformations could be observed. The majority of skeletal malformations were rudimentary cervical ribs. In general, skeletal variants increased with increasing concentrations of t-butanol. The lowest concentration was not associated with any defects. Variations seen were typical of fetotoxicity, especially reduced ossification.

 

t-Butanol (ppm)

 

0

2000

3500

5000

Number pregnant/number bred

15/16

18/20

15/15

13/15

 

 

 

 

 

Corpora lutea/litter a)

16 +/- 2

16 +/- 2

16 +/- 2

16 +/- 2

 

 

 

 

 

Resorptions/litter a)

1.1 +/- 1.2

1.2 +/- 1.1

0.9 +/- 1.0

1.1 +/- 0.9

 

 

 

 

 

Live fetuses/litter a)

13 +/- 2

13 +/- 4

15 +/- 2

14 +/- 2

 

 

 

 

 

Fetal weight (g) a)

 

 

 

 

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)

 

 

 

 

No. Examined

15 (96)

17 (104)

14 (103)

12 (83)

No. Malformations

0

0

0 (2)

2 (4)

No. Variations

10 (18)

14 (35)

14 (53 *)

12 (76 *)

% Fetuses normal

100 +/- 0

100 +/- 0

98 +/- 1

95 +/- 4

 

 

 

 

 

Visceral observations, litter (fetuses)

 

 

 

 

No. Examined

15 (100)

17 (116)

14 (102)

12 (83)

No. Malformations

1 (1)

1 (1)

2 (4)

1 (1)

No. Variations

6 (6)

4 (4)

6 (6)

12 (27)

% Fetuses normal

99 +/- 1

99 +/- 1

96 +/- 3

99 +/- 1

Table 1: Fetal observations after maternal exposure to t-butanol.(*: p< 0.05 when compared with appropriate control; a = mean +/- SD)

Conclusions:
The developmental toxicity of the read-across substance t-butanol was determined. The NOAEC (rats) based on teratogenicity was determined to be 5000 ppm.
Executive summary:

The developmental toxicity was tested in a read-across study with t-butanol. The publication presents the results of teratology assessment of industrial alcohols including t-butanol administered by inhalation to rats. For that, approx. 15 rats were exposed to 0, 2000, 3500 or 5000 ppm of t-butanol (estimated as 200, 350 and 500 mg/kg per day) for 7h/day on gestation days 1-19. Dams were sacrificed on gestation day 20, and fetuses were individually weighted and examined. The inhalation exposures were conducted in 0.5 m3 Hinners-type exposure chambers. The vapor generation equipment was housed above the exposure chambers in glove boxes which were maintained under negative pressure. Reagent-grade t-butanol was placed into a fIask. A low-flow pump (RP Model lab pump; Fluid Metering Inc., Oyster Bay, NY, USA) circulated liquid from the reservoir flask into a 10 -mL syringe contained within the flask such that the syringe was constantly overflowing. Thus, the syringe provided a constant head of chemical for a second pump (controlled by a micrometer adjustment) 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 ofthe three-way valve. Alcohol evaporation was controlled by regulating the preheating ofcompressed air. The impinger provided increased contact time between the air and the liquid to ensure total evaporation. In generation of high concentrations, glass beads were also placed at the bottom ofthe 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 down-stream ofthe vapor and air before the mixture entered the chamber. Airflow through the chambers provided approximately one air change per minute. Females with sperm (Day 0 of gestation) were placed individually into 30 X 34 x 17 cm polycarbonate cages having autoclavable polyester filter covers. Bedding consisted of cleaned, heat-treated sawdust. Weekly food and water intake, along with maternal weights, were measured on Gestation Days 0, 7, l4, and 20. Females were also weighed each morning for the first week of exposure. 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 13 X 25 X I8-cm compartments in stainless-steel wire-mesh caging within thc exposure chambers. Controls were placed in similar caging within an adjacent exposure chamber for the same hours as the exposed animals. Exposures were conducted 7 h/day, and the animals were left in the chambers for degassing tor approximately 1/2 hr after vapor generation was terminated. They were then removed and returned in their homecages to the animal quarters where the water bottles were replaced. The concentration in the chamber was monitored continuously with a Miran 1A general-purpose infrared analyzer (Wilks/Foxboro Analytical, South Norwalk, CT). The Miran 1A was connected to a strip chart recorder to record continously the concentration throughout the day. Hourly, the chamber concentration, temperature, and humidity were recorded on a daily observation sheet. At the end of each day, the mean, range, and time-weighted average concentrations were calculated. Purity of the bulk chemical was analyzed by gas chromatography. Maternal examinations were done by performing cage side observations, measuring the body weight and by performing post-mortem examinations. The ovaries and uterine content was also examined. Fetal examinations were done by determining extrernal malformations, fetus weight, external determination of sex, skeletal malformations and visceral malformations. As a result the LOAEC based on maternal toxicity was determined to be 2000 ppm. The LOAEC based on fetotoxicity was determined to be 2000 ppm. The NOAEC based on teratogenicity was determined to be 5000 ppm. Exposure to 5000 ppm induced narcosis of all animals, an unsteady gait, reduced body weight gain (significant at 5000 ppm), reduced food consumption, and impaired locomotor activity. Exposure to 3500 and 2000 ppm also resulted in an unsteady state and impaired locomotor acitity. Body weight and body weight gain were reduced in all treatment groups. No external malformations could be observed. The majority of skeletal malformations were rudimentary cervical ribs. In general, skeletal variants increased with increasing concentrations of t-butanol. The lowest concentration was not associated with any defects. Variations seen were typical of fetotoxicity, especially reduced ossification.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 556 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Well described publication and acceptable for assessment.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 000 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Well described publication and acceptable for assessment.
Additional information

