Isoprene

Carcinogenicity

Description of key information

There is clear evidence of carcinogenicity of isoprene in mice, with increases in the incidence of malignant neoplasms in the liver, lung, Harderian gland and forestomach. Histiocytic sarcomas and haemangiosarcomas were also observed, together with benign tumours observed in the liver, lung, Harderian gland, forestomach and pituitary.
 
In rats, there were no significant increases in the incidence of malignant tumours, although isoprene exposures were associated with increases in benign tumours of the testes, kidney and mammary gland.
 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, near guideline study, published in peer reviewed literature, fully reliable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Deviations:

not specified

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River's Portage Laboratory
- Age at study initiation: no older than 10 weeks
- Housing: individually housed in stainless steel wire-mesh inhalation cages that fitted into the exposure chambers
- Diet: Certified Purina Rodent Chow in pellet form ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: 25 days
- Prior to the study, sera were collected from at least 5 mice/sex and were found free of antibody titers to common murine pathogens.

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C (chamber)
- Humidity: 40-70% (chamber)
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data

Route of administration:

inhalation: vapour

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers (Hazelton H1000 or H2000, 1000 or 2000 L in volume)
- Method of holding animals in test chamber: stainless steel wire-mesh cages
- Test atmospheres were generated from the liquid as an aerosol by ultrasonic nebulization followed by evaporation in a stainless steel aging plenum
- Filtered compressed air was supplied to the ultrasonic spray nozzle. Isoprene was fed to the spray nozzle from the storage cylinder which was pressurized with N2 at 5 psig.
- Room air was drawn into the aging plenum through charcoal and high efficiency particulate air (HEPA) filters and carried isoprene vapour to the exposure chambers through a common distribution manifold with subsequent dilution at each chamber to the target concentrations.
- Temperature: 22±2°C (range:22.1 to 22.7°C); humidity 40-70% (range: 52.3 to 55.0%)
- Chamber flow was a nominal 15 air changes per h
- Chamber pressure and flowrate were monitored continuously during exposures

TEST ATMOSPHERE
- Brief description of analytical method used: A Miran-980 infrared spectrometer (IR) was used to measure isoprene concentrations in the chambers and exposure room.
- An hourly sampling sequence was used to minimize the step change in concentration among chambers.
- Some chamber samples were also analyzed with a gas chromatograph with a Flame Ionization Detector, primarily to determine the concentrations of limonene (isoprene dimer).
- Uniformity within the exposure chambers was verified by measuring the isoprene concentration at the front, middle, and back of each chamber level.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Buildup and decay of isoprene in the chambers were rapid with T90 achieved in 4 to 10 min. Using an aerodynamic particle size (APS 338; TSI, Inc., St. Paul, MN, USA), no residual aerosol was detected during vapour generation.
All values were within 4% of target concentrations.

Duration of treatment / exposure:

4 or 8 hours/day

Frequency of treatment:

variable - 5 days/week for 20, 40 or 80 weeks

Remarks:

Doses / Concentrations:
0, 10, 70, 140, 280, 700 or 2200 ppm
Basis:
nominal conc.
4 hr

Remarks:

Doses / Concentrations:
0, 28, 195, 390, 780, 1950, 6129 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
10.4, 70.1, 141, 280, 700 or 2173 ppm
Basis:
analytical conc.
grand means (4 h)

Remarks:

Doses / Concentrations:
2203 ppm
Basis:
analytical conc.
grand mean (8 h)

No. of animals per sex per dose:

50

Control animals:

yes, sham-exposed

Details on study design:

Post-exposure period: variable - animals held following exposures until week 96 or 105

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for 13 weeks and then monthly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for 13 weeks and then monthly

HAEMATOLOGY: Yes
- Blood samples were collected using EDTA-containing tubes from the retro-orbital sinus from each animal reaching scheduled necropsy.
- The following parameters were measured: red blood cell count, haematocrit, haemoglobin, total white blood cell count and white blood cell differential and morphology evaluation.

OTHER: Micronucleus evaluation:
- Evaluations of peripheral blood were performed on 10 animals per exposure group using samples obtained the day after the last exposure for those groups ending exposure after 40 and 80 weeks.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A complete gross necropsy was performed on all animals that died during the study or at scheduled necropsy
- Animals were anaesthetized using pentobarbital and exsanguinated
- Tissues from all major organs were removed and preserved in 10% neutral buffered formalin, except for the eyes and testes which were fixed in Bouin's solution.

HISTOPATHOLOGY: Yes
- Histopathological evaluations were conducted on all tissues from all animals, including early death or moribund-sacrificed animals.
- Tissues were embedded in paraffin, sectioned at approximately 5 µm, stained with haematoxylin and eosin and the slides evaluated by light microscopy.

