Butane-1,4-diol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-03-16 to 1992-04-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA-TSCA New and Revised Health Effects Test Guidelines [Developmental Toxicity Study]

Version / remarks:

1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission directive 87/302/EEC for 9th adaption of directive 67/548/EEC, pages 24 - 26,1988 (fEC 1988)

Version / remarks:

1987-11-18

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

12 May 1981

Deviations:

no

Remarks:

Yes, compared to current version (25 June 2018): number of dams: 15 instead of 20, inhalation route instead of requested oral route, however testing up to 5 mg/L, which is the acute limit concentration for aerosols.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

γ-butyrolactone

EC Number:

202-509-5

EC Name:

γ-butyrolactone

Cas Number:

96-48-0

Molecular formula:

C4H6O2

IUPAC Name:

oxolan-2-one

Details on test material:

Stated as "GBL (CAS Ser No 96-48-0), puriss. grade, was purchased from Fluka AG, Switzerland"

Test animals

Species:

rabbit

Strain:

Himalayan

Remarks:

Chbb:HM (outbred strain)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-W7950 Biberach/Riss, FRG
- Age at study initiation: 18 - 26 weeks
- Weight at study initiation: Approx. 2.356 kg
- Housing: Singly in wire cages (type K 300/8, EBECO, Becker & Co., Castrop-Rauxel, FRG).
- Diet: Ad libitum (during the exposure-free observation period)
- Water: Ad libitum (during the exposure-free observation period)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): Not indicated, but rooms were fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From March 25, 1992 to April 16, 1992

Administration / exposure

Route of administration:

other: inhalation: mix of vapour and aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.4 µm

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass-steel inhalation chambers (manufacturer: BASF Aktiengesellschaft)
- Method of holding animals in test chamber: They were fixed in modified fixation cages wrapped with aluminium foil.
- Source and rate of air: See table 1 under "Any other information on materials and methods, incl. tables"
- Method of conditioning air: The test substance was supplied to a two-component atomizer by means of a continuous metering pump. By means of compressed air, the aerosol was generated into an aerosol mixing vessel. In the mixing vessel the aerosol was mixed with conditioned supply air and passed through a cyclonic separator into the inhalation chamber.
- Temperature, humidity, pressure in air chamber: 22-23.3 °C, rel. humidity 47.8-67.1 %, pressure [Pa]: test group 0: 10.2+/-0.3, test group 1: -10+/- 0.2, test group 2: -9.2+/- 1.3, test group 3: -6.8+/- 5.2
- Air flow rate: See table 1 under "Any other information on materials and methods, incl. tables"
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor in test groups 2 and 3. One sample was taken from test group 2 and 3 respectively with a cascade impactor at a sampling velocity of 1.25 m/sec. In addition to the cascade impactor measurements, a laser photometer (Malvern API) was used for detection of aerosols.
- Treatment of exhaust air: The exhaust air system flow was set higher than the supply air system flow (negative pressure). This ensured that no contamination of the laboratory could occur due to any leakages from the inhalation chambers.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the test atmospheres were analysed by gas chromatography after absorption into 2-propanol. The gas chromatograph was calibrated with weighed amounts of the test substance.
- Samples taken from breathing zone: The samples were taken from the exposure chambers, from the breathing zones of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test atmospheres were analyzed by gas chromatography after absorption into 2-propanol. The gas chromatograph was calibrated with weighed amounts of the test substance.

Details on mating procedure:

- Impregnation procedure: artificial insemination
0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Reeeptal®, trademark of HOECHST AG, Frankfurt/Main) were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
- The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).

Duration of treatment / exposure:

From day 7 to day 19

Frequency of treatment:

Daily for 6 hours

Duration of test:

From day 0 (day of insemination) to day 29 (day of sacrifice)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

1.4 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

0.5 mg/L air (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/L air (nominal)

Remarks:

Control (fresh air)

No. of animals per sex per dose:

15 female animals per dose (divided into 3 replicates - 3x5 animals per dose)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The doses were selected based on a dose-range finding study where no adverse treatment-related effects occured up to the limit dose of 5 mg/L.
- Rationale for animal assignment: Animals were allocated to the test groups according to a computergenerated randomization plan on the basis of their body weights.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The behavior and state of health of the test animals were checked each day at least 3 times on exposure days and, as a rule, once during the preflow period and the post-exposure observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was checked on day 0 (= day of insemination) and on days 3, 7, 10, 13, 16, 19, 21, 24, 27 and 29 p.i..