Faulkner (1989)

The developmental toxicity was tested in a read-across study with tertiary butanol. Pregnant mice were given tertiary butanol by gavage twice daily from day 6 through day 18 of gestation. Examinations were made on day 18. The females were sacrificed on day 18 by decapitation. The following concentrations were used: 20 mL/kg bw/day of a 10 % (v/v) solution equivalent to 1556 mg/kg bw/day (Basis=nominal conc. 10.5 mmoles/kg bw (=778 mg/kg bw every 12 hours)). Two different strains were used to determine the toxicity: CBA/J (7 females control and 12 females test item) and C57BL/6J (5 females control and 9 females test item). The ovaries and uterine content was examined by determining the number of implantations, the number of resoprtions and number of live fetuses. Also fetal examinations were performed by determining soft tissue examinations, skeletal examinations and the mean fetal weight. As a result the NOAEL was determined to be >= 1556 mg/kg bw/day based on teratogenicity. The LOAEL was determined to be 1556 mg/kg bw/day based on embryotoxicity. No maternal toxicity was observed during the study. Tertiary butanol produced a significant increase in the number of resorptions per litter, F(1,29)=7.361, p.=0.011, and a significant decrease in the number of live fetuses per litter, F(1,29)=10.060, p.=0.004. There were no significant effects of strain on either the number of resorptions per litter, F(1,29)=0.07, p.=0.787, or the number of live fetuses per litter, F(1,29)=0.322, p.=0.575. Eight of the 21 litters in the t-butanol treated groups had all of the fetuses resorbed vs.none in the control groups (X2=4.10, p.<0.05). Significantly more of the resorptions in the treated groups (33/75) required ammonium sulfide for visualization than in the control groups (2/14) (X2=4.66, p.<0.05), suggesting an early effect on fetal viability. No inter-strain differences in fetal mortality were observed. There was a slight but statistically insignificant decrease in the weight of the surviving fetuses in both strains. The results of morphological examination of the surviving offspring: Soft tissue examination revealed no teratogenic effects of t-butanol in either strain. There was no evidence that t-butanol produced the dilated ventricles, open eyelids, exencephaly, heart defects, or gastroschisis reported for ethanol in the CBA/J strain (27), even though fetuses were carefully examined for these defects. Skeletal examinations showed defects limited to the skull and sternum. Minor variations (misaligned or underossified sternebrae and underossified supraoccipital bones) occurred more frequently, but were not significantly different between treatments or strains.

Nelson (1989)

The developmental toxicity was tested in a read-across study with t-butanol. The publication presents the results of teratology assessment of industrial alcohols including t-butanol administered by inhalation to rats. For that, approx. 15 rats were exposed to 0, 2000, 3500 or 5000 ppm of t-butanol (estimated as 200, 350 and 500 mg/kg per day) for 7h/day on gestation days 1-19. Dams were sacrificed on gestation day 20, and fetuses were individually weighted and examined. The inhalation exposures were conducted in 0.5 m3 Hinners-type exposure chambers. The vapor generation equipment was housed above the exposure chambers in glove boxes which were maintained under negative pressure. Reagent-grade t-butanol was placed into a fIask. A low-flow pump (RP Model lab pump; Fluid Metering Inc., Oyster Bay, NY, USA) circulated liquid from the reservoir flask into a 10-mL syringe contained within the flask such that the syringe was constantly overflowing. Thus, the syringe provided a constant head of chemical for a second pump (controlled by a micrometer adjustment) 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 ofthe three-way valve. Alcohol evaporation was controlled by regulating the preheating ofcompressed air. The impinger provided increased contact time between the air and the liquid to ensure total evaporation. 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 down-stream of the vapor and air before the mixture entered the chamber. Airflow through the chambers provided approximately one air change per minute. Females with sperm (Day 0 of gestation) were placed individually into 30 X 34 x 17 cm polycarbonate cages having autoclavable polyester filter covers. Bedding consisted of cleaned, heat-treated sawdust. Weekly food and water intake, along with maternal weights, were measured on Gestation Days 0, 7, l4, and 20. Females were also weighed each morning for the first week of exposure. 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 13 X 25 X I8-cm compartments in stainless-steel wire-mesh caging within thc exposure chambers. Controls were placed in similar caging within an adjacent exposure chamber for the same hours as the exposed animals. Exposures were conducted 7 h/day, and the animals were left in the chambers for degassing tor approximately 1/2 hr after vapor generation was terminated. They were then removed and returned in their homecages to the animal quarters where the water bottles were replaced. The concentration in the chamber was monitored continuously with a Miran 1A general-purpose infrared analyzer (Wilks/Foxboro Analytical, South Norwalk, CT). The Miran 1A was connected to a strip chart recorder to record continously the concentration throughout the day. Hourly, the chamber concentration, temperature, and humidity were recorded on a daily observation sheet. At the end of each day, the mean, range, and time-weighted average concentrations were calculated. Purity of the bulk chemical was analyzed by gas chromatography. Maternal examinations were done by performing cage side observations, measuring the body weight and by performing post-mortem examinations. The ovaries and uterine content was also examined. Fetal examinations were done by determining extrernal malformations, fetus weight, external determination of sex, skeletal malformations and visceral malformations. As a result the LOAEC based on maternal toxicity was determined to be 2000 ppm. The LOAEC based on fetotoxicity was determined to be 2000 ppm. The NOAEC based on teratogenicity was determined to be 5000 ppm. Exposure to 5000 ppm induced narcosis of all animals, an unsteady gait, reduced body weight gain (significant at 5000 ppm), reduced food consumption, and impaired locomotor activity. Exposure to 3500 and 2000 ppm also resulted in an unsteady state and impaired locomotor acitity. Body weight and body weight gain were reduced in all treatment groups. No external malformations could be observed. The majority of skeletal malformations were rudimentary cervical ribs. In general, skeletal variants increased with increasing concentrations of t-butanol. The lowest concentration was not associated with any defects. Variations seen were typical of fetotoxicity, especially reduced ossification.


Justification for selection of Effect on developmental toxicity: via oral route:
Well described publication and acceptable for assessment.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Well described publication and acceptable for assessment.

Justification for classification or non-classification

Self-Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the results the substance is not considered to be classified for developmental toxicity under Regulation (EC) No 1272/2008.