Isoprene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study, published in peer reviewed literature, no restrictions, fully adequate for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD® (Sprague-Dawley) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age/weight at study initiation: Approximately 12 weeks/342-409 grams (males) and 230-283 grams (females) at initiation of exposures (P) x 15 days. Selected F1 males and females (one male and one female from each litter) were exposed for 7 consecutive days (Postnatal days [PND] 21-27 or 28-34)
- Fasting period before study: None
- Housing: Upon receipt (F0) or selection (F1) and until pairing (F0 only), all F0 and F1 test animals were individually housed in clean, wire-mesh suspended cages. The F0 animals were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages with nesting material. F1 offspring that were weaned on PND 28 for the PND 28-34 exposures were individually housed in suspended wire-mesh cages.
- Diet: Certified Rodent Lab Diet® 5002 (PMI Nutrition International, Inc.) ad libitum except during exposure
- Water: ad libitum ad libitum except during exposure
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.7-22.3°C
- Humidity: 35.4-53.4%
- Air changes: Approximately 10 per hr (animal room); at least 12 to 15 air changes per hour (chamber)
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: Feb 1, 2002 To: Apr 26, 2002

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted in 1.0 m3 stainless steel and glass inhalation chambers; operated under dynamic conditions at a slight negative pressure
- Method of holding animals in test chamber: Individually in cages
- One chamber was dedicated for each group for the duration of the study.
- Chamber air supplied by an Inhalation facility HVAC system, conditioned by humidification (if required) and filtration through a HEPA filter and an activated charcoal bed prior to delivery to the chambers.
- System of generating particulates/aerosols: The generation of BTD gas test atmospheres involved delivery of BTD gas directly from the headspace of a single liquefied gas cylinder, followed by distribution and dilution to produce the three required target concentrations.
- Temperature, humidity, pressure in air chamber: 20-26°C, 30-70%, 33 psig
- Air flow rate: 300 mL/minute
- Treatment of exhaust air: Charcoal and HEPA filtration.

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations determined approximately every 35 minutes using a gas chromatographic method. Exposure concentrations determined at least 10 times for each chamber during each 6-hour exposure
- Samples taken from breathing zone: yes
- Oxygen content was approximately 19% for all chambers.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: copulatory plug or sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually, in a plastic maternity cage with nesting material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of the test article was collected from each cylinder at the beginning and end of use (during the exposure phase). The samples were analyzed by a validated gas chromatographic method. The area percent purity assessment for 1,3-butadiene and 4-vinyl-cyclohexene, (CAS no. 10-40-3; lot no. 0730CU) as a potential contaminant was determined. No other significant contaminants were observed.
Actual concentrations were 301, 1507 and 6006 ppm

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

7 days/week

Details on study schedule:

Daily 6-hour exposures, beginning 14 days prior to initiation of the breeding period (15 exposures prior to breeding); F0 males exposed for 83-84 consecutive days; F0 females exposed through gestation day 20 and from lactation day 5 through to the day prior to euthanasia (60-70 total days; F0 females which did not deliver were exposed until one day prior to euthanasia (post-mating day 25). For the lactation exposures, the dams were removed from their litters during each daily six-hour exposure period. Selected F1 males and females (one male and one female from each litter) were exposed for 7 consecutive days (Postnatal days [PND] 21-27 or 28-34).

Remarks:

Doses / Concentrations:
0, 300, 1500 and 6000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 301, 1507 and 6006 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

Exposure levels were selected based on the results of previous studies. The exposure concentrations selected for this screening study were designed to bracket the range of linear and saturable metabolism for 1,3-butadiene (saturation of epoxybutene concentrations in blood occurs at ~1250 ppm). The control group of identical design was exposed to clean, filtered air on a comparable regimen.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- In addition, F0 and F1 animals were observed for appearance, behavior and pharmacotoxic signs prior to exposure, during exposure (for animals visible through the chamber windows) and within one hour after completion of exposure.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weeky for both sexes. Bodyweights were also recorded at the midpoint of study week 1.
- Once evidence of mating was observed, female body weights were recorded on gestation days 0, 7, 14 and 20 and on lactation days 1, 4, 7, 14, 21 and 28.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Female food consumption was recorded on gestation days 0, 7, 14 and 20 and lactation days 1, 4, 7, 14, 21 and 28. Food consumption was calculated and reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in all F0 males:
- epididymis weight, sperm motility, sperm morphology
- The left testis and epididymis from all F0 males from all exposure groups were stored frozen, homogenized and evaluated for determination of homogenization-resistant spermatid count and sperm production rate, using the method described by Blazak et al and the Hamilton-Thorne CASA system.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were weighed, killed and discarded.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
- Detailed physical examinations and body weights were recorded for each pup on PND 1, 4, 7, 14, 21 and 28; food consumption was not recorded for F1 pups.

GROSS EXAMINATION OF DEAD PUPS: yes
- Pups dying from PND 0-4: necropsied using a fresh dissection technique described by Stuckhardt and Poppe (1984).
- Pups that died after PND 4 but prior to weaning were given an external examination and discarded.

Postmortem examinations (parental animals):

SACRIFICE
- All surviving F0 animals killed as soon as possible after the F1 litters were weaned.

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, lymph nodes, thyroid glands, mammary glands, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- the following F0 parental tissues and organs were collected and placed in 10% neutral buffered formalin (except as noted): Coagulating gland, testes with epididymides and vas deferens, prostate, seminal vesicles, lungs, mammary glands, uterus with oviducts and cervix, ovaries, vagina, pituitary and all gross lesions.
- The following organs from all F0 parental animals euthanized at scheduled termination and for all F0 females that failed to deliver offspring were weighed: brain, prostate, epididymides (total and cauda), seminal vesicles with coagulating glands (with accessory fluids), pituitary, testes, ovaries, uterus with oviducts and cervix.

OTHER
- Microscopic examination of the following tissues was conducted for all F0 parental animals from the control and high-exposure groups and from all parental animals dying spontaneously or euthanized in extremis or that failed to breed, conceive, or deliver offspring, if applicable: epididymides (right; caput, corpus, and cauda), ovaries, testes (right).
In addition, due to decreases in absolute and relative seminal vesicle weights observed in the 1500 and 6000 ppm groups, seminal vesicles from all males in all dose groups were examined microscopically.

Postmortem examinations (offspring):

SACRIFICE
- Pups exposed to the test article on PND 21-27 were euthanized on PND 28. Pups exposed to the test article on PND 28-34 were euthanized on PND 35.
- Pups that were stillborn or those that died between birth and PND 4, any pups considered moribund and euthanized in extremis during the lactation period were subjected to macroscopic pathological evaluation.
- All remaining pups were euthanized on PND 28. Pups were examined externally, euthanized by carbon dioxide inhalation, and discarded without macroscopic internal evaluation.

Statistics:

Parametric one-way analysis of variance (ANOVA) – bodyweight, body weight gain, food consumption, gestation length, precoital interval, number of pups born, live litter size, pup weights, organ weights (absolute and relative to final body weight), epididymal and testicular sperm numbers and sperm production rate; Chi-square test with Yates correction factor– mating and fertility indices; Kruskal Wallis with Mann-Whitney U test – sex ratios, postnatal survival, percentage of motile sperm with normal morphology.

Reproductive indices:

Reproductive performance

Offspring viability indices:

Clinical observation, body weight

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Clinical observations indicative of chromodacryorrhea, chromorhinorrhea, and salivation were observed in F0 males and females at 6000 ppm. Similar, but less severe observations were reported at 300 and 1500 ppm. The observations were not considered adverse at these lower levels because the signs were always transient and only reported during the one-hour post-exposure observations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Persistent reductions in body weight parameters were seen in F0 and F1 males and females (see below)..

FOOD CONSUMPTION (PARENTAL ANIMALS)
Transient reductions in food consumption (week 0-1) for F0 males and females were observed.

REPRODUCTIVE PERFORMANCE
Male and female mating indices were 100% for all groups on study. Male and female fertility indices were 83.3%, 100%, 100%, and 91.7%, for the control, 300, 1500, and 6000 ppm groups, respectively.

GESTATION LENGTH
The mean gestation lengths for the control, 300, 1500, and 6000 ppm groups were 21.7, 21.8, 21.6, and 22 days, respectively.