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 29 p.i.
- Organs examined: ovaries and uterine content (see below)

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 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: [all per litter]

Statistics:

Examination of the dams and fetuses
DUNNETT's Test (DUNNETT, 1955, 1964) was used for statistical evaluation of body weight, body weight change, corrected body weight gain (net matern al body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses.
FISHER's Exact Test (SIEGEL, 1956) was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Significances resulting from these tests have been indicated in the tables (a for p < 0.05, b for p < 0.01).

Indices:

conception rate and pre- and postimplantation losses

Historical control data:

Historical control data are available for the maternal body weights and reporduction data including fetal weights and fetal examinations.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight and body weight change of the test groups, compared with the control group, was not influenced in a statistically significant manner over the study period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy none of the does of test groups 1 - 3 (0.5, 1.4 and 5.0 mg/L) showed any substance-induced findings. One spontaneous necropsy finding was recorded for single animals of all dose groups including the controls. This finding, lungs with edema, has to be related to the sacrifice of the animals.
Lungs with edema in test group 0 (control): 5 (33 %), test group 1 (0.5 mg/L): 4 (27 %), test group 2 ( 1.4 mg/L): 5 (33 %), test group 4 ( 5 mg/L): 2 (13 %).

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related and/or statistically significant differences in conception
rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre and the postimplantation losses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.
test group 0 (0 mg/L): 5 resorptions (4.7 % +/- 7.12)
test group 1 (0.5 mg/L): 10 resorptions (9.5 % +/- 16.9)
test group 2 (1.4 mg/L): 11 resorptions (10.5 % +/- 12.67)
test group 3 (5 mg/L): 11 resorptions (8.3 % +/- 11.71)

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The conception rate was 100% in all groups. Concerning all groups, there were no substance-related and/or statistically significant differences in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEC

Effect level:

5 mg/L air

Based on:

test mat.

Basis for effect level:

other: absence of adverse effects

Remarks on result:

other: no adverse effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1 - 3 (0.5; 1.4 and 5.0 mg/L) was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance.

