Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EPA Guideline, In vivo mammalian bone marrow cytogenetic tests: micronucleus assay. HG. - Chromo-Micronuc, August 1982.
GLP compliance:
yes (incl. certificate)
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): 1,1,1,2-tetrafluoroethane
- Substance type: pure active substance
- Physical state: colourless gas at room temperature
- Analytical purity: >98.3% according to specification.
- Impurities (identity and concentrations): Toxicology test grade specification defined by joint panel 10-20-1987

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males mean: 28.0g (25 - 30 g); females mean: 23.0g (21 - 37g)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ad libitum - rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe)
- Water (e.g. ad libitum): ad libitum - tap water in plastic bottles
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours darkness


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation
Vehicle:
none
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass cylinder with a volume of 80 litres, standing in a vent pipe with a volume of approximately 4m3.
- Method of holding animals in test chamber: placed individually in cylindrical plastic tubes.
- Source and rate of air: 800 litres / hour
- Method of conditioning air: oxygen was added as necessary to prevent anoxia
- Treatment of exhaust air: drawn off and neutralised by gas-cleaning equipment


TEST ATMOSPHERE
- Brief description of analytical method used: every 30 minutes using a single beam photometer (Foxboro Analytical, South Northwalk, USA)
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
6 hours. In the highest dose level (50000ppm) the inhalation period of the females was only 5 hours due to low supplies of test substance.
Frequency of treatment:
single exposure
Post exposure period:
Animals were terminated 24, 48 or 72 hours after treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50000, 150000, 500000 ppm
Basis:
nominal conc.
see table A for measured mean concentrations.
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent no treatment
Positive control(s):
cyclophosphamide - Endoxan®
- Justification for choice of positive control(s):
- Route of administration: inhalation (nose only)
- Doses / concentrations: 2.5 hours (2.5 mg/l air)

Examinations

Tissues and cell types examined:
Bone marrow, erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels were selected on the basis of acute toxicity studies, which yielded a 4 hour LC 50 greater than 50 vol % in rats. Therefore a top dose of 50 vol % HFC 134a was chosen.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):


DETAILS OF SLIDE PREPARATION: In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after the end of the inhalation period. For each animal, about 3ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. Teh proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. On drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number, and air dried for about 24 hours.

Staining procedure:
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
-10 minutes staining in 1 part Giesma solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellen (rapid mounting media for microscopy)


METHOD OF ANALYSIS:


OTHER:
Evaluation criteria:
One thousand polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group to which they belonged was unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occuring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occuring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon ( paired, one sided increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and sacrifice interval were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).
Statistics:
The statistical evaluations were performed using the "Diamant" computer program version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95% level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
5.0 vol % - increased spontaneous activity, irregular breathing; 15.0 vol % - increased spontaneous activity, irregular breathing; 50.0 vol % - increased spontaneous activity, irregular breathing, ataxic gait, reduced pawreflex to pinching, reduced placin
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The bone marrow smears were examined for the occurance of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the dose groups of HFC 134a was within the range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The number of normochromatic erythrocytes with micornuclei did not differ significantly from the values of the simultaneous control animals for each of the three sacrifice intervals investigated. The ratio of polychromatic erythrocytes remained essentially unaffected by the test compound.

Treatment with Cyclophosphamide (Endoxan®) for 2.5 hours induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.

The test ws performed according to the methods described. No unforseen circumstances were observed which may have affected the the quality and integrity of the the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In a micronucleus assay, NMRI mice were exposure for 6 hours to concentrations of up to 500000ppm HFC 134a. The incidence of micronucleated polychromatic erythrocytes in the bone marrow was not statistically significantly different from controls. The ratio of poly- to normo-chromatic cells remained unaffected by the treatment with HFC 134a. HFC 134 was therefore demonstrated to be not mutagenic in the micronucleus test.
Executive summary:

HFC 134a was tested in the micronucleus test. The test compound was administered in a single inhalation period to male and female NMRI mice. The following doses were tested: 0, 5.0, 15.0 and 50.0% vol/vol.

Single acute toxicity studies yielded a 4 -hour L50 greater than 50% v/v HFC 134a and this concentration was chosen the top dose level.

The animals were treated over an exposure period of 6 hours with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after inhalation. In the highest dose level the inhalation period of the females was only 5 hours instead of 6 hours due low substance resources.

Endoxan® was used as a positive control substance and was administered in a single inhalation period over 2.5 hours (2.5 mg/l) in air.

The incidence of micronucleated polychromatic erythrocytes in the animals treated with HFC 134a was within the normal range of negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by treatment with HFC 134a and was statsically not changed from control values.

Endoxan® induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, confirming the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to any significant extent.

The results show that, under the conditions of the study, HFC 134a is not mutagenic in the micronucleus test.