Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Deviations did not affect the validity or the integrity of the study. Refer any other information on materials and methods for deviations noted.
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
yes
Remarks:
Deviations did not affect the validity or the integrity of the study. Refer any other information on materials and methods for deviations noted.
GLP compliance:
yes
Type of assay:
other: in vivo micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Details on test material:
- Purity: 99%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc
- Weight at study initiation: Male: mean of 226.0 g (range: 204.1 to 247.4 g) and Female: mean of 194.4 g (range: 183.2 to 208.2 g)
- Age: 54 or 55 days old
- Assigned to test groups randomly: yes
- Housing: 1 per cage in standard wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23±2°C
- Humidity: 50±10%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
Air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass (NYU style) exposure chambers were used with a nominal internal volume of approximately 150 litres.
- Method of holding animals in test chamber: whole-body
- Source and rate of air: Directly inside the chamber inlet, a baffle was positioned to provide uniform distribution of the vapour within the chamber.
- Method of conditioning air:
- System of generating particulates/aerosols: Vapour atmospheres of test substance were generated by metering the gaseous test substance from a cylinder located in an outside cylinder shed. The vapour flowed through 1/4 inch O.D. stainless steel and Teflon tubing and a rotometer prior to entry into the mixing flask. Filtered houseline air added to the mixing flask carried the test substance/air mixture into the 150 litre exposure chamber. Desired atmospheric concentrations of the test substance vapour were produced by regulating the flow of test substance vapour through the rotometer. Test atmospheres were generated dynamically.
- Temperature, humidity, pressure in air chamber: 24-29°C, 15-54%; pressure not reported; chamber oxygen concentration of 20-21%. concentrations. These airflows resulted in 13-14 air changes per hour in the 150 litre exposure chambers. The chamber oxygen concentration measurements were between 20 and 21%. The relative humidity in the chambers was between 15 and 54% and the chamber temperatures were between 24 and 29°C. Although the relative humidity was consistently below the targeted range of 40-60% and the temperature was consistently higher than the targeted range of 21-25°C, the environmental conditions in the exposure chambers were considered adequate for the conduct of the study.
- Air flow rate: 32 to 35 litres/min.
- Air change rate: 13-14 air changes per hour in the 150 litre exposure chambers
- Method of particle size determination:
- Treatment of exhaust air: Chamber atmospheres were exhausted directly into the fume hoods with the exception of the high concentration, which was mixed with nitrogen prior to discharge into the fume hood.

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of test substance was determined by gas chromatography at approximately 20 minute intervals during each 6 hr exposure. Gas samples were drawn by vacuum pump from representative areas of the chamber where rats were exposed. Chamber atmosphere samples were directly injected into a Hewlett Packard Model 5880A Series Gas Chromatograph equipped with a flame ionization detector for analysis of test substance concentration. All samples were chromatographed isothermally at 85°C on a 3% OV-17 Chromosorb column. The atmospheric concentration of test substance was determined from a standard curve derived from gas standards. The gas standards were prepared prior to each exposure by injecting known volumes of test substance into gas bags that contained known volumes of air.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
2 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
4 500 ppm (analytical)
Remarks:
± 120 ppm
Dose / conc.:
9 000 ppm (analytical)
Remarks:
± 250 ppm
Dose / conc.:
18 000 ppm (analytical)
Remarks:
± 410 ppm
No. of animals per sex per dose:
Test substance or negative control: 10
Positive substance: 5
Control animals:
yes, concurrent no treatment
Positive control(s):
The positive indicator was cyclophosphamide (CP). The positive indicator was assumed to be stable during this study and no evidence of instability was observed. Any impurities in the positive indicator were not expected to have interfered with the study. CP was dissolved in sterile water and administered by oral intubation in a volume of 10 mL/kg. A 4.0 mg/mL solution was used, yielding a dose of 40 mg/kg.

