Registration Dossier

There is a clear species difference in response to exposure to isoprene, with mice being more sensitive than rats. In rats findings were essentially limited to splenic fibrosis, renal tubule hyperplasia, and interstitial cell hyperplasia of the testis. In contrast, in mice non-neoplastic toxicities include spinal cord degeneration, partial rear limb paralysis, testicular atrophy, olfactory epithelial degeneration, forestomach epithelial hyperplasia and macrocytic anaemia. The overall repeat dose NOAEC for rats is 220 ppm (613 mg/m3) based on splenic fibrosis in males. There was no overall NOAEC for mice, with the LOAEC being 70 ppm (195 mg/m3) based on increased incidence of spinal cord degeneration

Endpoint conclusion:

no study available

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY, USA
- Age at study initiation: At least 6 weeks
- Housing: Individually in wire mesh cages
- Diet: NIH-07 diet ad libitum except during the exposure periods
- Water ad libitum
- Acclimation period: 11-13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 75±3°C
- Humidity: 55 ± 15%
- Air changes: 15 per hr
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Stainless steel Hazelton 2000 chambers (total volume 2.3 m3)
- Exposure apparatus: Vapours were generated in a Buchi Rotavapor system at 50°C
- Chamber concentrations of isoprene were regulated by adjusting metering valves into the exposure chamber
- The daily mean concentrations of isoprene was 99-100% of target concentration

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations in the chambers were measured continuously during the exposures with a Hewlett-Packard gas chromatograph equipped with a flame ionization detector.
t90 (time to reach 90% of target concentration) was approximately 12 mins.

Duration of treatment / exposure:

6 hours/day for 13 weeks

Frequency of treatment:

5 days/week for 13 weeks

Remarks:

Doses / Concentrations:
0, 70, 220, 700, 2200, or 7000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 195, 613, 1950, 6129 and 19,503 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Post-exposure period: not applicable

Observations and examinations performed and frequency:

BODYWEIGHTS / CLINICAL OBSERVATIONS
- Weekly

HAEMATOLOGY: Yes, Blood samples were collected for clinical pathology evaluations on days 4, 24 and at study termination
- Blood samples collected from supraorbital sinus of CO2 anaesthetised animals
- The following parameters were measured:
erythrocyte, leukocyte, and platelet counts, haemoglobin concentration, haematocrit, reticulocyte count, mean cell volume, mean cell haemoglobin and leukocyte differential counts

CLINICAL CHEMISTRY: Yes, Blood samples were collected for clinical pathology evaluations on days 4, 24 and at study termination
- Blood samples collected from supraorbital sinus of CO2 anaesthetised animals
- The following parameters were measured:
urea nitrogen, creatinine, alanine aminotransferase, glutamate dehydrogenase and sorbitol dehydrogenase

URINALYSIS: Yes, All animals in study week 12
- Urinalysis of overnight urine samples. The following parameters were measured: glucose, creatinine, alkaline phosphatase and aspartate aminotransferase concentrations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Complete necropsies were performed on all animals.

ORGAN WEIGHTS: Yes. Brain, heart, right kidney, liver, lung, spleen, right testis, and thymus were weighed prior to fixation.

HISTOPATHOLOGY: Yes. Tissue samples, preserved in 10% neutral buffered formalin, were embedded in paraffin, sectioned, and stained with haematoxylin and eosin. -Tissues examined: gross lesions and tissue masses, adrenal glands, brain, oesophagus, femur and marrow, heart, small intestine, large intestine, kidneys, larynx, liver, lungs, lymph nodes (bronchial, mediastinal, mandibular, and mesenteric), mammary gland with adjacent skin, nasal cavity and turbinates, ovaries, pancreas, parathyroid glands, pituitary gland, preputial gland, prostate gland, salivary glands, spinal cord, and sciatic nerve, spleen, forestomach, glandular stomach, testes with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Analysis of survival and incidence of neoplastic and nonneoplastic lesions was performed. Clinical chemistry, haematology, and urine data were analyzed by nonparametric methods (Shirley (1977) or Dunn (1964)).

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):

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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Following 13 week inhalation exposure to 7000 ppm (19,503 mg/mg3) isoprene, no treatment-related effects were observed in rats.

Key result

Dose descriptor:

NOAEC

Effect level:

19 503 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: Based on lack of toxicological effects in rats exposed up to 7000 ppm (19,503 mg/m3) isoprene for 13 weeks.

Dose descriptor:

LOAEC

Effect level:

> 19 503 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: Based on lack of toxicological effects in rats exposed up to 7000 ppm (19,503 mg/m3) isoprene for 13 weeks.

Critical effects observed:

not specified

Conclusions:

No toxicological effects were evident in rats exposed up to 7000 ppm (19,503 mg/m3) isoprene for 13 weeks.

Executive summary:

In a sub-chronic inhalation toxicity study, Fischer 344 rats (10/sex/concentration) were exposed via whole body inhalation to vapours of isoprene at concentrations of 0, 70, 220, 700, 2200, or 7000 ppm (0, 195, 613, 1950, 6129 and 19,503 mg/m³) 6 hours/day, 5 days per week for a period of 13 weeks.

No toxicological effects were evident in rats exposed up to 7000 ppm (19,503 mg/m³) isoprene for 13 weeks.

The NOAEC was determined to be 19,503 mg/m³.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

195 mg/m³

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY, USA
- Age at study initiation: At least 6 weeks
- Housing: Individually in wire mesh cages
- Diet: NIH-07 diet ad libitum except during the exposure periods
- Water ad libitum
- Acclimation period: 11-13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 75±3°C
- Humidity: 55 ± 15%
- Air changes: 15 per hr
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Stainless steel Hazelton 2000 chambers (total volume 2.3 m3)
- Exposure apparatus: Vapours were generated in a Buchi Rotavapor system at 50°C
- Chamber concentrations of isoprene were regulated by adjusting metering valves into the exposure chamber
- The daily mean concentrations of isoprene was 99-100% of target concentration

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations in the chambers were measured continuously during the exposures with a Hewlett-Packard gas chromatograph equipped with a flame ionization detector.
t90 (time to reach 90% of target concentration) was approximately 12 mins.

Duration of treatment / exposure:

6 hours/day for 13 weeks

Frequency of treatment:

5 days/week for 13 weeks

Remarks:

Doses / Concentrations:
0, 70, 220, 700, 2200, or 7000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 195, 613, 1950, 6129 and 19,503 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Post-exposure period: not applicable

Observations and examinations performed and frequency:

BODYWEIGHTS / CLINICAL OBSERVATIONS
- Weekly

HAEMATOLOGY: Yes, Blood samples were collected for clinical pathology evaluations on days 4, 24 and at study termination
- Blood samples collected from supraorbital sinus of CO2 anaesthetised animals
- The following parameters were measured:
erythrocyte, leukocyte, and platelet counts, haemoglobin concentration, haematocrit, reticulocyte count, mean cell volume, mean cell haemoglobin and leukocyte differential counts

CLINICAL CHEMISTRY: Yes, Blood samples were collected for clinical pathology evaluations on days 4, 24 and at study termination
- Blood samples collected from supraorbital sinus of CO2 anaesthetised animals
- The following parameters were measured:
urea nitrogen, creatinine, alanine aminotransferase, glutamate dehydrogenase and sorbitol dehydrogenase

URINALYSIS: Yes, All animals in study week 12
- Urinalysis of overnight urine samples. The following parameters were measured: glucose, creatinine, alkaline phosphatase and aspartate aminotransferase concentrations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Complete necropsies were performed on all animals.

ORGAN WEIGHTS: Yes. Brain, heart, right kidney, liver, lung, spleen, right testis, and thymus were weighed prior to fixation.

HISTOPATHOLOGY: Yes. Tissue samples, preserved in 10% neutral buffered formalin, were embedded in paraffin, sectioned, and stained with haematoxylin and eosin. -Tissues examined: gross lesions and tissue masses, adrenal glands, brain, oesophagus, femur and marrow, heart, small intestine, large intestine, kidneys, larynx, liver, lungs, lymph nodes (bronchial, mediastinal, mandibular, and mesenteric), mammary gland with adjacent skin, nasal cavity and turbinates, ovaries, pancreas, parathyroid glands, pituitary gland, preputial gland, prostate gland, salivary glands, spinal cord, and sciatic nerve, spleen, forestomach, glandular stomach, testes with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Analysis of survival and incidence of neoplastic and nonneoplastic lesions was performed. Clinical chemistry, haematology, and urine data were analyzed by nonparametric methods (Shirley (1977) or Dunn (1964)).

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):

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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Following 13 week inhalation exposure to 7000 ppm (19,503 mg/mg3) isoprene, no treatment-related effects were observed in rats.

Key result

Dose descriptor:

NOAEC

Effect level:

19 503 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: Based on lack of toxicological effects in rats exposed up to 7000 ppm (19,503 mg/m3) isoprene for 13 weeks.

Dose descriptor:

LOAEC

Effect level:

> 19 503 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: Based on lack of toxicological effects in rats exposed up to 7000 ppm (19,503 mg/m3) isoprene for 13 weeks.

Critical effects observed:

not specified

Conclusions:

No toxicological effects were evident in rats exposed up to 7000 ppm (19,503 mg/m3) isoprene for 13 weeks.

