Pent-1-ene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined repeated exposure toxicity, reproduction and neurotoxicity screening in rats via whole-body inhalation exposures

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant, guideline study, available as unpublished report, fully adequate for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

Combined repeated exposure toxicity, reproduction and neurotoxicity screening in rats via whole-body inhalation exposures

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 July to 25 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant, guideline study, available as unpublished report, fully adequate for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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:

- Source: Charles River Laboratories, Kingston, New York, USA.
- Age at study initiation: c.a. 8 weeks
- Weight at study initiation: Males 200-300g, females 150-250 g
- Housing: Individually in stainless steel, wire mesh cages within a 1.0 m3 glass and stainless steel whole body exposure chamber except during the mating period when one male and one female were housed together in a cage of suitable size.
- Diet: Certified Rodent Diet No. 5002 (PMI Nutrition International, St. Louis, MO, USA) ad libitum except during exposure
- Water: Mains water ad libitum except during exposure.
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 36-82%
- Air changes: Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1 July 2002 To: 25 August 2002

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD: 0.822 ± 0.1, 0.893 ± 0.2, 0.7986 ± 0.0343 and 3.6503 ± 7.0 for 0, 500, 2000 and 8000 ppm respectively.
GSD: 2.019 ± 0.4, 1.869 ± 0.3, 1.893 ± 0.3 and 2.181 ± 0.8 for 0, 500, 2000 and 8000 ppm respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The whole-body exposure chambers each had a volume of approximately 1000 L. The chambers were operated at a minimum flow rate of 200 L/minute. The final airflow was set to provide at least one air change in 5.0 minutes (12 air changes/hour) and a T99 equilibrium time of at most 23 minutes. At the end of the 6-hour exposure, all animals remained in the chambers for a minimum of 30 minutes. During this time, the chambers were operated at approximately the same flow rate using clean air only. The chambers were exhausted through the in housed filtering system, which consisted of a coarse filter, a HEPA filter and activated charcoal.
For the treated groups, the test article was delivered from the cylinder via a regulator with a backpressure gauge via 1/4" tubing through a flowmeter via 1/4" tubing. The outlet of the flowmeter was regulated by a built-in metering valve. The test article laden airstream was directed into the turret of a 1.0 m3 glass and stainless steel exposure chamber via 1/4" tubing, where it was diluted with room air as it was drawn into the chamber. For controls, room air was drawn into the turret of the 1.0 m3 glass and stainless steel exposure chamber.

TEST ATMOSPHERE
During each exposure, measurements of airborne concentrations were performed in the animals' breathing zone at least 4 times using an appropriate sampling procedure and on-line GC analytical method. During each week of exposure, particle size determinations were performed using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean (± standard deviation) analytical (GC) concentrations for the control and the exposure groups were as follows: 0 ± 0, 524 ± 40, 2062 ± 126 and 8271 ± 683 ppm. The analytically measured exposure levels of the airborne test article were reasonably close to the targeted exposure levels.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 7 days/week

Dose / conc.:

500 ppm (nominal)

Remarks:

524 ± 40 ppm (analytical concentration)

Dose / conc.:

2 000 ppm (nominal)

Remarks:

2062 ± 126 ppm (analytical concentration)

Dose / conc.:

8 000 ppm (nominal)

Remarks:

8271 ± 683 ppm (analytical concentration)

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

The study design included the main study for repeated dose toxicity end points and reproductive/ developmental toxicity satellite groups (summarized separately). The reproductive and developmental toxicity satellite groups (12 females per exposure level) were exposed for two weeks prior to breeding, during breeding, and continuing through day 19 of gestation. Males from the main study were used to breed these females.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to randomisation and then once/week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: Yes
- How many animals: 12/sex/group
- Parameters examined: erythrocyte count, haematocrit, haemoglobin, MCV, MCHC, leucocyte count (total and differential), platelet count, reticulocyte count, erythrocyte morphology, prothrombin time, activated partial thromboplastin

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: Yes
- How many animals: 12/sex/group
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, urea nitrogen, creatinine, glucose, total cholesterol, triglycerides, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, sodium, potassium, chloride, calcium, phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-test and during final week of exposure
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / rectal temperature

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all animals including those dying spontaneously or killed moribund). The nasal pharynx was preserved but not examined microscopically.

