Norflurane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 1989 - 14 November 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Norflurane

EC Number:

212-377-0

EC Name:

Norflurane

Cas Number:

811-97-2

Molecular formula:

C2H2F4

IUPAC Name:

1,1,1,2-tetrafluoroethane

Details on test material:

- Name of test material (as cited in study report): HFC 134a
- Substance type: pure active substance
- Physical state: gas - suppled as a liquefied gas
- Analytical purity: at least 99.8%
- Purity test date: various between August 1989 and August 1991
- Lot/batch No.: Batches assigned the unique CTL referenc nos. Y00035/019, 020, 022, 024, 025, 026, 027, 029, 031, 032, 034 and 035.
- Stability under test conditions: stable
- Other: HFC 134a was supplied in cylinders by the sponsor as a highly volatile liquid with a boiling point of -27°C. The sponsor also provided information to demonstrate the stability of HFC 134a over the period of the study. Each batch of HFC 134a was assigned a unique CTL Reference Number (Y00035/019, 020, 022, 024, 025, 026, 027, 029, 031, 032, 034 and 035). The purity in each cylinder was at least 99.8%. The certificates of analysis are available in the full report.

Test animals

Species:

rat

Strain:

other: Wistar-derived Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Barriered Animal Breeding Unit at Zeneca Pharmaceuticals
- Age at study initiation: 21 days
- Weight at study initiation: males: 157.9 - 161.6g; females: 139.6 - 144.2g
- Housing: following randomisation the animals were housed 8 per cage (sexes seperately)
- Diet (e.g. ad libitum): Ad libitum (except during exposure period)
- Water (e.g. ad libitum): Ad libitum (except during exposure period)
- Acclimation period: 6-9 days

Male and female Alpk: APfSD rats were obtained from the Barriered Animal Breeding Unit at Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire UK. The rats were delivered in 3 batches on each of three consecutive days fo the same week as follows:

29 litters of 4 males per litter & 29 litters of 4 females per litter: Batch 1, delivered 4 October 1989
29 litters of 4 males per litter & 29 litters of 4 females per litter: Batch 2, delivered 5 October 1989
39 litters of 4 males per litter & 39 litters of 4 females per litter: Batch 3, delivered 6 October 1989

All rats were supplied as weanlings (approximately 21 days old) segregated by sex and litter of origin. They were transported to the Long Term Inhalation Unit, Zeneca Central Toxicology Laboratory in sealed containers to maintain their SFP status. For 10 days following delivery of the rats, personnel access to the unit was restricted as a quarantine procedure.
The rats were housed (sexes seperately) by litter in cages in stainless steel holding chambers of approximately 3.4m3 capacity for 6 - 9 days prior to randomisation. Each chamber contained 12 cages and each cage (45x41x20 cm) housed 2 litters and was made of wire mesh with a central wire mesh divider.
Each half of the cage had a removable water bottle and a food jar of approximately 300g capacity. Following randomisation, the animals were housed 8/cage (sexes separately) with the central wire mesh divider removed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 50-75% RH with occassional excursions of relative humidity down to 45% and up to 85%
- Air changes (per hr): 450 l/min. From week 24 onwards this was increased during non exposure periods to 600 l/min
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 25 October 1989 To: 22 October 1991

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: rotary inhalation chamber
- Method of holding animals in test chamber: 8 rats per wire mesh cage
- Source and rate of air: 450 l / min
- System of generating particulates/aerosols: n/a
- Temperature, humidity, pressure in air chamber: 21.9 - 23.6°C, 51.9 - 71.4% RH.


TEST ATMOSPHERE
- Brief description of analytical method used: automatic air sampling automatically analysed by gas chromatograph equipped with a gas sampling valve and flame ionisation detector.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmospheres were sampled using an automatic air sampling system and were analysed automatically using a gas chromatograph equipped with a gas sampling valve and a flame ionisation detector. Each test atmosphere (including control and room air) was analysed at least hourly during each exposure period (apart from a small number fo exceptions when this was not possible). The analysis system was calibrated using an appropriate range of freshly prepared gaseous standards prior to the study, daily during the first week of exposures (apart from day 6, when there were no exposures due to technical difficulties with the chamber air flow) and then weekly thereafter (apart from a short period during which the system was calibrated fortnightly).
A summary of measured chamber HFC 134a concentrations, temperature, relative humidity and airflow during expsosure is given in Table 1.