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Only one type of external variation (pseudoankylosis) was found in one control and three high concentration fetuses; however, all values concerning fetal and litter incidences are fully in the historical control range and the differences between the groups are therefore considered random.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the fetal skeletons were recorded for one fetus of the control group (different thoracic vertebral bodies and/or arches severely malformed), one fetus of the 0.5 mg/L group and two fetuses of the 1.4 mg/L group (lumbar vertebra absent) and one 5.0 mg/L fetus (different cervieal vertebral bodies and/or arches severely malformed). The variations elicited were related to the skull (splitting of skull bones, epactal bone between nasal and frontal bones), the ribs (accessory 13th rib(s)), the vertebral column (accessory thoracic or lumbar vertebra) and the sternum (sternebra(e) of irregular shape, fused or accessory sternebra). All of these skeletal variations were found in single fetuses of all groups including the controls, occurred without a clear dose-response relationship, without any biologically relevant differences between the groups, and/or can be found at comparable fetal/litter incidences in the historical control. This includes the statistically significantly higher values concerning the finding epactal bone between nasal and frontal bones and the statistically significantly lower values concerning the finding sternebra(e) of irregular shape in the 1.4 mg/L group. Moreover, the lower number of low dose fetuses with total skeletal variations is considered random. In all groups signs of retardations (incomplete or missing ossification of skull bones, vertebral column, os pubis, talus and/or sternebra(e)) were found; they occurred in a comparable frequency in the control and the substance-treated groups. All differences between the groups concerning fetal skeletal retardations are without any biological relevance. This includes the marginally, but statistically significant higher incidence of total skeletal retardations at 0.5 mg/L.
The number of 0.5 mg/L fetuses with skeletal variations is statistically significantly lower in comparison to the actual control group; in the same test group the occurrance of skeletal retardations is statistically significantly higher in comparison to the control group. Both, the lower incidence of skeletal variations and the higher incidence of skeletal retardations in the 0.5 mg/L group, however, are considered random because the relevant values recorded for this group are fully in the range of the historical control data and no dose-response relationship exists.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in all groups including the controls without any relation to the treatment. One low dose fetus showed hydrocephaly. Septal defect occurred in one control and one intermediate dose fetus each and 2 low dose fetuses. Hypoplastic lungs were found in 2 intermediate dose fetuses; in one of these fetuses it was associated with a diaphragmatic hernia. Moreover a hypoplastic spleen was recorded for one low dose and agenesia of gallbladder for one control, one 1.4 and one 5.0 mg/L fetus. Due to the fact that no dose-relationship exists and nearly all above mentioned soft tissue mal formations can be found at a low frequency in the historical control data, these malformations are regarded as being of spontaneous origin and not associated with the test substance exposure of the does. Variations were detected in each group including the control, all occurred without a clear dose-response relationship and/or can be found at a comparable incidence in the historical control data. Aside from a separated origin of carotids, a very common finding in the rabbit strain used, another soft tissue variation (heart with traces of interventricular foramen/septum membranaceum) was also found quite frequently. Furthermore, one fetus of the high dose group (5.0 mg/L) exhibited hypoplasia of gallbladder. One control and one intermediate dose fetus showed a so-called unclassified observation (focal liver necrosis). Blood coagulum around the bladder was seen in one high dose fetus.
There are only very few statistically significant differences between the control and the test substance exposed fetuses concerning soft tissue variations. The variation rate is not increased in the fetuses of the substance-exposed groups in comparison to the control group. All differences between the groups are without any biological relevance, do not show a clear dose-response relationship and/or appear to about the same extent in the historical control data.

Details on embryotoxic / teratogenic effects:

The malformation rate is not increased in the fetuses of the substance-exposed groups in comparison to the control group. All differences between the groups are without any biological relevance. The number of 0.5 mg/L fetuses with skeletal and total variations is statistically significantly lower in comparison to the actual control group; in the same test group the occurrance of skeletal (and consequently total) retardations is statistically significantly higher in comparison to the control group. Both, the lower incidence of skeletal and overall variations and the higher incidence of skeletal and overall retardations in the 0.5 mg/L group, however, are considered random because the relevant values recorded for this group are fully in the range of the historical control data and no dose-response relationship exists. For all other differences between the actual control group and the substance-exposed groups concerning fetal external, soft tissue and/or skeletal observations it can be said, that their occurrence is without any biological relevance, do not show a clear dose-response relationship and/or appear to about the same extent in the historical control data.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

5 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no adverse effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: supernumerary rib

skeletal: vertebra

other: Soft tissue malformations occured in all groups including the control without relation to treatment

Description (incidence and severity):

The malformation rate is not increased in the fetuses of the substance-exposed groups in comparison to the control group. All differences between the groups are without any biological relevance. The number of 0.5 mg/L fetuses with skeletal and total variations is statistically significantly lower in comparison to the actual control group; in the same test group the occurrance of skeletal (and consequently total) retardations is statistically significantly higher in comparison to the control group. Both, the lower incidence of skeletal and overall variations and the higher incidence of skeletal and overall retardations in the 0.5 mg/L group, however, are considered random because the relevant values recorded for this group are fully in the range of the historical control data and no dose-response relationship exists. For all other differences between the actual control group and the substance-exposed groups concerning fetal external, soft tissue and/or skeletal observations it can be said, that their occurrence is without any biological relevance, do not show a clear dose-response relationship and/or appear to about the same extent in the historical control data.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

