(R)-(-)-butane-1,3-diol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This endpoint study record is the experimental source record for the registered target substance.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Data for butane-1,3-diol (CAS No. 107-88-0) was used to address the toxicological data requirements for (R)-(-)-butane-1,3-diol (CAS No. 6290-03-5) in an analogue read-across approach. The basis for this read-across approach is the extreme structural similarity of the source and target substances, in that the source substance is a racemic mixture of a pair of enantiomers, whereas the target substance is solely the R-enantiomer of that source pair. Two compounds that are enantiomers of each other have the same physical properties, except for the direction in which they rotate polarized light and how they interact with different optical isomers of other compounds (ECHA, 2008). Passive absorption of a substance into a test species and distribution through its tissues are governed by the physical-chemical properties of the substance, particularly its molecular size, log P, and water solubility (ECHA, 2014), and are therefore expected to be exactly the same for both enantiomers. The R-enantiomer half of the source substance and all of the target substance have been shown to metabolise in a mammalian system to a physiological ketone body, whereas the S-enantiomer of that ketone body derived from the other half of the source substance has been shown to metabolise into a compound that is not naturally present, but which can still be utilized by a less direct pathway (Desrochers et al., 1992). On the premise that a less direct metabolic pathway must be more energy-expensive, and therefore may be more likely to perturb the system and potentially produce an adverse effect, toxicity data on the source substance will represent a very similar or slightly worse case than, and provide a sound basis for a slightly conservative assessment of, the toxicity of the target substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target Chemical: (R)-(-)-butane-1,3-diol (228-532-0; 6290-03-5)
Source Chemical: butane-1,3-diol (203-529-7; 107-88-0)
For further details refer to attached Justification For Read-Across Of Toxicity Data

The target substance is known to be of high purity (≥99 % w/w), so the low levels of impurities it could contain are not expected to substantially affect its physical-chemical properties. The purities of the samples of source material that were tested are not specifically known, but it is assumed that they would not have been sufficiently impure as to substantially affect the study results. On this basis, the applicability of the data on the source substance to the target substance is not expected to be compromised by the presence of impurities in either substance.

3. ANALOGUE APPROACH JUSTIFICATION
The basis for this read-across approach is the extreme structural similarity of the source and target substances. Specifically, the source substance is a racemic mixture of a pair of enantiomers, whereas the target substance is solely the R-enantiomer of that source pair. The source substance is therefore nominally comprised 50% of the target substance itself (the R-enantiomer), and 50% of its mirror image (the S-enantiomer), which differs from the target substance only in the chirality of one carbon atom. The selection of this source substance is justified on the basis that there is no other source substance that could possess a greater degree of structural similarity to the target substance.

Enantiomers are two stereoisomers that are related to each other by a reflection: they are mirror images of each other. Every stereocentre in one has the opposite configuration in the other. Two compounds that are enantiomers of each other have the same physical properties, except for the direction in which they rotate polarized light and how they interact with different optical isomers of other compounds (ECHA, 2008). Passive absorption of a substance into a test species and distribution through its tissues are governed by the physical-chemical properties of the substance, particularly its molecular size, log P, and water solubility (ECHA, 2014), and are therefore expected to be exactly the same for both enantiomers.

In a mammalian system, both enantiomers have been shown to be taken up by the liver and converted to their respective 3-hydroxybutyrate (beta-hydroxybutyrate; BHB) at identical rates. The target substance and one half of the source substance are converted into R-BHB, and the other half of the source substance is converted into S-BHB. R-BHB is a physiological ketone body, whereas S-BHB is not naturally present, but can still be utilized by a less direct pathway (Desrochers et al., 1992). On the premise that a less direct metabolic pathway is more energy-expensive, and may therefore be more likely to perturb the system and potentially produce an adverse effect, toxicity data on the source substance will represent a very similar or slightly worse case than, and provide a sound basis for a slightly conservative assessment of, the toxicity of the target substance.

4. CONCLUSION
Values generated on the source substance will represent a very similar or slightly worse case than the target substance

REFERENCES
Desrochers S, David F, Garneau M, Jetté M, Brunengraber H (1992). Metabolism of R- and S-1,3-butanediol in perfused livers from meal-fed and starved rats. Biochem J 285:647-653.

