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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1997-05-02 to 1997-07-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
MacGregor, J. Wehr, C., Henika, P., Shelby, M. 1990. The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundamental and Applied Toxicology 14:513–522.
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): MRD-96-575; n-pentane
- Substance type: C5 aliphatic
- Physical state: liquid
- Analytical purity: 97.4% (considered the pure substance as received)
- Lot/batch No.: 2036924
- Expiration date of the lot/batch: June 2001; December 2001 (arbitrarily assigned by EBSI per Standard Operating Procedures)
- Storage condition of test material: Room temperature
- Other: Analyses for the stability, identity, strength, purity, and composition or other characteristics that identify the test material were performed by the testing laboratory.

Test animals

Species:
rat
Strain:
other: Crl:CDBR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Stone Ridge, New York
- Age at study initiation: 7 to 8 weeks (n-pentane treated and control groups); 15 to 16 weeks (positive control group)
- Weight at study initiation: 230 to 256 grams for n-pentane treated and control males; 433 to 470 grams for positive control males; 185 to 213 grams for n-pentane treated and control females; 270 to 295 grams for positive control females
- Assigned to test groups randomly: yes; using a computer-generated body weight sorting program that placed animals in groups that were within ±20% of the mean body weight of their sex
- Fasting period before study: no; however animals were fasted prior to clinical laboratory studies
- Housing: Individually housed in suspended stainless-steel and wire mesh cages
- Diet (e.g. ad libitum): ad libitum during non-exposure periods
- Water (e.g. ad libitum): ad libitum during non-exposure periods
- Acclimation period: 12 days for n-pentane treated and control animals; 13 days for positive control animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 18 to 22°C
- Humidity (%): 30 to 70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 1996-11-18 (n-pentane treated and control animals); 1997-05-05 (positive control animals) To: 1997-02-21 (n-pentane treated and control animals); 1997-05-08 (positive control animals)

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1.5 cubic meter stainless steel and glass whole body inhalation exposure chambers
- Source and rate of air: 300 liters per minute under a slight negative pressure to the room
- Temperature, humidity, pressure in air chamber: approximately 19 to 25°C with 40 to 70% humidity; chamber airflow, temperature, and relative humidity were continuously monitored throughout the exposure and recorded approximately every 30 minutes
- Other: Additional information regarding the generation of the test atmosphere is in the 90-day inhalation study (Exxon 157518).
Duration of treatment / exposure:
n-pentane: 6 hours plus chamber equilibrium (theoretical T99 = 23 minutes)
Cyclophosphamide (positive control): three days
Frequency of treatment:
n-pentane: 6 hours per day, 5 days per week for 13 weeks
Cyclophosphamide (positive control): once daily for three days
Post exposure period:
Not applicable
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
n-pentane
Basis:
nominal conc.
5000; 10,000; and 20,000 mg/m3
Remarks:
Doses / Concentrations:
n-pentane
Basis:
analytical conc.
5097±97; 10,203±151; and 20,483±734 mg/m3
Remarks:
Doses / Concentrations:
positive control
Basis:
nominal conc.
20 mg/kg; dosing volume did not exceed 1.0 mL/100 grams per body weight
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent no treatment
Positive control(s):
cyclophosphamide (diluted with Reagent grade water)

- Justification for choice of positive control(s): cyclophosphamide is a known clastogen
- Route of administration: gavage
- Doses / concentrations: 20 mg/kg with a dosing volume of 10 mL/kg; individual doses were based on the animal’s day 0 body weight

Examinations

Tissues and cell types examined:
It was noted that selected tissues were weighed, preserved, and examined microscopically in n-pentane treated and control animals. However, additional details, including what tissues were selected, are not provided. This information is presented in the 90-day inhalation study (Exxon 157518). Micronucleated polychromatic erythrocytes (MNEs), polychromatic erythrocytes (PCEs), and normochromatic erythrocytes (NCEs) were examined.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: One femur per animal was removed per animal immediately after sacrifice. The bone marrow was aspirated, pooled, flushed in fetal bovine serum, and centrifuged. Resulting cell pellet was resuspended in any remaining supernatant after supernatant was decanted. Smears (2 slides per animal) were prepared from the resuspended cell pellet. Slides were stained using Acridine Orange, wet mounted, and labeled with the study and animal number.

METHOD OF ANALYSIS: Two thousand PCEs from each animal were examined for the presence of micronuclei. The ratio of polychromatic to total erythrocytes (by percent), number of polychromatic erythrocytes with micronuclei, and number of polychromatic erythrocytes scored were recorded. The percent of PCEs in the total population of erythrocytes was determined for each animal by counting the number of PCEs and NCEs per 1000 erythrocytes. Means and standard deviations of micronuclei data were calculated, and a statistical analysis to compare group means was conducted.
Evaluation criteria:
An assay was considered valid if (1) the mean incidence of MNEs per 1000 PCEs (calculated from the evaluation of 2000 PCEs) did not exceed 4 (or 0.4%) in the control group; and (2) the mean incidence of MNEs for the positive control group was significantly greater (p<0.05) than the control group.

