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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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 assessement.

Data source

Reference
Reference Type:
publication
Title:
Lack of Selective Developmental Toxicity of Three Butanol Isomers Administered by Inhalation to Rats
Author:
B.K. Nelson
Year:
1989
Bibliographic source:
FUNDAMENTAL AND APPLIED TOXICOLOGY 12,469-479 ( 1989)

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): t-butanol
- Analytical purity: > 99%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system 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 (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26 °C
- Humidity (%): 40-60 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2000, 3500, 5000 ppm estimated as 200, 350, and 500 mg/kg per 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

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
ca. 3 500 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEC
Effect level:
<= 2 000 ppm
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEC
Effect level:
ca. 2 000 ppm
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion