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Administrative data

two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Study was performed prior to GLP or OECD guidelines and followed rats for two-generations through to weaning. The F2 pups were not necropsied but instead placed into a chronic, carcinogenicity study. F1b generation was produced for teratologic examination.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
F2 pups were not necropsied
Principles of method if other than guideline:
The P animals were re-mated and F1b fetuses were produced and examined in a teratology study. The F2 pups were produced and followed through weaning, but not necropsied. F1a pups were reared and mated and then necropsied as adults. The F1b fetuses were necropsied for a complete teratological examination.
GLP compliance:
study was prior to GLP
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Material was stated to be secondary butanol, and assumed to be 100%.

Test animals

Details on test animals or test system and environmental conditions:
Rats (30/sex/group) were housed individually in mesh-bottom cages in an air-conditioned room and provided food and water ad libitum. Food and fluid intake, and body weights were measured on a weekly basis.

Administration / exposure

Route of administration:
oral: drinking water
Details on exposure:
Animals were provided with drinking water via stainless steel sipper tubes containing 0.3, 1.0, or 3.0% (approximately 538, 1644, or 5089 mg/kg/dayfor males; and 594, 1771, and 4571 mg/kg/day for females) secondary butanol. On day 10 post-partum of the F1 pups, the top dose was lowered to 2.0% (approximately 3000 mg/kg/day) due to clear loss in body weight of the dams and lower birthweight of the pups.

The average daily intake in mg/kg/day at the 2% exposure level was not reported by the study investigators, but was reported by the USEPA in their 2003 IRIS assessment of MEK as average daily intakes of 3,384 mg/kg/day in males and 3,122 mg/kg/day in females based on a linear regression analysis of the reported average intakes for males and females at drinking water concentrations of 0, 0.3, 1, and 3%.

Negative controls were provided with tap water.
Details on mating procedure:
For the P1 mating, after 9 weeks on test, one male was co-housed with one female until a vaginal sperm plug was observed or sperm were evident in a vaginal smear. Females were transferred to separate cages on gestation day 17 or 18, and allowed to carry their litters to term. Dams having more than 8 pups had the number of pups randomly culled to a maximum litter size of 8. Pup and dam weights were recorded on day 4 and 21 of lactation.

For the P2 mating, after separation from their weaned F1 litters, dams were allowed a two-week rest interval (no treatment), then placed back on treatment and re-mated as before until the first 20 dams showed evidence of pregnancy.

For the F1 mating, at 12 weeks of age, matings were accomplished as per the P1 method described above.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
F0 animals: 9 weeks premating exposure, 18 days gestation, 21 days lactation
F1 animals: 21 days lactation, 8 weeks premating exposure, 18 days gestation, 21 days lactation
F2 animals: 21 days lactation.

Second mating of the P-generation:
Two-week "rest period" then remating. Exposure through day 20 gestation.
Frequency of treatment:
Continuous (P, F1)
A two week rest interval occurred between the first and the second matings of the P generation.
Details on study schedule:
Rats (30/sex/group) were exposed continuously via drinking water to SBA concentrations of 0, 0.3, 1.0, or 3.0% for 9 weeks and then mated until evidence of pregnancy achieved. After the birth of the F1a generation, on day 10 of lactation, the top dose was lowered to 2.0% for all subsequent stages of the study. The F1a pups were reared to day 28, and then transferred to individual cages and continued on treatment until 12 weeks of age, when they were mated to form an F2 generation, which was brought to term and followed through day 21 weaning.