Statistics:

Body weights, organ weights and haematology data were evaluated by analysis of variance (ANOVA) followed by Duncan's new multiple range test. Incidences of tumour types were analyzed using Fischer's exact test applied to each combination of exposure group and tumour type.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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 was an isoprene concentration-related effect on survival. At approximately Week 80, animals exposed to higher concentrations (280 to 2200 ppm, Groups 7 through 12) had lower survival rates (< 50%) than the controls or animals exposed to less than 280 ppm for 80 weeks.

GROSS PATHOLOGY
Gross lesions considered to be directly or in directly related to isoprene exposure were observed in the eyes, forestomach, Harderian gland, liver, lung, spleen, mesenteric lymph node, and testis of male and, in general, correlated with the histopathological findings.

HISTOPATHOLOGY:
Eyes were opaque and often protruded due to enlarged Harderian glands. The forestomach, when affected, had small nodules on the mucosal surface and two isoprene-exposed mice had forestomach masses. The incidences of liver nodules and masses were increased in higher isoprene exposure-level concentration groups compared to controls, as were the incidences of lung nodules and masses. The number of enlarged spleens was slightly increased at higher isoprene exposure-levels, as was the incidence of enlarged mesenteric lymph nodes. The incidence of small testes was increased at exposures above 5600 ppm-weeks (concentration times exposure-weeks).

ORGAN WEIGHT
Absolute testis, testis-to-body weight ratio and testis-to-brain weight ratio values were significantly less (20 to 30%) in Groups 7, 8, 10, and 12 compared to controls.
Heart weight was significantly less in 10 ppm females but not at 70 ppm. Mean ovary weight was less, although not significantly, in 10 and 70 ppm
females compared to controls. The lower gonadal weights may have been exposure-related.

NON-NEOPLASTIC CHANGES
Numerous non-neoplastic lesions were observed. There was a slight increase in alveolar epithelial lining cell hyperplasia at the higher doses in male mice. Focal areas of epithelial hyperplasia of the forestomach mucosa were observed more often in males at the higher levels of isoprene. The nose/nasal cavity had a significant non-neoplastic lesion that was more prominent in the four highest doses in males and the highest dose in females. The lesion was in the dorsal meatus and consisted of focal areas of mild metaplasia of the olfactory epithelium to respiratory epithelium. The metaplastic epithelium often in-vaginated to form glandular patterns.

NEOPLASTIC CHANGES
Several types of neoplasms occurred only, or in greater frequency, in mice exposed to isoprene compared to controls.

Dose descriptor:

NOEC

Effect level:

28 mg/m³ air (nominal)

Sex:

male/female

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOEC

Effect level:

1 950 mg/m³ air (nominal)

Sex:

male

Basis for effect level:

other: lung tumour and haemangiosarcoma

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOEC

Effect level:

780 mg/m³ air (nominal)

Sex:

male

Basis for effect level:

other: malignant forestomach tumours and histiocytic sarcomas

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOEC

Effect level:

390 mg/m³ air (nominal)

Sex:

male

Basis for effect level:

other: liver tumours

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOEC

Effect level:

195 mg/m³ air (nominal)

Sex:

male

Basis for effect level:

other: Harderian gland tumours

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOEC

Effect level:

195 mg/m³ air (nominal)

Sex:

female

Basis for effect level:

other: possible haemagiosarcomas

Remarks on result:

other: Effect type: carcinogenicity

The carcinogenic potential of isoprene was evaluated as a function of concentration, length of daily exposure, and weeks of exposure as independent variables. Exposure of mice to the varied concentrations and schedules did not produce any significant signs of general toxicity.

There was a concentration-related effect on survival due to increases in selected tumour development and associated mortality. Survival was near or below 50% after 95 weeks for mice exposed >280 ppm for 80 weeks - surviving mice in these groups were necropsied during week 96.

Isoprene exposure caused an increase in neoplasms of the lung, liver, Harderian gland, forestomach, lymphoreticular system of male mice and in the Harderian gland and pituitary gland of female mice at concentrations of 70 ppm and higher.

The product of concentration and length/duration of exposure was not a sufficient basis for prediction of tumour risk.

