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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Frog embryo teratogenesis assay: Xenopus (FETAX) (Dawson, 1996)
In a frog embryo teratogenesis assay performed according to a standard guide of ASTM (E1439-91) using isobutyric acid as test substance, a LC50 of 7925 mg/L and an EC50 (malformation) of 2567 mg/L was determined.
Supp. subst. isobutanol
Andrological measures (Fail/Res. Triangle Inst., 1996)
In one study, three anthrological measures were investigated (stage - the cycle of seminiferous epithelium - frequency and normalcy, number and concentration of testicular spermatides, number and concentrations of epididymal spermatozoa; Res. Triangle Inst. 1996). The results are defective, as the testes were damaged (compressed and flattend by pressure) during transport. Observations were not significant or invalid due to the distortion of the three-dimensional structure of the testes.
Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from iso-butanol to iso-butyric acid information on toxicokinetics for isobutanol showing a readily absorbtion from the gastrointestinal as well as from the respiratory tract. Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid is formed (Saito 1975, Poet and Corley (OPP/ACC) 2004). Because the metabolization takes place rapidly it is scientifically relevant to assume that possible genotoxic effects will depend most likely on the metabolites (isobutyraldehyde and isobutyric acid) than on the original substance isobutanol

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals Isobutanol was investiagate as a pure compound without significant impurities. The target substance iso-butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.

3. ANALOGUE APPROACH JUSTIFICATION
For isobutyric acid, no study concerning reproductive toxicity could be identified. As substitute, data for isobutanol will be used based on following reasons (for detaild information on toxicokinetics/metabolism and justification of supporting substances see Section 7.1, endpoint summary).
 
After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic effects of isobutyric acid.
 
Effect levels derived for the supporting substance isobutanol will be converted to effect levels for isobutyric acid using the respective molecular weights. These values are used to evaluate the toxic potential of isobutyric acid.
 
Toxicity to reproduction has been evaluated in one valid study of high reliability, which is used as key study (OPP/ACC, 2003).
 
In a valid 2-generation reproduction inhalation study under GLP, isobutanol (99.9%) was administered to groups of male and female Crl:CD(SD)IGS BR rats (30/sex/dose) by whole body exposure at dose levels of 0, 500, 1000 and 2500 ppm (0, 1476, 2952, and 7380 mg/m³). The animals were exposed 6 hours per day 7 days per week from the commencement of the study (F0 generation) or postnatal day 28 (F1 generation) until sacrifice after weaning of the pups (F0 generation postnatal day 28, F1 generation postnatal day 21). For dams, exposure was discontinued after day 20 of gestation until lactation day 5.
 
F0 and F1 parental survival were unaffected by isobutanol exposure in all exposure groups. No exposure-related effects were observed on F0 and F1 reproductive performance, body weights, food consumption and food efficiency in males or females. Spermatogenic endpoints were unaffected by exposure to isobutanol in all F0 and F1 exposure groups. There were no exposure-related macroscopic findings or changes in mean organ weights in the F0 or F1 males and females in all treatment groups. Microscopic evaluation of the F0 and F1 males and females revealed no isobutanol-related histopathologic lesions, including for animals that failed to breed or produce a litter.
 
No treatment-related effects on primordial follicle counts and corpora lutea counts were observed in the F1 2500 ppm group females (only group examined).
 
F1 and F2 pup survival and the general physical condition of the pups were unaffected by exposure to isobutanol. No treatment-related effects on mean pup body weights were observed in F1 or F2 pups. In addition, no macroscopic findings were observed in F1 or F2 pups that were found dead or euthanized at the scheduled necropsy. There were no isobutanol-related changes in mean organ weights for the F1 or F2 pups and no effects were seen on the maturation of the F1 pups.
 
The NOAEC of isobutanol for parental systemic, reproductive and neonatal toxicity is 2500 ppm (7700 mg/m³, maximum dose applied) in males and in females of the F0, F1 and F2 generation (OPP/ACC, 2003).
 
Deduction of the NOAEC for isobutyric acid
The NOAEC for isobutyric acid will be calculated on basis of the NOAEC of isobutanol (OPP/ACC, 2003) using the mass concentration/m³ and the respective molecular weights (88.11 and 74.12).
 
The deduced NOAEC (parentel systemic, reproductive and neonatal toxicity) for isobutyric acid is ca. 9150 mg/L (2500 ppm).
Short description of key information: For isobutyric acid, no data on toxicity to reproduction could be located. To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure. In a 2-generation reproductive inhalation study in rats, isobutanol did not show any effects on fertility up to a concentration of 2500 ppm (7700 mg/m³) for the F0, F1 and F2 generation. The NOAEL for isobutyric acid (conversion using the respective molecular weight) is 9150 mg/m³.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC, USA
- Age at study initiation: (P) 7 wks; (F1) x wks
- Weight at study initiation: (P) Males: 200 - 305 g; Females: 150 - 199 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no data
- Housing: individyally in wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): Certified Rodent LabDiet® 5002, PMI Nutrition International, Inc.,
- Water (e.g. ad libitum): municipal water, reverse-osmosis-treated (on-site), delivered by an automatic watering system
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 24.0
- Humidity (%): 30.0 - 57,9
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2001-08-14 To: 2002-05-31
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: nitrogen
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass whole-body inhalation chamber, volume 2.0 m³
- Method of holding animals in test chamber: individually housed in stainless steel, wire-mesh cages
- Source and rate of air: chamber supply air provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source
- Method of conditioning air: no data
- System of generating particulates/aerosols: TS was delivered to heated bead columns (100°C and 170°C for the 2500 ppm atmosphere respectively) using different caiibrated metering pumps (models QG-6 and QG-20 for the 2500 ppm atmosphere respectively, FMI - Fluid Metering Inc., Oyster Bay, NY, USA)
- Temperature, humidity, pressure in air chamber: 19-26ºC, 30-56%, negative pressure
- Air flow rate: approx.450 L/minute
- Air change rate: 12 - 15 air changes/hour
- Method of particle size determination: no data
- Treatment of exhaust air: charcoal- and HEPA-filtration