ECHA (2008). Guidance on information requirements and chemical safety assessment. Chapter R.6: QSARs and grouping of chemicals. May 2008. Available at: https://echa.europa.eu/documents/10162/13632/information_requirements_r6_en.pdf

ECHA (2014). Guidance on information requirements and chemical safety assessment. Chapter R.7c: Endpoint specific guidance. Volume 2.0, November 2014. Available at: https://echa.europa.eu/documents/10162/13632/information_requirements_r7c_en.pdf/e2e23a98-adb2-4573-b450-cc0dfa7988e5

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

(R)-(-)-Butane-1,3-diol value is read-across from supporting (R/S)-butane-1,3-diol (203-529-7; 107-88-0) data.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

only slight depression of body weight gain

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other:

Remarks:

Values generated on the source substance will represent a very similar or slightly worse case than the target substance

Additional information on results:

Dietary concentrations of 5, 10 and 24% (R/S)-1,3-butanediol correspond with body doses of 2000, 4000 and 9600 mg/kg bw for males and 2500, 5000 and 12000 mg/kg bw for females (based on a daily food consumption of 40 and 50 g/kg bw for males and females, respectively, according to the Guidance on Information Requirements R.8).

The percentage of pregnancies as well as the percentage of viable fetuses per implantation site were not significantly different between treatment and control groups. The mutagenic index did not show a trend with increasing doses. Values generated on the source substance will represent a very similar or slightly worse case than the target substance.
	Control	5%	10%	24%
No. pregnancies total	106	97	130	117
% Pregnancies (20 matings)	66.3	60.6	81.3	73.1
Implant sites	1165	1024	1452	1310
Viable fetuses total	1101	962	1389	1269
% Viable fetuses/implant sites	94.5	94.0	95.7	96.9
Resorptions total	64	62	63	41
% Resorptions/implant sites*	5.5	6.1	4.3	3.1

*: mutagenic index

Conclusions:

The test substance (R/S)-13,-butanediol did not induce dominant lethal effects after oral exposure of rats with dietary concentrations of up to 24%. Values generated on the source substance will represent a very similar or slightly worse case than the target substance.

Executive summary:

Rats were fed (R/S)-butane-1,3-diol in concentrations up to 24% of the diet and paired to produce F1A and F1B litters. Males of the F1B generation were used to examine dominant lethal effects after mating them with virgin females. The exposure did not cause a significant effect with respect to fertility, viable fetuses per implantation sites and percentage of resorption per implantation sites (mutagenic index). A dose-related trend was not evident.

Values generated on the source substance will represent a very similar or slightly worse case than the target substance. It is predicted that oral consumption of (R)-1,3 -butanediol would not induce dominant lethal effects.

 

This study was performed at high doses, which produced body weight gain. Deficiencies of this study are the incomplete data reporting (e.g. with respect to substance purity, statistical analysis of the results). In summary, this study is judged to be reliable with restrictions.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

multi-generation reproductive toxicity

Remarks:

four generation study with embedded continuous breeding study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This endpoint study record is the experimental source record for the registered target substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: Four generation study with embedded continuous breeding study

Version / remarks:

extended EPA OTS 798.4700 (Reproduction and Fertility Effects) study design

Deviations:

not applicable

Principles of method if other than guideline:

Four generation study with embedded continuous breeding study

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

The test compound, 1,3-butanediol, was obtained from the Celanese Chemical Company, New York

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats (FDRL-stock)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 14-15 weeks
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Photoperiod: 12 hrs dark/12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

SEMIPURIFIED DIET
Casein: 20 %
Refined corn oil: 8%
Salt mix: 4%
Vitamin mix: 1%
Corn starch 33.5%
Dextrose: 33.5%

DIET PREPARATION
- test diets were prepared by substituting 1,3-butanediol for equal amounts by weight of corn starch and dextrose

Details on mating procedure:

- M/F ratio per cage: one male/ one female
- Length of cohabitation: 7 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

F0 rats were treated 4 weeks before the mating period. Female rats of the F0 were fed diets containing 1,3-butanediol throughout the mating, gestation and lactating period. After 11 weeks of feeding, 25 males and 25 females from each dosage group of F1A animals were randomly selected and paired to produce the F2 generation (no further information).

