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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with GLP Guidelines.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: USEPA OPPTS 870.3650 (2000)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diisobutyl Carbinol
- Molecular formula (if other than submission substance): C9H20O
- Molecular weight (if other than submission substance): 144.15
- Physical state: Clear, Liquid
- Analytical purity: 99.1 ± 0.06%, as the sum of two isomers
- Lot/batch No.: AA0155T3YY

Test animals

Species:
rat
Strain:
other: CRL:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: Approximately eight weeks of age at initiation of treatment
- Weight at study initiation: Males: ave 271.1 ± 10.8 g; Females: ave 197.6 ± 12.2 g;
- Housing: animals were housed singly in stainless steel cages, except during breeding (one male and one female) and during the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material from
approximately GD 19 until completion of lactation.
- Diet (e.g. ad libitum): Animals were provided LabDietâ Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form,
provided ad libitum.
- Water (e.g. ad libitum): Drinking water obtained from the municipal water source, provided ad libitum.
- Acclimation period: At least one week prior to teh start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The average room temperature was maintained at 22 ± 1°C (with a maximum permissible excursion of ± 3°C).
- Humidity (%): The relative humidity was maintained within a range of 40-70%, with the exception of one deviation of 32%.
- Air changes (per hr): Room air was exchanged approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m.

IN-LIFE DATES: Gavage dosing began two weeks prior to breeding on July 20, 2005 for both males and females. Adult males were necropsied on
August 22, 2005 and adult females were necropsied on September 10, 2005.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: 0.5% Methyl Cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Dose solutions were prepared in a 0.5% METHOCEL solution to achieve dose levels of 0, 50, 150 and 500 mg/Kg/day, body weight adjusted.

Oral gavage is the preferred route of exposure according to OECD Guideline 422.Male rats were dosed daily for 14 days prior to mating and
continuing throughout the mating period for a total of 33 days. Female rats were dosed once daily for 14 days prior to breeding, and continuing
through breeding (two weeks), gestation (three weeks), and lactation (four days).

The test material was administered in a 0.5% METHOCEL A4M vehicle, such that a dose volume of 4 ml/kg body weight yielded the targeted dose.
Dose volumes were adjusted using the most current body weight. Dose suspensions were prepared periodically throughout the study period based upon stability.
Details on mating procedure:
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level
(1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the
presence of sperm as an indication of mating. Theday on which sperm was detected or a vaginal copulatory plug was observed in situ was
considered GD 0. The sperm- or plug-positive (presumed pregnant) females were thenseparated from the males and returned to their home cages. If
mating had not occurred after two weeks, the animals were separated without further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of all dosing suspensions from the initial mix revealed mean concentrations ranging from 99.2 to 104% of targeted concentrations.
Analyses of aliquots for the low- and high-dose suspensions indicated that the test material was homogeneously distributed. DIBC in 0.5% methylcellulose was found to be stable for 25 and 64 days at concentrations of 2.50 and 250 mg/ml.
Duration of treatment / exposure:
Oral gavage is the preferred route of exposure according to OECD Guideline 422.
Male rats were dosed daily for 14 days prior to mating and continuing throughout the
mating period for a total of 33 days. Female rats were dosed once daily for 14 days prior
to breeding, and continuing through breeding (two weeks), gestation (three weeks), and
lactation (four days).
Frequency of treatment:
Once Daily
Details on study schedule:
0 (control), 50, 150, or 500 mg/kg/day
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
Gavage
No. of animals per sex per dose:
12 male and 12 female.
Control animals:
yes
Details on study design:
- Dose selection rationale: A preliminary range-finding study was performed to aid in dose level selection for the main study. In this study, five
rats/sex were administered DIBC by gavage for 14 days at dose levels of 0, 250, 500,
750, or 1000 mg/kg/day. All animals survived to the scheduled necropsy. Transient increased salivation (perioral soiling clear) was recorded in
both males and females at doses >/= 500 mg/kg/day. Feed consumption and body weights were not obviously altered at any dose level. The most
noteworthy effect was an increase in liver weights in both sexes at all dose levels. The liver weight increases ranged from 19.7-41.8% in males and
4.8-37.6% in females. Kidney weights were also increased, but to a lesser extent. There were no treatment-related gross observations recorded at
necropsy.

The high-dose level was based upon data obtained from a preliminary range-finding study (see above) and was expected to induce some toxic
effects, but not death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity that may have been
observed among the high-dose group rats and to establish a NOEL.

- Rationale for animal assignment (if not random): Prior to test material administration, animals were stratified by body weight and thenrandomly
assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard
deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations: This examination was
typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a
limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed
necessary. Animals were examined for abnormalities such as, but were not limited to: decreased/increased activity, repetitive behavior,
vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale
skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and waterat least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes See TABLE 1
- Time schedule: Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated females
received DCO examinations on GD 0, 7, 14, and 20, and LD 3. The DCO was conducted at approximately the same time each examination day
prior to dosing, according to an established format. The examination included cage-side, hand-held and open-field observations, which are
recorded categorically or using explicitly defined scales (ranks).

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:


OTHER:
Oestrous cyclicity (parental animals):
Data not collected however, There were no treatment-related effects at any dose level on reproductive indices such as time to mating.
Sperm parameters (parental animals):
Parameters examined in adult male parental generations: testis weight and epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
Litters were not standarized, during littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material
from approximately GD 19 until completion of lactation.

PARAMETERS EXAMINED
The following parameters were examined in offspring: All litters were examined as soon as possible after delivery. The following information was
recorded on each litter: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on LD 0, 1, and 4 , and the
sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily In-Life Observations). In addition, pup clinical observations were recorded on each litter on days 0
through 4 postpartum.

GROSS EXAMINATION OF DEAD PUPS: Any pups found dead were sexed and examined grossly, if possible, for external and visual defects and then
discarded.
Postmortem examinations (parental animals):
SACRIFICE - All Animals survived through necropsy.
- Male animals: Adult males (fasted) were submitted for necropsy after at least four weeks (actual: TD 34) of exposure.
- Maternal animals: Adult females (fasted) were terminated on LD 5, or at least 24 days after the end of the mating period for females not producing a litter.

GROSS NECROPSY
- A complete necropsy was conducted on all animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and
then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.
Statistics:
See Table 1 Below.
Reproductive indices:
There were no treatment-related effects at any dose level on reproductive indices, time to mating, gestation length, postimplantation loss, pup
survival or pup sex ratio.
Offspring viability indices:
Not evaluated

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See Body Wt in Table 1 below
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See Body Wt in Table 1 below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 1 below
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

All aminals lived through necropsy

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No systemic effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings:
not examined

Details on results (F1)

All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Final Body Weight and Organ Weight Effects

 mg/Kg/day:  0  Historical1  50  150  500
 

MALES

 Final Body Weight (g)  375.0  354.1 -447.2  385.5  401.2  383.7
 Relative Adrenals (g / 100g bw)  0.017  0.014 -0.021  0.014*  0.015  0.016
 Relative Kidneys (g / 100g bw) 0.763   0.713 -0.801  0.740  0.773  0.882
 Absolute Liver (g)  10.519  10.108 -13.420  11.469  12.278*  13.421*
 Relative Liver (g / 100b bw)  2.800  2.719 -3.298  2.972  3.062*  3.493*
 

FEMALES         

 Final Body Weight (g)  277.5 251.2 -290.4   268.0  278.3 258.8*
 Absolute Adrenals (g)  0.089  0.070 -0.094  0.084  0.079  0.132
 Relative Adrenals (g / 100g bw) 0.032   0.027 -0.037  0.031  0.029  0.051$
 Relative Kidneys (g / 100g bw)  0.686  0.696 -0.742  0.741  0.755*  0.773*
 Absolute Liver (g)  9.882  8.185 -10.433  9.714  10.315  10.860
Relative Liver (g / 100g bw)   3.557  3.155 -3.748  3.628  3.696  4.195*

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

$Statistically Different from Control Mean by Wilcoxon’s Test, Alpha = 0.05

1Historical controls group mean range from recent OECD 422 studies.

Bold typeindicates the effects judged to be treatment related.

Applicant's summary and conclusion

Conclusions:
Oral gavage administration of 500 mg/kg/day of DIBC resulted in decreased feed consumption and body weights in females only. The liver was the primary target organ for systemic toxicity. Treatment-related statistically significant increases in absolute liver weights were noted in males of the middle (17%) and high dose (28%) groups as well as the high dose females (10%). Corresponding increases in relative liver weights were statistically identified in these groups. The higher liver weights corresponded with very slight hypertrophy of centrilobular hepatocytes in males given 150 or 500 mg/kg/day and females given 500 mg/kg/day. Males given 500 mg/kg/day and females given 150 or 500 mg/kg/day had higher relative kidney weights that were interpreted to be treatment related. There were no corresponding clinical pathologic or histopathologic alterations for the higher kidney weights. There was no evidence of systemic toxicity in rats given 50 mg/kg/day. Additional treatment-related effects that were interpreted to be nonadverse consisted of transient salivation noted only around the time of dosing in the highdose males and females, slightly decreased urine pH in males at all dose levels, as well as increased serum total protein and cho lesterol in males or females given 500 mg/kg/day. Degeneration and/or inflammation of the olfactory and respiratory epithelium were noted in 1, 2-3, and 4-6 rats/sex in the 50, 150, and 500 mg/kg/day groups, respectively. These nasal effects were interpreted to be the result of local irritation of the test material
associated with the gavage procedure. There were no adverse effects of DIBC on neurological or reproductive function at any dose level.
Based on these data, the no-observed-adverse effect level (NOAEL) for general toxicity was considered to be 150 mg/kg/day. The NOEL for reproductive and neurological effects was 500 mg/kg/day, the highest dose level tested.