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (Hewlett Packard, model 5890 series with FID detection)
- Samples taken from breathing zone: in a pre-study examination the chamber atmophere was proven to be nearly homogenous (deviations (means) for repeated meaurements between different sample points within the chamber compared to a reference point: -0.1 to -3.6%)

VEHICLE (if applicable)
- Justification for use and choice of vehicle: vaporization vehicle
- Composition of vehicle: pure nitrogen
- Concentration of test material in vehicle: 0.005, 0.009, 0.012%
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually in plastic a cage with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
9 to 10 samples were taken from the reference point in the chamber during each daily exposure (approx. every 35 min).
Duration of treatment / exposure:
males:
F0: from week 7 until euthanasia
F1: from postnatal day 28 until euthanasia
females:
F0: from week 7 throughout the breeding period and post-mating interval (excluding the period from gestation day 21 to lactation days 4) until the day prior to euthanasia
F1: from postnatal day 28 throughout mating, gestation and lactation (with the exception of gestation day 21 through lactation day 4) until the day prior to necropsy:
Frequency of treatment:
6 hours per day, 7 days a week
Details on study schedule:
- F1 parental animals not mated until 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 15-16 weeks
Remarks:
Doses / Concentrations:
f(0) generation: 643 ppm, 1131 ppmand 2791 ppm
Basis:
nominal conc.
overall mean
Remarks:
Doses / Concentrations:
f(0) generation: 500 ppm, 1008 ppm and 2522 ppm
Basis:
analytical conc.
overall mean
Remarks:
Doses / Concentrations:
f(1) generation: 668 ppm, 1140 ppm and 2867 ppm
Basis:
nominal conc.
overall mean
Remarks:
Doses / Concentrations:
f(1) generation: 494 ppm, 1012 ppm and 2521 ppm
Basis:
analytical conc.
overall mean
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: preliminary range finding study and results of preceding studies
- Rationale for animal assignment (if not random): random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
males: on days 0, 1, 4 and 7, weekly thereafter throughout the study and prior to the scheduled necropsy
females: on days 0, 1, 4 and 7 and weekly thereafter; after mating on gestation days 0, 4, 7, 11, 14 and 20 and on lactation days 1, 4, 7, 14 and 21 (in additon day 28 for F0); After weaning (lactation day 28 (F0) or 21 (F1)) weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:
Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of estrus for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, smearing was continued until termination of the mating period.
The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] beginning 21 days prior to initiation of the mating period and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 male parental generations: Yes
epididymis weight, sperm production rate, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
other: progressive motility
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, detailed clinical observation
other: developmental landmarks: balanopreputial separations, vaginal patency

GROSS EXAMINATION/GROSS NECROPSY OF DEAD PUPS:
yes, for external and internal abnormalities (pups dying after PND 4 and prior to weaning)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after postnatal day 28 (F0) and postnatal day 21 (F1) respectively
- Maternal animals: All surviving animals after postnatal day 28 (F0) and postnatal day 21 (F1) respectively
All animals were euthanized by isoflurane inhalation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.
At the time of necropsy, the following F0 and F1 parental tissues and organs were collected and were placed in 10% neutral-buffered formalin:
Adrenals (2), Aorta, Bone with marrow (sternebrae), Brain (forebrain, midbrain, hindbran), Coagulating gland, Eyes with optic nerve (2), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of two lobes), Lungs (including bronchi), Lymph node (mesenteric), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary, Prostate, Salivary gland (submandibular (2)), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymidesa (1)( fixed in Bouin’s solution ) and vas deferens, Thymus, Thyroids (with parathyroids if present (2)), Trachea, Urinary bladder (fixed by inflation with fixative), Uterus with cervix and vagina

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic evaluations were performed on the following tissues for 10 parental animals/sex/group from the control and high exposure groups and for all F0 and F1 adult animals that were euthanized in extremis or failed to breed, conceive or deliver offspring:
Adrenal glands (cortex and medulla), Brain, Cervix, Epididymis (right,caput, corpus and cauda) a), Kidneys, Liver, Ovaries b), Pituitary, Prostate, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testis (right) a), Thymus, Uterus (with oviducts), Vagina, All gross (internal) lesions c)
a) = PAS and hematoxylin staining were used for the right testis and epididymis. Transverse sections of 2 to 4 microns of the testes and
longitudinal sections of the epididymides were made.
b) = One section from each ovary from the selected F0 females were examined. Five sections from each ovary from all F1 females in the control and high exposure groups were examined. Quantitative histopathological evaluation from multiple sections (including enumeration of primordial follicles10,11 and corpora lutea) was conducted on all F1 females from the control and high exposure groups. Due to the size of the corpora lutea (much larger than primordial follicles), each corpus luteum was possibly sectioned and counted multiple times, resulting in a value that was larger thenwould be expected.
c = All gross lesions were examined from all F0 and F1 adults.

The following organs from all F0 and F1 parental animals euthanized at scheduled termination were weighed:
Adrenals, Brain, Epididymes a) (total and cauda), Kidneys, Liver, Ovaries, Pituitary , Prostate, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes a), Thymus gland, Uterus with oviducts and cervix
a) = These paired organs were weighed separately.
Except as noted, paired organs were weighed together. Absolute weights and organ-to-final-body-weight ratios were reported.



Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 28, 34, 36 or 37 days of age.
all F2 offspring were sacrificed at 21 days of age
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- complete necropsy performed on 1 pup/sex/litter (F0 and F1 generation), brrain, spleen and thymus gland weight were recorded
- All remaining non-selected F1 and F2 weanlings were euthanized by CO2 inhalation and necropsied on PND 28 and PND 21, respectively, with emphasis on developmental and reproductive system morphology

HISTOPATHOLOGY / ORGAN WEIGTHS
- All gross lesions from F1 and F2 weanlings were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded.
Statistics:
Statistical methods used
Two-tailed tests for minimum significance levels of 5%, comparing each test substance-exposed group to the control group by sex.
Parental mating and fertility indices were analyzed using the Chi-square test with Yates’ correction factor.
parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test or Student’s T-test was used to compare the test substance-exposed groups to the control group.
Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, the Mann-Whitney U-test was used to compare the test substance-exposed groups to the control group.
Histopathological findings in the test substance-exposed groups were compared to the control group using a two-tailed Fisher’s Exact test.





