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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
Peer-reviewed published report of a study mandated by the US Environmental Protection Agency as part of the TSCA Section 4 Test Rule

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1995
Reference Type:
publication
Title:
Calculation of benchmark dose for reproductive and developmental toxicity observed after exposure to isopropanol
Author:
Allen B, Gentry R, Shipp A, van Landingham C
Year:
1998
Bibliographic source:
Regul Tox Pharmacol, 18, 38-44

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Version / remarks:
Guideline not specified in this report.
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Propan-2-ol
EC Number:
200-661-7
EC Name:
Propan-2-ol
Cas Number:
67-63-0
IUPAC Name:
propan-2-ol
Constituent 2
Reference substance name:
Isopropanol
IUPAC Name:
Isopropanol
Details on test material:
- Name of test material (as cited in study report): Isopropanol
- Source; Exxon Chemical Company
- Substance type: aliphatic alcohol
- Physical state: colourless liquid
- Analytical purity: 99.9%

- Stability under test conditions: Stable
- Storage condition of test material: refrigerated
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Kingston, NY USA)
- Housing: wire mesh caging
- Diet: commercial lab feed ad libitum
- Water: tapwater ad libitum:
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%): 18-70%
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Solution dosed at rate of 5mL/kg.
Details on exposure:
Single daily doses p.o. for at least 10 weeks prior to mating and 7 days during mating period repeated if mating not successful. After confirmation of mating dosing continued through gestation and lactation until euthanasia. Parental males were dosed until day prior to euthansia

Neonates F1 were dosed from postnatal day 21.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 7 days; repeated x 2 if mating not successful
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 ] of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no ]
- After successful mating each pregnant female was caged (how): singly
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
At least 10 weeks prior to mating and 7 days during mating period repeated if mating not successful. After confirmation of mating dosing continued through gestation and lactation until euthanasia. Parental males were dosed until day prior to euthansia
Frequency of treatment:
Daily
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age (two per sex per litter). Treatment commenced at this point. Experimental procedures for F1 generation as for P.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
30
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on range finder study. Severe parental toxicity seen at doses of 1750 and 2500mg/kg. 2/20 animals died at 1000mg/kg suggesting this was the MTD. It is now also regarded as the limit dose for reproductive toxicity testing.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gestation days 0, 7, 14 and 21

BODY WEIGHT: Yes
- Time schedule for examinations: male weekly; female weekly before mating then on days 0, 7, 14 and 21 and on post-partum days 0, 4, 7, 14 and 21
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: culled to 4 of each sex as far as possible. Culled pups that appeared abnormal were subject to gross postmortem.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
-number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: following delivery of the last litter they sired.
- Maternal animals: At post natal day 21 (5 per sex per treatment group.)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. All adult males used for mating examined.

ORGAN WEIGHTS : Liver and kidneys (all animals)

HISTOPATHOLOGY
The tissues indicated in below from the control and 1000 mg/kg groups, and all parental groups were prepared for microscopic examination and weighed, respectively: Pituitary, Testes, Epididymides, Prostate, Seminal Vesicle, Vagina, Uterus, Ovaries, Gross lesions. (Control and top dose animals only and intermediate doses when adverse findings seen.
Postmortem examinations (offspring):
GROSS NECROPSY
- External examination only: all pups.
- Gross postmortem examination: all abnormal fetuses and 5 pups of each sex from treatment group on PND4

HISTOPATHOLOGY: Not on pups but on F1 generation after littering (as adults) as per parental histopathology description above.
Statistics:
Bartlett's test for homogeneity leading to parametric analyses (one-way ANOVA with Dunnett's Test) if homogeneous at 1%. If heterogeneous, Kruskall-Wallis and Dunn';s Rank Sum tet and Jonckheere's test for ordered response. Incidence data by Chi-squared followed by Armitage test for trend or FFisher's Exact test.
Reproductive indices:
Male mating index%
Female fertility and fecundity indices%
Gestational index %
Mean gestation length (days)

---for both P1 and P2
Mean litter size; live/litter; dead/litter


Offspring viability indices:
Live birth index%
Survival indices(5) on days 1, 4, 7, 14 and 21
Lactation index % (F1 and F2)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality: 2 females from high dose group only
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Increased bodyweight gain during lactation in high-dose parental females (40.4+/-24.4 vs 15.1+/-30.5g for control)
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys of mid and high dose males characterised by increased number of hyaline droplets in proximal cells of convoluted tubules, increased incidence and severity of epithelial degeneration and hyperplasia, increased increased incidence of proteinaceous casts in the renal tubules and increased incidence of focal interstitial mononuclear clear cell infiltration. No treatment related observations were made in any reproductive tissues.
Other effects:
not examined

Reproductive function / performance (P0)

