(Z)-2-butene-1,4-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Remarks:

BASF AG

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 11-12 weeks (males), 10-11 weeks (females)
- Housing: individually, DKII stainless stell cages/ Makrolon type M II cages
- Diet (ad libitum): Kliba maintenance diet mouse/rat
- Water (ad libitum):drinking water
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: doubly distilled water

Details on mating procedure:

At least 13 days after the beginning of treatment, males and females from the same dose group were mated overnight in a ratio of 1:1. As soon as sperm was detected in the vaginal smear, the mating was discontinued.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The male rats were treated for 29 days
The female rats were treated for 50 days.

Frequency of treatment:

once daily

Details on study schedule:

Males: approx. 2 weeks premating, 2 weeks mating and post mating
Females: approx. 2 weeks premating, during mating and gestation through day 4 after delivery.

Remarks:

Doses / Concentrations:
20, 60, and 200 mg/kg-bw
Basis:
other: amount of test substance administered by oral gavage

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The daily volume administered was 10 ml/kg of bodyweight. The calculation of the volume administered was based on the most recent individual body weight. Food consumption of the F0 parents was determined regularly during premating, after the mating period and, in dams, during gestation and lactation periods.

Positive control:

None

Parental animals: Observations and examinations:

GENERAL OBSERVATIONS:
- A detailed clinical observation was performed in all animals once before the initial test substance administration and there after at weekly intervals. The animals were examined for signs of toxicity or mortality at least once a day. The F0 parental animals were examined for their mating and reproductive performances.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before first dose and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Regularly during premating, after the mating period and, in dams, during gestation and lactation periods.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males on study day 29, Females on study day 50
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 5

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males on study day 29, Females on study day 50
- Animals fasted: No data
- How many animals: 5

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males on study day 28, Females on study day 42
- Dose groups that were examined: low, medium, and high dose groups
- Battery of functions tested: functional battery and motor activity measurement

Litter observations:

The pups were sexed on day 0 p.p. and weighed one day after their birth and on day 4 post partum. Their viability was recorded.

Postmortem examinations (offspring):

All pups were sacrificed four days after birth under CO2 anesthesia and examined macroscopically for external and visceral findings at necropsy.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation after treatment was observed in several males of the high dose from week 2 to the end of the treatment period as well as in females of the high dose during study weeks 6 and 7. This finding, reported for these high dose groups, was considered as been related to the treatment.
No substance-related clinical findings were observed in any other treated group.

After test substance administration, at least one female from the high dose group showed salivation during the lactation period.
Several female animals from the high dose groups did not nurse their F1 pups properly in the beginning of the lactation period (5/10 females - on day O p.p. and 1/10 female - on day 1 p.p.). The pups from these animals were found dead between days 1 and 2 p.p ..
Pups from two females which did not show any nursing problem, were also found dead on days 1 and 2 p.p ..
All these findings were related to the administration of the high dose of the test substance.

No substance-related findings during lactation were observed in any other treated group.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant impairment in body weights was observed in males from the high dose group during the treatment period (max. -9%). The body weight change of the same treated group was found 59% decreased, when compared to controls, from study week 0 to 4.
From gestation day 7 onwards, the body weights of the high dose females were markedly lower (max. -14%) when compared to concurrent control. If calculated for the entire gestation period (days 0-20), the high dose animals gained statistically significantly less weight (-29%) than controls.
During the lactation period, body weights of females from the high dose group were observed significantly decreased (from day O to 4; max -16%) as well as body weight gain, for the same dose group, was found 77% reduced when compared to control animals.
Body weight from females treated with the high dose of the test substance was reported significantly reduced on study weeks 6 and 7 (max. -16% and -8%, respectively). Due to a marked reduction found in the body weight of animals from the high dose group after weaning (study week 6), the body weight change of this group of animals was found significantly higher than control animals.
The reduction in body weights observed after the high dose of the test substance administration in males and females groups are considered as being related to treatment.

Body weights/body weight gains of FO parental animals in low and mid test groups were not influenced by the test substance administration. All differences in body weights and body weight gains observed for these rats were without any biological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A significant reduction in food consumption was observed in male (-12%) and female
(-17%) animals treated with the high dose of the test substance during the first week of administration. Food consumption of the same female dose group was also found below than controls in the beginning of gestation period (-10%; days 0-7) and during lactation (-31%; days 0-4).
All these findings were related to the high dose of the test substance treatment.
No significant differences in food consumption were observed in any other treated group.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological examinations revealed no treatment-related changes in males. In females, slight, but statistically significant decreases in hemoglobin and hematocrit were observed in the peripheral blood of the mid and high dose animals.
No treatment-related effects were seen in the other hematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Compound-related differences in serum enzyme activities were not evident at any dose level in either males or females.
Blood chemistry investigations showed significantly increased potassium, inorganic phosphate and albumin concentrations in the circulation of the high dose animals of either sex. Decreased creatinine and glucose levels as well as increased total bilirubin concentrations were also found in the high dose males. Moreover, increased calcium and magnesium concentrations and decreased chloride levels were recorded in the serum of the high dose females. In the males mid dose potassium was increased. This finding, however, was not of sufficient magnitude to be considered adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Males treated with the high dose of the test substance showed a reduced number of rearings when compared to controls (-50%). Although this finding was not statistically significant, it was related to the test substance treatment.
Regarding the overall motor activity a reduction of beams numbers was observed in male animals treated with the high dose of the test substance from interval 1 to interval 6. This finding is considered as being related to the treatment.