Examinations

Tissues and cell types examined:
Bone marrow; Erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The high concentration of test substance for the micronucleus study was selected on the basis of an acute inhalation median lethal concentration (LC50) study and knowledge about the flammability of the test substance. In the LC50 study, 5 male and 5 female rats were exposed to a concentration of 21000 ppm for a single 4 hr period. During exposure, subjective assessments of the alerting response to an auditory stimulus and the activity level of animals were decreased. Immediately after exposure, clinical signs of toxicity included irregular respiration in 2 male rats. No clinical signs of toxicity or significant body weight losses were evident the day after exposure. These results, and the safety issues associated with generation of the test substance vapours in 150 litre exposure chambers, resulted in a targeted high concentration of 18000 ppm for the micronucleus study. The high concentration was approximately 60% of the lower explosive limit (30000 ppm). The testing laboratory safety professionals judged this value to be the maximum concentration that permitted an adequate safety margin for laboratory personnel operating inhalation chambers of the size and type used in this study. In addition, selection of 60% of the lower explosive limit was based on safety standards set by the National Fire Protection Association. Two lower concentrations, at 1/2 (9000 ppm) and 1/4 (4500 ppm) the high concentration level, were also selected for the micronucleus study.

TREATMENT AND SAMPLING TIMES: Groups of 10 male and 10 female rats were exposed to the test substance or negative control 1 day after release from quarantine. Exposures lasted 6 hrs/day and were performed on 2 consecutive days. The positive indicator rats (5 rats/sex) were dosed 2 days after release from quarantine, which coincided with the end of the second exposure period for the test substance and negative control groups. The test substance and negative control groups were sacrificed approximately 24 and 48 hrs after the end of the second exposure period. The positive indicator rats were sacrificed approximately 24 hrs post-dosing, which coincided with the 24 hr sacrifice for the test substance and negative control groups.

DETAILS OF SLIDE PREPARATION: Immediately after sacrifice, marrow from 1 femur of each animal was aspirated and flushed into 1.5 or 2.0 mL of prewarmed foetal bovine serum. Marrow was collected by centrifugation. Most of the supernatant was removed and the cells were resuspended in the remaining 1-2 drops of serum. A Miniprep® automatic blood smearing instrument was used to prepare marrow smears. At least 2 slides per animal were prepared and fixed in absolute methanol for 5 min. Slides were stained for 2.5 min in 0.0125 mg/mL acridine orange in phosphate buffer (pH 7.2). Prior to scoring, a coverslip was floated on each slide using phosphate buffer.

METHOD OF ANALYSIS: Representative slides were examined blindly using incident light fluorescence microscopy. Only cells with good morphology and staining were scored. Colour was used to distinguish PCEs (reddish) from NCEs (dark green). PCEs (2000 per animal) were scored for the presence of micronuclei (round, bright yellow-green fluorescing bodies). Cellular inclusions that were irregularly shaped or stained, or out of the focal plane of the cell were considered artifacts. The unit of scoring was the micronucleated cell; PCEs with more than 1 micronucleus were scored as a single MNPCE. Micronucleated NCEs seen in the optic fields scored to obtain 2000 PCEs were also counted. Additionally, the number of PCEs among 1000 erythrocytes was recorded for each animal.
Statistics:
Data for the proportion of MNPCEs among 2000 PCEs and the proportion of PCEs among 1000 erythrocytes (MNPCE frequency and PCE frequency, respectively) were transformed prior to analysis using the arcsin square root function. This transformation is appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion (Biometry, W.H. Freeman and Company, San Francisco, pp. 386-387, 1969). Transformed data for PCE and MNPCE frequencies were analyzed separately for normality of distribution and equal variance using the Shapiro-Wilk and Bartlett’s tests, respectively. Positive indicator data were not included in evaluating normality of distribution. Results indicated that the transformed values for MNPCE frequency were not normally distributed. Therefore, nonparametric statistics (viz., Kruskal-Wallis and Mann-Whitney U tests) were performed using the untransformed data. For analysis of PCE frequency, results indicated that, with the exception of the 9000 ppm female group at the 48 hr time point, the data were normally distributed and had equal variance. Therefore, parametric statistics (viz., analysis of variance (ANOVA) and Dunnett’s test) were performed using the transformed data. For analysis of PCE frequency in the 9000 ppm female group at the 48 hr time point, nonparametric statistics utilizing nontransformed data were employed. Body weight data were assumed to be normally distributed and were analyzed by ANOVA. Data from each concentration or dose level, sex, and sacrifice time were analyzed separately. All analyses were conducted at a significance level of 5%, except Bartlett’s test, which was conducted at a significance level of 0.5%. The animal was considered the experimental unit.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The high concentration of test substance for the micronucleus study was selected on the basis of an acute inhalation median lethal concentration (LC50) study and knowledge about the flammability of the test substance. In the LC50 study, 5 male and 5 female rats were exposed to a concentration of 21000 ppm for a single 4 hr period. During exposure, subjective assessments of the alerting response to an auditory stimulus and the activity level of animals were decreased. Immediately after exposure, clinical signs of toxicity included irregular respiration in 2 male rats. No clinical signs of toxicity or significant body weight losses were evident the day after exposure. These results, and the safety issues associated with generation of the test substance vapours in 150 litre exposure chambers, resulted in a targeted high concentration of 18000 ppm for the micronucleus study. The high concentration was approximately 60% of the lower explosive limit (30000 ppm) of test substance. In addition, selection of 60% of the lower explosive limit was based on safety standards set by the National Fire Protection Association. Two lower concentrations, at 1/2 (9000 ppm) and 1/4 (4500 ppm) the high concentration level, were also selected for the micronucleus study.

