Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Remarks:
The study was conducted according to the guideline dated 1998.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.99%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 70 days
- Weight at study initiation: 317-445 grams (males) and 201-269 grams (females)
- Fasting period before study: No reported
- Housing: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30%–70%
- Air changes (per hr): 10-13
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
- Vehicle used: inline house air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A stainless steel baffle below the chamber turret inside the chambers promoted uniform chamber distribution of the test atmosphere
Exposure #s 1 to 10, stainless steel and glass (NYU style) with a nominal internal volume of 350 L
Exposure #s 11 to 71, control animals were exposed to air in the same chamber while test substance exposures were performed in stainless steel and glass (NYU style) chamber with a nominal internal volume of 150 L
- Method of holding animals in test chamber: individually placed in stainless steel, wire-mesh modules and exposed whole-body in chamber
- Source and rate of air: high pressure house line air and oxygen
- System of generating particulates/aerosols: test substance vapour and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without test substance.
- Temperature, humidity, pressure in air chamber: 21-25ºC, 45-67%, and 21% oxygen, no data on pressure
- Air flow rate: Exposure #s 1 to 10 - chamber flow 60-64 L/min; Exposure #s 11 to 20 - chamber flow 29-33 L/min
- Air change rate: 10-13 per hour
- Treatment of exhaust air: exhausted from the bottom of each chamber through MSA filters using vacuum pumps and discharged into the fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: vapour concentration was directly injected into a gas chromatograph
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
Approximately 20 days of exposure
Frequency of treatment:
6 hours per day, 5 days per week
Post exposure period:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2500, 10000, and 50000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.0; 2500 ± 11; 10000 ± 74; and 51000 ± 650 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle

Examinations

Tissues and cell types examined:
RETs plus normochromatic erythrocytes [NCEs] from rat tail vein
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502000 ppm. Based on these results, exposure concentrations of 0, 2500, 10000, and 50000 ppm were selected for the current study.

TREATMENT AND SAMPLING TIMES: the evaluation was conducted by flow cytometry. Peripheral blood samples were collected by tail vein venipuncture from the 5 animals per sex per dose. There were 2 blood collections, one following the fourth exposure (test day 3), and on the last exposure day before scheduled sacrifice.

DETAILS OF SLIDE PREPARATION: Approximately 6 to 8 drops (i.e. 60-120 µL) of blood was collected from the tail vein of each animal directly into a labelled microcentrifuge tube containing 350 µL anticoagulant/diluent (anticoagulant) provided in the In Vitro Micro Flow Plus® Rat Micronucleus assay kit. The tubes were capped and inverted several times to mix the blood with the anticoagulant. The blood/anticoagulant mixture was either stored for up to 6 hours at room temperature or for up to 24 hours refrigerated, prior to fixation. The blood samples were fixed in duplicate. Approximately 180 µL of blood/anticoagulant mixture was fixed in 2 mL ultra-cold reagent grade methanol and stored below -75ºC until processed.

METHOD OF ANALYSIS: Samples were analysed by flow cytometry on a BD FACSCalibur™ running Cell Quest Pro Software. At least 20000 reticulocytes were analysed per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus normochromatic erythrocytes [NCEs]). The samples were analysed and evaluated using the reagents and templates provided in the In Vitro MicroFlow Plus® Rat Micronucleus assay kit. The frequency of micronucleated reticulocytes (%MN-RETs) were used as a measure of induction of aneugenic or clastogenic alterations by the test substance. Initially, only the samples from the control and high-concentration group were analysed. Samples from the lower exposure groups were also analysed from the day 3 female blood collection to confirm the findings from the high concentration group. The positive and negative control standards, provided in the assay kit, were used as a methods control.
Evaluation criteria:
Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis by the principal investigator for the micronucleus evaluation or the study director.
The test substance was judged positive if the following conditions were met:
• A statistically significantly increase mean MN-RETs was observed at one or more concentrations of the test substance compared to the concurrent negative control values.
• An accompanying statistically significant dose-response increase in MN-RETs was observed.