No substance-related findings were observed in any other treated group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treated female animals had a slightly higher incidence of focal pelvic mineralization in kidney (0/2/2/6). This finding was regarded to be incidental as in almost every animal the kidney was only unilaterally affected, the finding was only minimal to slight and there was no clear dose-response relationship.
All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at a low incidence or at a biologically comparable incidence and graded severity in control and high dose males and/or females. They were all regarded to be of spontaneous origin and to have developed unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The male mating index reached 100% in all substance treated groups including the control group.
The male fertility index reached 100% in all substance treated groups including the control group.
The female mating index calculated after the mating was 100% in all test groups.
The female fertility index was 100% in all test groups.
The gestation index was 100% in all groups of females.
The mean duration of gestation period was similar in all test groups (between 21.9 and 22.2 days).
The mean number of implantation sites per dam was found statistically significantly decreased in the high dose group (11.2 implantations against 14.0 in the control group). Furthermore, post implantation loss was 20% in this dose group, indicating intrauterine embryo-fetolethality.
A significant reduction of 30% in the number of pups delivered in the high dose group was observed.
The live birth index was slightly but not significantly impaired (94% compared to controls: 98%) in animals from the high dose group.
The female reproduction and delivery parameters assessed in this study were not affected in groups of animals treated with the low and mid dose of the test substance.

CLINICAL EXAMINATIONS
Test group 3 (200 mg/kgbw/day):
salivation in males after treatment from second week onwards
slight impairment of motor activity in males from interval 1 to 6
significant reduction in food consumption during the first week of treatment in both sexes (-12% males and -17% females)
significant impairment of body weights and body weight gain (max.-9% and -59%, respectively) in males
significant impairment of females body weights and body weight gain in studyweeks 6 and 7 (max.-16% and -8%, respectively)
Test groups 1 and 2 (20 & 60 mg/kg bw/day):
- no treatment-related effects were observed

FEMALES CLINICAL EXAMINATIONS DURING GESTATION
Test group 3 (200 mg/kgbw/day):
salivation before and after test substance administration
significant reduction in food consumption within the first week of gestation (-10%),
significant impairment of body weights and body weight gain (max.-14% and-29%, respectively)
Test groups 1 and 2 (20 & 60 mg/kg bw/day):
- no treatment-related effects were observed

FEMALE CLINICAL EXAMINATIONS DURING LACTATION (DAYS 0-4 POSTPARTUM)
Test group 3 (200 mg/kgbw/day):
salivation after test substance administration
significant reduction in food consumption (-31%)
significant impairment of body weights and body weights gain (max.-16% and-77%, respectively)
Test groups 1 and 2 (20 & 60 mg/kg bw/day):
- no treatment-related effects were observed

CLINICAL PATHOLOGY
Test group 3 (200 mg/kgbw/day):
increased potassium, inorganic phosphate and albumin in both sexes
increased total bilirubin in males and calcium and magnesium in females
decreased creatinine and glucose in males and hemoglobin, hematocrit andchloride in females
Test groups 2 (60 mg/kg bw/day):
- decreased hemoglobin and hematocrit in females
Test group 1 (20 mg/kg bw/day):
- no treatment-related effects were observed

PATHOLOGY
Test group 3 (200 mg/kgbw/day):
significant decrease in terminal body weight in males and females
significant increase of relative kidney weights in male animals
significant increase of absolute and relative liver weights in male and female animals
minimal to moderate storage of alpha 2u protein in epithelial cells of proximal tubules in all male animals
minimal to moderate centrolobular hypertrophy in the liver of nine male and nine female animals
minimal to slight hypertrophy/hyperplasia of the thyroid follicular cells of three female animals
significant increase of absolute and relative kidney weights in male animals
significant increase of absolute and relative liver weight in males
significant increase of relative liver weights in females
minimal to slight storage of alpha 2µl protein in epithelial cells of proximal tubules in five male animals
Test group 2 (60 mg/kg bw/day):
- minimal centrolobular hypertrophy in the liver of four female animals
Test group 1 (20 mg/kg bw/day):
- no treatment-related effects were observed

FERTILITY/REPRODUCTIVE PERFORMANCE
Test group 3 (200 mg/kgbw/day):
significantly reduced implantation sites numbers per dam (11.2/14)
significantly increased postimplantation loss (20%)
reduced number of pups delivered (-30%)
higher number of stillborn pups (6 pups from 4 litters vs. 2 pups from 2 litters in thecontrol)
impaired nursing (5/10 dams) and insufficient maternal care (1/10 dams)
complete loss of pups (7/10 dams)
Test groups 1 and 2 (20 & 60 mg/kg bw/day):
- no treatment-related effects were observed

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Whereas the cohabitation, fertility and integrity of the reproductive organs of both male and female rats were not affected, a substantially impaired pre- and postnatal development of the offspring was observed at maternally toxic dose (200 mg/kg b.w.).

