Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Data obtained from peer-reviewed publication. Read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture complete hydrolysis will take place with no significant reaction products other than alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the toxicokinetics of target substance. Because of the rapid hydrolysis, the influence of the mode of administration through inhalation, dermal and oral is related to the hazardous degradation product (alcohol) released from the target substance. The identification of degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance, which might change the hazardous properties of the target substance compared to the properties of the pure alcohol. As there is a mechanistic reasoning to the read-across, the unnecessary animal testing is avoided by using the read-across data from the degradation product (relevant alcohol) to evaluate irritation, sensitization and the short term and long-term toxicological effects and mutagenicity of the target substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): IPA, 2-propanol
- Physical state: liquid

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sparague Dawley Inc., Frederick, MD
- Age at study initiation: 8-11 weeks of age
- Assigned to test groups randomly: yes
- Housing: group-housing up to 5 mice / group
- Diet (e.g. ad libitum): Purina Certified Laboratory Chow #5002 ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%):30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
Details on exposure:
For IP injection isopropanol was dissolved in 0,9% sodium chloride. The stability of the mixture was verified at the beginning and the end of the study. The vehicle control substance was sterile 0,9% sodium chloride (Kendall Mc graw, CAS # 7440-23-5).
Duration of treatment / exposure:
Range finding test
A single exposure dose selection study was conducted to establish an appropriate range of doses for the definitive mouse bone marrow micronucleus assay. Six randomly assigned ICR mice/group (3 males/3 females) were treated with IPA in 0,9% sterile saline at dose levels of 500, 1 625, 2 750, 3 875, 5 000 mg IPA/kg by IPA injection. The mice were observed immediately after dosing and twice daily for the duration of the 72 hr observation period.
Frequency of treatment:
Vehicle control, positive control and the test group animals were injected at a rate of 10 ml/kg
Post exposure period:
Selected vehicle and test group animals were sacrificed at 24, 48 or 72 h after treatment. Positive control animals were euthanized by CO2 inhalation 24hr post-administration.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
350mg/kg bw
Basis:
other: IP injection
Remarks:
Doses / Concentrations:
1 173mg/kg bw
Basis:
other: IP injection
Remarks:
Doses / Concentrations:
3 500mg7kg bw
Basis:
other: IP injection
No. of animals per sex per dose:
15 mice / sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
The positive control substance was cyclophosphamide (Sigma Chemical Co, CAS # 50-18-0), prepared by dissolving the chemical in sterile deionized water to yield a concentration of 8.0mg/ml. Positive control substance was orally gavaged in 5 animals per sex with 80 mg cyclophosphamide / kg.

Examinations

Tissues and cell types examined:
Bone marrow polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
Bone marrow harvest from each pair of tibia was performed by remoal of soft tissue and epiphyses, then flushing with 3ml of FBS to remove the marrow. Folllowing centrifugation, most of the supernatant was drawn off: the cells were resuspended and the suspension was spread on slides. The slides were air dried and then fixed in methanol and stained in May-Grunwald solution followed by Giemsa. After air drying, each slide was coverslipped using Depex mounting medium.
The coded slides were then scored for micronuclei and the PCE to normochromatic erythrocyte (NCE) cell ratio was determined. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based upon the total PCEs present in the scored optic field. The normal frequency of micronuclei in the ICR strain is approximately 0-0.4%
Evaluation criteria:
The micronucleus data were evaluated by an analysis of variance on the square root arscine transformation which was performed on the proportion of micronucleated cells per mouse. Once the analysis of variance had been performed, Tukey's Studentized range test with adjustment for multiple comparisons was used at each harvest time to determine which dose groups , if any, were significantly different from the vehicle control. Analysis was performed for each harvest time / sex, and also for each harvest time with sexes combined. A test substance did not induce either a statistically significant dose-related increase in micronucleated PCEs or a reproducible and statistically significant positive response for at least one dose level was considered negative. The PCE/NCE ratios were statistically evaluated using non-parametric Kruskall-Wallis or Wilcoxon (rank sum) analysis procedures.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
the frequency of micronuclei was not elevated and that there were no significant changes in the PCE/NCE ratio
Toxicity:
yes
Remarks:
mortality was observed in the treatment groups of 2500mg/kg bw or higher
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The following mortality occurred by 72hr:
500mg/kg, 0/6; 1625mg/kg, 0/6; 2750mg/kg, 1/6; 3875mg/kg, 2/6; 5000mg/kg, 4/6.
Single dose IP LD50 value for mice was 4384mg/kg. Based on these results, the dose levels selected for study were set at 350, 1173, 3500 mg isopropanol/kg.

RESULTS OF DEFINITIVE STUDY
However, 35 of the 40 mice treated at 3500mg/kg died within 22 hr of injection. The mice at 350 and 1173mg/kg groups were sacrificed and and evaluated as scheduled. In order to confirm that IPA was not mutagenic at a maximally attainable dose, a second trial was conducted at a dose level of 2500mg/kg. Within 72 hr of dosing, 6 mice had died.

In the 2500, 1173 and 350mg/kg treatment groups the frequency of micronuclei was not elevated and that there were no significant changes in the PCE/NCE ratio. Thus, IPA was judged to be non-mutagenic in the micronucleus assay. See attached result table in the background material.

Any other information on results incl. tables

Read-across justifications and data matrices are presented in IUCLID section 13.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Mutagenic potential of Isopropyl alcohol was assessed in mouse micronucleus study. No increased incidence of micronuclei was observed in bone marrow polychromatic erythrocytes (PCE) harvested at 24, 48, or 72 hr post-dosing of IPA.
Executive summary:

IPA was administered as a single intraperitoneal (IP) injection at the concentration of 350, 1173 or 2500 mg/kg in male and female mice. The bone marrow was extracted at 24, 48 and 72 hr post-injection, smeared onto glass slides and stained. The slides were then scored for micronuclei and the PCE to normochromatic erythrocyte (NCE) cell ratio was determined. It was found that the frequency of micronuclei was not elevated and there were no significant changes in in the PCE/NCE ratio.

This study was regarded as reliable, since the publication is based on the study performed according to US EPA guideline and in compliance with GLP. The result of this publication would indicate that there is no evidence of mutagenic activity of IPA. Furthermore, IPA has not been classified for germ cell mutagenicity according to EU regulation No. 1272/2008 (CLP) Annex VI Table 3.1.