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 hours / day; 5 days / week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2500, 10000, 50000 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

85/sex

Control animals:

yes, concurrent no treatment

Details on study design:

The study comprised one control and three treatment groups, with 75 male and 75 female rats per group, to be exposed to the appropriate atmosphere for up to 104 weeks. In addition, a further 10 males and 10 females per group were designated for 52 weeks exposure only provide data at the interim sacrifice. The experimental design is shown in Table 2.
The groups of test rats were designated for exposure for six hours per day, 5 days/week for up to 104 weeks, to target concentrations as shown in Table 2. The control group was exposed to air only but otherwise treated in a similar manner to test animals. The animals were housed and exposed in the same chambers.
The rats were housed 8/cage at the start of the study. When the scheduled numbers were less than 8/cage (1 cage per chamber, containing 5 designated rats), and additional 3 rats (un-numbered) were added to maintain this density and act as microbiological sentinels. In addition, another cage containing 8 rats was allocated to each chamber for a similar purpose. The allocation of animals to cages and cages to chambers is detailed in Appendix A of the study report. After 1 year, the last numbered animal in each of the first 10 cages/sex/group (if surviving) was taken for interim sacrifice.
At randomisation, the rats were randomly allocated to cages by the method described in Appendix D of the full study report. This procedure ensured that each litter was equally represented in all groups (including controls). Each rat was uniquely identified by ear punching with the number assigned to it by the experimental design. Rats not required for the study were discarded.
Each exposure chamber was indentified by treatment group, study number, individual numbers of the contained animals and positions of the cages within the chamber (study report Appendix A).
On the front of each cage was a card identifying the contained animals by sex, individual number, study number, test substance, date of start of study, project licence number, Home Office Licensee and procedure number. The rats and their identification cards were transferred to clean cages as necessary during the study.

Microbiological sentinals were included in the study design to provide information about the microbiological status of the experimental animals. There was no evidence of disease or infection which might have compromised interpretation of the findings.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly during exposure, including the observation of "no abnormalities detected".


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily.



BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first exposure (week 1) and then on the same day of each subsequent week for the first 14 weeks of the study (weeks 2 - 15) and thereafter fortnightly (weeks 17 - 33 and then from week 34) for the remainder of the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data



OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals were examined during the pre-treatement period. Control group and 50000ppm group examined at week 52 and week 104.
- Dose groups that were examined: all pre-exposure, 0 and 50000ppm at 52 and 104 weeks.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 14, 27, 53 and 79. In addition blood samples were taken from these animals at week 104 (terminal kill) and for those animals designated for interim kill at 53 weeks.
- Anaesthetic used for blood collection: Yes: halothane at terminal and interim kills.
- How many animals: Pre-designated ten male and ten female rats per exposure group.
- Parameters checked in table [No.3] were examined


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 14, 27, 53 and 79. In addition blood samples were taken from the terminal kill animals at 104 weeks and from the interim kill animals at 53 weeks.
- How many animals: pre-designated ten male and ten female rats perexposure group (these were different animals from those selected for haemotology,
- Parameters checked in table [No.4] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: weeks 13, 26, 52, 78, 104
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes: 12 - 14 hours while urine samples were collected
- Parameters checked in table [No.5] were examined.


NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

At 52 weeks interim sacrifice of 10 males and 10 females from each exposure group. The remaining rats were killed after 104 weeks.