Reproductive indices:
Male and Female Mating Index (%) = (No. of Males (Females) with Evidence of Mating (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating) x 100
Female Fertility Index (%) = (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) x 100
Male Fertility Index (%) = (No. of Males Siring a Litter / Total No. of Males Used for Mating) x 100
Offspring viability indices:
Live Litter Size = Total Viable Pups Day 0 / No. Litters With Viable Pups Day 0
Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = (Σ (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. of Litters Per Group) x 100
Postnatal Survival for All Other Intervals (% Per Litter) = (Σ (Viable Pups Per Litter of Interval N/Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group) x 100 Where N = PND 0-1, 1-4 (Pre-Selection), 4 (Post-Selection)-7, 7-14, 14-21, 21-28 or 4
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test article-related effects (F0 and F1 generation)

OTHER FINDINGS (PARENTAL ANIMALS)
No test article-related effects on primordial follicle counts and corpora lutea counts (F1 generation)
Dose descriptor:
NOAEL
Effect level:
2 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: analytical 7444 mg/m3; overall effects; highest dose tested
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:
not examined
VIABILITY (OFFSPRING)
No test article-related effects (F0 and F1 generation)

CLINICAL SIGNS (OFFSPRING)
No test article-related effects (F0 and F1 generation)

BODY WEIGHT (OFFSPRING)
No test article-related effects (F0 and F1 generation)

SEXUAL MATURATION (OFFSPRING)
No test article-related effects (F1 generation)

ORGAN WEIGHTS (OFFSPRING)
No test article-related effects (F0 and F1 generation)

GROSS PATHOLOGY (OFFSPRING)
No test article-related effects (F0 and F1 generation)

HISTOPATHOLOGY (OFFSPRING)
Not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: analytical 7442 mg/m3; overal effects; highest dose tested
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
2 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: birth index; live birth index; litter size; pup weight; sex ratio; survival index; viability index
Reproductive effects observed:
not specified
Conclusions:
Isobutanol did no show any adverse effects on parental systemic, reproductive and neonatal toxicity when administered for two generations via whole-body inhalation at the highest dose applicated (2500 ppm ≜ 7400 mg/m³)
inhalation exposure to rats.
Executive summary:

In an 2-generation reproduction inhalation study isobutanol (99.9%) was administered to 30 rats (strain Crl:CD(SD)IGS BR)/sex/dose by whole body exposure at dose levels of 0, 500, 1000 and 2500 ppm (0, 1476, 2952, and 7380 mg/m³). The animals were exposed 6 hours per day 7 days per week from the commencement of the study (F0 generation ) or postnatal day 28 (F1 generation) until sacrifice after weaning of the pups (F0 generation postnatal day 28, F1 generation postnatal day 21). For dams, exposure was discontinued after day 20 of gestation until lactation day 5.

F0 and F1 parental survival were unaffected by isobutanol exposure in all exposure groups. No exposure-related effects were observed on F0 and F1 reproductive performance, body weights, food consumption and food efficiency in males or females. Spermatogenic endpoints were unaffected by exposure to isobutanol in all F0 and F1 exposure groups. There were no exposure-related macroscopic findings or changes in mean organ weights in the F0 or F1 males and females in all treatment groups. Microscopic evaluation of the F0 and F1 males and females revealed no isobutanol-related histopathologic lesions, including for animals that failed to breed or produce a litter.

No treatment-related effects on primordial follicle counts and corpora lutea counts were observed in the F1 2500 ppm group females (only group examined).

F1 and F2 pup survival and the general physical condition of the pups were unaffected by exposure to isobutanol. No treatment-related effects on mean pup body weights were observed in F1 or F2 pups. In addition, no macroscopic findings were observed in F1 or F2 pups that were found dead or euthanized at the scheduled necropsy. There were no isobutanol-related changes in mean organ weights for the F1 or F2 pups and no effects were seen on the maturation of the F1 pups.

The NOAEL of isobutanl for parental systemic, reproductive and neonatal toxicity is 2500 ppm (7380 mg/m³, maximum applied dose)  in males and in females of the F0, F1 and F2 generation (OPP/ACC 2003). 

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study (OPPTS 870.3800; OECD 416) in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
9 150 mg/m³
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For isobutyric acid, no study concerning reproductive toxicity could be identified. As substitute, data for isobutanol will be used based on following reasons (for detaild information on toxicokinetics/metabolism and justification of supporting substances see Section 7.1, endpoint summary).

 

After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic effects of isobutyric acid.

 

Effect levels derived for the supporting substance isobutanol will be converted to effect levels for isobutyric acid using the respective molecular weights. These values are used to evaluate the toxic potential of isobutyric acid.

Supporting substance: isobutanol

 

Toxicity to reproduction has been evaluated in one valid study of high reliability, which is used as key study (OPP/ACC, 2003).

 

In a valid 2-generation reproduction inhalation study under GLP, isobutanol (99.9%) was administered to groups of male and female Crl:CD(SD)IGS BR rats (30/sex/dose) by whole body exposure at dose levels of 0, 500, 1000 and 2500 ppm (0, 1476, 2952, and 7380 mg/m³). The animals were exposed 6 hours per day 7 days per week from the commencement of the study (F0 generation) or postnatal day 28 (F1 generation) until sacrifice after weaning of the pups (F0 generation postnatal day 28, F1 generation postnatal day 21). For dams, exposure was discontinued after day 20 of gestation until lactation day 5.