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One pup of the FO high dose group showed hypothermia. This single occurrence was considered as being incidental and spontaneous in nature.
The F1 pup clinical observations did not reveal any additional abnormalities in all test groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

An increase in the number of pups, which died (42%) or were cannibalized (16%), in the FO high dose group was observed. Thus, the viability index, as an indicator for pup mortality between days O and 4 post partum, was found significantly lower (38%) when compared to control group (99%).
One pup from the FO low and one from the FO mid dose treatments were cannibalized. These single occurrences were assessed as being spontaneous in nature having no relationship with the test substance administration. The viability index was not affected for both dose groups (99%) when compared to controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant impairment in body weights and body weight gain (-23% and -34%, respectively) of the pups from the FO high dose females were reported from days 1 to 4 post partum.
The mean pup body weights/pup body weight gain from the low and mid dose animals did not show any statistically significant differences when compared to control groups.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male of the examined F1 pups of the high dose group and one male pup from the control group showed hemorrhagic testis at necropsy.
This finding, observed also in the control group, occurred without any clear relation to the test substance treatment. Thus, this occurrence was considered incidental and not biologically relevant.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL EXAMINATIONS (DAYS 0-4 POST PARTUM)
Test group 3 (200 mg/kgbw/day):
- markedly increased number of pups found dead or cannibalized, viability index 38% (vs. 99% in the control);
- significant impairment of pups body weights and body weight gain (-23% and-34%, respectively)
Test groups 1 and 2 (20 & 60 mg/kg bw/day):
- no treatment-related effects were observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

60 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Viabiity and body weight.

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Reproductive effects observed:

not specified

Conclusions:

In conclusion, the gavage administration of 200 mg/kg body weight/day 2-Butene-1,4- diol to male and female Wistar rats caused distinct systemic toxicity such as salivation, markedly reduced food consumption and retarded body weight development, liver enzyme induction, storage of alpha 2u protein and mild impairment of motor activity in the males as well as mild anemia in the females. Whereas the cohabitation, fertility and integrity of the reproductive organs of both male and female rats were not affected, a substantially impaired pre- and postnatal development of the offspring was observed at this overtly maternally toxic dose.
A dose of 60 mg/kg of body weight/day still evoked liver enzyme induction in both genders, storage of alpha 2u protein in males and mild anemia in females but had no influence on reproduction.
Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance is 60 mg/kg of body weight/day for the FO parental rats.
The NOAEL of the test substance for general, systemic toxicity is 20 mg/kg of body weight/day for the FO parental rats of both sexes.
The NOAEL of the test substance for developmental toxicity in the F1 progeny is 60 mg/kg of body weight/day.

Reference 2

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed publication of studies performed with scientifically sound methods.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: weanling rats
- Weight at study initiation: not specified
- Fasting period before study: not applied
- Housing: individually housed, except during mating and lactation, in wire mesh cages or plastic cages with corn-cob bedding
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Purina Rodent Chow, Ralston-Purina, St. Louis, MO.
- Water (e.g. ad libitum): tap water
- Acclimation period: All rats were acclimated to the laboratory for at least 10 days prior to initiating the study.

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of mating period : 15 days (twice)
- Proof of pregnancy: by vaginal plugs or sperm-positive vaginal smears
- After successful mating each pregnant female was transferred to an individual plastic cage containing nesting material. Males were returned to their wire-mesh cages at the end of the mating period.
- The day on which evidence of copulation was observed was identified as Day 0 of gestation.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The desired dose was administered orally in a volume of 10 ml/kg. Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated. Treatment began when F0 rats were 5 to 6 weeks and F1 animals were 22 days of age, and continued until the generation was terminated.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 20, 55, or 150 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

F0: 10 males and 20 females

Control animals:

yes, concurrent vehicle

Positive control:

Not specified.

Parental animals: Observations and examinations:

Rats were observed for signs of toxicity and body weights were recorded at intervals during the study.

Oestrous cyclicity (parental animals):

No data.

Sperm parameters (parental animals):

No data.

Litter observations:

STANDARDISATION OF LITTERS
Performed on day 4 postpartum: yes, litters with more than 10 pups were reduced to five males and five females, if possible.
PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: number and sex of pups, live births, postnatal mortality, presence of gross and detailed anomalies/malformations

Postmortem examinations (parental animals):

A histopathological evaluation was performed on approximately 30 tissues from each parent that died during the study, selected parents from all groups in the F0 generation, selected parents from the control and mid-dose groups in the F1 generation.