Table 2: Summary of isoprene exposure-related neoplasms in male mice

(based on Placke M, Griffis L, Bird M, Bus J, Persing R and Cox L Jr.1996, Toxicology, Vol. 113, pp. 253-262, Table 1)

Concentration (ppm)	0	10	70		140	280		700	2200
Daily exposure (h)	8	8	8	8	8	8	8	8	4	4	8	8
Weeks exposed	80	80	40	80	40	20	80	80	20	80	40	80
Incidence of alveolar/bronchiolar adenomas and carcinomas
no tissues examined	50	50	50	50	50	50	50	50	50	50	49	50
adenoma	11	16	8	4	10	16	13	23	14	15	29	30
carcinoma	0	1	0	2	1*	3*	1*	7*	2*	3*	3*	7*
Incidence of hepatocellular adenomas and carcinomas
no tissues examined	50	50	49	50	50	49	50	48	50	50	47	50
adenoma	11	12	14	15	22	18	24	27	22	21	28	30
carcinoma	9	6	11	9	10*	12*	16	17*	12*	15*	18*	16*
Incidence of Harderian gland adenomas and carcinomas
no tissues examined	47	49	48	50	50	49	50	49	49	50	50	50
adenoma	4	4	13	9	12	16	17	26	19	28	31	35
carcinoma	0	0	0	0	2	3	1	3	1	2	0	2
Incidence of heart haemangiosarcomas
no tissues examined	49	50	49	50	50	50	50	50	50	50	49	50
haemangiosarcomas	0	0	0	0	0	0	2	1	4	1	1	1
Incidence of spleen haemangiosarcomas
no tissues examined	49	48	47	50	50	47	50	48	48	50	47	49
haemangiosarcomas	1	3	1	2	3	2	1	2	2	2	0	1
Incidence of forestomach squamous papillomas (SP) and squamous carcinomas (SC)
no tissues examined	50	48	47	50	49	46	50	47	48	50	47	50
SP	0	0	0	0	0	0	0	1	0	1	2	1
SC	0	0	0	0	0	0	1	0	1	1	0	3
Incidence of histiocytic sarcomas
no tissues examined	50	50	50	50	50	50	50	50	50	50	50	50
histiocytic sarcoma	0	2	2	2	1	8*	4	2	5	7*	7*	2
Incidence of lymphomas
no tissues examined	50	50	50	50	50	50	50	50	50	50	50	50
lymphomas	2	1	2	4	1	7	5	4	4	4	5	6
*incidences significantly greater than in control (p<0.05)

 

Table 3: Summary of isoprene exposure-related neoplasms in female mice

(based on Placke M, Griffis L, Bird M, Bus J, Persing R and Cox L Jr.1996, Toxicology, Vol. 113, pp. 253-262, Table 2)

Concentration (ppm)	0	10	70
Incidence of alveolar/bronchiolar adenomas and carcinomas
no tissues examined	50	50	50
adenoma	5	6	5
carcinoma	0	0	0
Incidence of hepatocellular adenomas and carcinomas
no tissues examined	50	50	50
adenoma	4	6	3
carcinoma	2	4	2
Incidence of Harderian gland adenomas and carcinomas
no tissues examined	49	49	49
adenoma	2	3	8*
carcinoma	0	0	0
Incidence of heart haemangiosarcomas
no tissues examined	50	50	50
haemangiosarcomas	0	0	0
Incidence of spleen haemangiosarcomas
no tissues examined	50	49	50
haemangiosarcomas	1	1	4
Incidence of forestomach squamous papillomas (SP) and squamous carcinomas (SC)
no tissues examined	50	49	50
SP	0	0	0
SC	1	0	0
Incidence of pituitary adenoma
no tissues examined	49	46	49
adenomas	1	6	9*
Incidence of histiocytic sarcoma
no tissues examined	50	50	50
sarcomas	4	5	6
Incidence of any lymphomas
no tissues examined	50	50	50
lymphomas	9	10	12
*incidences significantly greater than in control (p<0.05)

 

Conclusions:

The results of this study indicated that concentration, length of daily exposure, and weeks of exposure did not affect tumour incidence equivalently and total cumulative exposure was not sufficient for predicting oncogenic risk from isoprene exposure in mice. There appeared to be threshold for oncogenic effects in mice, which varied by organ and tumour type. For male mice, the LOEC was 700 ppm (1950 mg/m3) for lung tumour and haemangiosarcoma, 280 ppm (780 mg/m3) for malignant forestomach tumours and histiocytic sarcomas, 140 ppm (390 mg/m3) for liver tumours, and 70 ppm (195 mg/m3) for Harderian gland tumours. For female mice, the LOEL was 70 ppm for total non-liver, non-lung adenomas and possibly for haemagiosarcomas.

Executive summary:

Groups of mice were exposed to isoprene 8 hr/day, 5 days/week at concentrations of 0, 10, 70, 70, 140, 280, 280, 700, 2200 and 2200 ppm for 20-80 weeks. Two groups were exposed 4 hr/day, 5 days/week at 2200 ppm for 20 or 80 weeks. The concentration x time (duration of exposure) values provided a series of theoretically equivalent exposure hazards.

