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:
guideline study
Remarks:
The study named “H-30385: 28-Day Inhalation Toxicity Study in the Wistar Rat”, report number DuPont-19179-782 is not a separate in vivo micronucleus test, but rather 28-day repeated dose inhalation toxicity study (OECD 412), in which the genetic toxicity endpoint (i.e., micronucleus – OECD 474) was evaluated as well by collecting and evaluating blood samples at day 4 and 28 of exposure. The same study is also referenced in sections 7.5.2 (repeated dose toxicity: inhalation). The micronucleus study was performed to comply with product stewardship as well as other global regulatory requirements.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear gas
Details on test material:
- Purity: >99.964%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at arrival: Males: 7 weeks; Females: 8 to 10 weeks
- Weight at study initiation: Males ranged from 263 - 269 g; Females ranged from 181 - 186 g
- Fasting period before study: no data
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment
- Diet: ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations
- Water: ad libitum in water bottles except during the periods when the animals were restrained in the exposure tubes
- Acclimation period: 12 or 13 days under test conditions after health examination. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
Inhalation Exposure System
Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The animals were confined separately in restraint tubes. The atmosphere was discharged constantly through the exposure system and exhausted using a tubing/filter system. The exposure system ensured a uniform distribution and provided a constant flow of test material to each
exposure tube. The flow of air at each tube was 1 L/min, which was sufficient to minimize re-breathing of the test atmosphere as it was more than twice the respiratory minute volume of a rat. Before commencement of the exposure of the groups, technical trials were conducted (without animals) using the inhalation system foreseen for the study. Technical trial data are documented in the raw data after review but not reported.

Test Atmosphere Generation
The concentration of the test item in the inhalation chamber was controlled by regulating the flow of the test item to the inhalation tower and by the addition of dilution air.

Exposure System Monitoring
Atmosphere concentration, relative humidity, temperature and oxygen concentration were measured on test atmosphere samples taken at a representative exposure port. See 28-day report summary for chamber information. All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters.

Determination of Nominal Atmosphere Concentration
Nominal concentrations were determined in groups 2 to 4 by weighing the cylinders before and after each exposure to determine the quantity of test item used. This was done weekly for group 2 and once per exposure for groups 3 and 4. The weight used for gas generation was then divided by the total airflow volume to give the nominal concentration.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000, 10000, 20000/15000 ppm
Remarks:
Doses / Concentrations:
1000 ± 3, 10010 ± 40, 20239 ± 557 / 15076 ± 145 ppm
No. of animals per sex per dose:
5/sex/concentration
Control animals:
yes

Examinations

Tissues and cell types examined:
Anti-CD71 antibodies (transferrin receptor 1) differenciate young erythrocytes (reticulocytes, CD71-positive) from normochromatic erythrocytes (CD71-negative).
Details of tissue and slide preparation:
- Dose selection rationale: The initial target atmosphere concentrations were selected by the Sponsor based on a previously performed acute inhalation study that demonstrated a threshold for seizure activity in rats of 33000 ppm. The target atmosphere concentration in group 4 was reduced due to the premature death of two male animals during the 3rd exposure and moderate body weight loss in several males in this group from day 1 to day 4 of exposure.
Evaluation criteria:
A test item was classified as mutagenic if it induced either a concentration-related increase in the number of micronucleated reticulocytes or a statistically significant positive response for at least one of the test points. A test item producing neither a concentration-related increase in the number of micronucleated reticulocytes nor a statistically significant positive response at any of the test points was considered non-mutagenic in this system.
Statistics:
Statistical significance was confirmed with the nonparametric Mann-Whitney test. However, both biological and statistical significance were considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The test item did not induce micronuclei as determined by the micronucleus test in peripheral blood cells. Therefore, the test item was considered to be non-mutagenic in this micronucleus assay under the experimental conditions of this study.
Executive summary:

The purpose of this inhalation study was to assess the cumulative toxicity of the test substance when administered 6 hours daily and 5 days per week to rats by nose-only, flow-past inhalation exposure for a period of 28 days. The reversibility or progression of any test substance related effects or any delayed toxicity was assessed during a 2-week exposure free recovery period. In addition, potential genotoxicity was investigated by the evaluation of micronuclei level in the blood. Groups of 10 male and 10 female Wistar rats each were exposed by nose-only flow-past inhalation to target concentrations of 1000, 10000 and 20000 / 15000 ppm test substance in each of groups 2 to 4, respectively. The rats of the control group were exposed to filtered air only (group 1). An additional 5 male and 5 female rats were kept for a 2-week recovery period. Mortality, clinical signs, functional observation battery, grip strength, body temperature, locomotor activity, body weights, food consumption, clinical laboratory investigations, micronucleus evaluation, organ weights, macroscopic and microscopic findings were recorded.

Exposure to chemically determined atmosphere concentrations of 1000, 10010 and 20239 / 15076 ppm test substance were achieved in groups 2 to 4, respectively, and were close to the respective targets. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

The test substance did not induce micronuclei as determined by the micronucleus test in peripheral blood cells on day 4 or at the end of exposure. Therefore, the test substance was considered to be non-mutagenic in this micronucleus assay under the experimental conditions of this study. No effects on the organ weights and no gross or microscopic findings were observed that were considered to be related to exposure.

The test item did not induce micronuclei as determined by the micronucleus test in peripheral blood cells. Therefore, the test item was considered to be non-mutagenic in this micronucleus assay under the experimental conditions of this study.