P animals were re-mated (20 dams/group) to generate F1b fetuses which were necropsied for teratological examination.
Doses / concentrationsopen allclose all
Doses / Concentrations:
nominal in water
Doses / Concentrations:
Low dose
nominal in water
0.3 % (538 - 594 mg/kg/day)
Doses / Concentrations:
Mid dose
nominal in water
1.0 % (1644 - 1771 mg/kg/day)
Doses / Concentrations:
High dose - P1 generation only
nominal in water
3.0 % (5089 - 4571 mg/kg/day)
Doses / Concentrations:
High dose - P2 and F1 generations only
nominal in water
2.0 % (approximately 3000 mg/kg/day)
No. of animals per sex per dose:
30 dams + 30 males (reproduction)
20 dams (teratology)
Control animals:
Details on study design:
An F1b generation was produced in a second mating of 20 of the dams/group from the P generation, exposed to secondary butanol at concentrations of 0, 0.3, 1.0, or 2.0 %. This F1b generation was necropsied for teratological evaluation (1/3 of the pups for visceral, and 2/3 of the pups for skeletal examinations).

F1a Litters were weaned at 21 days and housed together for an additional 6 days under the same treatment as the dams. Pups from these litters (30 males and females aged 28 days) were selected for the F1 mating.
Positive control:
Not applicable


Parental animals: Observations and examinations:
Body weight (weekly) and daily food and water consumption up to 8 weeks.
Calculated test material intake.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
% viable at birth, 4 days, and 21 days. (viability and lactation indices)
Body weight day 4 and day 21.
Number of live/dead pups were ascertained as soon as possible following delivery, and completed by day 4.
Postmortem examinations (parental animals):
All animals received a gross necropsy. Uteri were examined. Ten animals per sex/group received histopathological evaluations and organ weight measurements on spleen, adrenals, pituitary, gonads, and heart. Twenty animals/sex/group were examined histologically for lesions and organ weights measured for the liver and kidney. Any gross lesions noted were examined histologically.

Teratology portion reproductive parameters:
* Corpora lutea
* Implant sites
* Resorptions
* Live fetuses
* Dead fetuses

Clinical Chemistry: fasting blood sugar, urea nitrogen, serum glutamic oxaloacetic transaminase (SGOT), serum alkaline phosphatase, serum ornithine carbamyl transferase (SOCT), total serum proteins.

Haematology: hematocrit, hemoglobin, erythrocyte count, total and differential leucocyte counts, and prothrombin time.

Urinalysis: Semi-quantitative for sugar, protein, ketone bodies, specific gravity, and microscopic characterization of the centrifuged sediment.
Postmortem examinations (offspring):
* Live/dead fetuses
* Sex of live fetuses

1) Soft tissue (visceral) examination:

2) Skeletal examination:
* Sternebrae
* ribs
* vertebrae
* extremities
* skull
* hyoid
Not reported
Reproductive indices:
Offspring viability indices:

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

Body weight gain was depressed at 3.0% (4571 - 5089 mg/kg/day) secondary butanol, but efficient food utilisation was not affected, indicating the effect was due to depressed appetite.

Fertility rate for the P animals treated with 3.0% SBA was 73%, which is below historical norms for the rat colony. After lowering the top dose to 2.0% secondary butanol (3384 and 3122 mg/kg/day in males and females, respectively), no effect on fertility was observed in the F1 matings.

No hematologic, urinary, or clinical biochemical alterations were seen in the sacrificed adult animals of either generation.

Males had slightly elevated liver and kidney weights, and females had elevated liver weights at 2.0% secondary butanol, but these increases were not statistically significant using a t-test.

In all dose groups, a high rate of respiratory and renal disease were noted upon microscopic evaluation. These findings were assessed as being typical of rat colonies not barrier maintained.

In the lungs of animals in all groups, a progressive hypertrophy of lymphoid tissue around bronchi, bronchioles, and often blood vessels, and in the mucosa of the trachea and bronchi were typically seen. Later changes include chronic interstitial inflammation of the alveolar walls, and loci of acute and subacute inflammation of the bronchial and tracheal mucosa, which precedes the development of bronchiectasis and abscess formation or atelectasis.

A low incidence of worms and the parasite (Trichosomoides crassicauda) was found in the renal pelvis of two animals (not related to treatment).