Executive summary:

In a sub-chronic inhalation toxicity study, Fischer 344 rats (10/sex/concentration) were exposed via whole body inhalation to vapours of isoprene at concentrations of 0, 70, 220, 700, 2200, or 7000 ppm (0, 195, 613, 1950, 6129 and 19,503 mg/m³) 6 hours/day, 5 days per week for a period of 13 weeks.

No toxicological effects were evident in rats exposed up to 7000 ppm (19,503 mg/m³) isoprene for 13 weeks.

The NOAEC was determined to be 19,503 mg/m³.

Endpoint conclusion:

no adverse effect observed

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Inhalation

Animal data

A series of well conducted, repeat dose inhalation studies with durations of 2, 13, 26 (with 26 week recovery) and 105 weeks have been conducted on isoprene in both rats and mice. These studies have demonstrated that there is a clear species difference in the susceptibility of rats and mice to isoprene exposure, with mice being more sensitive. This difference is likely related to differences in the rate of metabolism of isoprene to its epoxide metabolites. For instance, it has been estimated using a toxicokinetic model that at high exposure concentrations, metabolism leading to monoepoxides is up to 3.8 times faster in mice than in rats, whereas at lower exposure concentrations this difference is reduced (Csanady et al., 2001). For this reason the repeat dose data for the two species is discussed separately below.

In rats findings were essentially limited to splenic fibrosis, renal tubule hyperplasia, and interstitial cell hyperplasia of the testis. In contrast, in mice non-neoplastic toxicities include spinal cord degeneration, partial rear limb paralysis, testicular atrophy, olfactory epithelial degeneration, forestomach epithelial hyperplasia and macrocytic anaemia. This summary discusses non-neoplastic findings with neoplastic findings being addressed under the carcinogenicity endpoint summary.

Rats

In a 2-week (Melnick et al., 1990) and a 13-week repeated dose whole-body inhalation study (Melnick et al., 1994), F344 rats were exposed by whole-body inhalation to isoprene at concentrations up to a maximum of 7000 ppm (19,503 mg/m3) for 6 hours/day, 5 days/week for two weeks. There were no exposure related effects in either study the NOAEC was at the top dose of 7000 ppm (19,503 mg/m3).

In the 26-week whole-body inhalation exposure study (Melnick et al., 1994), groups of 40 male Fischer 344 rats were exposed to 0, 70, 220, 700, 2200, or 7000 ppm (0; 195; 613; 1950; 6129; 19,503 mg/m3) isoprene vapour by inhalation for 6 hours/day, 5 days/week for 6 months. The NOAEC was 2200 ppm (6129 mg/m3) based on the only non-neoplastic effect observed following 26 weeks of exposure which was an increase in the incidence and relative severity of interstitial cell hyperplasia of the testis at the top dose.

In a chronic oncogenicity study (NTP, 1999), groups of 50 male and female F344/N rats were exposed to 0, 220, 700, or 7000 ppm (0, 613; 1950 or 19,503 mg/m3) isoprene vapour by inhalation, 6 hours per day, 5 days per week, for 104 weeks. Survival of all exposed groups was similar to the chamber controls. There were no differences in survival, mean body weights, or clinical findings in male and female rats. Renal tubule hyperplasia was significantly greater and kidney nephropathy was slightly increased in males exposed at the top dose. Also in males, a significantly greater incidence of splenic fibrosis was reported at =700 ppm (=1950 mg/m3), which defines the LOAEC for this study.

The overall repeat dose NOAEC in rats is therefore 220 ppm (613 mg/m3) from the 2 year NTP (1999) study.

Mice

In the 2-week studies (Melnick et al., 1990; NTP, 1994), B6C3F1 mice were exposed by whole-body inhalation to isoprene at concentrations of 0, 438, 875, 1750, 3500, or 7000 ppm (0; 1220; 2438; 4876; 9751; or 19,503 mg/m3) for 6 hours/day, 5 days/week for two weeks. No mice died, but the mean body weight of males in the 7,000 ppm group was less than that of the controls. Exposure to isoprene caused decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts, atrophy of the testis and thymus, cytoplasmic vacuolization of the liver, olfactory epithelial degeneration in the nasal cavity, and epithelial hyperplasia in the forestomach. The LOAEC for mice was 438 ppm (1220 mg/m3).

