Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a five generation study with embedded continuous breeding study no effects on fertility were observed in 4 generations up to the highest concentration tested (24% in diet; 12000 mg/kg bw/d). The pregnancy rate of F1A rats decreased during 5 successive mating cycles, no pubs were obtained at the highest concentration of the fifth series of litters (F2E). Therefore, the mid dose group (10% in diet; 5000 mg/kg bw/d) was selected as NOAEL. No effects on fertility were observed in additional studies of lower reliability at comparable dose-levels.

Link to relevant study records
Reference
Endpoint:
multi-generation reproductive toxicity
Remarks:
five generation study with embedded continuous breeding study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: publication with some shortcomings in documentation (purity of test substance not stated, exposure duration not clearly stated, no statistical evaluation)
Qualifier:
no guideline followed
Principles of method if other than guideline:
five generation study with embedded continuous breeding study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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
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 are indicated by the chart in the orginal paper to be 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.
Remarks:
0, 5, 10 and 24 % nominal in diet, corresponding to 0, 2500, 5000 and 12000 mg/kg bw/day, calculated with food factor 0.05 according to Guidance on Information Requirements R.8
No. of animals per sex per dose:
25 rats per sex per dose group in the F0, F1A, F1B, F2A, F3A
Control animals:
yes, plain diet
Positive control:
none
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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
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. Except the P0-Generation, body weight gains of male rats in all four consecutive generations were slightly depressed with an apparent dose relationship (for details see below). The efficiency of food utilization through 10 weeks of post-wening remained constant for all generations of both sexes and was not affected by 1,3-BD treatment.

CLINICAL STUDIES
Hematology, blood chemistry and urinalysis showed no trend associated with treatment for F0, F1, F2 and F3 generation animals

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.
Key result
Dose descriptor:
NOAEL
Effect level:
12 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Dose descriptor:
NOAEL
Effect level:
12 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: absence of effects on reproduction
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
12 000 mg/kg bw/day (nominal)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Female animals showed no significant abnormal growth rates. Body weight gains of adult male rats in four F1 generations were slightly depressed with an apparent dose relationship (for details see bel
ow). The efficiency of food utilization through 10 weeks of post-wening remained constant for all generations of both sexes and was not affected by 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 t
he 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 signifi
cant 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.
Key result
Dose descriptor:
LOAEL
Effect level:
12 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Dose descriptor:
LOAEL
Effect level:
12 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
12 000 mg/kg bw/day (nominal)
System:
other: unspecific effects
Organ:
other: body weight gain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
12 000 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
other: reduced fertility
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
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 specified
Developmental immunotoxicity:
not specified
- no treatment related effects in F1 offspring (details for the effects of P1 generation (F2 litters) are presented above and in the attached document)
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
12 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no developmental toxicity
Remarks on result:
other: maternal dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
12 000 mg/kg bw/day (nominal)
System:
other: no developmental effects observed
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
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
Key result
Dose descriptor:
NOAEL
Generation:
other: F2A-F2E
Effect level:
12 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no developmental effects
Remarks on result:
other: maternal dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
12 000 mg/kg bw/day (nominal)
System:
other: no developmental effects observed
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
12 000 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

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  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 five 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.
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 four of five generations of dams and pups. The pregnancy rate of F1A rats decreased during five successive mating cycles: no pups were obtained at the high-dose level group of the fifth series of litters (F2E generation). Excluding this 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 (Hess et al., 1981).

The study indicates that fertility is not impaired through 1,3 -butylene glycol exposure up to 10% in diet (5000 mg/kg bw/d).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
sufficient for evaluation
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No toxicity data on fertility is available for the submission substance. However, adequate and reliable data are reported here for a structural analogue (source substance 1,3 -butanediol). Please also refer to the read-across justification document.


Effects on fertility were investigated in rats which received up to 24% 1,3 -butylene glycol in the diet (12000 mg/kg bw/d). No effects were observed in 4 generations of a five generation study, but the pregnancy rate of F1A rats decreased during 5 successive mating cycles and no pubs were obtained at the highest concentration of the fifth series of litters (2FE). Additionally, in this study a dose dependent influence of the test item on the body weight gain of male rats was observed (Hess et al., 1981). The highest dose (24%, 12000 mg/kg bw/d) was therefore regarded as LOAEL and the mid dose (10% in diet, about 5000 mg/kg bw/d) as NOAEL. In spite of some shortcomings in the documentation, this well planned and performed study was judged to be reliable and was selected as key study for this endpoint.


