Isobutyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 AUG 2001 to 13 NOV 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

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:

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:

NOAEC

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

Dose descriptor:

NOAEC

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

Dose descriptor:

NOAEC

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

Read-across justification: for details please see read-across report in section 13.

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) to rats.

Executive summary:

In a 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.

Reference 2

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Data on toxicokinetics and metabolism, acute inhalation toxicity, skin, eye and respiratory irritation, inhalation repeated dose toxicity, neurotoxic effects and bacterial gene mutation can be read-across from n-butyl acetate to iso-butyl acetate, because the substances are structurally very similar and possess similar physicochemical properties.
Data on bacterial or mammalian cell gene mutation, as well as for effects after repeated exposure (i.e. oral and inhalation repeated dose toxicity, 2-generation Toxicity study, developmental toxicity studies) can be read-across from isobutanol to isobutyl acetate, because isobutyl acetate is rapidly hydrolysed to isobutanol and acetic acid.
Data on repeated oral dose toxicity can be read-across from isobutyl isobutyrate to isobutyl acetate, because isobutyl isobutyrate is rapidly hydrolysed to isobutanol and isobutyric acid. Isobutyric acid itself is a metabolite of isobutanol. Thus isobutyl isobutyrate shares isobutanol as common intermediate with isobutyl acetate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read-across to cover end point requirements is performed only from studies with pure substance. The analytical purities reported are > 98 % in case of isobutyl isobutyrate or even ≥ 99 % in case of isobutanol and n-butyl acetate.
Therefore no relevant impurities are included and thus no consequences on the relevant endpoints are to be expected.
3. ANALOGUE APPROACH JUSTIFICATION
Read-across justification 1:
Data from n-butyl acetate to isobutyl acetate can be read-across, because the source substance is structurally very similar to isobutyl acetate. Both compounds are only different in the terminal methyl group position of the butyl alcohol part of the molecule. Physical and chemical properties are only slightly influenced by this difference. Physical and chemical reactivity will basically be the same and only differ gradually. Relevant physico-chemical properties like e.g. water solubility, partition coefficient, and vapour pressure are very similar as well as acute toxicity data (details see data matrix in section 1.5). Both substances are rapidly (within minutes) hydrolysed to acetic acid and the respective alcohols (see below).
Therefore read-across of toxicity data for human health endpoints is considered justified.
Read-across justification 2:
After being absorbed the parent compound isobutyl acetate is hydrolysed rapidly to metabolites isobutanol and acetic acid (by ubiquitous esterases in various tissues including liver and lung). Isobutanol is further oxidised to isobutyraldehyde and subsequently to isobutyric acid, whereas the acetate is introduced in the intermediary metabolic pathway (acetyl coenzyme A) or excreted. There will be no further discussion on this endogenous metabolite. As stated in IUCLID section 7.1.1 on Toxicokinetics, metabolism and distribution data verify this assumption on metabolic pathway for isobutyl acetate and the supporting substance n-butyl acetate (cf. IUCLID section 7.1.1 study records OPP/ACC, 2004 and Supp. subst.: n-butyl acetate; Teeguarden, 2005). In these experiments peak levels of parent compound are reached after approximately 10 minutes. Peak levels of respective alcohols and acids are achieved at 20 minutes. Higher levels of the respective alcohols (metabolite)