Postmortem examinations (offspring):

A histopathological evaluation was performed on 10 pups/sex from the control and mid-dose groups from the F2b litters.

Statistics:

Several different statistical methods were used to compare measurements made on test animals to the corresponding values determined for controls. The methods and the measurements to which they were applied are analyis of variance and Dunnett’s test (Steel and Tonie, 1960) for adult body weights, litter size, and pup body weights; Fisher’s exact probability test (Siegel, 1956) for mortality and fertility data; Mann-Whitney U test (Siegel, 1956) for fetal body weights; and x2 test with Yates’ correction or Fisher’s exact probability test (Siegel, 1956) for litters with anomalies. In all instances, p < 0.05 was selected as the level of significance.

Reproductive indices:

Implants/, viable fetuses/ and Resorptions/dam

Offspring viability indices:

Survival and body weights

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of respiratory irritation and significant adult mortality in the high dose group for F0 and F1. For F1 significant mortality was attributed to gavage-related injuries.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Adult body weights were not significantly affected in the low- or mid-dose groups. In the high-dose group, mean body weights of both sexes of F0 were significantly reduced by Week 11 and of F1 by week 30, in both persisting for the remainder of the test.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Adult body weights were not significantly affected in the low- or mid-dose groups. In the high-dose group, mean body weights of both sexes of F0 were significantly reduced by Week 11 and of F1 by week 30, in both persisting for the remainder of the test.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Renal cortical necrosis in 60% F0-males and in 15% F0-females, no microscopic changes in F1- kidneys.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Significant mortality due to gavage-related injuries.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was neither a dose-related reduction nor a pattern within a generation that suggested the presence of a treatment-related effect.

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality

Dose descriptor:

NOAEL

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality, fertility, litter size

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality, fertility, pup survival and weight, littersize and weight, histopathology.

Reproductive effects observed:

not specified

Conclusions:

Maleic anhydride, a metabolic analogue of 2-butene-1,4-diol, does not appear to present a reproductive hazard in the workplace.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

In an OECD 422 Guideline study, Male and female rats were exposed to 20, 60, and 200 mg/kg 2 -Butene-1,4-diol (B2D) by oral gavage (BASF AG, 2006).  The gavage administration of 200 mg/kg body weight/day 2-Butene-1,4- diol to male and female Wistar rats caused distinct systemic toxicity such as salivation, markedly reduced food consumption and retarded body weight development, liver enzyme induction, storage of alpha 2u protein and mild impairment of motor activity in the males as well as mild anemia in the females. Whereas the cohabitation, fertility and integrity of the reproductive organs of both male and female rats were not affected, a substantially impaired pre- and postnatal development of the offspring was observed at this overtly maternally toxic dose. A dose of 60 mg/kg of body weight/day still evoked liver enzyme induction in both genders, storage of alpha 2u protein in males and mild anemia in females but had no influence on reproduction. Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance is 60 mg/kg of body weight/day for the FO parental rats. The NOAEL of the test substance for general, systemic toxicity is 20 mg/kg of body weight/day for the FO parental rats of both sexes. The NOAEL of the test substance for developmental toxicity in the F1 progeny is 60 mg/kg of body weight/day.

Additionally, the dossier is extended with a teratology and a 2-generation reproductive toxicity study in rats exposed to maleic anhydride (Short et al., 1986). Maleic anhydride is considered to be analogue to 2-Butene-1,4-diol as it is also metabolically hydrolyzed to maleic acid. Maleic anhydride did not induce any reproductive effects at doses up to 55 mg/kg/day over 2 generations and thus clearly supports the results of the screening tests with 2-Butene-1,4-diol. Further supporting data are collected from a Prenatal Developmental Toxicity Study (OECD Guideline 414) of B2D with rabbits where NOAEL was 20 mg/kg b.w. with no changes in pregnancy performance, and the Prenatal Developmental Toxicity Study with rats exposed to but-2-yne-1,4-diol (B3D), where NOAEL was 40 mg/kg bw. Since B2D is considered to be a less active molecule than B3D, this result supports the key value for B2D.

In keeping with Regulation (EC) 1907/2006 Annex X Column 2 and for reasons of animal welfare, the dossier does not need additional results of an extended one generation reproductive toxicity study, since there are no serious concerns about the potential for adverse effects on fertility based on available data and read across data from analogue substances. Further, the NOAEL for systemic effects (20 mg/kg bw) is below the NOAELs for reproductive endpoints.

Effects on developmental toxicity

Description of key information

Under the conditions of OECD 414 study in rabbits, the NOAEL for maternal and developmental toxicity is 20 mg/kg of bw/day. Developmental toxicity at the dose level of 60 mg/kg b.w. was secondary to maternal toxicity at the same dosage level. 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

By error, the brains of control fetuses A004-6 and A005-12 were not assessed by mid-coronal slice. Evaluation: Sufficient control data was available for a proper evaluation. The study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females were used at initiation of the study.
Stock male NZW rabbits were used for mating with the females. These males were adult and proven fertile. After mating these males were placed back in their stock and might be used for future studies.