The findings of the key study are supported by the data from one other multigeneration studies, which did not report an impairment of fertility by 1,3 -butylene glycol concentrations or 24% in diet (2000 mg/kg bw/d; Celanese, 1974). However, due to their incomplete documentation these studies were judged to be of lower reliability.


Taking into account all information available for the source substance 1,3 -butylene glycol, there is only single evidence from the continuous breeding study in rats which revealed adverse effects on fertility in the fifth litter of the F1 in the highest dose group (Hess et al. 1981). As the effects were only observed under extreme conditions (continuous breeding) in the highest dose group -- at a dose which is irrelevant under normal use conditions and far (about ten times) above the limit dose usually used for such kind of studies -- these data are regarded as irrelevant with respect to a possible classification. Therefore, it is concluded that no classification for effects on fertility is required.

Effects on developmental toxicity

Description of key information

No teratogenic effects were observed at doses up to 12000 mg/kg bw/d. Fetotoxicity was observe at doses of 12000 and 5000 mg/kg bw/d (missing or incomplete ossificated sternebrae). The NOAEL for fetotoxicity was 2500 mg/kg bw/d.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Detailed publication with some shortcomings in documentation (purity of test substance not stated, no statistical evaluation for the majority of end points)
Qualifier:
no guideline followed
Principles of method if other than guideline:
study on developmental toxicity
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals 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
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 multigeneration 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
Duration of test:
Part of multigeneration study
Remarks:
0, 5, 10 and 24 % nominal in diet, corresponding to 0, 2500, 5000 and 12000 mg/kg bw/day, calculated with food factor 0.05 according to Guidance on Information Requirements R.8
No. of animals per sex per dose:
14-15 females per dose group
Control animals:
yes, plain diet
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
Indices:
Fertility, gestation, gestation and lactation in remaining pups, not sacrificed for teratological examination
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: neoplastic:
not specified
Number of abortions:
no effects observed
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:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
12 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- viability of pups, number of implantation and resorption sites and the mean fetal weight were unaffected by feeding diets with 1,3-butylene glycol up to 24% (12000 mg/kg bw/d), for details see below
- statistically significant increase of incomplete ossification of sternebrae for the middle and high level fetuses as compared with the control fetuses, and a statistically significant increase of missing sternebrae for high dose fetuses, for details see below
Key result
Dose descriptor:
NOAEL
Effect level:
12 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Remarks on result:
other: maternal dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Dose descriptor:
LOAEL
Effect level:
5 000 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity
Remarks on result:
other: maternal dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
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: maternal dose calculated (24% in diet, food factor 0.05 according to Guidance on Information Requirements R.8)
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
Description (incidence and severity):
missing and incomplete ossification of sternebrae
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
5 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

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
  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) 1,3-butylene glycol in the diet. But fetotoxic effects occurred in concentrations at or above 10% (5000 mg/kg bw/d) 1,3-butylene glycol in the diet.
Executive summary:

Teratogenic effects of 1,3-butylene glycol were investigated as part of a multigeneration study in rats receiving 0, 5, 10 and 24% 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 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. The NOAEL for fetotoxicity was 2500 mg/kg bw/d of 1,3-butylene glycol in the diet (Hess et al., 1981).