 

F0 and F1 parental survival were unaffected by isobutanol exposure in all exposure groups. No exposure-related effects were observed on F0 and F1 reproductive performance, body weights, food consumption and food efficiency in males or females. Spermatogenic endpoints were unaffected by exposure to isobutanol in all F0 and F1 exposure groups. There were no exposure-related macroscopic findings or changes in mean organ weights in the F0 or F1 males and females in all treatment groups. Microscopic evaluation of the F0 and F1 males and females revealed no isobutanol-related histopathologic lesions, including for animals that failed to breed or produce a litter.

 

No treatment-related effects on primordial follicle counts and corpora lutea counts were observed in the F1 2500 ppm group females (only group examined).

 

F1 and F2 pup survival and the general physical condition of the pups were unaffected by exposure to isobutanol. No treatment-related effects on mean pup body weights were observed in F1 or F2 pups. In addition, no macroscopic findings were observed in F1 or F2 pups that were found dead or euthanized at the scheduled necropsy. There were no isobutanol-related changes in mean organ weights for the F1 or F2 pups and no effects were seen on the maturation of the F1 pups.

 

The NOAEC of isobutanol for parental systemic, reproductive and neonatal toxicity is 2500 ppm (7700 mg/m³, maximum dose applied) in males and in females of the F0, F1 and F2 generation (OPP/ACC, 2003).

 

Deduction of the NOAEC for isobutyric acid

The NOAEC for isobutyric acid will be calculated on basis of the NOAEC of isobutanol (OPP/ACC, 2003) using the mass concentration/m³ and the respective molecular weights (88.11 and 74.12).

 

The deduced NOAEC (parentel systemic, reproductive and neonatal toxicity) for isobutyric acid is ca. 9150 mg/L (2500 ppm).


Short description of key information:
For isobutyric acid, no data on toxicity to reproduction could be located.
To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure.
In a 2-generation reproductive inhalation study in rats, isobutanol did not show any effects on fertility up to a concentration of 2500 ppm (7700 mg/m³) for the F0, F1 and F2 generation.
The NOAEL for isobutyric acid (conversion using the respective molecular weight) is 9150 mg/m³.

Effects on developmental toxicity

Description of key information
For isobutyric acid, no data on developmental toxicity/teratogenicity could be located.
To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure.
In a developmental toxicity inhalation study in rats, isobutanol did not exhibit treatment-related effects on maternal toxicity up to the highest dose applied (10,000 mg/m³). Similarly, no signs of treatment-related embryo-/fetotoxicity or teratogenicity were observed. Thus, the maternal and developmental NOAEC in rats was 10,000 mg/m³ (Klimisch/BASF, 1990).
For rabbits, the maternal NOAEC was determined to be 2500 mg/m³ while the developmental/teratogenic NOAEC was the same as for rats (10,000 mg/m³; Klimisch/BASF, 1990).
The developmental NOAEC for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from iso-butanol to iso-butyric acid information on toxicokinetics for isobutanol showing a readily absorbtion from the gastrointestinal as well as from the respiratory tract. Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid is formed (Saito 1975, Poet and Corley (OPP/ACC) 2004). Because the metabolization takes place rapidly it is scientifically relevant to assume that possible genotoxic effects will depend most likely on the metabolites (isobutyraldehyde and isobutyric acid) than on the original substance isobutanol

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals Isobutanol was investiagate as a pure compound without significant impurities. The target substance iso-butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.


3. ANALOGUE APPROACH JUSTIFICATION

For isobutyric acid, no data on developmental toxicity/teratogenicity could be located. To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure. In a developmental toxicity inhalation study in rats, isobutanol did not exhibit treatment-related effects on maternal toxicity up to the highest dose applied (10,000 mg/m³). Similarly, no signs of treatment-related embryo-/fetotoxicity or teratogenicity were observed. Thus, the maternal and developmental NOAEC in rats was 10,000 mg/m³ (Klimisch/BASF, 1990). For rabbits, the maternal NOAEC was determined to be 2500 mg/m³ while the developmental/teratogenic NOAEC was the same as for rats (10,000 mg/m³; Klimisch/BASF, 1990). The developmental NOAEC for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.

After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic effects of isobutyric acid.

Developmental toxicity/teratogenicity has been evaluated in one valid study of high reliability, which is used as key study (Klimisch/BASF 1990). This study was performed using two species (rat and rabbit).
Data are presented for rats only because results for developmental toxicity are not different for the two species and rat is the species preferred according to OECD TG 414. Species differences were found for maternal toxicity (NOAEC 10000 mg/m³ for rats and 2500 mg/m³ for rabbits).
The developmental toxicity of isobutanol (purity 99.8%) was tested in an inhalation study in pregnant wistar rats. 25 female animals were exposed to vapors of isobutanol at concentrations of 0, 0.5, 2.5 and 10 mg/L for 6 hours/day from day 6 through 15 of gestation. All animals were killed on day 20 of gestation. The fetuses were removed and examined for compound-related effects.
There were no treatment-related effects on maternal toxicity (no mortality, no significant differences between controls and treated groups in clinical signs, body weight development, and gross pathology) even at the highest dose.
The maternal NOAEC in rats is 10 mg/L.

The fetuses did not exhibit any signs of treatment-related embryo-/fetotoxicity or teratogenic effects.
The developmental NOAEC in rats is 10 mg/L.