indicate the fast metabolic cleavage of the iso- or n-butyl acetate (parent compound). Half-lifes in vivo have only been determined for n-butyl acetate, but not for iso-butyl acetate (Teeguarden, 2005; Essig, 1989). N-butyl acetate disappeared completely from blood within 1 min after termination of intratracheal exposure of rats (cf. IUCLID section 7.1.1 study record Essig, 1989). After intravenous dosing of rats (30 mg/kg) the elimination half-life for n-butyl acetate was similar in blood and brain, estimated to be 0.4 min. Hydrolysis of n-butyl acetate was calculated to be 99% complete in 2.7 min (cf. IUCLID section 7.1.1 study record Deisinger, 1997). For isobutyl acetate, it is estimated that hydrolytic cleavage in blood and tissues is comparably fast falling in the range of minutes. This is supported by the fact that hydrolysis rates e.g. in S-9 preparations from ethmoturbinates are nearly the same for iso- and n-butyl acetate (i.e. 67 and 77 nmol/mg S-9 protein/min for isobutyl acetate and n-butyl acetate, respectively; cf. IUCLID section 7.1.1 study record Dahl, 1987). Reaction rates of alcohol dehydrogenase and aldehyde dehydrogenase for members of the isobutyl series of metabolites are in the same order as for members of the n-butyl series. This is evidenced by similar concentration patterns of ester, alcohol and acid in blood (cf. OPP/ACC, 2004; Teeguarden, 2005).
Based on these findings, isobutyl alcohol can be considered the key metabolite of isobutyl acetate. Thus isobutanol will be used as supporting substance in the evaluation of the systemic toxicity of isobutyl acetate in cases when data for systemic effects of isobutyl acetate are not available.
As can be seen in ECHA Chem (ECHA, 2015b) all available genotoxicity studies using isobutanol gave negative results. The in vitro reverse mutation assay in bacteria and in vitro mammalian gene mutation assay using isobutanol are regarded relevant for evaluating the genotoxicity of isobutyl acetate and are supported by the results from the in vivo MNT study using isobutanol. Moreover, valid studies using isobutanol for repeated oral and inhalation toxicity are read-across, as well as the results from a 2-generation toxicity study and developmental toxicity studies with rats and rabbits.
Read-across justification 3:
Isobutyl acetate is first metabolized in blood and in tissues by esterases to isobutanol and acetate (cf. Dahl, 1987; OPP/ACC, 2004; Römmelt, 1986). In following steps, isobutanol is enzymatically oxidized to isobutyraldehyde and isobutyric acid by alcohol dehydrogenase and aldehyde dehydrogenase respectively (cf. OPP/ACC, 2004; Römmelt, 1986). Isobutyric acid will subsequently be utilized in intermediary metabolic pathways (tricarboxylic acid cycle) and ultimately excreted as CO2. It has been demonstrated with 14C labelled isobutyric acid (label at C-1), that between 90 and 97% of an orally administered dose was excreted as CO2 in expired air within 48 hours, In urine, 3.2 to 4.6% of radioactivity was excreted. Fecal radioactivity was less than 1% of the dose (DiVincenzo and Hamilton, Toxicol Appl Pharmacol 1978, 47, 609). Based on the commonly known metabolism for carboxylic acid esters, isobutyl isobutyrate is rapidly hydrolysed to isobutanol and the isobutyric acid, with the latter being itself a metabolite of isobutanol. Thus isobutyl isobutyrate shares isobutanol as common intermediate with isobutyl acetate, and therefore reading across data on repeated oral dose toxicity from isobutyl isobutyrate to isobutyl acetate is justified and is used as supporting evidence in the evaluation of the systemic toxic effects of isobutyl acetate.
4. DATA MATRIX
Please refer to attached document in chapter 13.