The test substance was judged negative if the following conditions were met:
• No statistically significant increases in the mean MN-RETs above the concurrent negative control value occurred at any concentration of the test substance.
• The MN-RET values of the test substance-treated animals were within reasonable limits of the recent laboratory negative historical control range.
Statistics:
Micronucleus data were evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. For each treatment group, the mean and standard deviation of % RETs and % MN-RETs were calculated. Data were transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. The individual animal was considered the experimental unit for the micronucleus data. All data analyses were one-tailed and conducted at a significance level of 5%. See Table 1 below.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Negative
- Ratio of PCE/NCE (for Micronucleus assay): No statistically significant test substance-related decreases in %RET. No statistically significant increases in the frequency of MN-RETs compared to the negative control group; the negative control group exhibited a response consistent with the %MN-RETs historical control data.
- Statistical evaluation: yes

Any other information on results incl. tables

OTHER: These results are taken from the 28-Day Inhalation Study in Section 7.5.2.

CLINICAL SIGNS AND MORTALITY

Unscheduled deaths did not occur in the animals assigned to the Subchronic and Subchronic with Recovery Subsets. No test substance-related clinical sigs of toxicity were observed in males or females at any exposure concentration. Incidental clinical signs common to the age and strain of rat were occasionally observed during the exposure and/or recovery periods.

BODY WEIGHT AND WEIGHT GAIN

No adverse, test substance-related effects on body weight or weight gain were observed for males or females exposed to any concentration of the test substance during the exposure or recovery periods. Body weight of the 10000 ppm males was statistically significantly greater compared to the control group on test days 3 and 7. However, this statistical difference was considered to be spurious since a dose-response relationship was not evident. Weight gain for 2500 ppm females was statistically significantly lower compared to the control group during test days 7-14. However, this statistical difference was considered to be spurious since a dose-response relationship was not present.

FOOD CONSUMPTION & FOOD EFFICIENCY

No test substance-related effects or statistically significant differences in food consumption or food efficiency were observed for males or females exposed to any concentration of the test substance during the exposure of recovery periods.

OPHTHALMOSCOPIC EXAMINATION

No test substance-related ophthalmological observations occurred in males or females exposed to any concentration of the test substance.

HAEMATOLOGY

There were no exposure-related changes in any group mean haematology parameter in male or female rats at any concentration tested.

The following statistically significant difference was not considered to be related to exposure to the test substance:

• Absolute large unstained cells (ALUC) were minimally higher in female rats exposed to 50000 ppm (90% above the control). There were no other statistically significant differences in other white blood cell parameter in any subsets of male or female rats at any concentration tested. Therefore, this change was considered unrelated to exposure and non-adverse.

• Coagulation - There were no statistically significant or exposure-related changes in any group mean coagulation parameter in male or female rats at any concentration tested.

CLINICAL CHEMISTRY

There were no exposure-related changes in group mean clinical chemistry parameters in male or female rats at any concentration tested.

The following statistically significant difference was not considered to be related to exposure to the test substance:

• Blood urea nitrogen (BUN) was minimally lower in female rats exposed to 50000 ppm (18% below the control). However, there were no statistically significant differences in other kidney-related clinical pathology parameters and no exposure-related changes in renal histopathology in this group. In addition, there were no changes in BUN in any other male or female group, including the 50000 ppm females from the reproduction subset. Therefore the lower BUN in the 50000 ppm females from the subchronic subset was considered unrelated to exposure and non-adverse.

URINALYSIS

There were no statistically significant differences in group mean urinalyses parameters in male or female rats at any concentration tested.

• Plasma and Urine Fluoride - There were no statistically significant changes in plasma and urine fluoride parameters in male or female rats.

NEUROBEHAVIOUR (only done on Subchronic with Recovery Subset)

• Forelimb and Hindlimb Grip Strength - No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength for males or females exposed to any concentration of the test substance.

• Open Field Observations - No test substance-related or statistically significant effects were observed for any behavioural parameter evaluated in males or females exposed to any concentration of the test substance. The incidences for all groups were similar to the values for the control groups.

A wound observed during the FOB at week 6 in one male in the 50,000 ppm group was considered to be incidental.