Source: Charles River Deutschland, Sulzfeld, Germany.
Age at delivery: Females were approximately 17 weeks.
Acclimatization: At least 5 days prior to pairing.
Health inspection: upon receipt of the animals.


Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
The temperature range in the animal room of this Test Facility (18-24°C) was slightly higher than the range proposed in the OECD 414 guideline (18±3°C). Laboratory historical control data did not indicate any effect of this slightly higher range on the animals condition.
Accommodation: Females were individually housed in labelled cages with perforated floors and shelters.
Diet: Free access to pelleted diet for rabbits. In addition, pressed hay and wooden sticks were provided during the study period.
Water: Free access to tap-water.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Accuracy and homogeneity of formulations were confirmed by chemical analyses.

Prior to start of the main study, stability of the test item in the vehicle was demonstrated as part of the method development and validation study.

Details on mating procedure:

One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

From Days 6 to 28 post-coitum, inclusive

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

7 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels
In a dose range finding study in which dose levels of 7, 20 and 60 mg/kg were tested, slight toxicity was noted at 60 mg/kg (reduced maternal body weight gain and food consumption, reduced faeces production, and reduced fetal body weight). In the preceding tolerability study mortality occurred at 120 mg/kg. Based on these findings dose levels of 7, 20 and 60 mg/kg were selected for the main study.

Maternal examinations:

Mortality / Viability: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights: Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

Food consumption: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

Water consumption: Subjective appraisal was maintained during the study.

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Animals found dead or sacrificed before planned necropsy were subjected to relevant examinations of the ovaries and uterine horns.

Fetal examinations:

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (=60mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from all groups.

The specimens were archived in glycerin with bronopol as preservative.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Pre-implantation loss, post-implantation loss, viable fetuses affected/litter

Historical control data:

Yes, general pregnancy performance, fetal malformations and variations (attached)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Reduced faeces production was noted in all groups, including the control group, but a dose related increase in incidence and severity was seen in all groups treated with the test item.
Additional treatment-related findings at the highest dose level (60 mg/kg) consisted of piloerection in 3/22 animals and a lean appearance in 5/22 animals. At the lower dose levels (7 and 20 mg/kg) no additional toxicologically relevant clinical signs were noted.

In the low dose group, one female was noted with red staining of the vagina on Days 16 and 17 post-coitum. She and another females from this group had also red fluid on the manure tray on Days 25 and 28 post-coitum. As these findings were limited to the low dose group, and since these 2 females had normal litters, no toxicological significance was attached to this observation. Both findings are seen more often for rabbits in this type of study.

Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study, did not show any apparent dose-related trend, were limited to isolated cases and/or were also observed during the pre-treatment period. Therefore, these signs were considered to be of no toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 60 mg/kg four females were killed in extremis on Days 16 or 17 post-coitum. All females had a lean appearance and slightly to severely reduced production of faeces (which was pale in one animal). Additionally, two animals showed piloerection, one was lethargic and another one had a pale skin. All females showed severely reduced to no food intake, generally from postcoitum Day 6 onwards, resulting in significant body weight loss. Animals were affected with a body weight loss of 9-13% on Day 15 post-coitum.

At 20 mg/kg, one female died spontaneously on Day 25 post-coitum. This female showed no clinical signs of toxicity and her body weight development and food consumption were normal. Macroscopic findings in this female included lung lesions (many dark red foci, perforation(s) in the right caudal lobe) and dark red fluid in the thoracic cavity, both indicative for an oral gavage accident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain at 60 mg/kg was severely reduced from the start of treatment onwards, resulting in statistically significantly reduced mean body weights from post-coitum Day 12 onwards. Corrected body weight gain (for uterus) was also statistically significantly reduced
at 60 mg/kg (-10.5%, versus -3.3% in the control group). Body weight loss occurred in more than half of the animals, most severely in the females that were killed in extremis or delivered early. Body weight gain of the 60 mg/kg females that did not lose weight was generally at the lower end or slightly below the concurrent control range.

At 7 and 20 mg/kg, mean body weight gain was slightly (not statistically significantly) reduced from post-coitum Day 15 onwards. Changes as compared to the concurrent control group were relatively slight, reaching statistical significance on post-coitum Days 18
(20 mg/kg) and 21 (7 and 20 mg/kg) only. A few females of these groups occasionally showed slight body weight loss (generally between 1 and 5%, up to 9% in one female at 7 mg/kg). Corrected body weight gain (for uterus) at 7 and 20 mg/kg was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females treated at 60 mg/kg had statistically significantly reduced food consumption between Days 6-24 post-coitum (absolute values) and Days 6-21 post-coitum (relative to body weight). Changes were most pronounced in the second week of treatment (Days 12-15 post-coitum), when
mean food intake in the high dose group was approximately 60% lower as compared to the mean of the concurrent control group. Thereafter, food consumption at 60 mg/kg remained low but the mean differences from controls were smaller and no longer statistically significant. This could be explained by the lower food consumption of (a few) control females between Days 21-29 post-coitum (compared with their food consumption in the preceding periods), which resulted in lower control group mean values.