In a 13-week repeated dose whole-body inhalation study conducted as part of a 26-week carcinogenicity study (Melnick et al., 1994), B6C3F1 mice were exposed to 0, 70, 220, 700, 2200, and 7000 ppm (i. e., 0; 195; 613; 1950; 6129; 19,503 mg/m3) isoprene, 6 hours/day, 5 days/week for 13 weeks. There were no effects on survival, body weight gain, or clinical signs of toxicity. Cytoplasmic vacuolization of hepatocytes due to glycogen accumulation was also observed in male mice, being statistically significant at the 7000 ppm dose level. Degeneration of the olfactory epithelium was also reported in male mice at this top dose.

In male and female mice exposed to =700 ppm (=1950 mg/m3) hematologic effects indicative of a nonresponsive, macrocytic anaemia were reported at day 24 and after thirteen weeks. As in the 2 week study, focal epithelial hyperplasia of the forestomach was observed in both males and females exposed to =700 ppm. The NOAEC for this study in mice was 220 ppm (613 mg/m3).

In the 26-week whole-body inhalation exposure study (Melnick et al., 1994), groups of 40 male B6C3F1 mice were exposed to 0, 70, 220, 700, 2200, or 7000 ppm (0; 195; 613; 1950; 6129; 19,503 mg/m3) isoprene vapour by inhalation for 6 hours/day, 5 days/week for 6 months. Following the 26-week exposure period, survival was reduced in the top dose group.

Although no treatment-related histopathological changes were detected in the lungs of isoprene exposed mice at the end of the 26-week exposure period, an increased incidence of alveolar epithelial hyperplasia was observed =700 ppm following the 26-week recovery period. In addition, at the end of the 26-week exposure and after the recovery period, focal hyperplasia of the forestomach epithelium was observed at =700 ppm.

Exposure-related decreases in testis weight and testicular atrophy were observed in mice following 26 weeks of exposure to isoprene, but not after the recovery period. Mild to minimal olfactory epithelial degeneration in the nasal cavity was also observed in all mice in the 7000 ppm exposure group after 26 weeks of exposure and, at the end of the 26-week recovery period, the incidence of mild to moderate olfactory epithelial degeneration was significantly elevated at =220 ppm.

Mainly at the top dose and towards the end of the exposure period, there was incidence of abnormal posture and impaired hindlimb function (grip strengths significantly less at =220 ppm). During the 26 week recovery period clinical signs subsided and affected animals gradually returned to normal state. Spinal cord degeneration, likely associated with this hindlimb dysfunction, was evident but minimal in mice exposed to 7000 ppm for 26 weeks; but incidence was significantly increased in all exposure groups by the end of the recovery period. In contrast with this study (Melnick et al, 1994), there were no apparent effects on motor function and no exposure-related lesions in the spinal cord in a chronic oncogenicity study in mice (Placke et al, 1996).

In the chronic oncogenicity study (Placke et al., 1996), 12 groups of male B6C3F1mice were exposed to 0, 10, 70, 140, 280, 700, or 2200 ppm (i. e., 0, 28, 195, 390, 780, 1950, 6129 mg/m3) of isoprene vapour for 4 or 8 hours/day, 5 days/week for 20, 40, or 80 weeks followed by a holding period, leading to a total planned study length of 104 weeks. Female mice were exposed to 0, 10, and 70 ppm of isoprene, 8 hours/day for 80 weeks and also held for observation through week 104. There were no treatment-related effects on body weights. A slight increase in alveolar epithelial lining cell hyperplasia was observed in the higher doses in male mice. Focal areas of epithelial hyperplasia of the forestomach mucosa were observed more often in males exposed to higher levels of isoprene. At >390 and 6129 mg/m3 in the males and females, respectively, there was a significant increase in nose/nasal cavity lesions. These lesions were in the dorsal meatus and consisted of focal areas of mild metaplasia of the olfactory epithelium to respiratory epithelium. Several degenerative lesions were seen at higher incidence in exposed mice, and included chronic degeneration of the interventricular septal myocardial muscle, seminiferous cell atrophy in the testes, increased sperm granulomas in the epididymis, and chronic-active inflammation of the preputial glands in the higher dose males. These degenerative lesions may be the result of the relatively poorer condition of exposed mice due to other complicating health conditions (tumours). In addition, hematopoietic cell proliferation in the spleen and myeloid hyperplasia of the bone marrow was increased slightly in all isoprene-exposed animals.

The overall repeat dose LOAEC in mice is 70 ppm (195 mg/m³) based on increased incidence of spinal cord degeneration after 26 weeks exposure and 26 weeks recovery period, with no NOAEC identified in the Melnick et al (1994) study. However, it is of note that this effect was not reproduced in the chronic oncogenicity study, which also showed a clear NOAEC at 10 ppm (Placke et al, 1996).

Human data

No data are available.

No classification for repeat dose effects is proposed under CLP as no significant toxic effects were reported within the concentration guidance values specified for such classification.