(Klimisch/BASF, 1990)
The following information is taken into account for any hazard / risk assessment:
For isobutyric acid, no data on developmental toxicity/teratogenicity could be located.
To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure.
In a developmental toxicity inhalation study in rats, isobutanol did not exhibit treatment-related effects on maternal toxicity up to the highest dose applied (10,000 mg/m³). Similarly, no signs of treatment-related embryo-/fetotoxicity or teratogenicity were observed. Thus, the maternal and developmental NOAEC in rats was 10,000 mg/m³ (Klimisch/BASF, 1990).
For rabbits, the maternal NOAEC was determined to be 2500 mg/m³ while the developmental/teratogenic NOAEC was the same as for rats (10,000 mg/m³; Klimisch/BASF, 1990).
The developmental NOAEC for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.
Value used for CSA (route: inhalation): NOAEC: 11890 mg/m³
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 24 - 29 weeks
- Weight at study initiation: 2.5 - 2.7 kg
- Fasting period before study: no data
- Housing: individually in wire-mesh cages, type K 300/8 (EBECO, Becker and Co., Castrop Rauxel, Germany), air conditioned rooms
- Diet (e.g. ad libitum): KLIBA rabbit laboratory diet 24-341-4, 10 mm pellets (Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: horizontal-flow whole-body exposure chamber (glas/steel construction, volume 1.1.m³ (BASF AG, Ludwigshafen, Germany)
- Method of holding animals in test chamber: individually in wire cages
- Source and rate of air: clean air
- Method of conditioning air: no data
- System of generating particulates/aerosols: evaporator maintained at 50-70°C, TS was delivered by a continuously operating pump
- Temperature, humidity, pressure in air chamber: 21-24°C, 49-64%, minimal negative pressure
- Air flow rate: 275 L/minute
- Air change rate: 15 air changes per hour
- Method of particle size determination: no particle size determination
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (Hewlett-Packard GC, Model 5840 A with FID detector)
- Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the inhalation atmosphere were analyzed hourly during exposure.
Mean concentrations ± SD measured in inhalation chamber: 0.50 ± 0.010 mg/L, 2.51 ± 0.091 mg/L, 10.00 ± 0.370 mg/L

Details on mating procedure:
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: day of insemination referred to as day 0 of pregnancy, the following day was defined as day 1 of pregnancy or postinsemination (pi) respectively
Duration of treatment / exposure:
day 7 - 19 of pregnancy (pi)
Frequency of treatment:
6 hours / day
Duration of test:
29 days
Remarks:
Doses / Concentrations:
0.5; 2.5; 10 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
15 female rabbits per group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: preliminary range-finding study
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 3, 7, an from then on at 3-day intervals until day 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 by intravenous application of pentobarbitone
- Organs examined: uterus and ovaries

OTHER: Yes
- gross pathology for all animals
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: all per litter
- Soft tissue examinations: Yes: all per litter; fixation in Bouin's solution according to the method of Barrow and Taylor (1969)
- Skeletal examinations: Yes: all per litter; fixation in ethyl acohol and staining according to a modified method of Dawson (1926)
- Head examinations: Yes: No data
Statistics:
The Dunnett test (Dunnett, 1955, 1964) was used to statistically compare body weight, body weight changes, the number of corpora lutea, implants, resorptions, live fetuses, and pre- or postimplantation losses. The Fisher's exact test (Dixon, 1981) was used for evaluating the conception rate, maternal mortality, and all fetal findings.
Historical control data:
Historical control data were used to further evaluate findings of uncertain relevance.

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One rabbit of the 0.5 mg/L group was killed on day 21 of gestation due to abortion. One rabbit exposed to 2.5 mg/L was found dead on day 24 of gestation. Both cases were considered not to be related to isobutanol inhalation.

10 mg/L group
Slight retardation of body weight gain of the dams, especially during the first of the exposure periods.
Indication of an irritant eye effect (reddish, lid closure, slight discharge)

2.5 mg/L and 0.5 mg/L group
no substance-related effects were observed.

Gross-pathologiy examination revealed no effects that could be attributed to the exposure with isobutanol.

Dose descriptor:
NOAEL
Effect level:
2.97 mg/L air
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: calculatated via mole weight ratio to isobutyric acid
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
All treatment groups
The fetuses did not showed any substance-related effects (no indications of any embryo-/fetotoxicity or any teratogenic effect).

The uterine weights of the treated animals were not significantly different from the controls. Compound related effects occured neither for conception rate, mean number of corpora lutea, and implantation sites nor in the values calculated for the pre- and postimplantation loss and the number of resorptions as well as viable fetuses.
Dose descriptor:
NOAEL
Effect level:
11.89 mg/L air
Basis for effect level:
other: fetotoxicity
Remarks on result:
other: calculatated via mole weight ratio to isobutyric acid
Dose descriptor:
NOAEL
Effect level:
11.89 mg/L air
Basis for effect level:
other: embryotoxicity
Remarks on result:
other: calculatated via mole weight ratio to isobutyric acid
Dose descriptor:
NOAEL
Effect level:
11.89 mg/L air
Basis for effect level:
other: teratogenicity
Remarks on result:
other: calculatated via mole weight ratio to isobutyric acid
Abnormalities:
not specified
Developmental effects observed:
not specified
Table 1     Data on Reproduction, Maternal Body Weight Change and Uterine Weight

Dose [mg/L]

0 (sham)

0.5

2.5

10.0

Number of animals

15

15

15

15

Number of dams (pregnant animals)

15

15

14a,b

14a

Corpora lutea/dam

8.4

8.6

7.7

7.6

Implants/dam

7.5

6.7

6.5

7.0

Live fetuses/dam

7.0 (1.20)

6.2 (1.72)

6.0 (2.20)

7.1 (1.32)

Dead implants/dam

0.5

0.5

0.5

0.4

Sex ratio (male / female

48.6 / 51.4

51.7 / 48.3

44.9 / 55.1

50.0 / 50.0

Mean fetal weight [g]

39.0 (3.74)

39.7 (4.01)

41.0 (3.46)

39.4 (1.61)

Mean placental weight [g]

4.3 (0.48)

4.6 (0.56)

4.8* (0.66)

4.4 (0.40)

Mean corrected maternal body weight change [g] **

-71.9 (65.77)

-45.3 (63.91)

-47.1 (41.43)

-67.0 (77.51)

Intact uterine weight [g]

367.0 (52.48)

329.3 (69.99)

330.1 (110.81)

344.3 (111.43)

Numbers in parentheses indicate standard deviations

* significantly different from control, p < 0.05

** corrected maternal body weight change = (body weight on day 20 of pregnancy) - (body weight on day 6 of pregnancy) - (uterine weight)a    one not pregnant animalb    one animal aborted

Table 2               Fetal Anomalies

Dose [mg/L]

0 (sham)

0.5

2.5

10

Number of litters evaluated

15

14

13

13

Number of fetuses evaluated

105

87

78

92

Total fetal external malformations

Fetal incidence

0

0

0

0

Litter incidence

0

0

0

0

Total fetal soft tissue malformation

Fetal incidence

0

0

0

0

Litter incidence

0

0

0

0

Total fetal soft tissue variations

Fetal incidence

21 (20.0)

13 (14.9)

17 (21.8)

30 (32.6)

Litter incidence

11 (73.3)

9 (64.3)

7 (53.8)

12 (92.3)

Total fetal skeletal malformations

Fetal incidence

2 (1.9)

1 (1.1)

0

1 (1.1)

Litter incidence

2 (13.3)

1 (7.1)

0

1 (7.7)

Total fetal skeletal variations

Fetal incidence

9 (8.6)

14 (16.1)

8 (10.3)

16 (17.4)

Litter incidence

5 (33.3)

10 (71.4)

5 (38.5)

8 (61.5)

Numbers in parentheses indicate percentage of fetuses/litters affected

* significantly different from control; p < 0.05

** significantly different from control; p < 0.01

The examination of external changes showed no malformations or variations.

For fetal soft tissue no malformations were observed.

Variations were seen in all groups including controls. An increased incidence of separated origin of carotids in the 10 mg/L group was statistically significant (p < 0.01). But historical control data showed that the incidence of fetuses showing this variation was in the range of biological variation. The significantly increased occurence of this variation at the highest concentration was therefore assessed to be incidental.

The skeletal examination revealed various malformations of the sternebrae and/or the vertrebral column. Variations seen in skull, the vertrebal column, the sternum and the ribs were found in all groups without apparent concentration relationships or statistically significant differences between the groups.

Any observed differences between groups were within the range of biological variation and/or occurred without a clear concentration dependency.

Conclusions:
A slight maternal toxicity was seen at the highest exposure (10 mg/L, borderline concentration) in this inhalation study. No signs of developmental toxicity or teratogenicity were noted even with the highest exposure concentration.
Executive summary:

The developmental toxicity of isobutanol (purity 99.8%) was tested in an inhalation study in pregnant Himalayan rabbits. 15 female animals were exposed to vapors of isobutanol at concentrations of 0, 0.5, 2.5 and 10 mg/L for 6 hours/day from day 7 through 19 of gestation. All animals were killed on day 29 of gestation. The fetuses were removed and examined for compound-related effects.

In the high concentration exposure group (10 mg/L) retardation in body weight change was observed especially during the first phase of the exposure. This is an indication of maternal toxicity. The exposure to 10 mg/L seems to be a borderline concentration for causing maternal toxicity. Thus the maternal NOAEL is 2.5 mg/L . 

The maternal NOAEL for isobutyric acid (conversion using the respective molecular weight) is 2.97 mg/m³.

The fetuses did not exhibit any signs of treatment-related embryo-/fetotoxicity or teratogenic effects at all exposure levels. The developmental NOAEL is 10 mg/L.

(Klimisch 1990)

The developmental NOAEL for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.

This inhalation developmental toxicity study in the rabbit is classified acceptable and satisfies the guideline requirements for a developmental toxicity study (OECD 414).

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from iso-butanol to iso-butyric acid information on toxicokinetics for isobutanol showing a readily absorbtion from the gastrointestinal as well as from the respiratory tract. Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid is formed (Saito 1975, Poet and Corley (OPP/ACC) 2004). Because the metabolization takes place rapidly it is scientifically relevant to assume that possible genotoxic effects will depend most likely on the metabolites (isobutyraldehyde and isobutyric acid) than on the original substance isobutanol

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals Isobutanol was investiagate as a pure compound without significant impurities. The target substance iso-butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.


3. ANALOGUE APPROACH JUSTIFICATION

For isobutyric acid, no data on developmental toxicity/teratogenicity could be located. To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure. In a developmental toxicity inhalation study in rats, isobutanol did not exhibit treatment-related effects on maternal toxicity up to the highest dose applied (10,000 mg/m³). Similarly, no signs of treatment-related embryo-/fetotoxicity or teratogenicity were observed. Thus, the maternal and developmental NOAEC in rats was 10,000 mg/m³ (Klimisch/BASF, 1990). For rabbits, the maternal NOAEC was determined to be 2500 mg/m³ while the developmental/teratogenic NOAEC was the same as for rats (10,000 mg/m³; Klimisch/BASF, 1990). The developmental NOAEC for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.

After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic effects of isobutyric acid.

Developmental toxicity/teratogenicity has been evaluated in one valid study of high reliability, which is used as key study (Klimisch/BASF 1990). This study was performed using two species (rat and rabbit).
Data are presented for rats only because results for developmental toxicity are not different for the two species and rat is the species preferred according to OECD TG 414. Species differences were found for maternal toxicity (NOAEC 10000 mg/m³ for rats and 2500 mg/m³ for rabbits).
The developmental toxicity of isobutanol (purity 99.8%) was tested in an inhalation study in pregnant wistar rats. 25 female animals were exposed to vapors of isobutanol at concentrations of 0, 0.5, 2.5 and 10 mg/L for 6 hours/day from day 6 through 15 of gestation. All animals were killed on day 20 of gestation. The fetuses were removed and examined for compound-related effects.
There were no treatment-related effects on maternal toxicity (no mortality, no significant differences between controls and treated groups in clinical signs, body weight development, and gross pathology) even at the highest dose.
The maternal NOAEC in rats is 10 mg/L.

The fetuses did not exhibit any signs of treatment-related embryo-/fetotoxicity or teratogenic effects.
The developmental NOAEC in rats is 10 mg/L.

(Klimisch/BASF, 1990)
The following information is taken into account for any hazard / risk assessment:
For isobutyric acid, no data on developmental toxicity/teratogenicity could be located.
To compensate for this lack of data, information resulting from isobutanol as supporting substances will be used as substitute. Data are available for inhalation exposure.
In a developmental toxicity inhalation study in rats, isobutanol did not exhibit treatment-related effects on maternal toxicity up to the highest dose applied (10,000 mg/m³). Similarly, no signs of treatment-related embryo-/fetotoxicity or teratogenicity were observed. Thus, the maternal and developmental NOAEC in rats was 10,000 mg/m³ (Klimisch/BASF, 1990).
For rabbits, the maternal NOAEC was determined to be 2500 mg/m³ while the developmental/teratogenic NOAEC was the same as for rats (10,000 mg/m³; Klimisch/BASF, 1990).
The developmental NOAEC for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.
Value used for CSA (route: inhalation): NOAEC: 11890 mg/m³
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: mean body weight approx. 216 g
- Fasting period before study: no data
- Housing: individually in wire-mesh cages, type DK III (EBECO, Becker and Co., Castrop Rauxel, Germany), air conditioned rooms
- Diet (e.g. ad libitum): KLIBA rat/mouse laboratory diet 24-343-4, 10 mm pellets (Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: horizontal-flow whole-body exposure chamber (glas/steel construction, volume 1.1.m³ (BASF AG, Ludwigshafen, Germany)
- Method of holding animals in test chamber: individually in wire cages
- Source and rate of air: clean air
- Method of conditioning air: no data
- System of generating particulates/aerosols: evaporator maintained at 50-70°C, TS was delivered by a continuously operating pump
- Temperature, humidity, pressure in air chamber: 21-24°C, 49-64%, minimal negative pressure
- Air flow rate: 275 L/Minute
- Air change rate: 15 air changes per hour
- Method of particle size determination: no particle size determination
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (Hewlett-Packard GC, Model 5840 A with FID detector)
- Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the inhalation atmosphere were analyzed hourly during exposure.
Mean concentrations ± SD measured in inhalation chamber: 0.49 ± 0.012 mg/L, 2.50 ± 0.084 mg/L, 10.10 ± 0.330 mg/L
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/4, no further data on mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy or postcoitum (pc) respectively
Duration of treatment / exposure:
day 6- 15 day of pregnancy
Frequency of treatment:
6 hours / day
Duration of test:
20 days
Dose / conc.:
0.5 mg/L air (nominal)
Dose / conc.:
2.5 mg/L air (nominal)
Dose / conc.:
10 mg/L air (nominal)
No. of animals per sex per dose:
25 females per group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: preliminary range-finding study
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 3, 6, an from then on at 3-day intervals until day 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries

OTHER: Yes
- gross pathology for all animals
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: all per litter
- Soft tissue examinations: Yes: half per litter; fixation in Bouin's solution according to the method of Barrow and Taylor (1969)
- Skeletal examinations: Yes: half per litter; fixation in ethyl acohol and staining according to a modified method of Dawson (1926)
- Head examinations: No data
Statistics:
The Dunnett test (Dunnett, 1955, 1964) was used to statistically compare body weight, body weight changes, the number of corpora lutea, implants, resorptions, live fetuses, and pre- or postimplantation losses. The Fisher's exact test (Dixon, 1981) was used for evaluating the conception rate, maternal mortality, and all fetal findings.
Historical control data:
Historical control data were used to further evaluate findings of low significance.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No differences in body weight/body weight gain were observed between controls and treated groups.
In addition no clinical signs of toxicity were seen.
Gross-pathologiy examination revealed no effects that could be attributed to the exposure with isobutanol.
Key result
Dose descriptor:
NOAEL
Effect level:
11 890 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The fetuses did not showed any substance-related effects (no indications of any embryo-/fetotoxicity or any teratogenic effect).

The uterine weights of the treated animals were not significantly different from the controls. Compound related effects occured neither for conception rate, mean number of corpora lutea, and implantation sites nor in the values calculated for the pre- and postimplantation loss and the number of resorptions as well as viable fetuses.
Dose descriptor:
NOAEL
Effect level:
11.89 mg/L air
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
11.89 mg/L air
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
11.89 mg/L air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
In no test group (0.5 mg/l - 10 mg/l) substance-related effects neither on the dams nor on the fetuses were obtained. There were no indications of substance-related maternal toxicity and embryo-/fetotoxicity or any teratogenic effect.
    
Table 1     Data on Reproduction, Maternal Body Weight Change and Uterine Weight

Dose [mg/L]

0 (sham)

0.5

2.5

10.0

Number of animals

25

25

25

25

Number of dams (pregnant animals)

21

23

23

19

Corpora lutea/dam

15.2

15.5

15.3

14.5

Implants/dam

13.6

14.6

13.1

13.8

Live fetuses/dam

12.3 (2.89)

13.7 (1.99)

12.3 (3.35)

13.2 (1.86)

Dead implants/dam

1.2

1.0

0.8

0.6

Sex ratio (male / female

53.3 / 46.7

48.7 / 51.3

53.0 / 47.0

50.8 / 49.2

Mean fetal weight [g]

3.9 (0.29)

3.9 (0.28)

3.9 (0.21)

3.9 (0.15)

Mean placental weight [g]

0.46 (0.07)

0.43 (0.06)

0.45 (0.06)

0.46 (0.05)

Mean corrected maternal body weight change [g] *

47.7 (9.21)

50.3 (6.86)

50.4 (8.25)

50.9 (6.96)

Intact uterine weight [g]

71 (15.28)

78.1 (10.12)

70.0 (17.36)

75.6 (11.59)

Numbers in parentheses indicate standard deviations

* corrected maternal body weight change = (body weight on day 20 of pregnancy) - (body weight on day 6 of pregnancy) - (uterine weight)

Tabelle 2 Fetal Anomalies


Dose [mg/L]

0 (sham)