At 7 and 20 mg/kg, mean food consumption was slightly lower as compared to the concurrent control group, reaching statistical significance for the 20 mg/kg group from Day 12-15 postcoitum (absolute and relative) and the 7 mg/kg group from Day 18-21 post-coitum (absolute
only). As changes were relatively slight and in the absence of a clear dose-related trend, no toxicological relevance was attached to this finding.

In general, relatively large variations in food consumption occurred among individual animals of all groups (including controls). This is more often seen for rabbits of this age and strain which are housed and treated under the conditions in this study.

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only test item -related gross finding at necropsy was the emaciated appearance noted in two high-dose females treated at 60 mg/kg (related to the test item-induced growth retardation).

One female in the low dose group had agenesis of her right uterus horn. This was a congenital abnormality and by no means related to treatment that started after the animal had reached adolence.

The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show any apparent dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

The numbers of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and test groups were similar and in the range of normal biological variation.

There were 1, 2, 3 and 0 non-pregnant females in the control, 7, 20 and 60 mg/kg groups, respectively. All remaining females were pregnant. As in the high dose group four females were euthanized preterm, and in the mid dose group one female died due to an oral gavage accident, the number of litters available for fetal evaluation was 21, 20, 18 and 18, respectively. This includes the high dose female that had an early delivery on Day 29 post-coitum. She delivered 9 dead fetuses.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One female at dose 60 mg/kg b.w. had an early delivery on Day 29 post-coitum (the morning of planned necropsy). The female had a body weight loss of 21% and 23% before and after correction for gravid uterus weight, respectively. She delivered 9 dead fetuses.

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

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weight was adversely affected at 60 mg/kg as indicated by the significantly lower weights of male and female fetuses (both sexes combined: 34.5 gram at 60 mg/kg versus 41.7 gram in controls).

At 7 and 20 mg/kg, mean fetal body weights (males and females separately and both sexes combined) were unaffected by treatment. All values remained within the available historical range.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal body weight was adversely affected at 60 mg/kg as indicated by the significantly lower weights of male and female fetuses (both sexes combined: 34.5 gram at 60 mg/kg versus 41.7 gram in controls).

At 7 and 20 mg/kg, mean fetal body weights (males and females separately and both sexes combined) were unaffected by treatment. All values remained within the available historical range.

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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on external morphology following treatment up to 60 mg/kg.

The only external malformation observed in this study was noted in fetus at the high dose level (60 mg/kg). This fetus had a carpal flexure for which no skeletal cause was found.
Due to its single occurrence, this malformation was considered a chance finding.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related skeletal malformations were noted following treatment up to 60 mg/kg.

Skeletal malformations were observed in 2 (2), 4 (4), 1 (1) and 2 (2) fetuses (litters) in Groups 1, 2, 3 and 4, respectively. Both fetuses in Group 4 (60 mg/kg) had fused sternebrae, but as this sternal malformation was also noted in two control fetuses and two Group 2 (7 mg/kg) fetuses it was considered not to be treatment-related.

The other skeletal malformations in this study were a vertebral anomaly with or without associated rib anomaly in 2 Group 2 (7 mg/kg) fetuses and a rib anomaly in Group 3 (20 mg/kg) fetus. However, because of their incidence and group distribution these were considered chance findings.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment up to 60 mg/kg.

In Group 4 (60 mg/kg), four fetuses had a visceral malformation and in three of them the accessory lung lobe was missing. However, as this malformation also occurred in two control fetuses and was seen at a higher mean litter incidence in historical controls (1.7% versus 1.4% per litter), this was
considered not to be treatment related. The fourth fetus was delivered dead and appeared to have internal hydrocephaly at serial sectioning of the head, which was considered a chance finding because of its single occurrence.

Other viscerally malformed fetuses of treated dams were observed in Group 2 (7 mg/kg) and in Group 3 (20 mg/kg) Because of their single occurrence these malformations were considered chance findings. Apart from a control fetus with multiple malformations, there were no other visceral malformations observed in this study.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg, delayed fetal ossification (classified as skeletal variation) was noted. There were no test item-related skeletal variations following treatment at 7 and 20 mg/kg.

Among the skeletal variations, there were several ossification parameters indicating delayed fetal ossification in Group 4 (60 mg/kg). This was demonstrated by higher incidences compared to the controls for unossified metatarsals (14.2% versus 4.9% per litter), unossified hyoid bodies (7.1% versus 1.0% per litter), unossified tarsals (6.3% versus 0.4% per litter) and unossified pubis (2.0% versus 0.4% per litter). Only the increase of unossified hyoid body was statistically significant, but all the above mentioned incidences of Group 4 were higher than the historical control maximum values (10.1% for unossified metatarsals, 3.2% for unossified hyoid bodies, 2.1% for unossified tarsals and 0.8% for unossified pubis).
Therefore, it was considered that these signs of delayed fetal ossification in Group 4 were related to treatment. Moreover, the delayed fetal ossification in Group 4 was in line with the lower mean fetal body weights in that group (34.5 versus 41.7 grams in Group 1).