RESULTS OF DEFINITIVE STUDY
CLINICAL OBSERVATIONS, BODY WEIGHT CHANGES, AND MORTALITY:
There were no clinical signs of toxicity observed in any treatment group during exposure. Within 1 hr after the first exposure, 2/10 male and 2/10 female rats in the 4500 ppm groups exhibited ocular or nasal discharge, or facial stains. Similar signs were observed at a low frequency in the 9000 ppm groups, with the additional observation of wet perineum recorded for 4 female rats. The incidence of clinical observations was greater in the 18000 ppm groups, with 6/10 male rats and 7/10 female rats exhibiting clinical signs post-exposure. In addition to the signs observed at the lower concentrations, rats in the 18000 ppm exposure groups exhibited wet inguen, and/or stained chin, perineum, nose, or legs. Clinical signs persisted overnight in the 9000 ppm females and the 18000 ppm groups of both sexes. Prior to the second exposure, clinical signs of toxicity were observed in 1/10 female rats in the 9000 ppm group and in 5/10 males and 4/10 females at the high concentration. In addition to those signs observed previously, additional stains were noted in some rats on the tail, back, or inguen. After the second exposure, clinical signs were observed with approximately the same frequency in the 4500, 9000, and 18000 ppm groups of both sexes as had been observed after the first exposure. The same or similar clinical signs of toxicity, including wet underbody region, discharge, and stains, were displayed. One control male exhibited nasal discharge. The majority of clinical signs were transient in the 4500 and 9000 ppm groups following exposure. Prior to the 24 and 48 hr sacrifice times, clinical signs in these groups occurred with low frequency and were limited to stains or alopecia. Stains, wetness, or discharge were observed at a relatively greater frequency in the 18000 ppm groups prior to sacrifice. No unscheduled deaths occurred during the study. Male positive indicator rats (3/5) exhibited stained perineum or lethargy on the day following dosing.

STATISTICAL EVALUATION
Statistically significant, concentration-dependent decreases in body weight gain or body weight loss were measured in the 9000 and 18000 ppm male rats prior to the 24 and 48 hr sacrifice times. Statistically significant body weight loss or decrease in body weight gain was also observed in the 18000 ppm female rats prior to the 24 and 48 hr sacrifice times, respectively. Although not statistically significant, decreases in body weight gain or body weight loss which also appeared to be part of a dose-dependent trend were measured in the 4500 ppm females prior to the 24 hr sacrifice and in the 4500 ppm males and 9000 ppm females prior to both sacrifice times. In addition to the body weight alterations in the treatment groups, a statistically significant body weight loss was measured in the positive indicator male rats.