Reason / purpose for cross-reference:

read-across source

Vehicle:

other: nitrogen

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.

Key result

Dose descriptor:

NOAEC

Remarks:

converted value via mole weight ratio

Effect level:

3 918 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: analytical 7444 mg/m3; overall effects; highest dose tested; cossresponds to 11666 mg/m³ of read across substance isbutyacetate

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

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

3 918 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: analytical 7442 mg/m3; overal effects; highest dose tested, corresponds to 10666 mg/m³ of isobutylacetate

Dose descriptor:

NOAEC

Remarks:

converted via mole weight ratio

Generation:

F2

Effect level:

3 918 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

Read-across justification: for details please see read-across report in section 13.

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) to rats. Corresponds to 3918 ppm or 10666 mg/m³ of isobutyl acetate.

Executive summary:

In a 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

11 565 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Results from reliable, GLP conform Two-Generation toxicity study with supporting substance (immediate metabolite of submission substance) in rats is of high reliability (Klimisch score 2).

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For isobutyl acetate, no study concerning reproductive toxicity could be identified. As substitute, data for isobutanol will be used. For details on read-across hypothesis and justification please see read-across report in IUCLID section 13.

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 (7380 mg/m³, maximum dose applied) in males and in females of the F0, F1 and F2 generation (OPP/ACC, 2003, RL2).

Deduction of the NOAEC for isobutyl acetate

The NOAEC for isobutyl acetate will be calculated on basis of the NOAEC of isobutanol (OPP/ACC, 2003) using the mass concentration/m³ and the respective molecular weights (116.16 and 74.12)

The deduced NOAEC (parentel systemic, reproductive and neonatal toxicity) for isobutyl acetate is ca. 11.57 mg/L (11565 mg/m³; 2500 ppm).

Short description of key information:
For isobutyl acetate, 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.
In a 2-generation reproductive inhalation study in rats, isobutanol did not show any effects on fertility up to a concentration of 2500 ppm (7380 mg/m³) for the F0, F1 and F2 generation. The NOAEC for isobutyl acetate (conversion using the respective molecular weight) is 11566 mg/m³

Justification for selection of Effect on fertility via oral route:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Justification for selection of Effect on fertility via inhalation route:
Only one Two-Generation study available for

Justification for selection of Effect on fertility via dermal route:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Effects on developmental toxicity

Description of key information

For isobutyl acetate, 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. 
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 mg/L). Similarly no signs of treatment-related embryo-/fetotoxicity or teratogenicity were observed. Thus the maternal and developmental NOAEC in rats was 10 mg/L (corresponds to 5671 mg isobutyl acetate/m³; Klimisch/BASF, 1990).
For rabbits signs of maternal toxicity were observed in the highest dose group (10 mg/L). In contrast, the fetuses did not show any signs of developmental toxicity. Thus the maternal and the developmental NOAEC in rabbits are 2.5 mg/L and 10 mg/L, respectively (corresponds to 3919 or 15671 mg isobutyl acetate/m³, respectively; Klimisch/BASF, 1990).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study, but as read-across from supporting substance maximum reliability is 2. Read-across hypothesis: for details please see read-across report in section 13.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

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

Remarks:

Doses / Concentrations:
0.5; 2.5; 10 mg/L
Basis:
nominal conc.

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.

Dose descriptor:

NOAEC

Effect level:

10 mg/L air

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:

NOAEC

Effect level:

10 mg/L air

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEC

Effect level:

10 mg/L air

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOAEC

Effect level:

10 mg/L air

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Read-across justification: for details please see read-across report in section 13. 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.

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 NOAEC is 10 mg/L . 

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

(Klimisch/BASF, 1990).

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

Reference 2

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Data on toxicokinetics and metabolism, acute inhalation toxicity, skin, eye and respiratory irritation, inhalation repeated dose toxicity, neurotoxic effects and bacterial gene mutation can be read-across from n-butyl acetate to iso-butyl acetate, because the substances are structurally very similar and possess similar physicochemical properties.
Data on bacterial or mammalian cell gene mutation, as well as for effects after repeated exposure (i.e. oral and inhalation repeated dose toxicity, 2-generation Toxicity study, developmental toxicity studies) can be read-across from isobutanol to isobutyl acetate, because isobutyl acetate is rapidly hydrolysed to isobutanol and acetic acid.
Data on repeated oral dose toxicity can be read-across from isobutyl isobutyrate to isobutyl acetate, because isobutyl isobutyrate is rapidly hydrolysed to isobutanol and isobutyric acid. Isobutyric acid itself is a metabolite of isobutanol. Thus isobutyl isobutyrate shares isobutanol as common intermediate with isobutyl acetate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read-across to cover end point requirements is performed only from studies with pure substance. The analytical purities reported are > 98 % in case of isobutyl isobutyrate or even ≥ 99 % in case of isobutanol and n-butyl acetate.
Therefore no relevant impurities are included and thus no consequences on the relevant endpoints are to be expected.
3. ANALOGUE APPROACH JUSTIFICATION
Read-across justification 1:
Data from n-butyl acetate to isobutyl acetate can be read-across, because the source substance is structurally very similar to isobutyl acetate. Both compounds are only different in the terminal methyl group position of the butyl alcohol part of the molecule. Physical and chemical properties are only slightly influenced by this difference. Physical and chemical reactivity will basically be the same and only differ gradually. Relevant physico-chemical properties like e.g. water solubility, partition coefficient, and vapour pressure are very similar as well as acute toxicity data (details see data matrix in section 1.5). Both substances are rapidly (within minutes) hydrolysed to acetic acid and the respective alcohols (see below).
Therefore read-across of toxicity data for human health endpoints is considered justified.
Read-across justification 2:
After being absorbed the parent compound isobutyl acetate is hydrolysed rapidly to metabolites isobutanol and acetic acid (by ubiquitous esterases in various tissues including liver and lung). Isobutanol is further oxidised to isobutyraldehyde and subsequently to isobutyric acid, whereas the acetate is introduced in the intermediary metabolic pathway (acetyl coenzyme A) or excreted. There will be no further discussion on this endogenous metabolite. As stated in IUCLID section 7.1.1 on Toxicokinetics, metabolism and distribution data verify this assumption on metabolic pathway for isobutyl acetate and the supporting substance n-butyl acetate (cf. IUCLID section 7.1.1 study records OPP/ACC, 2004 and Supp. subst.: n-butyl acetate; Teeguarden, 2005). In these experiments peak levels of parent compound are reached after approximately 10 minutes. Peak levels of respective alcohols and acids are achieved at 20 minutes. Higher levels of the respective alcohols (metabolite)
indicate the fast metabolic cleavage of the iso- or n-butyl acetate (parent compound). Half-lifes in vivo have only been determined for n-butyl acetate, but not for iso-butyl acetate (Teeguarden, 2005; Essig, 1989). N-butyl acetate disappeared completely from blood within 1 min after termination of intratracheal exposure of rats (cf. IUCLID section 7.1.1 study record Essig, 1989). After intravenous dosing of rats (30 mg/kg) the elimination half-life for n-butyl acetate was similar in blood and brain, estimated to be 0.4 min. Hydrolysis of n-butyl acetate was calculated to be 99% complete in 2.7 min (cf. IUCLID section 7.1.1 study record Deisinger, 1997). For isobutyl acetate, it is estimated that hydrolytic cleavage in blood and tissues is comparably fast falling in the range of minutes. This is supported by the fact that hydrolysis rates e.g. in S-9 preparations from ethmoturbinates are nearly the same for iso- and n-butyl acetate (i.e. 67 and 77 nmol/mg S-9 protein/min for isobutyl acetate and n-butyl acetate, respectively; cf. IUCLID section 7.1.1 study record Dahl, 1987). Reaction rates of alcohol dehydrogenase and aldehyde dehydrogenase for members of the isobutyl series of metabolites are in the same order as for members of the n-butyl series. This is evidenced by similar concentration patterns of ester, alcohol and acid in blood (cf. OPP/ACC, 2004; Teeguarden, 2005).
Based on these findings, isobutyl alcohol can be considered the key metabolite of isobutyl acetate. Thus isobutanol will be used as supporting substance in the evaluation of the systemic toxicity of isobutyl acetate in cases when data for systemic effects of isobutyl acetate are not available.
As can be seen in ECHA Chem (ECHA, 2015b) all available genotoxicity studies using isobutanol gave negative results. The in vitro reverse mutation assay in bacteria and in vitro mammalian gene mutation assay using isobutanol are regarded relevant for evaluating the genotoxicity of isobutyl acetate and are supported by the results from the in vivo MNT study using isobutanol. Moreover, valid studies using isobutanol for repeated oral and inhalation toxicity are read-across, as well as the results from a 2-generation toxicity study and developmental toxicity studies with rats and rabbits.
Read-across justification 3:
Isobutyl acetate is first metabolized in blood and in tissues by esterases to isobutanol and acetate (cf. Dahl, 1987; OPP/ACC, 2004; Römmelt, 1986). In following steps, isobutanol is enzymatically oxidized to isobutyraldehyde and isobutyric acid by alcohol dehydrogenase and aldehyde dehydrogenase respectively (cf. OPP/ACC, 2004; Römmelt, 1986). Isobutyric acid will subsequently be utilized in intermediary metabolic pathways (tricarboxylic acid cycle) and ultimately excreted as CO2. It has been demonstrated with 14C labelled isobutyric acid (label at C-1), that between 90 and 97% of an orally administered dose was excreted as CO2 in expired air within 48 hours, In urine, 3.2 to 4.6% of radioactivity was excreted. Fecal radioactivity was less than 1% of the dose (DiVincenzo and Hamilton, Toxicol Appl Pharmacol 1978, 47, 609). Based on the commonly known metabolism for carboxylic acid esters, isobutyl isobutyrate is rapidly hydrolysed to isobutanol and the isobutyric acid, with the latter being itself a metabolite of isobutanol. Thus isobutyl isobutyrate shares isobutanol as common intermediate with isobutyl acetate, and therefore reading across data on repeated oral dose toxicity from isobutyl isobutyrate to isobutyl acetate is justified and is used as supporting evidence in the evaluation of the systemic toxic effects of isobutyl acetate.
4. DATA MATRIX
Please refer to attached document in chapter 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