An exposure-related increased incidence of liver, lung, Harderian gland and forestomach tumours and haemangiosarcomas and histiocytic sarcomas was reported with the overall LOEC apparently at 70 ppm (195 mg/m3).

Following statistical analyses, it was concluded that the product of isoprene concentration and length/duration of exposure was not a sufficient basis for predicting tumour risk. In addition, extrapolation of tumour probability between the high and low doses, based on cumulative exposure, was not appropriate or justified by the statistical models. It was concluded that, for this study, a threshold effect level and strong non-linearities with respect to concentration appeared to exist for tumour development.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

28 mg/m³

Study duration:

chronic

Species:

mouse

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Isoprene is classified as Cat 1B H350 'May cause cancer' and, since there is clear evidence of carcinogenicity of isoprene in mice but no data available to indicate carcinogenicity in humans, this position is unchanged based on review of the data.

Additional information

There are sufficient animal studies available on isoprene in order to assess for possible carcinogenic activity. 

Isoprene has been examined for carcinogenic activity in both the mouse and rat.

Placke et al (1996) exposed groups of 50 male B6C3F1 mice to 0, 10, 70, 140, 280, 700, or 2200 ppm (equivalent to 0, 28, 195, 390, 780, 1950 or 6129 mg/m³) isoprene vapour by inhalation for 4 or 8 h/day, 5 days/week for 20, 40 or 80 weeks. Following exposure, animals were maintained without exposure to an intended termination time at 104 weeks. The combination of a range of doses and exposure times followed by a holding period and then termination at a standard time was undertaken to allow an investigation into the effect of integrated exposure x time in producing effects. Groups of 50 female B6C3F1 mice were also exposed to isoprene vapour at levels of 0, 10 or 70 ppm for 8 h/day for 80 weeks, and then held through to termination at 104 weeks.

There was a concentration-related effect on survival with approximately 50% or less of those animals exposed to concentrations of 280 ppm and higher surviving for 80 weeks. In male mice, isoprene exposure produced an increase in histiocytic sarcomas and in neoplasms of the liver, lung, Harderian gland and forestomach. The incidence of hepatocellular adenomas and carcinomas was increased as were haemangiosarcomas and histiocytic sarcomas of the liver. Primary alveolar/bronchiolar adenomas and carcinomas were significantly increased. The incidence of Harderian gland adenomas was significantly increased. Harderian gland carcinomas were not as numerous as adenomas but were present in the higher concentration groups. Neoplasms in the stomach included squamous cell carcinomas of the stomach. Exposed mice also had a slight increase in the incidence of haemangiosarcomas in the spleen and heart compared to controls. In female mice, which were only exposed to lower levels of isoprene, increases were observed in haemangiosarcomas in the spleen, Harderian gland adenomas and adenomas of the pituitary gland. No increased in tumours were seen at 10 ppm in either male or female mice.

 

Melnick et al (1994) exposed male Fischer F344 rats and male B6C3F1 mice to 0, 70, 220, 700, 2200, or 7000 ppm (equivalent to 0, 195, 613, 1950, 6129 or 19503 mg/m³) isoprene vapour by inhalation for 6h/day, 5 days/week for 26 weeks. After exposure, 10 animals per group were killed and examined, with the remaining 30 animals per group continuing without exposure for a further 26 weeks before termination. 

In mice, incidences of malignant lesions in the liver, lung, forestomach, and Harderian gland were significantly increased following both the 26 week exposure and 26 week recovery periods at doses of 700 ppm and above. There was no clear dose response seen with most tumours and the carcinogenic effects of isoprene produced at 700 or 2200 ppm were not much different from those at 7000 ppm. The survival was reduced in the 7000 ppm group with early deaths attributed to various neoplastic lesions and terminations due to hindlimb paralysis.

In rats, interstitial cell hyperplasia of the testis was observed after 26 weeks of exposure to 7000 ppm isoprene. Following the 26-week recovery period, the only effect reported was a marginal increase in benign testicular interstitial cell tumours in the 7000 ppm group.

 

The NTP exposed groups of male and female F344/N rats to 220, 700, or 7000 ppm (equivalent to 613, 1950, 19503 mg/m³) isoprene by inhalation, 6/ day, 5 days/week, for 105 weeks (NTP 1999).

Isoprene exposures were associated with increases in rates of benign tumours in the testes and kidney of males, and in the mammary gland of male and females. No significant increases were seen for malignant tumours in this study. Several rare neoplasms were observed in the brain of exposed female rats. However, the fact that they were from different cell types makes it difficult to conclude that they are treatment related.