Frequency of treatment:

daily

Details on study schedule:

At 1-2 weeks after weaning of the first litters (F1A), each female of the F0 generation was mated with a different male and a second series of litters was produced (F1B). All animals of the F1B generation were discarded at weaning except for ten males per group, which were reared to sexual maturity and used in a dominant lethal test. Pubs of the F1A were reared to maturity. After 11 weeks of feeding, 25 males and 25 females from each dosage group of F1A animals were randomly selected and paired to produce the F2 generation. Five successive mating cycles were achieved with the F1A rats within a period of 77 weeks (F2A, F2B, F2C, F2D, F2E). The F2B, F2C, F2D and F2E were examined and sacrificed as part of the continuous breeding phase of the study, while the F2A litter was mated to produce the F3A and F3B litters. The F3A litter was used for the cytogenetic portion of the study and was mated to produce the F4A and F4B litter, which form part of the cytogenicity study. The pregnant dams of the F2A litters (producing the F3B) were divided in two groups: 1/4 were allowed to give birth normally and 3/4 were used for teratological examination on day 19 of gestation.

Dose / conc.:

0 other: % Basis: nominal in diet

Dose / conc.:

5 other: % Basis: nominal in diet

Dose / conc.:

10 other: % Basis: nominal in diet

Dose / conc.:

24 other: % Basis: nominal in diet

No. of animals per sex per dose:

25 rats per sex per dose group in the F0, F1A, F1B, F2A, F3A

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

After 4 weeks of feeding of the F0 the respective diets, blood samples were collected from ten rats per sex per group for determination of alkaline phosphatase, glucose, hematocrit, hemoglobin and total and differential leucocyte counts. Urine analysis of the same animals provided measurements of albumin, glucose, ketones, occult blood, pH, specific gravity and microscopic examination. For F1A rats which survived at least 66 weeks, the gonads and pituitary glands were examined microscopically. During the eleventh week of feeding of F1A animals blood and urine samples were collected from ten rats per sex per group and evaluated as mentioned above.

Body weight: yes

Reproductive performance: yes

Litter observations:

viability, mean pub weight at day 4 and 21 post partum

Postmortem examinations (parental animals):

histopathologic examination of the testes or ovaries and pituitary glands of the F1A

Reproductive indices:

fertility (percent matings resulting in pregnancies) and gestation indices (percent pregnancies resulting in litters cast alive)

Offspring viability indices:

- percent pubs cast alive that survived to 4 days
- percent pups alive at 4 days that survived to 21 days

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Female animals showed no significant abnormal growth rates. Body weight gains of male rats in all four generations were slightly depressed with an apparent dose relationship. The efficiency of food utilization through 10 weeks of post-weaning remained constant for all generations of both sexes and was not affected by (R/S)-1,3-butanediol treatment.

No significant treatment-related differences were noted on histopathologic examination of testes or ovaries and pituitary glands.

Dose descriptor:

NOAEL

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects in F0 generation

Remarks on result:

other:

Remarks:

24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced weight gain F1A, F1B generations

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Female animals showed no significant abnormal growth rates. Body weight gains of male rats in all four generations were slightly depressed with an apparent dose relationship. The efficiency of food utilization through 10 weeks of post-weaning remained constant for all generations of both sexes and was not affected by (R/S)-1,3-butanediol treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During five successive mating cycles of F1A rats, a gradual decrease in the pregnancy rate was seen. Both the number of pregnant females and the fertility index appeared to be dose-related for several series of F2 litters, especially F2D and F2E. For the fifth series of litters, no pups were obtained in the highest-dose group. However, the gestation, viability and lactation indexes, as well as the mean pup body weights at 4 and 21 days showed no significant differences between specific litter series or between control and test groups (excluding high-dose animals of the fifth series of litters).

No significant treatment-related differences were noted on histopathologic examination of testes or ovaries and pituitary glands as a possible explanation of the observed reproductive failure during the fifth cycle.

Dose descriptor:

LOAEL

Generation:

other: F1A, F1B

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

all generations (F1A, F1B, F2A, F3A) except F0

Dose descriptor:

NOAEL

Generation:

other: F1A, F1B

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no treatment related effects

Remarks on result:

other:

Remarks:

calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Dose descriptor:

NOAEL

Generation:

other: F1A, F1B

Effect level:

5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

calculated (10% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced weight gain F2A, F3A generations

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Female animals showed no significant abnormal growth rates. Body weight gains of male rats in all four generations were slightly depressed with an apparent dose relationship. The efficiency of food utilization through 10 weeks of post-weaning remained constant for all generations of both sexes and was not affected by (R/S)-1,3-butanediol treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During five successive mating cycles of F1A rats, a gradual decrease in the pregnancy rate was seen. Both the number of pregnant females and the fertility index appeared to be dose-related for several series of F2 litters, especially F2D and F2E. For the fifth series of litters, no pups were obtained in the highest-dose group. However, the gestation, viability and lactation indexes, as well as the mean pup body weights at 4 and 21 days showed no significant differences between specific litter series or between control and test groups (excluding high-dose animals of the fifth series of litters).