not specified

Limit test:

no

Dose descriptor:

NOAEC

Effect level:

10 mg/L air

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:

NOAEC

Effect level:

10 mg/L air

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEC

Effect level:

10 mg/L air

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOAEC

Effect level:

10 mg/L air

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Read-across justification: for details please see read-across report in section 13. 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.

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 NOAEC is 10 mg/L . Converted via mole weight ratio this corresponds to 15.7 mg/L.

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

Converted via mole weight ratio this corresponds to 15.7 mg/L.

(Klimisch/BASF, 1990).

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

10 000 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Results are available from two reliable, GLP conform prenatal developmental toxicity studies with supporting substance (immediate metabolite of submission substance) in different species (rats and rabbits; high reliability Klimisch score = 2).

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

For isobutyl acetate, no study concerning developmental toxicity/teratogenicity could be identified. As substitute, data for isobutanol will be used.

For details on read-across hypothesis and justification please see read-across report in IUCLID section 13.

 

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 present 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.

 

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 is 10 mg/L for rats and 2.5 mg/L for rabbits.

 

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

 

The developmental NOAEC in both species is 10 mg/L.

 

(Klimisch/BASF, 1990)

Deduction of the NOAEC for isobutyl acetate

The NOAEC for isobutyl acetate will be calculated on basis of the NOAEC of isobutanol using the respective molecular weights (116.16 and 74.12)

The deduced maternal NOAEC is 15.671 mg/L for rats and 3.918 mg/L for rabbits.

The deduced developmental NOAEC in both species is 15.671 mg/L.

Justification for selection of Effect on developmental toxicity: via oral route:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Justification for selection of Effect on developmental toxicity: via inhalation route:
There are two reliable read-across studies available for different species (rats and rabbits) revealing no adverse developmental toxic effects up to the highest concentrations tested. The study with rats was clicked as rats are the preferred species according to OECD TG 414.

Justification for selection of Effect on developmental toxicity: via dermal route:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Justification for classification or non-classification

Based on the lack of adverse findings on fertility and developmental endpoints obtained from valid reproduction and developmental toxicity studies with supporting substance isobutanol (immediate metabolite of submission substance), no classification for reproductive toxicity is required for isobutyl acetate according to Regulation (EC) No 1272/2008.