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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): isobutanol
- Physical state: clear colorless liquid
- Analytical purity: 99.9%
- Purity test date: prestudy 2000-12-19, poststudy 2002-07-30
- Lot/batch No.: 15867
- Expiration date of the lot/batch: no data
- Stability under test conditions: yes
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals 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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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)

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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

Effect levels (F1)

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

Results: F2 generation

Effect levels (F2)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.