No significant treatment-related differences were noted on histopathologic examination of testes or ovaries and pituitary glands as a possible explanation of the observed reproductive failure during the fifth cycle.

For the other three generations of dams and pups, no significant dose-related trends were observed for the reproduction and lactation parameters, as described above.

Dose descriptor:

LOAEL

Generation:

other: F2A, F3A

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Dose descriptor:

LOAEL

Generation:

other: F2E

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no pregnant females to produce the F2E generation; reduced number of females in former litters of the F2 generation

Remarks on result:

other:

Remarks:

calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Dose descriptor:

NOAEL

Generation:

other: FE2

Effect level:

5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no pregnant females to produce the F2E generation; reduced number of females in former litters of the F2 generation

Remarks on result:

other:

Remarks:

calculated (10% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Dose descriptor:

NOAEL

Generation:

other: F2A, F3A

Effect level:

5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

(10% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Reproductive effects observed:

not specified

Body weight gain of male rats is presented in the following table:

Generation	Dietary level (%)	Weeks	Mean weight gain (g)
F0	0	23	153
	5	23	149
	10	23	141
	24	23	149
F1A	0	77	481
	5	77	429
	10	77	410
	24	77	383
F1B	0	11	298
	5	11	278
	10	11	263
	24	11	257
F2A	0	11	305
	5	11	282
	10	11	278
	24	11	272
F3A	0	9	296
	5	9	270
	10	9	263
	24	9	222

 

Conclusions:

1,3-butylene glycol did not influence fertility in a four generation study with an embedded continuous breeding study in concentrations up to 10% in the diet (5000 mg/kg bw/d). In the highest concentration tested (24%, 12000 mg/kg bw/d) no offspring in the fifth litter of the F2 generation were produced (likely secondary to parental toxicity and stress).

Executive summary:

Twenty five animals of both sexes were fed either control diet or diet supplemented with 1,3-butylene glycol at dose levels of 5, 10 or 24% of the diet (2500, 5000 or 12000 mg/kg bw/d). Treatment with the test item had no influence on reproduction and lactation parameters for all but one group of dams and pups. The pregnancy rate of F1A rats decreased during five successive mating cycles (likely secondary to parental toxicity and stress): no pups were obtained at the high-dose level group of the fifth series of litters (F2E generation). However, the added stress of a highly ketogenic, semi-synthetic diet may have contributed significantly to this effect. A significant increase in the concentration of ketone bodies in blood, urine and liver tissues was noted in rats fed diets contain 1,3-butanediol at the levels of 20 or 25% (study referenced in journal article). Therefore, physiological stress may be associated with increased ingestion of 1,3-butanediol.

Excluding F2E group, the viability of F2 generation pups revealed no significant differences between litters or between control and test groups. Body weight gains of male rats in all four generations were slightly depressed with an apparent dose relationship. Body weight gain of females was not affected.

 

Documentation of this well-planned study is insufficient: Exposure duration is not clearly stated, purity of the test item is not given. Examination of the parental generation is insufficient (e.g.no histopathological examination of sexual organs), test results were not evaluated statistically. Despite these shortcomings the study was judged to be reliable with restrictions as it indicates that fertility is not impaired through 1,3-butylene glycol exposure up to 10% in diet (5000 mg/kg bw/d).

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This endpoint study record is the experimental source record for the registered target substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

study on teratogenicity

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

The test compound, 1,3-butanediol, was obtained from the Celanese Chemical Company, New York

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 14-15 weeks
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Photoperiod: 12 hrs dark/12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

SEMIPURIFIED DIET
Casein: 20 %
Refined corn oil: 8%
Salt mix: 4%
Vitamin mix: 1%
Corn starch 33.5%
Dextrose: 33.5%
DIET PREPARATION
- test diets were prepared by substituting 1,3-butanediol for equal amounts by weight of corn starch
and dextrose

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Investigation of teratogenicity was performed with part of the second litter of the F3 generation of a multi generation study