The following organs were weighed: lungs (with trachea attached but larynx removed), testes, adrenal glands, kidneys, liver, heart, spleen, ovaries and brain. Paired organs were weighed together.
Samples of the following tissues from intercurrent, interim and terminal animals were preserved in fixative: adrenal gland, aorta, bladder, bone (femur including stifle joint), bone marrow (femur), brain, caecum, cervical lymph node, cervix, colon, duodenum, epididyis, eye, Harderian gland, heart, ileum, jejunum, kidney, larynx, liver, lung, mammary gland (females only), mesenteric lymph node, nasal passages, oesophagus, oral cavity, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, rectum, salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord, spleen, sternum, stomach, testis, thymus, thymic lymph node, thyroid gland, tongue, trachea, uterus, vagina, voluntary muscle and any macroscopically abnormal tissue.

All tissues were submitted for histology except for the oral cavity which was stored. Microscopic findings were peer reviewed by an external consultant appointed by the sponsor.

Statistics:

Initial bodyweights (day 1) were considered by analysis of variance and bodyweights for all subsequent weeks by analysis of covariance on initial bodyweight, separately for males and females.
Weekly food consumption, and food utilisation during the periods weeks 1-4, 5-8, 9-14 and 1-14 were considered by analysis of variance separately for males and females.

Haemotology and clinical blood and urine chemistry were considered by analysis of variance at each week of sampling, separately for males and females.

Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females.
All analyses were carried out using the GLM procedure in SAS (1985). Least square means for each group were calculated using the LSMEANS opiton in SAS PROC GLM. Unbiased estimates of differences from control were provided by the difference between each treatment group least square mean and the control group least square mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Plasma Gamma-glutamyl Transferase activity was not analysed statistically as the majority of the values were zero.
Kaplan-Meier survival estimates (Kaplan and Meier, 1958) were calculated seperately for each sex and treatment group. Mortalities following accidents or at scheduled termination were considered censored observations.
Intergroup comparisons of mortality comparing each treated group with the control group were performed using the logrank test (Peto and Pike, 1973)
The incidence of selected neoplastic findings were analysed using Fisher's Exact Test, the incidence in each group being compared with the control group incidence. The incidence of these selected findings were tested for dose-related trends using the logrank test. Both analyses were one-sided, looking for a positive relationship with dose.

Where unusual data were detected analyses were repeated omitting these values to determine their influence.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical abnormalities observed during exposure and none observed during the study which were attributable to exposure to HFC 134a. All groups had a low mortality rate which was similar in control and treated groups of both sexes.


BODY WEIGHT AND WEIGHT GAIN
Bodyweights of males exposed to 10000 and 50000ppm were marginally lower than controls during the first six to eight months of the study. Thereafter, these treatment groups were comparable with controls. Female groups exposed to 2500ppm, 10000ppm and 50000ppm had consistently significantly higher bodyweights throughout the 104 week period. However, this increase showed no coherent dose response and was considered not to be treatment related. None of the bodyweight changes seen is considered to have any toxicological significance.


FOOD CONSUMPTION
In males, the food consumption of all treated groups was marginally lower than controls during the first four months. Thereafter, these treatment groups were, in general, comparable to control animals.
In females, food consumption for all treated groups was marginally higher over the 104 week period.

The majority of male and femal groups showed a small reduction in food consumption at week 44. This coincided with the period of increased ambient temperature and is considered not to be treatment related.

FOOD EFFICIENCY
In males, food utilisation was similar to the control group for all treatment groups. However, there were small but consistent statistically significant increases in efficiency for those exposed to 2500ppm.
For females, all treatment groups showed greater (between 6.5 to 11%) approximately, over the entire measurement period) efficiency of food utilisation, although this was not concentration-related.


WATER CONSUMPTION: no data


OPHTHALMOSCOPIC EXAMINATION
Incidence of occular change was small in treated and control rats. None was considered to be treatment-related and the nature and incidence of the changes were consistent with the expected background for this strain of rat.


HAEMATOLOGY
No consistent changes of toxicological or haematological significance were seen at any of the analysis time points.
Males from all exposure groups at week 14 showed minior decreased in haemoglobin, haematocrit and red cell count. These were attributed to initial samples clotting and the animals being re-bled the following day. These were not evident at week 27. No controls were re-bled for comparative purposes.
At termination, the white blood cell count of femals exposed to 2500ppm was considerably increased. This was attributable to one individual having a very high count count which correlated with the pathological diagnosis of large granular lymphocyte leukaemia. Where this value was excluded the mean value was similar to other groups.