Endpoint:
developmental toxicity
Remarks:
WEC - Whole-Embryo Culture Assay
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
other: WEC - Whole-Embryo Culture Assay
Version / remarks:
The European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy: Statement on the Scientific Validity of the Postimplantation Rat Whole-Embryo Culture Assay - an In Vitro Test for Embryotoxicity / 2001
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Albino Wistar rats
Details on test animals and environmental conditions:
Albino Wistar rats were supplied by the Animal Resources Centre, Murdoch, W.A. Rats were mated overnight and presence of sperm in the vagina was taken as indication of pregnancy (day 0). Embryos were explanted on day 10.
Route of administration:
other: via culture medium (rat post-implantation embryo culture model)
Vehicle:
other: culture medium
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 hours
Frequency of treatment:
static exposure
Duration of test:
2 hours
Dose / conc.:
25 other: mM
Control animals:
yes, concurrent no treatment
Details on study design:
Rat embryos were explanted on day 10. Uteri were aseptically removed and the decidua dissected free. The deciduum and underlying trophoblast and parietal yolk sac were carefully removed leaving the visceral yolk sac intact. Culture medium consisted of 70% rat serum, Dulbecco’s modification of Eagles minimal essential medium (EDMEM) to which was added 100 IU/ml benzyl penicillin and 100 µf/ml streptomycin sulphate. The culture medium was equilibrated with gases O2, CO2 and N2. The embryos floated in the medium in 60 ml reagent bottles and rotated on a modified haematological roller. After exposure, embryos were evaluated for their morphological development using a dissecting microscope. The number of somites were counted. Embryos were homogenised by ultrasonication and the protein content measured by the method of Lowry and DNA by the fluorometric method of Hinegardner.
Fetal examinations:
Endpoints investigated in post-implantation embryo: embryonic protein, DNA, somite development, gross morphology, or viability.

There was no significant effect on the total embryonic protein, DNA or the number of pairs of somites found in post-implanted embryos exposed to butane-2,3-diol via culture medium at a concentration of 25 mM. Furthermore, there were no alterations in the gross morphology or viability of the embryos observed with a dissecting microscope. No effects were seen on growth and development at up to 25 mM.
Statistics:
Treatments groups were assessed for statistically significant differences by Students t-test.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Endpoints observed in post-implantation embryo: embryonic protein, DNA, somite development, gross morphology, or viability

Dose descriptor:
NOAEC
Effect level:
>= 2 253 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: no embryotoxicity observed
Remarks on result:
other: 25 mM culture medium concentration (2253 mg/L)
Abnormalities:
no effects observed
Developmental effects observed:
no
Lowest effective dose / conc.:
2 253 ppm (nominal)
Treatment related:
yes

Effects of acetaldehyde and butane-2,3-diol on 10 -day rat embryo development in vitro (WEC - Whole-Embryo Culture Assay)

Treatment No. Somites Protein (µG) DNA (µG) No with heartbeat Normal gross morphology (%)
Control 18 33.6 ± 0.3 738 ± 49 73 ± 4 18 18 (100)
Acetaldehyde [µM]            
102 ± 6.7 17 33.8 ± 0.3 662 ± 31 73 ± 4 17 16 (94)
260 ± 26.3 21 33.6 ± 0.2 628 ± 20 73 ± 4 21 19 (91)
815 ± 24.6 6 Uncountable * 67 ± 19 * < 5 * 0 * 0 (0) *
2,3-butanediol [µM]            
25 12 33.6 ± 0.3 698 ± 24 78 ± 3 12 12 (100)

* P < 0.5 compared with control values.

Conclusions:
No effects were observed on embryonic protein, DNA, somite development, gross morphology, or viability in post-implantation embryos after 2 hours of exposure to 25 mM butane-2,3-diol (2253 mg/L).
Executive summary:

Priscott (1984) investigated the effects of acetaldehyde and 2,3-butanediol on rat embryos developing in vitro. The study used rat post-implantation embryo culture model (WEC - Whole-Embryo Culture Assay). Embryo toxicity of 2,3-butanediol were examined in cultured 10-day Albino Wistar rat embryos over a 2-day period (n=12). After exposure to 25 mM (2253 mg/L) of 2,3-butanediol, no differences in embryonic protein, DNA, somite development, gross morphology and viability were observed. 2,3 -butanediol had no significant effect in any of the examined parameter at a culture medium concentration of 25 mM in comparison to the control.