All of the above changes were considered to be expected findings in rat colonies from this era and not treatment-related.

Findings that were unexpected, and potentially treatment-related included the following four:

i) Nonreactive tubular degeneration in the outer medullary zone, probably involving mainly the thick ascending limb of the loop of Henle. The
epithelium shows loci of pycnosis, cytoplasmic granulation, increased eosinophilia, and, sometimes, desquamation with downstream
intraluminal clusters of free cells. In this study the change observed in female animals showed equal extent and intensity in animals of all groups. However, among the males, the change is two to three times more prominent in the high level test group (2% secondary butanol) than in the water control.

2) Tubular casts were found in five of the 2% group only. This excludes (a) the marked scarring and cast formation in the two animals with polyarteritis nodosa; and (b) a solitary small cast that might be seen in an occasional animal of any group.

3) Foci of tubular regeneration to a notable extent in eight 2% secondary butanol animals, and in only one water control animal, one low dose secondary butanol animal and in two mid dose secondary butanol animals. Some of these foci apparently are regenerating epithelium, and others have recently regenerated.

4) Microcysts in the tip of the renal papilla were found in 2% secondary butanol animals but in no water controls. These presumbably are dilatations of collecting ducts just before they open into the renal pelvis. Some of these "cysts" were empty, others were more or less filled with homogeneous eosinophilic material that resembles plasma, and may have been fluid during life, with component proteins that were fixed in place by formalin.

Of the above unexpected renal findings, none were regarded as having clear pathologic significance.

Compared to water, 2% secondary butanol evoked changes in kidneys of rats (males more than females) including an accelerated
appearance of tubular casts and focal tubular regeneration, development of microcysts in the apices of renal papillae, and
possibly epithelial degeneration in tubules in the outer medullary zone.

Effect levels (P0)

open allclose all
Dose descriptor:
Effect level:
10 000 mg/L drinking water
Basis for effect level:
other: No changes in clinical signs, organ weights, histopathology, or fertility indices. Equivalent to 1644 and 1771 mg/kg/day for males and females, respectively.
Dose descriptor:
Effect level:
20 000 mg/L drinking water
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Fetotoxicity as measured by a significant reduction in pup viability at birth, a reduced birthweight, and reduced survival to day 21 was seen at 3.0% SBA in the F1a generation. No treatment-related effects were seen on corpora lutea or resorptions.

No skeletal or soft tissue abnormalities or terata were observed with SBA treatment. Fetotoxicity as reduced birthweight was also seen in the F2 pups exposed to 2.0% SBA.

Effect levels (F1)

open allclose all
Dose descriptor:
Effect level:
10 000 mg/L drinking water
Basis for effect level:
other: Based on no observed fetotoxicity, viability or teratogenicity. Equates to 1644 and 1771 mg/kg/day for males and females, respectively
Dose descriptor:
Effect level:
20 000 mg/L drinking water
Basis for effect level:
other: Decreased fetal weights

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Summary of Reproduction Parameters in the P/F1b generation

 Parameter  Water  0.3% SBA  1.0% SBA  2.0% SBA
 Number of pregnancies  29  28  30  29
 Number of pregnancies to term  29  28  30  29
 Corpora lutea (total)  392  357  422  383
 Corpora lutea (per dam)  13.1  11.9  14.1  12.8
 Live litters (total)  29  27  30  29
 Implant sites (total)  344  312  372  322
 Implant sites (per dam)  11.9  11.1  12.4  11.1
 Resorptions (total)  5  11  1  10
 Resorptions (1 or more per dam)  5  8  1  6
Resorptions (complete litter)  0  0  0  0
 % partial resorptions  17.2  28.6  3.33  20.7
 Live fetuses (total)  339  300  371  312
 Live fetuses (per dam)  11.7  10.7  12.4  10.8
 Sex ratio (M/F)  0.99  1.08  0.91  0.89
 Dead fetuses (total)  0  1  0  0
 Dams with all dead fetuses  0  1  0  0
 % all dead  0  3.57  0  0
 Average fetal weight (g)  4.14  4.16  4.38  3.74
 Net body weight gain (P males)  269  274  261  229
 Net body weight gain (P females)  154  158  155  130

Table 2. Summary of Reproduction and Lactation Parameters (P generation)

 Parameter  Water  0.3% SBA  1.0% SBA  3.0% SBA
 Number of matings  30  30  30  30
 Number of pregnancies  29  27  29  27
 Number of litters:        
 Born alive  29  27  29  26
 Alive at 4 days  29  27  29  25
 Alive at 21 days  28  24  29  23
 Number of pups:        
 Born alive  300  289  311  220
 Born dead  6  3  23
 Number of pups:        
Alive at 4 days  300  283  309  203
 Alive at 21 days  215  180  225  153
Number of pups/litter:        
 Born alive  10.3  10.7  10.7  8.5
 Alive at 4 days  10.3  10.5  10.7  8.1
 Culled to at 4 days  7.8  7.6  7.9  6.8
Alive at 21 days   7.7  7.5  7.8  6.8
 Mean pup body weight:        
 at 4 days  10.3  10.2  10.0  8.2
 at 21 days  49.5  47.2  44.4  28.4
 Fertility  96.7  90.0  96.7  90.0
 Gestation  100.0  100.0  100.0  96.3
 Viability  100.0  97.9  99.4  92.3
 Lactation  95.6  88.2  97.8  89.5

Table 3. Summary of Reproduction and Lactation Parameters (F1/F2 generation)

 Parameter  Water  0.3%  1.0%  2.0%
 Number of matings  30  30  30  30
 Number of pregnancies  29  29  28  27
 Number of litters:        
 Born alive  29  29  28  27
 Alive at 4 days  28  28  27  24
 Alive at 21 days  27  28  25  23
 Number of pups:        
 Born alive  296  302  267  272
 Born dead  4  3  2  4
 Number of pups:        
 Alive at 4 days  282  293  236  241
 Alive at 21 days  200  209  170  166
 Number of pups/litter:        
 Born alive  10.2  10.4  9.54  10.1
 Alive at 4 days  10.1  10.5  8.74  10.0
 Culled to at 4 days  7.54  8.00  7.00  7.46
 Alive at 21 days  7.41  7.46  6.80  7.22
 Mean pup body weight (g)        
 At 4 days  10.0  9.74  9.56  9.48
 At 21 days  40.1  39.2  39.1  34.9
 Fertility  96.7 96.7  93.3  90.0
 Gestation  100  100  100  100
 Viability  95.3  97.0  88.7  88.6
 Lactation  94.8  93.3  90.0  92.7

Applicant's summary and conclusion

Secondary butyl alcohol, administered in drinking water to rats over two generations did not affect reproductive parameters or cause fetotoxicity up to a concentration of 1.0% (approximately 1700 mg/kg/day). A decrease in pup viability was seen at 3.0% and a slight decrease in fetal body weight was seen at 2.0% (3122 mg/kg/day in females). Kidney pathology in adult rats exposed to the highest doses of 3.0 and 2.0% were typical of kidney lesions seen in rats with aging.
Executive summary:

Secondary butyl alcohol, administered in drinking water to rats over two generations did not affect reproductive performance or cause developmental toxicity up to a concentration of 1.0% (1644 mg/kg/day). Adult rats exposed to 2.0% SBA (3122 mg/kg/day) showed significant kidney histopathology in the form of renal tubular degeneration/regeneration, renal tubular casts, microcysts on the tip of the papilla. Pup viability was reduced at 3.0% (4571 mg/kg/day). Fetal body weights were slightly lower at 2.0%. The NOAEL in the study for general systemic, reproductive, and fetotoxic effects was 10000 mg/L (1644 mg/kg/day).