0.5

2.5

10

Number of litters evaluated

21

23

23

19

Number of fetuses evaluated

124

164

149

128

Total fetal external malformations

Fetal incidence

0

0

0

2 (0.8)

Litter incidence

0

0

0

2 (10.5)

Total fetal soft tissue malformation

Fetal incidence

0

0

0

1 (0.8)

Litter incidence

0

0

0

1 (5.3)

Total fetal soft tissue variations

Fetal incidence

60 (48.0)

47** (31.2)

47* (35.1)

49 (40.2)

Litter incidence

19 (90.5)

18 (78.3)

20 (87.0)

17 (89.5)

Total fetal skeletal malformations

Fetal incidence

5 (3.7)

9 (5.5)

2 (1.3)

1 (0.8)

Litter incidence

5 (23.8)

7 (30.4)

2 (8.7)

1 (5.3)

Total fetal skeletal variations

Fetal incidence

59 (44.0)

75 (45.7)

72 (48.3)

51 (39.8)

Litter incidence

19 (90.5)

22 (95.7)

22 (95.7)

18 (94.7)

Numbers in parentheses indicate percentage of fetuses/litters affected

* significantly different from control; p < 0.05

** significantly different from control; p < 0.01

The external changes showed two types of malformations. Anasarca was observed in two fetuses. In addition, one showed a cleft palate.

The only soft tissue malformation was dilatation of both ventricles (globular shaped heart).

Variations were seen in all groups including controls. They occurred independent of exposure or concentration.

The skeletal examination revealed various malformations of the sternebrae and/or the vertrebral column. Variations seen in the ribs and the sternum were found in all groups.

Any observed differences between groups were within the range of biological variation and/or occurred without a clear concentration dependency.

Conclusions:
No signs of maternal and developmental toxicity or teratogenicity were noted in this inhalation study, where the maximum exposure concentration was 10 mg/L (correspond to 11.890 mg/l for isobutyric acid)
Executive summary:

The developmental toxicity of isobutanol (purity 99.8%) was tested in an inhalation study in pregnant wistar rats. 25 female animals were exposed to vapors of isobutanol at concentrations of 0, 0.5, 2.5 and 10 mg/L for 6 hours/day from day 6 through 15 of gestation. All animals were killed on day 20 of gestation. The fetuses were removed and examined for compound-related effects.

There were no treatment-related effects on maternal toxicity (no mortality, no significant differences between controls and treated groups in clinical signs, body weight development, and gross pathology) even at the highest dose. The maternal NOAEL is 10 mg/L . 

The maternal NOAEL for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.

The fetuses did not exhibit any signs of treatment-related embryo-/fetotoxicity or teratogenic effects. The developmental NOAEL is 10 mg/L.

(Klimisch 1990).

The developmental NOAEC for isobutyric acid (conversion using the respective molecular weight) is 11,890 mg/m³.

This inhalation developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirements for a developmental toxicity study (OECD 414).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
11 890 mg/m³
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

For isobutyric acid, no study concerning developmental toxicity/teratogenicity could be identified. As substitute, data for isobutanol will be used based on following reasons.

 

After administration, isobutanol will rapidly be metabolized in vivo to isobutyraldehyde by alcohol dehydrogenases and subsequently to isobutyric acid by aldehyde dehydrogenases. In the course of metabolic transformation of isobutanol, isobutyric acid is generated rapidly and predominantly as intermediary metabolite. Thus, it is justified to use isobutanol as supporting substance in the evaluation of the systemic effects of isobutyric acid.

 

Effect levels derived for the supporting substance isobutanol will be converted to effect levels for isobutyric acid using the respective molecular weights. These values are used to evaluate the toxic potential of isobutyric acid.

 

Supporting substance: isobutanol

 

Developmental toxicity/teratogenicity has been evaluated in one valid study of high reliability, which is used as key study (Klimisch/BASF 1990). This study was performed using two species (rat and rabbit).

 

Data are presented for rats only because results for developmental toxicity are not different for the two species and rat is the species preferred according to OECD TG 414. Species differences were found for maternal toxicity (NOAEC 10000 mg/m³ for rats and 2500 mg/m³ for rabbits).

 

The developmental toxicity of isobutanol (purity 99.8%) was tested in an inhalation study in pregnant wistar rats. 25 female animals were exposed to vapors of isobutanol at concentrations of 0, 0.5, 2.5 and 10 mg/L for 6 hours/day from day 6 through 15 of gestation. All animals were killed on day 20 of gestation. The fetuses were removed and examined for compound-related effects.

 

There were no treatment-related effects on maternal toxicity (no mortality, no significant differences between controls and treated groups in clinical signs, body weight development, and gross pathology) even at the highest dose.

 

The maternal NOAEC in rats is 10 mg/L.

 

The fetuses did not exhibit any signs of treatment-related embryo-/fetotoxicity or teratogenic effects.

 

The developmental NOAEC in rats is 10 mg/L.

 

(Klimisch/BASF, 1990)

Toxicity to reproduction: other studies

Additional information

Frog embryo teratogenesis assay: Xenopus (FETAX) (Dawson, 1996)

In a frog embryo teratogenesis assay performed according to a standard guide of ASTM (E1439-91) using isobutyric acid as test substance, a LC50 of 7925 mg/L and an EC50 (malformation) of 2567 mg/L were determined. The DHI (Developmental Hazard Index) calculated to 3.1 indicating that the effective dose of isobutyric acid causing malformation was about 3-fold lower than the dose causing lethality. In general, the toxicity of isobutyric acid on frog embryos observed in the present test is relatively low.

 

The test system used is not comparable to in vivo developmental toxicity tests with mammals. Exposure, intake, distribution and metabolism do not match. It may be used for screening. But to decide under REACH about developmental toxicity effects of a test substance in humans, test procedures laid down in OECD or EU test guidelines on developmental toxicity have to be followed.

Justification for classification or non-classification

Based on read across results from the supporting substance Isobutanol, Isobutyric acid has not to be classified for reproductive toxicity.

Additional information