Duration of treatment / exposure:

day 0 to day 19 of gestation, additional to exposure of the parental (F2) and former generations (F0 and F1)

Frequency of treatment:

daily

Dose / conc.:

0 other: % Basis: nominal in diet

Dose / conc.:

5 other: % Basis: nominal in diet

Dose / conc.:

10 other: % Basis: nominal in diet

Dose / conc.:

24 other: % Basis: nominal in diet

No. of animals per sex per dose:

14-15 females per dose group

Control animals:

yes, concurrent no treatment

Maternal examinations:

- sacrifice at day 19 of gestation
- number of implantations, resorptions, viable and nonviable fetuses

Fetal examinations:

- data on growth abnormalities, weight and sex of fetuses were recorded
- one third of fetuses were examined for soft tissue abnormalities and remaining fetuses were used for skeletal examinations
- soft tissue examinations: fetuses of each group were fixed in Bouin's solution, sectioned according to the method of Wilson and examined in detail for abnormalities
- skeletal examinations: fetuses were fixed in ethyl alcohol and stained with alizarin red and examined for defects

Statistics:

Skeletal tissue examinations: evaluated by the approximate chi-square test

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No treatment related effects

Remarks on result:

other: calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not specified

External malformations:

not specified

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no definitive dose-related teratological findings in either soft or skeletal tissue

Dose descriptor:

NOAEL

Effect level:

12 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Remarks on result:

other: calculated (24% in diet, food factor 0.05 according to Guidance on Informati on Requirements R.8)

Dose descriptor:

LOAEL

Effect level:

5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Remarks on result:

other: calculated (10% in diet, food factor 0.05 according to Guidance on Informati on Requirements R.8)

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Remarks on result:

other: calculated (5% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)

Abnormalities:

not specified

Developmental effects observed:

not specified

Conducted as part of reproduction study;

no definitive dose-related teratological findings in either soft or skeletal tissue.

Fetotoxicity (e.g., delayed ossification of sternebrae) noted at 10% and 24% doses, 5000 and 12000 mg/kg bw/d, respectively.

Incidence of fetal skeletal abnormalities in F3B generation:

	Dietary level (%)
	0	5	10	24
No. of fetuses examined	124	103	120	103
Sternebrae
Incomplete ossification	31	31	48*	65*
Scrambled	1	0	0	0
Bipartite	1	1	0	3
Extra	1	0	0	0
Missing	10	3	13	37**
Ribs
More than 13	4	4	1	1
Vertebrae
Incomplete ossification	4	1	1	2
Scoliosis	1	0	0	0
Skull
Incomplete closure	9	0	3	10
Hyoid bone
Missing	2	0	0	2
Reduced	0	0	0	1

*: significantly different from respective control, p </= 0.025

**: significantly different from respective control, p </= 0.01

Resorption and implantation data for F3B generation:

		Mean no. of pups per litter
Dietary level (%)	No. of pregnant females	Viable	Non-viable	Implantations (mean per dam)	Resorptions (mean per dam)	Mean fetal weight (g)
0	15	11.9	0	12.5	0.6	3.5
5	15	10.1	0	10.4	0.3	4.0
10	14	12.1	0	12.6	0.5	4.1
24	14	10.9	0	11.4	0.5	3.4

 

Conclusions:

No teratogenic effects were seen in rats treated with up to 24% (12000 mg/kg bw/d) (R/S)1,3-butylene glycol in the diet. But fetotoxic effects occurred in concentrations at or above 10% (5000 mg/kg bw/d) (R/S)-1,3-butylene glycol in the diet.

Executive summary:

Teratogenic effects of (R/S)-1,3-butylene glycol were investigated as part of a multi-generation study in rats receiving 0, 5, 10 and 24% (R/S)-1,3-butylene glycol in the diet (0, 2500, 5000, 12000 mg/kg bw/d). No conclusive teratogenic effects were seen in pups of the F3B generation at levels up to 12000 mg/kg bw/d (R/S)-1,3-butylene glycol in the diet. Incomplete sternebral ossification at mid- and high-dose levels and missing sternebrae at high-dose level were noted, probably indicating slight delayed development of fetal skeletal tissue (likely secondary to parental toxicity and stress). The NOAEL for fetotoxicity was 2500 mg/kg bw/d of (R/S)-1,3-butylene glycol in the diet.

The study was judged to be reliable with some minor restrictions due to some shortcomings of the study (e.g. not according to guideline, missing data on test substance purity, no data on sex distribution of the offspring reported although this endpoint was recorded according to the method section).