CLINICAL CHEMISTRY
There was a concentration-dependent increase in the plasma glucose levels of males at week 14 and females at week 27. No increases were seen in the respective opposite sex at both time points. It is considered that in the absence of persistent changes in plasma glucose levels, these increases were incidental to treatment. One contributary factor to these increases may have been the collection of samples in numerical order at these time points since the increases were not apparent from week 53 when the first animal from each group in turn was bled, followed by the second animal etc, until all of the designated animals had been bled.
Plasma alanine aminotransferase and aspartate aminotransferase activities were raised in both sexes after exclusions at 10000 and 50000ppm in week 79 and there was some evidence for a similar increase in males at 50000ppmat the terminal kill. These raised values were relatively small, showed no consistent relationship to macroscopic or microscopic findings or other indicators of liver damage in individual animals and as such their relationship to treatment is unclear.
In the absence of other correlates of liver damage they are considered not to be of toxicological significance.
On occasions, high mean values in some parameters were seen to be accompanied by large standard deviations, for example, lactate dehydrogenase activity at week 27 in males exposed to 2500ppm and in males exposed to 50000ppm at termination; also at termination alkaline phosphatase activity in male and female control groups, creatine kinase activity in male and females exposed to 50000ppm and alanine transaminase activity in female controls. These were attributable to a few individuals with abnormally high values (which showed no consistent relationship to histopathological findings) and overall, were considered to be unrelated to treatment in those animals exposed to HFC 134a. Exclusion of these outlying values resulted in the group means being consistent wityh those other groups for the same plasma parameter.
A number of other statistically significant differences between the control and test groups were seen during the study, however, these were minor and/or not dose-related and as such, were considered to be unrelated to treatment.

URINALYSIS
There were small increases (which were not always concentration related) in the urinary fluoride levels of males exposed to 10000ppm in week 26, males exposed to 50000ppm in weeks 26, 52, and 104 and females exposed to 50000ppm in weeks 13, 26 and 104. Some of these increases achieved statistical significance. Other statistically significant differences between the control and test groups were minor and/or not concentration-related and considered to be of no biological significance.
There were no effects or trends on the qualitative tests measured.

NEUROBEHAVIOUR


ORGAN WEIGHTS
At the interim kill and at termination, all treated female groups had increased absolute liver weights when compared with controls. These increases were statistically significant at 2500ppm and 10000ppm at the interim kill and at 2500ppm and 50000ppm at the terminal kill. Increases were similar across the groups and are considered to reflect the higher bodyweights of the test groups since when expressed relative to bodyweight only minor intergroup differences are apparent.
At termination ovary weights, expressed as absolute or adjusted for bodyweight, were statistically significantly higher in the group exposed to 50000ppm as compared with controls. These increases were attributable to two animals with high ovary weights which correlated with the histopathological findings of a malignant granulosa/theca cell tumour and reactive hyperplasia. A small increase in mean ovary weight was apparent in females exposed to 2500ppm. This was attributable to a few animals with increased ovary weights and diagnosed histopathologically with cystic bursae. None of these increases in ovary weight are considered to be treatment related.
Males and females exposed to 50000ppm showed small increases in absolute and relative spleen weights at termination. These are considered not to be treatment related and were attributable to a few individuals with relatively large spleens, which showed some correlation with the histopathological diagnosis of large granular lymphocyte leukaemia of which there was a slightly higher incidence in this group.
Males exposed to 50000ppm showed a statistically significant increase in absolute and relative testes weights at termination.
Other statistically significant differences were small and were considered to be unrelated to exposure.