A response was considered positive if there was (1) a dose-related statistical increase in the mean number of MNEs, including at least one dose point that is statistically (p<0.05) from the mean number of MNEs of the control group, that was outside the normal range (i.e., greater than 4); or (2) at least two dose points were statistically different (p<0.05) from the mean number of MNEs in the control group and greater than the normal range of the mean number of MNEs of the control group. A response was considered negative if the mean value for a statistically significant increase in MNEs was within the normal range of the control; a result was considered inconclusive if a single dose was statistically different from the control but there was no dose response.
Statistics:
A statistical analysis was conducted to compare group means using a one-way analysis of variance for each time period. Significant results were than analyzed using Duncan’s Multiple Range Test to compare the treated and control groups. A standard regression analysis was conducted to test for dose response.

Normality for the ANOVA residual was analyzed by either Wilk's Criterion or Kolmogorov Smirnov Statistic. For those not normally distributed in more than 25% of the analyses, nonparametric analyses were conducted. Nonparametric analyses included the Kruskal Wallis one-way analysis of variance, followed by Dunn's Summed Rank Test if significant differences were noted. Dose response was evaluated by Jonckheere’s test of Ordered response.

Sexes were analyzed separately.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
There were no increases in micronuclei formation
Toxicity:
no effects
Remarks:
There were no treatment-related effects observed regarding systemic toxicity.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: A 5-day range finding study in rats was conducted, in which animals were exposed whole-body for 5 consecutive days. Additional detail regarding this range finding study is presented above under Details of tissue and slide preparation.

- Dose range: 5000; 10,000; and 20,000 mg/m3 (nominal); 5446; 10,680; and 21,418 mg/m3 (actual)
- Clinical signs of toxicity in test animals: There were no clinical signs of toxicity observed in any of the animals.
- Rationale for exposure: The high dose was selected because it half of the lower explosive limit and was the highest considered safe to test. Inhalation was chosen as the route of exposure because it is the mostly likely route for human exposure.

RESULTS OF DEFINITIVE STUDY (see table in Remarks on results including tables and figures)
- Induction of micronuclei, ratio of MNE/1000 PCE (for Micronucleus assay): N-pentane did not induce a statistically significant increase in micronuclei formation at any exposure level when compared to the control group. The positive control substance did induce a statistically significant (p<0.01) increase in the mean number of MNEs, indicating that this substance was clastogenic, the test system responded in the appropriate manner.
- Percent PCE (for Micronucleus assay): N-pentane did not induce a statistically significant decrease in the percent PCEs at any exposure level when compared to the control group. The positive control substance did induce a statistically significant (p<0.01) decrease in the percent PCEs, indicating that this substance was clastogenic, the test system responded in the appropriate manner.
- Appropriateness of dose levels and route: Dose levels and route of administration were appropriate.

Any other information on results incl. tables

There were no adverse effects with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. Only body weight information is presented in the study report. Additional information regarding clinical signs of toxicity, food consumption, pathology parameters, organ weights, postmortem observations, and microscopic changes are presented in the 90 -day inhalation study.

Summary of Micronucleus Data (mean ± s.d.)

 

Males - mg/m3(n)

0 (5)

5000 (5)

10,000 (5)

20,000 (5)

20 mg/kg Cyclophosphamide (5)

%PCE

42.76±2.37

43.92±2.75

47.68±3.17

39.94±6.67

39.94**±1.36

MNE/1000 PCE

2.6±2.6

2.0±2.3

1.0±0.6

2.7±1.8

35.9**±13.5

 

Females - mg/m3(n)

0 (5)

5000 (5)

10,000 (5)

20,000 (5)

20 mg/kg Cyclophosphamide (5)

%PCE

46.34±4.94

45.76±4.85

44.52±10.51

44.30±3.41

11.54**±4.74

MNE/1000 PCE

1.8±1.0

1.2±1.0

2.2±0.8

1.1±0.9

31.3**±2.3

a Data obtained from pages 25 to 26 in the study report.

** Statistically significantly higher than control (p<0.01)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative n-pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group.
There were no adverse effects observed in any of the n-pentane treated animals with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. n-Pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group. N-pentane was tested at an adequate dose because the high dose was half of the lower explosive limit and was the highest dose considered safe to test. The positive control induced the appropriate response. 
Executive summary:

In a Crl:CDBR rat bone marrow micronucleus assay, 5 animals/sex/dose were administered n-pentane via inhalation at nominal doses of 0; 5000; 10,000; or 20,000 mg/m3. Doses were selected based on a 5 -day range finding inhalation test in rats, and the test was part of the 90-day inhalation study. Rats were exposed to either n-pentane or air (control) 6 hours per day, 5 days per week for 13 weeks. A positive control group was administered 20 mg/kg of cyclophosphamide diluted in water at a constant volume of 1.0 mL/100 grams body weight for three treatments via gavage, 24 hours apart. Animals were examined for signs of toxicity during the study, and sacrificed on day after the last dose was administered. Bone marrow cells were harvested immediately after sacrifice. 

 

Actual doses received were 5097±97; 10,203±151; and 20,483±734 mg/m3. There were no signs of toxicity during the study. There were no adverse effects observed in any of the n-pentane treated animals with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. n-Pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group. n-Pentane was tested at an adequate dose because the high dose was half of the lower explosive limit and was the highest dose considered safe to test. The positive control induced the appropriate response. 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP.