Isoprene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Isoprene is considered to be genotoxic in vivo, having shown clear effects in the mouse

Link to relevant study records

Reference

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP status unknown, near guideline study, published in peer reviewed literature, adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

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

Vehicle:

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

Duration of treatment / exposure:

20, 40 or 80 weeks

Frequency of treatment:

4 or 8 hrs/day

Remarks:

Doses / Concentrations:
0, 10, 70, 140, 280, 700, 2200 ppm/ 4 or 8 hours/day, 5
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control(s):

none

Tissues and cell types examined:

Micronucleated polychromatophilic or orthochromophilic erythrocytes

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
- 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.
- Ten controls were also sampled when an exposed group was.

DETAILS OF SLIDE PREPARATION:
- A slightly modified method (micronucleated polychromatophilic or orthoch-romophilic erythrocytes were counted) as de-scribed by Miller (1973) was used.
- A total of 2000 erythrocytes from each animal were evaluated.

Statistics:

Results from the micronucleus evaluation were analyzed using one way analysis of variance or a nonparametric test (Kruskal-Wallis and Dunn's summed rank).

Sex:

male/female

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

After 40 weeks of exposure, controls, 70 ppm and 140 ppm groups had similar group mean values. 2200 ppm animals had micronucleus counts that were significantly increased (147%) compared to controls. After 80 weeks of exposure, controls, 10 ppm and 70 ppm animals had similar values. All mice exposed to higher concentrations of isoprene (=280 ppm) had significantly greater micronuclei values compared to controls. 280 ppm, 700 ppm, 2200 ppm/4 h and 2200 ppm animals were 92, 133, 167, and 125% higher, respectively, than the controls.

Conclusions:

Interpretation of results (migrated information): positive
Exposure to isoprene resulted in genotoxic effects with the mean incidence of micronuclei in peripheral blood significantly (p=0.05) greater at 700 ppm or higher after 80 weeks.

Executive summary:

The mean incidence of micronuclei in peripheral blood was significantly increased at 700 ppm and higher after 80 weeks. Similar effects were observed after 40 weeks, although only the 2200 ppm animals had significant elevations of micronuclei compared to the control, 70 and 140 ppm groups. (The 280 and 700 ppm groups were not sampled at 40 weeks). There was no apparent relationship to exposure duration or cumulative exposure on the incidence of micronuclei.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

Non-human information

 

In vitro data

Two key studies are considered to be a bacterial mutation assay (HLS, 2004) and a mammalian cell cytogenetic assay (NTP, 1995).  These are two recognised core assay types for investigating mutation in vitro. An additional key study is a recent mammalian cell DNA damage (comet) assay (Fabiani, 2007).  

Isoprene was tested using vapour phase exposure in a standard Ames test with S. typhimurium strains TA1535, TA1537, TA98, and TA100 and E. coli WP2uvrA (pKM101). Exposure concentrations were 0.25 to 25% isoprene monomer, and tests were carried out in the absence or presence of an auxiliary metabolic activation system of S9 or microsomes prepared from uninduced mice. Isoprene was negative under these conditions. Similar negative results have been reported from other bacterial mutagenicity studies including Mortelmans et al, 1986 and Madhusree et al, 2002. The conditions used in these latter studies included the pre-incubation protocol for the Ames assay with dose levels up to 10 mg/plate, and exposure together with S9 from Aroclor-induced rat and hamster liver. Isoprene has therefore been examined in a range of bacterial strains, with and without auxiliary metabolic activation and as a liquid or vapour phase, and is reported as non-mutagenic.

Isoprene was examined for the ability to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells, both in the presence and absence of Aroclor-induced rat liver S9 (NTP, 1995). Isoprene was tested at dose levels up to 5000 µg/ml. No significant increase in chromosomal aberrations was observed.  

Fabiani (2007) examined isoprene in cultured human promyelocytic leukemia cells (HL60) for DNA damage using the single cell gel electrophoresis method for examining DNA strand breaks (comet assay). Isoprene was tested in the absence or presence of liver S9 from rats that were uninduced or induced with phenobarbital or ethanol. High concentrations of isoprene were tested (up to 30mM in the absence of auxiliary metabolic activation and 15 mM in its presence), and isoprene was negative in the absence of S9, but positive in the presence of both uninduced and induced S9 fractions.

 

An additional endpoint examined in cultured cells was the sister chromatid exchange endpoint (NTP, 1995), and isoprene was negative.

 

Isoprene has been shown to be negative for the induction of bacterial mutagenicity and chromosomal damage in mammalian cells, but positive at high concentrations for the induction of DNA damage in mammalian cells, in the form of DNA strand breaks, only in the presence of S9.

 

 

In vivo data

 

The key studies are considered to be those examining the cytogenetic endpoint (Tice et al, 1988, NTP, 1999, Placke et al, 1996). This is a recognised core assay type for investigating mutation in vivo.

 

Male mice were exposed to isoprene by inhalation for 6 hours/day at concentrations of 438, 1750, or 7000 ppm for 12 days (Tice et al, 1988). Examination of the peripheral blood showed increased incidences of micronucleated polychromatic and normochromatic erythrocytes (MPE and MNE) over controls at all exposure levels. A dose related decrease in the percentage of polychromatic erythrocytes (PCE) in the erythrocyte population was also seen indicating an accompanying toxic effect on the bone marrow.

In separate studies, similar positive results (increase in MPE and/or MNE) were observed in both male and female mice exposed to isoprene at dosed levels up to 7000 ppm for 13 weeks (NTP, 1999) and in male and female mice exposed to isoprene at dose levels up to 2200 ppm for 40 or 80 weeks (Placke et al, 1996).

In contrast to the above positive results with the micronucleus endpoint, exposure of mice to isoprene for 6 hours/day at concentrations of 438, 1750, or 7000 ppm for 12 days did not induce a statistically significant increase in the frequency of chromosomal aberrations measured in metaphase cells from the bone marrow (Tice et al, 1988). However, in the same paper, an examination of the sister chromatid exchange endpoint in animals exposed to the same conditions resulted in a significant increase over controls at all the dose levels studied. A negative result was also reported for the micronucleus endpoint in a study that examined lung fibroblasts in rats exposed to isoprene at doses up to 7000 ppm for 4 weeks.

Isoprene is genotoxic in the mouse, inducing chromosomal damage in bone marrow cells.

Human information

There is no information indicating any adverse effects of isoprene.

 

Summary and Discussion of Mutagenicity

Isoprene has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown negative results for mutagenicity in vitro in the core endpoints of bacterial mutation and cytogenetic damage, both with and without S9. However, isoprene induced DNA damage (strand breaks) at high concentrations in a mammalian cell line in vitro. These effects were only observed in the presence of S9. 

There are several reports of isoprene inducing micronuclei in the bone marrow of mice exposed for different times from 12 days through to 80 weeks. A single examination in the rat reported a negative result for micronucleus induction, although this was in lung fibroblasts and not in bone marrow cells. A direct comment on the relative sensitivity to genotoxic effects of isoprene in the mouse and rat is therefore not possible.

Isoprene is considered to be genotoxic in vivo, having shown clear effects in the mouse. 

 

Justification for classification or non-classification

Since Isoprene is considered to be genotoxic in vivo it has been classified as a germ cell mutagen Cat. 2 H341 (Suspected of causing genetic defects) under GHS/CLP.