GROSS PATHOLOGY
There were no treatment related findings in animals at the interim kill at 12 months.
At termination, there were an increased incidence of enlarged/discoloured testes at 50000ppm and an increased incidence of enlarged testes at 10000ppm. There was also a decrease in the incidence of reduced and/or flaccid testes at 50000ppm. These findings correlated with the microscopic findings described below.
A low incidence of masses in the epididymides was reported in animals exposed to 2500, 10000 or 50000ppm. In the 2500 and 50000ppm exposure groups, some of these masses correlated to mesotheliomas which were considered not to be treatment related. None was seen in controls.
The seminal vesicles were reduced in size in some animals from all groups but the incidence was slightly greater at 50000ppm compared with controls. These observations correlated with the microscopic finding of reduced secretion in the seminal vesicles and is considered incidental to treatment.
There was a slight increase in the incidence of enlarged and mottled livers and of livers with roughened surfaces in male rats exposed to 50000ppm compared with controls and in females the incidence of livers with an accentuated lobular pattern was increased at all exposure levels compared with controls. These observations correlated generally with reported microscopic findings which were of a similar incidence in control and treated animals and are considered not to be treatment related.
The minimal increases in masses at necroscopy in male adrenals at 50000ppm correlated with phaeochromocytomas at microscopic examination.
There was also a slight increase in ovarian and vaginal masses at 50000ppm compared with controls. This was associated with a variety of histopathological changes and is considered not to be treatment related.
In males exposed to 50000ppm there was a slight increase in stained and thickened eyelids and staining around the nares. There was no increased incidence in females. The former showed no correlation with histopathologyof the eye and the latter occassionally correlated with findings in the nasal passages. All are considred to be non-specific changes and incidental to treatment.
There was a slight increase in red spots on the lungs of males exposed to 50000ppm compared to controls but this was probably an agonal change.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment related findings in interim animals killed at 12 months.
Non-neoplastic findings in intercurrent and terminal animals are described below. Treatment-related changes were confined to the testes. Leydig cell hyperplasia was recorded in controls and at all exposure levels, mainly in terminal animals. The total incidence of leydig cell hyperplasia are tabulated in Table A.
In three animals exposed to 2500ppm the lesion was diffuse. There was an increased incidence of focal Leydig cell hyperplasia in rats exposed to 50000ppm compared to controls.
There was an increase in the incidence of minimal unilateral atrophy in the testis of rats exposed to 2500ppm or 50000ppm. This was considered to be incidental to treatment as an increased incidence of this lesion was not apparent at 10000ppm, only one testis was affected and the incidence of bilateral atrophy was reduced at these exposure levels.
There was a slight increase in the numbers of rats with reduced secretion in the seminal vesicles at 50000ppm. This change in some animals correlated with animals which were reported macroscopically as having reduced seminal vesicles. However this change did not correlate with testicular atrophy and is seen spontaneously in older rats it was regarded as an incidental finding.
Microscopic lessions correlating with changes reported macroscopically in liver and ovary were of a similar incidence in control and treated animals.
The incidence of the following changes was slightly increased compared to controls in one or both of the 2500ppm and 10000ppm groups - mononuclear cell inflitration in the sciatic nerves and congestion in the spleen of males, and cardiac degenerative myopathy, mammary gland hyperplasia and inflammation of the vagina in females. The increased incidence of these changes was not seen at 50000ppm and was therefore considered to be of no pathological significance.
A wide variety of non-neoplastic changes was present with occasional differences between treated and control groups. Many of these differences were not related to exposure concentration and they were considered to be spurious and not treatment related.