CYTOGENETIC RESULTS
There were no statistically significant increases in MNPCE frequency at any time point in either male or female rats exposed to the test substance. As expected, statistically significant increases in MNPCE frequency were found in CP-treated rats of both sexes. No depressions in PCE frequency were detected in either male or female rats exposed to the test substance. Statistically significant depressions in PCE frequency were found in both male and female rats administered CP.

Any other information on results incl. tables

Data Summary – MNPCE Frequency

Test Substance Concentration (ppm)

Sample Time (hours)

Sex

N

Median %MNPCE (IQR)

0

24

M

5

0.25 (0.15)

0

24

F

5

0.10 (0.13)

4500

24

M

5

0.15 (0.33)

4500

24

F

5

0.10 (0.13)

9000

24

M

5

0.30 (0.13)

9000

24

F

5

0.15 (0.13)

18000

24

M

5

0.10 (0.18)

18000

24

F

5

0.05 (0.18)

0

48

M

5

0.25 (0.15)

0

48

F

5

0.00 (0.20)

4500

48

M

5

0.40 (0.28)

4500

48

F

5

0.10 (0.08)

9000

48

M

5

0.45 (0.33)

9000

48

F

5

0.20 (0.18)

18000

48

M

5

0.35 (0.30)

18000

48

F

5

0.10 (0.18)

CP, 40 mg/kg

24

M

5

4.00 (2.63)*

CP, 40 mg/kg

24

F

5

1.10 (1.10)*

*Statistically significant difference from control, alpha = 0.05.

 

Data Summary – PCE Frequency

Test Substance Concentration (ppm)

Sample Time (hours)

Sex

N

Median %PCE (SD)

Mean PCE/NCE Ratio (SEM)

0

24

M

5

65.4 (8.12)

2.02 (0.30)

0

24

F

5

69.1 (5.46)

2.31 (0.24)

4500

24

M

5

61.6 (4.41)

1.64 (0.16)

4500

24

F

5

58.8 (13.9)

1.64 (0.37)

9000

24

M

5

61.4 (10.0)

1.73 (0.31)

9000

24

F

5

61.9 (13.4)

1.88 (0.42)

18000

24

M

5

59.8 (9.85)

1.61 (0.28)

18000

24

F

5

64.4 (6.85)

1.90 (0.26)

0

48

M

5

664 (8.73)

2.12 (0.32)

0

48

F

5

69.6 (3.32)

2.32 (0.16)

4500

48

M

5

66.1 (7.40)

2.07 (0.30)

4500

48

F

5

69.1 (4.97)

2.30 (0.24)

9000

48

M

5

63.7 (4.22)

1.79 (0.15)

9000

48

F

5

66.2 (27.4)a

1.56 (0.35)

18000

48

M

5

58.8 (6.30)

1.48 (0.19)

18000

48

F

5

70.3 (2.79)

2.40 (0.15)

CP, 40 mg/kg

24

M

5

49.2 (10.3)*

1.03 (0.19)

CP, 40 mg/kg

24

F

5

40.3 (12.0)*

0.73 (0.15)

aMedian value and IQR reported.

*Statistically significant difference from control, alpha = 0.05.

 

Applicant's summary and conclusion

Conclusions:
The test substance did not induce a significant increase in micronuclei in bone marrow cells of rats when administered by the inhalation route of exposure. The material is negative in this in vivo assay.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) of rats in accordance with OECD Guideline 474. Concentrations of 0, 4500, 9000, and 18000 ppm were administered by inhalation, 6 hrs/day for 2 consecutive days, to groups of male and female rats. Bone marrow smears were prepared approximately 24 and 48 hrs after the end of the second exposure. Two thousand PCEs per animal were scored for micronuclei. No statistically significant increases in the frequency of micro nucleated PCEs were observed in test substance treated rats at any sampling time. Additionally, no statistically significant depressions in the proportion of PCEs among 1000 erythrocytes were observed in test substance exposed rats. In this assay, the test substance is negative.