Data source

Reference

Reference Type:

publication

Title:

Reproduction and teratology study of 1,3-butanediol in rats

Author:

Hess, FG, et al.

Year:

1981

Bibliographic source:

Hess, Frederick G., et al. "Reproduction and teratology study of 1, 3‐butanediol in rats." Journal of Applied Toxicology 1.4 (1981): 202-209.

Materials and methods

Principles of method if other than guideline:

genotoxicity test in vivo after subchronic oral exposure over 3 generations

GLP compliance:

not specified

Type of assay:

other: chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Butane-1,3-diol

EC Number:

203-529-7

EC Name:

Butane-1,3-diol

Cas Number:

107-88-0

Molecular formula:

C4H10O2

IUPAC Name:

butane-1,3-diol

Test material form:

liquid

Details on test material:

no impurities described

Specific details on test material used for the study:

The test compound, 1,3-butanediol, was obtained from the Celanese Chemical Company, New York

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats (FDRL-stock)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 14-15 weeks
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

SEMIPURIFIED DIET
20% casein
8% refined corn oil
4% salt mix
1% vitamin mix
33.5% corn starch
33.5% dextrose

DIET PREPARATION
- test diets were prepared by substituting 1,3-butanediol for equal amounts by weight of corn starch and dextrose

Duration of treatment / exposure:

Rats were treated 4 weeks before the mating period. Female rats of the F0 were fed diets containing 1,3-butanediol throughout the mating, gestation and lactating period of the F1A generation. Pubs of the F1A were reared to maturity. After 11 weeks of feeding, 25 males and 25 females from each dosage group of F1A animals were randomly selected and paired to produce the F2A generation and the F2A litter was mated to produce the F3A generation.

Frequency of treatment:

daily

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: % Basis: nominal in diet

Doses / concentrations 2

Dose / conc.:

5 other: % Basis: nominal in diet

Doses / concentrations 3

Dose / conc.:

10 other: % Basis: nominal in diet

Doses / concentrations 4

Dose / conc.:

24 other: % Basis: nominal in diet

No. of animals per sex per dose:

F0: 25 males, 25 females
At least two animals per sex per dose from the F1A, F2A and F3A generation were examined.

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Examinations

Tissues and cell types examined:

femur bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Animals were treated with 1 mg/kg bw colchicine intraperitoneally 3-4 h prior to examination (exact time point of examination not stated). The bone marrow was washed with 5 ml of Hank's balanced salt solution (HBSS). The isolated cells were washed with HBSS repeatedly, suspended in hypotonic fetal calf serum and incubated for 20 min at 37 °C. Fixation of the cells was performed in methanol/glacial acetic acid (3:1 mixture) overnight at 4 °C and stained on coverslips with 2% aceto-orcein.

METHOD OF ANALYSIS:
100-250 metaphase cells per dose group were examined for chromosomal aberrations at 900x magnification by phase-contrast microscopy.

Statistics:

Statistical analysis was not stated

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

only slight depression of body weight gain

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

Dietary concentrations of 5, 10 and 24% correspond with body doses of 2000, 4000 and 9600 mg/kg bw for males and 2500, 5000 and 12000 mg/kg bw for females (based on a daily food consumption of 40 and 50 g/kg bw for males and females, respectively, according to the Guidance on Information Requirements R.8).

Any other information on results incl. tables

The number of abnormal cells was not increased with respect to the normal range of aberrant cells in untreated F1A, F2A and F3A animals. No specific abnormalities were observed in the treated animals and no dose-related effects were noted.

Applicant's summary and conclusion

Conclusions:

The test substance did not induce chromosomal aberrations after subchronic oral exposure of rats over 3 generations with dietary concentrations of up to 24%.

Executive summary:

Rats were fed butane-1,3-diol in concentrations up to 24% of the diet and paired to produce F1A, F2A and F3A litters. Analysis of the femur bone marrow of at least two animals per sex and dose of these litters revealed no increase in chromosomal aberrations.

This study was well performed with doses high enough to cause a reduced body weight gain. Despite some conceptional deficiencies (no positive controls, low numbers of cells per dose group examined) as well as incomplete data reporting (e.g. with respect to substance purity, time point of examination, statistical analysis of the results) this study is judged to be reliable and sensitive, due to the repeated application of high doses over long time periods and several generations.