HISTOPATHOLOGY: NEOPLASTIC (if applicable)
There were no significant neoplastic findings in interim animals.
In intercurrent and terminal animals, there was a slight increase over controls in the number of tumour bearing males in the group exposed to 50000ppm. The number of animals in this group with malignant, benign, multiple or metastatic tumours was slightly increased compared to controls. In females exposed to 2500 there was a slight increase in the number of animals with multiple benign tumours. This increase was absent from rats at the higher exposure levels and was considered to be an incidental finding.
There was an increased incidence of benign testicular Leydig cell tumours at 50000ppm compared with controls. A criterion used for distinguishing focal Leydig cell hyperplasia from neoplasia was the size of the lesion. Lesions larger than the cross section of one seminiferous tubule were classified as neoplasias.
Most Leydig cell tumours, both unilateral and bilateral, were found in rats which survived to termination. The relative proportions of bilateral tumours to total numbers of tumours expressed as a percentage was similar in controls (33%) and rats exposed to 50000ppm (35%). (See Table B).The proportion of multiple tumours in the total was higher at 50000ppm (52%) than controls (22%). (See Table C).
The majority of animals with Leydig cell tumours also displayed Leydig cell hyperplasia. Some of the masses in the epididymis recorded at necropsy correlated with mesotheliomas. These were seen in both the 2500 and 5000 ppm exposed groups. These are not uncommon findings in the Alpk/APfSD rat, were not dose related and were considered not to be treatment related.
in females, five animals exposed to 50000ppm, four animals exposed to 10000ppm, four animals exposed to 2500ppm and one control developed aI
generalised large granular lymphocyte leukaemia (LGL leukaemia). This neoplasm was also present as a localised tumour in the spleens of two animals exposed to 50000ppm and one animal exposed to 2500ppm. Similar cells to those seen in the livers of animals with a generalised LGL leukaemia were present in the livers of two female controls but the leukaemia cells were not apparent in the spleen or any other tissue. These were diagnosed as incipient LGL leukaemias. In males, four animals exposed to 50000Ppm, two exposed to 10000ppm, three exposed to 2500ppm and one control had a generalised LGL leukaemia. LGL leukaemia was also localised in the spleens of one animal exposed to 5000ppm and one control. In two animals exposed to 50000ppm incipient LGL leukaemia was recorded in the liver. Total incidences of LGL leukaemia in males and females including generalised, localised and incipient leukaemias are tabulated in Table D.
There was a generalised lymphosarcoma in a few animals throughout the groups. Some of these lympnosarcomas were leukaenric. In one male exposed to 10000ppm there was a single incidence of lymphosarcoma - mycosis fungoides confined to the tongue and skin on the limb. The total incidence of lymphosarcoma are tabulated in Tables E and F.
The minimal increase in masses at necropsy in male adrenals at 50000ppm correlated with phaeochromocytomas recorded at microscopic examination. The incidence of phaeochromocytomas was not significantly different from concurrent controls and was within the limits of historical controls.
(See 7.7 Carcinogenicity / Hext 1993)

HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS
Factors contributing to death.
Factors contributing to death were similar in controls and treated animals with no evidence for a treatment related increased incidence.

Effect levels

Dose descriptor:

NOAEC

Effect level:

50 000 ppm

Sex:

male/female

Basis for effect level:

other: There was no evidence of any toxicity or compound related effects at any exposure level in the blood clinical chemistry and haematology parameters investigated.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 See 7.7 Carcinogenicity / Hext 1993 for tables on testicular hyperplasia and benign Leydig cell tumour occurance.

Applicant's summary and conclusion

Conclusions:

In a combined chronic toxicity-carcinogenicity study on 1,1,1,2-tetrafluoroethane (HFC 134a) in rats exposed up to 50000ppm, there was no evidence of any toxicity or compound related effects at any exposure level in the blood clinical chemistry and haematology parameters investigated. The NOAEC for non-neoplastic effects was judged to be 50000ppm(208000mg/m3).

Executive summary:

In a combined chronic toxicity-carcinogenicity study, Alpk/APfSD Wistar-derived rats (85/sex/group) were exposed (6 h/d, 5 d/wk) by whole-body inhalation to 0, 2,500, 10,000 or 50,000 ppm HFC-134a (0, 10,400, 41,700, 208,000 mg/m3) for 52 weeks, when 10 rats of either sex from each group were killed ad interim; the remaining rats were continued to be exposed until terminal kill after 104 weeks. The highest concentration was chosen as the limit dose (5% in air, i.e. 50,000 ppm). All groups had a similar survival rate. The observed differences in body weight and food consumption reflected only biological variation and were not compound related. There were no treatment-related changes in clinical chemistry and haematology parameters in any of the exposed animals. Small increases in urinary fluoride levels were seen on occasion in groups exposed to 10,000 and 50,000 ppm, but were considered to be of no biological significance.