The European Centre for the Validation of Alternative Methods (ECVAM) declared in 2001 that the post-implantation rat whole-embryo culture (WEC) assay was a scientifically validated test, which was ready to be considered for regulatory purposes (The Use of Scientifically-Validated In Vitro Tests for Embryotoxicity, ECVAM Institute for Health & Consumer Protection, Joint Research Centre, European Commission, 2002). Im ECHA REACH guidance R.7a it is stated that the whole embryo culture showed high predictivity for certain strongly embryotoxic chemicals (which is supported by the second test item acetaldehyde in the present study) but due to the nature of the methods and limitations in their predictivity, it could be used only as supporting information along with other more reliable data to predict the developmental toxicity. In conclusion, the present study was considered relevant for assessment of developmental / teratogenic toxicity of 2,3 -butanediol.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
Reagent Grade, 98% minimum assay, Lot No. 714739, Fisher Scientific Co.
Species:
rat
Strain:
Long-Evans
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Blue-Spruce Farms in Altamont, NY
- Age at study initiation:
- Weight at study initiation: 200-300 g
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1,3-butanediol solutions (35.30, 21.18, or 3.53% w/v) and distilled water were freshly prepared, and daily doses of 20 ml/kg were given by gavage to pregnant Long-Evans rats, based on their current bodyweights.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
Duration of treatment / exposure:
from day 6 to day 15 of gestation
The presence of sperm in the vaginal smear was defined as day 0 of gestation.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group I
Dose / conc.:
7 060 mg/kg bw/day
Remarks:
Group II
Dose / conc.:
4 236 mg/kg bw/day
Remarks:
Group III
Dose / conc.:
706 mg/kg bw/day
Remarks:
Group IV
No. of animals per sex per dose:
40 females (10 females per each group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were chosen as fractional doses (24, 14.4, and 2.4%) of the acute LDso value for butanediol, similar to previous studies with other alcohols.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was monitored by daily visual inspection of ground diet (Wayne Lab Blox) remaining in calibrated metal feed cups.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: total uterine weight
Ovaries and uterine content:
The ovaries and uterine content was examined after termination
Examinations included:
total litter weigh;, individual pup weights, crown-rump length, number of live pups, stillbirths and resorptions, implantation sites, sex distribution, and number of corpora lutea
Fetal examinations:
- External examinations(gross malformation): Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter ]
- skeletal and cartilaginous examinations: Yes: [all per litter]
- Head examinations: No
Statistics:
The parametric analysis of variance and Newman-Keuls posthoc analyses were used to compare maternal bodyweights, uterine weights, litter weights, pup weights, crown-rump lengths, corpora lutea, implantations, percent of males per litter, intrauterine deaths per litter, malformed pups per litter, and pup bodyweights by contiguity classification on an absolute and relative (percent of control) basis. Contingency table analyses (Chisquare and Fisher exact test) were applied to litters bearing malformed pups. Linear regression analysis of butanediol dose against pup bodyweight was performed.
Historical control data:
Not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Signs of mild maternal narcosis (lethargy, decreased response to light cage tap) were noted in rats of groups I1 and 111 within 1 hour of butanediol gavage. All animals appeared to be normal within 8 hours of dosing. Feed consumptions, total gestation weight gains, and maternal weight gains were similar for all groups.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dams given high doses of the ethanol dimer butanediol had a significant (P < 0.001, two-tailed) dose-related (r -0.58) birthweight depression in their live offspring at day 20 of gestation. This significant (one-way analysis of variance; P < 0.01, F 5.52, df3) birthweight depression affected pups of group II as compared to controls (Newman-Keuls posthoc analysis; P < 0.01).
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
No significant increase of malformed pups, attributable to butanediol exposure, was observed in any group. However, a number of minor, growth-related structural variations were observed in pups of group II. Runting (defined as a pup weighing less than 2.7 g) occurred in 6 of 95 group II pups (no control pups were runted).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Sternebral variations (missing, incomplete, or incompletely calcified) were noted in 15 of 95 group II pups, as compared to 7 of 112 group I pups.
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
No differences attributable to butanediol were detected in litter weights, crown-rump lengths, corpora lutea, implantations, litter size, sex prevalence, intrauterine deaths, malformed pups per litter, or litters with malformed pups (P > 0.05; analysis of variance with Newman-Keuls posthoc test).
In the present studies, embryotoxity (reduced birthweight) selectively affects Mo offspring exposed in utero to high levels of butanediol, and although no significant overall increase in malformations was observed, group II pups were more frequently runted and had a greater incidence of sternebral variations than controls, coincident with depressed birthweight.
Dose descriptor:
NOAEL
Effect level:
4 236 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
yes
Lowest effective dose / conc.:
7 060 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Embryotoxity (reduced birthweight) selectively affects Mo offspring exposed in utero to high levels (4236 mg/kg and 7060 mg/kg) of butanediol, and although no significant overall increase in malformations was observed, group II pups were more frequently runted and had a greater incidence of sternebral variations than controls, coincident with depressed birthweight. Fetal bodyweight and skeletal variations (retarded ossifications) are considered to reflect the birthweight reductions and are not indicative of a teratogenic effect of butanediol.
Executive summary:

The embryotoxic effects of high doses of the narcotizing ethanol dimer 1,3-butanediol were evaluated in pregnant Long-Evans rats during the “critical period” of organogenesis. Butanediol was given by gavage at levels of 0, 7060, 4236, or 706 mg/kg per day. Maternal sedation was observed at 7060 and 4236 mg/kg, but feed consumptions and maternal body weights were unaffected. Butanediol caused a significant, dose-dependent decrease in offspring birthweights. Embryotoxity (reduced birthweight) selectively affects Mo offspring exposed in utero to high levels (4236 mg/kg and 7060 mg/kg) of butanediol, and although no significant overall increase in malformations was observed, group II pups were more frequently runted and had a greater incidence of sternebral variations than controls, coincident with depressed birthweight. Fetal bodyweight and skeletal variations (retarded ossifications) are considered to reflect the birthweight reductions and are not indicative of a teratogenic effect of butanediol.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2 500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
sufficient for evaluation
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No key study is available on developmental / teratogenic toxicity for the submission substance. However, adequate and reliable data are reported here for a structural analogue (source substance 1,3 -butanediol). Furthermore, a supporting study is available investigate the effects of 2,3-butanediol (isomer mix) on rat embryos developing in vitro. Please also refer to the read-across justification document.

No teratogenic effects were observed in the key study on developmental toxicity (Hess et al. 1981). Fetotoxic effects (missing sternebrae, incomplete ossification of sternebrae) were recorded at concentrations of 10% and 24% of 1,3 -butylene glycole in the diet (5000 and 12000 mg/kg bw/d) in the study. The NOAEL was 5% 1,3 -butanediol in the diet (2500 mg/kg bw/d). In a nearly guideline conform test a NOAEL for fetotoxic effects (decreased birthweight) of 4236 mg/kg bw/d was observed (Mankes et al., 1986). Additionally, in this study a number of minor, growth-related structural variations were observed in pups, which were in accordance with the findings of the key study.

The effects observed in fetuses, especially the effects on sternebrae, are regarded as substance related and adverse. However, as these effects were only observed at doses which clearly exceed the limit values recommended in current guidelines. Therefore it is concluded that no classification for effects on development is required.

Priscott (1984) investigated the effects of acetaldehyde and 2,3-butanediol (isomer mix) on rat embryos developing in vitro (supporting study). The study used rat post-implantation embryo culture model (WEC - Whole-Embryo Culture Assay). Embryo toxicity of 2,3-butanediol were examined in cultured 10-day Albino Wistar rat embryos over a 2-day period (n=12). After exposure to 25 mM (2253 mg/L) of 2,3-butanediol, no differences in embryonic protein, DNA, somite development, gross morphology and viability were observed. 2,3 -butanediol had no significant effect in any of the examined parameter at a culture medium concentration of 25 mM in comparison to the control. The NOAEC of 2253 mg/L (ppm) of the supporting study can be expressed as 0.23% exposure solution of 2,3 -butanediol.

In ECHA REACH guidance R.7a it is stated that the whole embryo culture showed high predictivity for certain strongly embryotoxic chemicals (which was supported by the second test item acetaldehyde examined in the same study at far lower concentration) but due to the nature of the methods and limitations in their predictivity, it could be used only as supporting information along with other more reliable data to predict the developmental toxicity. In conclusion, the present study was considered relevant for assessment of developmental / teratogenic toxicity of 2,3 -butanediol.

Mode of Action Analysis / Human Relevance Framework

In the absence of information on species specific effects or metabolism the results are regarded as relevant for humans.

Justification for classification or non-classification

Based on the available data it is concluded that the submission has not to be classified for toxicity on reproduction according to Regulation (EC) No 1272/2008.