There were no substantial, substance-related effects on the dams concerning body weight, body weight change, uterine weights, corrected body weight change, clinical and necropsy observations up to and including concentration of 5 mg/L. There were no differences of biological relevance between the control and the substance-treated groups in conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre-and the postimplantation losses. No concentration- and/or substance-related differences were recorded for placental and fetal body weights. The external, soft tissue and/or skeletal examination of the fetuses revealed no differences. between the control and the substance-treated groups which might be related to the test substance administration. Number and type of the fetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations, recorded for the 0.5; 1.4 and 5.0 mg/L fetuses were substantially similar to actual and/or historical control values. Thus, under the conditions of this study, 1-Butyrolacton caused no signs of maternal toxicity and no signs of embryo-/fetotoxicity up to and including a concentration of 5 mg/L. There were no indications of teratogenic effects which could be causally related to the test substance administration. Although no signs of matern al toxicity were found even at the highest concentration (5 mg/L), no further prenatal inhalation toxicity studies are deemed to be necessary, because this concentration is in accordance with the requirement for the LIMIT TEST, e.g. in the OECD Guideline for testing of chemicals No. 403 (OECD 1981) for acute inhalation studies and the EPA-TSCA guideline “Inhalation developmental toxicity study” § 798.4350 (EPA 1984). For this prenatal toxicity study in rabbits, the no observed adverse effect concentration (NOAEC) for the maternal and for the fetal organism is 5.0 mg/L.

Applicant's summary and conclusion

Conclusions:

For this prenatal toxicity study in rabbits, the no observed adverse effect concentration (NOAEC) for the maternal and for the fetal organism is 5.0 mg/L.

Executive summary:

1-Butyrolactone was tested for its prenatal inhalation toxicity in rabbits. Groups of 15 inseminated female Himalayan rabbits were exposed to clean air (control) or 1-Butyrolacton vapour and vapour-aerosol-mixtures (test groups) for 6 hours/day on days 7 to 19 post insemination (p.i.). The target concentrations for the study were set to 0.5 (vapour), 1.4 and 5.0 mg/L (vapour-aerosol-mixture). The animals were treated in whole body inhalation chambers, sitting in specially shielded cages to avoid contamination of body surface (head-nose exposure). Clinical examination of the animals was performed at least once daily in the pre- and post-exposure period. In the treatment interval the health of the animals was checked before, during and after exposure. Body weight development was followed throughout the study. On day 29 p.i. all animals were sacrificed and the uteri removed. The fetuses were dissected from the uteri, sexed, weighed and examined for any external, soft tissue and skeletal findings.

Analyses of the atmospheres demonstrated that the target concentrations were fully met. Particle size measurement revealed the presence of aerosols at 5.0 mg/L with an MMAD of 2.4 µm and a respirable fraction of 92%. There were no substantial, substance-related effects on the dams concerning body weight, body weight change, uterine weights, corrected body weight change, clinical and necropsy observations up to and including concentration of 5 mg/L. There were no differences of biological relevance between the control and the substance-treated groups in conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre-and the postimplantation losses. No concentration- and/or substance-related differences were recorded for placental and fetal body weights. The external, soft tissue and/or skeletal examination of the fetuses revealed no differences between the control and the substance-treated groups which might be related to the test substance administration. Number and type of the fetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations, recorded for the 0.5; 1.4 and 5.0 mg/L fetuses were substantially similar to actual and/or historical control values. Thus, under the conditions of this study, 1-Butyrolacton caused no signs of maternal toxicity and no signs of embryo-/fetotoxicity up to and including a concentration of 5 mg/L. There were no indications of teratogenic effects which could be causally related to the test substance administration.

Although no signs of maternal toxicity were found even at the highest concentration (5 mg/L), no further prenatal inhalation toxicity studies are deemed to be necessary, because this concentration is in accordance with the requirement for the LIMIT TEST, e.g. in the OECD Guideline for testing of chemicals No. 403 (OECD 1981) for acute inhalation studies and the EPA-TSCA guideline "Inhalation developmental toxicity study" § 798.4350 (EPA 1984). For this prenatal toxicity study in rabbits, the no observed adverse effect concentration (NOAEC) for the maternal and for the fetal organism is 5.0 mg/L.