SPERMATOGENIC ENDPOINTS
There were no test article-related effects on spermatogenesis at any exposure level tested. Testicular and epididymal sperm count data were comparable across all study groups. Mean sperm motility was 86.3%, 82.8%, 83.3%, and 81.1% for the control, 300, 1500, and 6000 ppm groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At 6000 ppm there were increased F0 male brain weight relative to final body weight (p<0.05) and reduced F0 male seminal vesicle/coagulating gland weight relative to brain weight (p<0.05). At 1500 ppm there was reduced F0 male seminal vesicle/coagulating gland weight relative to brain weight (p<0.05). Although the reductions occurred in a dose-related manner, there were no microscopic correlates when seminal vesicles were examined and there were no functional deficits in reproductive outcome in this group. Therefore, the reductions in mean absolute and relative (to final body and to brain weight) seminal vesicle weights were attributed to biological variation.

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

300 other: ppm (663 mg/m3)

Sex:

male/female

Basis for effect level:

other: Effects on body weight parameters in F0 males.

Dose descriptor:

NOAEC

Remarks:

Reproductive

Effect level:

6 000 other: ppm (13,276 mg/m3)

Sex:

male/female

Basis for effect level:

other: Highest dose level tested

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

See below

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Generation:

F1

Effect level:

300 other: ppm (663 mg/m3)

Sex:

male/female

Basis for effect level:

other: Post-weaning exposure during PND 21-27 or PND 28-34

Reproductive effects observed:

not specified

Occasional occurrences of dried red material (perioral and perinasal regions) were observed in four 6000 ppm exposed F1 offspring (three males and one female).

Treatment- related decreases in body weights and body weight gains were observed in F0 males at 1500 and 6000 ppm. Body weight losses were observed during the first week and body weight gains were reduced over the next few weeks in a dose-related fashion. As a result of the treatment-related effects on weight changes, at 1500 ppm, mean male body weights were 1-7% lower than controls and at 6000 ppm, mean body weights were 5-8% lower than controls over the course of the study. Differences from the control group were statistically significant at 6000 ppm at study week 1 and from study week 3 to study week 8. Mean final body weights were 5% lower than controls at 6000 ppm.

There were no treatment-related effects on body weight parameters in F0 females at any dose levels or F0 males at 300ppm.

Treatment- related decreases in body weights and body weight gains were observed in F1 males and females at 1500 and 6000 ppm during the pnd 21-27 period. The reduced body weights of the males were not statistically significant but those of the females were. Body weight gains were statistically significantly reduced in both sexes. Reduced body weight gains were also evident for males and females at 1500 and 6000 ppm during the pnd 28-34 period

There were no treatment-related effects on body weight parameters in F1 males or females at 300ppm.

Conclusions:

Exposure to 1,3-butadiene at concentrations of up to 6000 ppm (13,276 mg/m3) for two weeks prior to mating, during mating and through gestation and lactation resulted in adverse clinical signs and reductions in body weight. NOAELs for F0 parental toxicity and F1 offspring toxicity were both 300 ppm (663 mg/m3), whilst the NOAEL for reproduction was 6000 ppm (13,276 mg/m3, the highest concentration tested).

Executive summary:

Male and female rats were exposed to 1,3-butadiene by inhalation at target concentrations of 300, 1500 or 6000 ppm (663, 3319 or 13,276 mg/m3) for two weeks prior to mating, during mating and through gestation and lactation. F1 males and females were exposed for 7 days post weanling (pnd 21-27 or 28-34). At 6000 ppm adverse clinical signs were noted. At 1500 and 6000 ppm there were persistent reductions in body weight parameters in F0 and F1 males and females and transient reductions in food consumption (week 0-1) for F0 males and females. There were no effects on gonadal function, mating behavior, conception, gestation, parturition, lactation of the F0 generation, and the development of F1 offspring from conception through weaning. 1,3 -Butadiene at 300 ppm had no effects on F0 or F1 animals. The NOAEL for F0 parental systemic toxicity was 300 ppm (663 mg/m3). The NOAEL for effects on gonadal function, mating behaviour, conception, gestation, parturition, lactation of the F0 generation, and the development of F1 offspring from conception through weaning was 6000 ppm. (13276 mg/m3) The NOAEL for systemic toxicity for F1 animals following post-weaning exposure was 300 ppm (663 mg/m3).

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is no robust study that assesses the reproductive toxicity of isoprene.

 

A repeat dose inhalation study conducted in F344 rats and B6C3F1 mice included histopathology of the reproductive organs, evaluation of sperm and vaginal cytology (Melnick et al., 1994). However, neither F344 rats nor B6C3F1 mice are generally used for the evaluation of effects on reproduction because they are not outbred strains of laboratory animal. In addition, the F344 rat is known to develop a high incidence of testicular lesions during its life time. On this basis, the results of this study are considered to be of questionable relevance for predicting the potential effects of isoprene on reproduction. 

 

Rats and mice were exposed to 0, 70, 700, or 7000 ppm (0; 195; 1950; 19,503 mg/m3) of isoprene for 6 hours/day, 5 days/week for 13 weeks. No compound-related effects were observed in rats after 13 weeks of exposure to isoprene.

 

In mice, testicular weight was reduced in the 7000 ppm group, and seminiferous tubular atrophy was detected in 2/10 mice. In addition, epididymal weights were lower in mice exposed to 700 or 7000 ppm isoprene. No data are included in the publication to substantiate this result.

 

The publication states that male mice in the 700 and 7000 ppm groups had lower spermatid head counts, lower sperm concentrations, and reduced sperm motility. However, no methodology or data are presented and it cannot be established if this evaluation is sufficiently robust to be relevant for the determination of the potential effects of isoprene on reproduction.

 

Vaginal cytology was evaluated for both rats and mice. The female mice exposed to 7000 ppm had oestrous cycle lengths statistically significantly longer than the control group (4.8 versus 4.2 days). However, the biological and toxicological relevance of this small numerical difference is questionable. Furthermore, no methodology or data are presented in the publication. 

 

No effect of isoprene on sperm or vaginal cytology is reported for the rat and it is the rat that is the species usually evaluated for these endpoints because the technical procedures involved are reliable, well established and there is a wealth of data available. The evaluation of sperm and vaginal cytology in mice is much less common and not without significant procedural difficulties which can impact on data interpretation. For oestrous cyclicity, the length of period of assessment is particularly relevant. The lack of methodology and data in the publication do not allow for the apparent effects in mice to be substantiated and the results must be considered insufficiently robust to be relevant to predict the effects of isoprene on reproduction.

 

The publication of Melnick et al. (1994) is therefore considered to be insufficiently robust with respect to those endpoints relevant to reproduction to provide any meaningful conclusions on the reproductive toxicity of isoprene.

 

Isoprene is 2-methyl-1,3-butadiene and therefore chemically related to 1,3-butadiene.  An OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test) study was reported for 1,3-butadiene (WIL, 2003). This study was well conducted and GLP compliant. It evaluated the effects in rats following inhalation exposure to 0, 300, 1500 or 6000 ppm (663, 3319 or 13276 mg/m3) for 6 hours/day. Males were exposed for 14 days prior to initiation of the breeding period and for a total of 83-84 consecutive days. Females were exposed for 14 days prior to initiation of the breeding period, through to gestation day 20 and from lactation day 5 through to the day prior to euthanasia (60-70 days in total). During lactation exposures, the females were separated from their litters. Selected offspring (one male and one female from each litter) were exposed for 7 consecutive days (postnatal days 21-27 or 28-34).

 

For males, the endpoints measured included the following organ weights: prostate, epididymides (total and cauda), seminal vesicles with coagulating glands (with accessory fluids) and testis. Microscopic examination of the right epididymis (caput, corpus, and cauda) and testis was also included. Sperm were evaluated using the methods described by Blazak et al and the Hamilton-Thorne CASA system.

 

For females, the endpoints measured included the following organ weights: ovaries, uterus with oviducts and cervix. The ovaries were examined microscopically.

 

Litters were standardised to10 pups (5 pups/sex/litter as far as possible) on day 4 post partum. The number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities were recorded.

 

At 6000 ppm clinical observations indicative of chromodacryorrhea, chromorhinorrhea, and salivation were observed in parent males and females. Treatment-related decreases in body weight were observed in parent males exposed to 1500 or 6000 ppm but not in parent females although both male and female offspring exposed to these dose levels during both post natal treatment periods, days 21-27 and days 28-34, had reduced body weight gain.

 

There were no effects of 1,3-butadiene on gonadal function, mating behaviour, conception, gestation, parturition or lactation and neither were there any effects on the development of the offspring. Similarly, there were no compound-related effects on the weight or histology of the reproductive organs and no effects on sperm.

 

The NOAEC of 1,3-butadiene for systemic toxicity was (663 mg/m3) and the NOAEC for reproduction was 6000 ppm (13,276 mg/m3, the highest concentration tested).

 

There is no robust study that assesses the reproductive toxicity of isoprene. However, the results of this GLP compliant, guideline screening study for the reproduction/developmental toxicity of 1,3-butadiene, are directly relevant to isoprene (2-methyl-1,3-butadiene) given the similarity in chemical class.  The lack of effect of 1,3-butadiene on reproduction at an exposure concentration of 6000 ppm (13,276 mg/m3) and the fact that isoprene is a mutagenic carcinogen precludes the need for any additional testing.

Short description of key information:
There is no robust study that assesses the reproductive toxicity of isoprene.  However, considering the results of a guideline screening study for the reproduction/developmental toxicity of 1,3-butadiene as being directly relevant to isoprene (2-methyl-1,3-butadiene), the lack of effect of at an exposure concentration of 6000 ppm (13,276 mg/m3) and the fact that isoprene is a mutagenic carcinogen precludes the need for any additional testing.

Effects on developmental toxicity

Description of key information

Developmental toxicity has been assessed in rats and mice.  Rats are less sensitive to exposure to isoprene than mice.   In rats there were no adverse effect on the dam or offspring at any dose level (maximum dose 19503 mg/m3) and there was no increase in malformations or variations.  In mice increases in skeletal variations were associated with maternal toxicity.  However, there was no NOAEC for a reduction in foetal weight (i.e. LOAEC of 780 mg/m3).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, published in peer reviewed literature, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, MI, USA
- Age at study initiation: 12 week
- Housing: Virgin females individually caged 8 days prior to exposure, mated females individually caged on 0 dg
- Diet: pelleted NIH-07 diet (Ziegler Bros ., Inc ., Gardner, PA, USA) ad libitum (except during exposure)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 75±3°F
- Humidity: 55±15%
- 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: 2 .3 m3 stainless-steel chamber (1 .7 m3 active mixing volume) contained 3 levels of caging, each of which was split into 2 offset tiers. The cage units accommodated individual animal cages, feed troughs and automatic water dispensers. Stainless-steel catch pans designed to aid in maintaining a uniform concentration of vapour throughout the chamber, as well as for the collection of urine and faeces, were suspended below each cage unit.
- Rate of air: 15 ft3/min. The uniform mixture was diverted along the inner surfaces of the chamber and the catch pans created mixing eddies
- Method of conditioning air: HEPA- and charcoal-filtered.
- Isoprene vapour was generated by a rotary evaporation system which assured uniformity of chemical-laden atmosphere within the chamber.
- Temperature and humidity in air chamber: 75±3°F, 55±15%
- Air flow rate: 12 to 18 CFM

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of isoprene vapour in the chambers were monitored at 20-30 min intervals by gas chromatography (minimum detectable limit = 0.04 ppm isoprene). The grand means of chamber concentrations for all exposure levels were 100% of the target with relative SDs of 2 to 6%. At least 99% of all individual concentration measurements were within ±10% of the specified operating limits for the exposure target level. There was no measurable concentration in the control and holding chambers. The maximum concentration observed in the room was 0 .8 ppm.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 2-3 females/male
- Length of cohabitation: overnight
- Further matings: none
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of gestation (0dg)

Duration of treatment / exposure:

12 consecutive days. gestation days 6-17

Frequency of treatment:

6 hours/day

Duration of test:

days 0-18 of gestation

No. of animals per sex per dose:

10 virgin females/group and 33 positively mated females /group.

Control animals:

yes, sham-exposed

Details on study design:

Virgin mice were exposed for 12 consecutive days concurrently with mated animals and were killed 1 day after their last exposure day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Mated mice 0, 6, 9, 12, 15 and 18 dg. Virgin mice 3 days prior to exposure and on exposure days 1, 5, 10 and at termination.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18 mated mice, 1 day after final exposure virgin mice
- Organs weighed: liver and kidneys
- Organs examined: ovaries, uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data (ovaries examined at another laboratory and results not included in this publication)
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- Live foetuses were weighed examined for gross defects, and their sex was determined by internal examination of the gonads
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter plus any with gross external abnormalities]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

Statistics:

All means and SDs for animal data were calculated with SAS statistical software. Mean body weights (as a mean of litter means for foetal data) were analyzed with an analysis of variance (ANOVA) model for unbalanced data. Response variables, either body weight or the arcsin transformations of proportional incidence data, were analyzed against the class variable, "treatment", in a one-way ANOVA model. A Tukey's t-test (two-tailed) was used to assess statistically significant differences between control and exposed groups. If appropriate, the dose-response relationship was determined by means of an orthogonal trend test. In the case of proportional data the t-tests and trend analyses were performed on transformed variables. The litter was used as the basis for analysis of foetal variables.

Historical control data:

reported

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were statistically significant reductions in maternal body weight and body weight gain and, in uterine weight at the highest dose. Liver to body weight ratios for pregnant dams were significantly increased in the 1400 and 7000 ppm groups compared to the control group, and kidney to body weight ratios were significantly increased in the 7000 ppm group.

Dose descriptor:

NOAEC

Effect level:

3 900 mg/m³ air (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There was an exposure-correlated reduction in foetal body weight (statistically significant for females only at 780 mg/m3, for males only at 3900 mg/m3 and for males and females at 19,503 mg/m3). There was no increase in the incidence of foetal malformations although two foetuses with cleft palate were found, one in each of the two highest exposure groups (1400 and 7000 ppm). An increased incidence of supernumerary ribs was observed in the exposed groups. This skeletal variation is generally considered to be a secondary effect of maternal toxicity or stress and its significance is unclear. The incidence of supernumerary ribs (percent of foetuses examined) was 20.1, 23.8, 33.6, and 40.3% at 0, 780, 3900, and 19,503 mg/m3.

Dose descriptor:

NOAEC

Effect level:

19 503 mg/m³ air (nominal)

Basis for effect level:

other: teratogenicity

Key result

Dose descriptor:

LOAEC

Effect level:

780 mg/m³ air (nominal)

Basis for effect level:

other: Developmental Toxicity. statistically significant differences in foetal weight in the absence of maternal toxicity; no NOAEC

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The NOAEC for maternal toxicity was 3900 mg/m3 (1400ppm). The NOAEC for teratogenicity was 19,503 mg/m3 (7000 ppm). The NOAEC for developmental toxicity based on foetal weight effect was not established and is therefore <780 mg/m3 (<280 ppm).

Executive summary:

Exposure of Swiss (CD-1) mice to 0, 780, 3900 or 19,503 mg/m³ (0, 280, 1400 or 7000 ppm) isoprene resulted in significant reductions in maternal body weight and uterine weight at the highest exposure. There was no NOAEC for developmental toxicity, which was evident as an exposure-correlated reduction in foetal body weight (statistically significant for females only at 780 mg/m³ , for males only at 3900 mg/m³ and for males and females at 19,503 mg/m³). The incidence of foetuses with supernumerary ribs was statistically significant at 3900 mg/m³. There was no significant increase in the incidence of malformations. Two foetuses with cleft palate were observed, one in each of the two highest exposure groups. This low incidence of a relatively common finding in mice, is considered not to be indicative of teratogenicity / developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

780 mg/m³

Study duration:

subacute

Species:

mouse

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The relevant study for developmental toxicity was conducted by the NTP (National Toxicology Program, 1989) using the inhalation route of exposure. In this study, female Swiss CD-1 mice were exposed to 0, 280, 1400 or 7000 ppm (i.e. 0, 780, 3900 or 19,503 mg/m3) isoprene for 6 hours/day, 7 days/week on gestational days 6-17. 

 

Exposure to 7000 ppm isoprene significantly reduced maternal weight gain and uterine weight.  Developmental toxicity was evident as a reduction in foetal body weight observed at 280 and 1400 ppm. Increased incidences of variations (i.e. supernumerary ribs) observed in the exposed groups, were considered a secondary non-specific consequence of maternal toxicity. Therefore, 1400 ppm (3900 mg/m3) was the NOAEC for maternal toxicity in mice. A NOAEC for developmental toxicity was not determined in this study because a reduction in foetal bodyweight was observed at the lowest exposure concentration tested, at 280 ppm (780 mg/m3).

 

In the same study, female Sprague-Dawley rats were exposed to 0, 280, 1400 or 7000 ppm (i.e. 0, 780, 3900 or 19,503 mg/m3) isoprene for 6 hours/day, 7 days/week on gestational days 6-19. There was no adverse effect on the dam or offspring at any dose level and there was no increase in malformations or variations and therefore 7000 ppm (19,503 mg/m3) was the NOAEC for both maternal and developmental toxicity.

Justification for classification or non-classification

No classification is warranted under CLP since consideration of existing data (isoprene and read-across) indicates a lack of reproductive effects and, in relation to developmental toxicity, there was no increase in malformations or variations which were not associated with maternal toxicity.