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

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
2 females each the high dose group, one female from mid dose group and 2 males from low dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Increased bodyweight gain during lactation in mid and high-dose parental females (19.2+/-15.9 and 25.3+/-23.2g respectively versus 3.1+/-17.5g for controls.
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver weight: males absolute in mid dose group (+13%) and relative in mid and high dose groups (+11% and +14% respectively), females absolute in high dose group (+8%) and relative n mid and high dose groups (+8% and +18% respectively).
Increased kidney weight relative only in high-dose group: males +7%, Females +8% Changes in kidney may be of a type not relevant to humans.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys of all dose group males characterised by increased number of hyaline droplets in proximal cells of convoluted tubules, increased incidence and severity of epithelial degeneration and hyperplasia, increased increased incidence of proteinaceous casts in the renal tubules and increased incidence of focal interstitial mononuclear clear cell infiltration. Centrilobulary hepatocyte hypertrophy was seen in 6/26 high dose males. No treatment related observations were made in any reproductive tissues.

Reproductive function / performance (P1)

Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Only parameter affected was a significant reduction in the male mating index in the high dose group (reduction from 93->73%). There was a trend towards reduced male and female fertility indices and female fecundity index but these were not statistically significant.

Effect levels (P1)

Dose descriptor:
NOAEC
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Increased mortality in high-dose pups during early postnatal period (PND0-4). Losses 14% at PND1 and 9% at PND4 but no difference from PND 7 and beyond. The reduction at PND4 was also evident in the mid dose group (-4%). Live birth index slight but significantly reduced (-3%). All other statistically significant survival differences were minor and not regarded as biologically meaningful in the early PN period. There were 18 offspring deaths in the high dose group PND21-41 prior to selection of the P2 generation. There were no remarkable postmortem findings in these animals. These deaths left the top dose group with only 26 males and 26 females in the top dose second generation parental group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant body weight reductions in the high dose males at days 0 and 1 (-7%) and an increase in the low dose males on day 21 (+10%). Significant (~10%) increase in body weight in the low dose females on days 14 and 21. The authors considered these changes biologically irrelevant.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Reduced offspring bodyweight in early postnatal period in high-dose males. Increased kidney (relative) weights in high-dose groups.
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular hepatocyte hypertrophy was seen in a few hgh dose male rats

Details on results (F1)

Offspring that survived to termination were free of treatment related abnormalities and had milk present in their stomachs during the early postnatal. period.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
other: The reduced PND survival on a single day 4 at 500mg/kg was not deemed biologicaly significant. Body weight gain seen in females towards the end of weaning and only in low dose group not deemed a toxicologically significant finding.

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Effects seen in the F1 generation were also seen here. Increased mortality in mid and high-dose pups during early postnatal period (PND0-7). Losses ~5% at PND1 in mid and high dose groups, 4% at PND4 in high dose group animals only, 4% and 9% in mid and high dose group animals only at PND7. No differences betweeen groups from PND 14 and beyond.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant body weight reductions in the high dose males and females at days 0 and 4 (~-10%). The authors considered these changes biologically insignificant as the reduction was temporary and the weights of the pups recovered by day 7.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Reduction in lactation index in mid and high dose group animals (-6% and -9% respectively) with a dose response relationship.

Details on results (F2)

Offspring that survived to termination were free of treatment related abnormalities and had milk present in their stomachs during the early postnatal. period.

Effect levels (F2)

Dose descriptor:
NOAEC
Generation:
F2
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
other: lactation index.
Remarks on result:
other: The authors of the study did not consider these effects sufficiently biologically relevant to take them into account when determining the NOAEL for reproductive toxicity from this study.

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

The changes in liver weight are likely the result of adaptive changes at the relatively high dose levels used. The relevance of the kidney findings in males is of characteristic of hydrocarbon nephropathy that male rats are known to be uniquely sensitive to and which is not relevant to humans.

Slightly increased mortality (up to a maximum of 10%) was statistically significant in the mid and more so high dose groups and more so in the generation. It was not seen consistently at all time points. The results from other drinking water studies suggest that palatability could have affected uptake of milk during weaning and that this may not be a direct toxic effect.

Allen (2008) published the results of a benchmark dose study on the results and concluded that the BMDL5 for the reduced male mating index was 420mg/kgbw/day.

Applicant's summary and conclusion

Conclusions:
No treatment-related microscopic changes in reproductive tissues or biologically meaningful differences in reproductive parameters were observed in adults of either generation. A reduced mating index was seen at 1000mg/kgbw/day
Executive summary:

A two generation reproduction toxicity study in rats examined oral gavage exposure of isopropanol at doses of 100, 500 and 1000mg/kgbw/day. There were signs of parental toxicity at the highest dose, including effects on body weight gain, liver and kidney weights and liver and kidney histology. There was some evidence of increased mortality in the pups in the early post natal period in the high dose animals and this dose group also showed a trend towards reduced pup weights. No treatment related post-mortem findings were seen in any offspring. The NOEL for reproductive effects is 500 mg/kg bw/day based on reduced male mating index in high-dose P2 males.