• Motor Activity - No test substance-related or statistically significant effects were observed on motor activity for males or females exposed to any concentration of the test substance. At week 4, the mean duration and number of movements for 50000 ppm females during the 2nd – 6th intervals as well as for the total session were decreased (not statistically significant) compared to control. However, these decreases, although they showed a trend, were not considered to be adverse because the decreases were not observed in males, were not statistically significant, and were within historical control. Mean values for duration and number of movements for the 2500 and 10000 ppm males and females and 50000 ppm males were similar to their respective controls.

ORGAN WEIGHTS

There were no test substance-related organ weight effects at inhalation exposures up to 50000 ppm.

All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.

• In males, two of the accessory sex organs (ASO) mean weight parameters were statistically (p < 0.05) decreased in the 2500 ppm exposure group, as compared to the respective control values. However, since there was no dose response, no microscopic correlate, and no similar finding in the Subchronic with Recovery Subset or Reproduction Subset, the statistical finding was interpreted to be spurious.

• In females, the mean absolute adrenal weight and mean absolute kidneys weight were both statistically decreased in the 2500 ppm group, as compared to the respective control values. These were also considered spurious since there was no dose response, no microscopic correlate, and no similar finding in the Subchronic Subset males, Subchronic with Recovery Subset, or Reproduction Subset.

In the Subchronic with Recovery Subset, there were no test substance-related organ weight effects.

All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.

• In males, both the mean relative (% body weight) heart weight and lung weight were statistically decreased in the 10,000 ppm exposure group compared to the respective control values. These were both considered spurious since there was no dose response, no microscopic correlate, and no similar finding in the main study, recovery females, or reproduction study subsets. A statistically significant decrease in the 50000 ppm mean absolute lung weight was also interpreted as spurious because there was no microscopic correlate and no similar finding in the Subchronic Subset, Subchronic with Recovery females, or Reproduction Subsets.

• In females, the mean relative (% brain weight) lung weight was increased in the 50000 ppm group, as compared to the respective control values. This was also considered spurious since there was no microscopic correlate and no similar finding in the Subchronic, Subchronic with Recovery males, or Reproduction Subsets.

GROSS PATHOLOGY

At the terminal sacrifice, there were no test substance-related gross observations in either sex. All gross observations were consistent with normal background lesions in rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were no test substance-related microscopic findings in male or female rats in the 50000 ppm inhalation exposure group. All microscopic findings in this study were consistent with normal background lesions in rats of this age and strain.

For additional details regarding Reproductive/Developmental test refer to Section 7.8.1 (DI.K1.1Gen.RD/REPRO/DEV.R.D-19695-1422.KD) and 7.8.2 (DI.K1.DEV.RD/REPRO/DEV.R.D-19695-1422.KD)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant increases in the frequency of MN-RETs compared to the negative control group were observed in any evaluated test substance treated group of male or female animals at any time point. There were no statistically significant test substance-related decreases in %RET.

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The purpose of this study was to determine the potential micronucleus evaluation from repeated inhalation exposure of male and female rats to 0, 2500, 10000, or 50000 ppm of the test substance.  

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were exposed to the test substance 6 hours per day, 5 days per week over a one-month period. Concentrations of test substance were generated by metering the test substance into the inhalation chambers and diluting it with conditioned, filtered air, supplemented with oxygen to achieve an oxygen concentration of 20% in the high level chamber. An air control group was also evaluated using a similar generation method. Vapour concentrations of the test substance were measured by gas chromatography (GC). Temperature, humidity, and airflow were also recorded periodically during each exposure day.  Body weights, clinical signs, and food consumption were recorded throughout the study. After 3 days and 28 days of exposure, blood samples were collected from 5 male and 5 female rats per group and evaluated for induction of micronuclei. The mean concentrations (± standard error of the mean) were 2500 ± 11, 10000 ± 74, and 51000 ± 647 ppm in chambers targeted at 2500, 10000, and 50000 ppm, respectively.

 

No statistically significant increases in the frequency of MN-RETs compared to the negative control group were observed in any evaluated test substance treated group of male or female animals at any time point. There were no statistically significant test substance-related decreases in %RET. The negative control group exhibited a response consistent with the %MN-RETs historical control data.