In Group 3 (20 mg/kg), it was noted that the mean litter incidences of unossified metatarsals (11.3% per litter), unossified tarsals (2.9% per litter) and unossified pubis (0.9% per litter) were above the respective historical control maximum values (10.1%, 2.1% and 0.8% per
litter) as well. However, in comparison with Group 4 (60 mg/kg), these values were only marginally higher than the maximum values and the Group 3 mean fetal body weights were about the same as those in the control group (39.6 versus 41.7 grams, respectively). Moreover, no statistical changes were found in comparison to the control group.
Therefore, these signs of delayed ossification in Group 3 were considered not to be toxicologically relevant.

Of the remaining skeletal variations, the finding of 13th full ribs showed a curious group distribution. Incidences were 30.9%, 56.1%, 44.9% and 53.8% in Groups 1, 2, 3 and 4, respectively, and statistical significance was reached for the higher values in Groups 2 and 4.
An explanation for this could not be given, but the distribution does not indicate a treatment relationship. Moreover, all incidences were within the historical control range (26.8 - 71.4% per litter) and a higher incidence for this finding is considered not have any detrimental effect. Therefore, the curious group distribution of 13th full ribs is considered to have occurred by chance.

The other skeletal variations that were noted occurred in the absence of a dose-related incidence trend, occurred infrequently or were observed in control fetuses only. Therefore, they were considered not to be related to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: skeletal variations at dose level of 60 mg/kg b.w.

Remarks on result:

other: effects related to maternal toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Conclusions:

Treatment of pregnant female New Zealand White rabbits by daily oral gavage with 1,4-Butenediol (B2D) from Day 6 to 28 post-coitum at doses of 7, 20 and 60 mg/kg revealed significant maternal toxicity at 60 mg/kg (mortality, clinical signs, lower body weights and reduced food consumption). Secondary to the maternal toxicity at the highest dose level, some developmental toxicity (reduced fetal body weight and delayed ossification) was observed at 60 mg/kg.

No developmental toxicity was observed at the lower dose levels tested (7 and 20 mg/kg) which were maternally non-toxic.

Based on these results, a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity of 20 mg/kg was derived.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: weanling rats
- Weight at study initiation: not specified
- Fasting period before study: not applied
- Housing: individually housed, except during mating and lactation, in wire mesh cages or plastic cages with corn-cob bedding
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Purina Rodent Chow, Ralston-Purina, St. Louis, MO.
- Water (e.g. ad libitum): tap water
- Acclimation period: All rats were acclimated to the laboratory for at least 10 days prior to initiating the study.

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

oral: gavage

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of mating period : 15 days (twice)
- Proof of pregnancy: by vaginal plugs or sperm-positive vaginal smears
- After successful mating each pregnant female was transferred to an individual plastic cage containing nesting material. Males were returned to their wire-mesh cages at the end of the mating period.
- The day on which evidence of copulation was observed was identified as Day 0 of gestation.

Duration of treatment / exposure:

The desired dose was administered orally in a volume of 10 ml/kg. Each generation was dosed a minimum of 80 days with maleic anhydride before its members were mated. Treatment began when F0 rats were 5 to 6 weeks and F1 animals were 22 days of age, and continued until the generation was terminated.

Frequency of treatment:

Daily

No. of animals per sex per dose:

F0: 10 males and 20 females

Control animals:

yes, concurrent vehicle

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment-related mortality in the Fo generation occurred primarily in the high-dose group. Mortality was observed at lower doses in the Fl generation without a dose-related pattern at these levels that suggested an effect related to treatment. Hence also in the Fl generation, mortality attributed to treatment occurred primarily at the high dose.

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Remarks:

maleic anhydride

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

55 mg/kg bw/day

Based on:

test mat.

Remarks:

maleic anhydride

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Litter size and pub survival.

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed publication of studies performed with scientifically sound methods.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: 12 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: wire mesh cages or plastic cages with corn-cob bedding. All animals were maintained in environmentally controlled rooms with 12-hr photoperiods
- Diet (e.g. ad libitum): Purina Rodent Chow, Ralston-Purina, St. Louis, MO
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 10 days

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

finely ground with a mortar and pestle and suspended in corn oil with the aid of a tissue homogenizer. All doses were prepared daily in corn oil in order to minimize problems with stability. A 1% (w:v) concentration was used to administer all doses.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

One male and one female were housed together for mating. The day of mating was determined by daily inspection for a copulatory plug or a sperm-positive vaginal smear. This day was designated Day 0 of gestation.

Duration of treatment / exposure:

from Day 6 through Day 15 of gestation

Frequency of treatment:

daily

Duration of test:

10 days

Control animals:

yes, concurrent vehicle

Details on study design:

Body weights were recorded at intervals during gestation.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Significant mortality above 140 mg/kg/d in a pilot.

Dose descriptor:

NOAEL

Effect level:

140 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

140 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 4

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Preliminary Study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analytical method: capillary gas chromatography with internal standard

Duration of treatment / exposure:

from day 6 to day 15 post coitum

Frequency of treatment:

daily

No. of animals per sex per dose:

5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female

Maternal examinations:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day #
- Organs examined:

OTHER: Hematology, clotting analyses, enzymes, blood chemistry, organ weights and necropsy findings

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

Fetal examinations:

- External examinations: Yes

Details on maternal toxic effects:

Maternal toxic effects:yes

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The oral administration of 2 -Butyne-1,4 -diol by gavage to pregnant rats caused no adverse effects on the fetuses but did cause the following substance-induced changes in the dams:

Test group 3 (60 mg/kg bw):

-statistically significantly decreased food consumption at the beginning of the treatment period (days 6 -8 p.c.)

-body weight loss at the beginning of the treatment period (days 6 -8 p.c.)

-statistically significant increase in total protein and albumin

-statistically significant decrease in thromboplastin time, serum-cholinesterase and triglycerides

-statistically significantly increased absolute and relative kidney and liver weights

Test group 2 (40 mg/kg bw)

-statistically significant increase in albumin and an increase in total protein as a trend

-statistically significant decrease in thromboplastin time and serum-cholinesterase

-statistically significantly increased absolute and relative liver weights

Test group 1 (20 mg/kg bw)

-statistically significant increase in albumin and an increase in total protein as a trend

-decrease in serum-cholinesterase as a trend

Executive summary:

From the results of the preliminary study, it can be seen that 2 -Butyne-1,4 -diol caused signs of maternal toxicity at 60 mg/kg body weight in the form of adverse effects on food consumption and body weight gain at the beginning of the treatment period, an impairment in liver function, which manifested itself in reduced clotting times, decreased cholinesterase activities, increased serum protein levels and increased absolute and relative liver weights. Furthermore, an increase in absolute and relative kidney weights was observed in the high-dose animals. Marginal indications of maternal toxicity occurred also at the intermediate (40 mg/kg body weight /day) and low dose (20 mg/kg body weight/day). Due to the stage of development of the implants on the day when the study was terminated the examination of the uterus content was very limited; taking this into consideration, no adverse effects on the fetuses occurred.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Species:

rabbit

Additional information

In the OECD 414 study, treatment of pregnant female New Zealand White rabbits by daily oral gavage with 1,4-Butenediol (B2D) from Day 6 to 28 post-coitum at doses of 7, 20 and 60 mg/kg revealed significant maternal toxicity at 60 mg/kg (mortality, clinical signs, lower body weights and reduced food consumption). Secondary to the maternal toxicity at the highest dose level, some developmental toxicity (reduced fetal body weight and delayed ossification) was observed at 60 mg/kg. No developmental toxicity was observed at the lower dose levels tested (7 and 20 mg/kg) which were maternally non-toxic. Based on these results, a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity of 20 mg/kg was derived.

In the OECD 422 Guideline study in rats, the number of implantation sites and, as a consequence the number of pups delivered, were reduced and the number of post-implantation losses and stillborns (indicating intrauterine embryo-fetolethality) had increased at 200 mg/kg bw/day. Under the conditions of this reproduction / developmental toxicity screening test, the NOAEL for embryonic survival and development is 60 mg/kg of bw/day.

Additionally, in the 2-generation reproductive toxicity studies in rats exposed to maleic anhydride (Short et al., 1986), no adverse effects on litter size and on pup survival were observed at doses up to 150 mg/kg/day in Fl litters or 55 mg/kg/day in F2 litters.

Although a few statistically significant changes in pup body weights were observed, they were too inconsistent to be considered

treatment-related. Microscopic examination of tissues from pups in the F2b litters revealed no treatment-related changes. Therefore,

treatment with up to 55 mg/kg/day for two generations had no adverse effects on pups.

Further supporting data are collected from a Prenatal Developmental Toxicity Study (OECD Guideline 414) with rats exposed to but-2-yne-1,4-diol. The maternal NOAEL was 40 mg/kg bw, while the NOAEL for the F1 was at 60 mg/kg bw.

In keeping with Regulation (EC) 1907/2006 Annex X Column 2 and for reasons of animal welfare, the dossier does not need additional results of a pre-natal developmental study since there are no serious concerns about the potential for adverse effects on fertility or development based on available data and read across data from analogue substances. The substance was tested both in rats (OECD 422) and rabbits (OECD 414). The NOAEL for systemic toxicity (20 mg/kg b.w.) is sufficiently protective for the developmental toxicity.

Justification for classification or non-classification

1,4 -Butenediol (B2D) should not be classified as a reproductive or developmental hazard under the EU CLP classification criteria (Regulation (EC) 1272/2008) on the basis of the findings observed in the GLP OECD 414 and OECD 422 guidelines study with rabbits and rats.