ORGAN WEIGHTS: Yes (testes, epididymides, ovaries with oviducts, uterus with vagina, adrenals, brain with brain stem, heart, kidneys, liver, lungs, spleen, thymus)

HISTOPATHOLOGY: Yes (control and high dose only initially. Tissues examined: adrenals, bone marrow (femur), brain (medulla/pons, cerebrum and cerebellum), epididymides, heart, kidneys, large intestine (caecum, colon and rectum), liver, lungs (with mainstem bronchi), lymph nodes (mesenteric and mediastinal), mammary gland, ovary with oviduct, prostate, seminal vesicles, small intestine (duodenum, ileum and jejunum), spinal cord, spleen, stomach, testes, thymus, thyroid with parathyroids, tibial nerve, trachea, urinary bladder, uterus with vagina, all macroscopic lesions and tissue masses.

Other examinations:

None

Statistics:

Mean values of all exposure groups were compared to the mean value for the control group at each time interval. For all parameters except for organ weights, the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance was used. If significant differences among the means were indicated, additional testing was performed using Dunnett's t-test to determine which means were significantly different from the control. Organ weight data was analyzed only by parametric methods. Bartlett's test was performed to determine if groups had equal variances. The standard one-way analysis of variance (ANOVA) using the F ratio to assess significance was used. If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett's t-test for homogeneous data, or Cochran and Cox's modified t-test for non-homogeneous data. All t-tests were conducted at the 5% and 1% significance levels. Motor Activity Data was analyzed using split-plot repeated measures ANOVA with model terms for group, animal within group, interval and group by interval interaction. If the group x interval interaction was statistically significant (p=0.05), indicating non-parallelism in the behavioural profile between groups, a separate one-way ANOVA for group effects was performed at each interval. If the response data passed on the parallel hypothesis, an ANOVA (using summed responses over intervals) was used to test for the overall treatment effect which constituted the level hypothesis. If any significant overall treatment group effect was found by any of the above ANOVAs, Dunnett's t-test was used to find groups that differed from control. Analyses were performed for sexes separately and combined. Treatment group effects were deemed significant at the p=0.05 level. Plots, tables, listings, and analyses were generated using SAS version 6.12 for WINDOWS.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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

Other effects:

not specified

Details on results:

There were no microscopic findings considered to be related to exposure to 1-butene. In comparison with controls, there was a slightly increased incidence and severity of mixed inflammatory cells in the caecal mucosa of rats exposed to 1-butene at exposure levels of 2000 ppm and above. The caecal mucosa normally contains a small population of mixed inflammatory cells, which acts as a natural defence mechanism against ingested substances or organisms. Increased numbers of inflammatory cells are sometimes seen as a normal spontaneous finding, and this was evident in a few males and females from the control group in this study. Since the finding was present in the control group and there was no clear exposure level response relationship in the treated groups, the increased incidence is considered to be fortuitous and unlikely to be related to treatment with 1 -butene. Other microscopic findings occurred sporadically or showed a similar incidence in control and 1-butene-treated animals. None were considered to be associated with exposure to 1-butene.

Key result

Dose descriptor:

NOAEC

Effect level:

8 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: there were no adverse effects at 8000 ppm, the highest concentration tested

Remarks on result:

other: 18359 mg/m3, 18 mg/L

Key result

Critical effects observed:

not specified

None

Conclusions:

Exposure of male and female rats to target concentrations of 500, 2000 and 8000 ppm of 1-butene resulted in no general systemic effects. Under the test conditions, the NOAEC was 8000 ppm (18359 mg/m3) in rats.

Executive summary:

In a repeated dose toxicity performed according to OECD Guideline 422 and in compliance with GLP, Crl:CD® (Sprague-Dawley) IGS BR rats (12/sex/dose) were exposed to 1-butene via whole body inhalation (gas) at target concentrations of 500, 2000, 8000 ppm (approximately 1147, 4589, 18359 mg/m³) for 28 days for subchronic evaluations (males and females) or in pregnant female rats exposed for 14 days pre-mating, through mating and gestation to day 19. Then, the dams and the pups were observed on day 1-4 of lactation. A similarly constituted Control group received “air” throughout the treatment period. Examinations during the study included: clinical signs, body weight, food consumption, haematology, clinical chemistry, neurobehavioural examination, organ weights, gross and histopathology.

Exposure of male and female rats to target concentrations of 500, 2000 and 8000 ppm of 1-butene resulted in no general systemic effects. No treatment-related effects on body weight, food consumption, haematology, clinical chemistry, organ weights or gross/histopathology were found. Neurotoxicity screening also showed no effects on motor activity or functional observation battery.

Under the test conditions, the NOAEC was 8000 ppm (18359 mg/m³) in rats.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Kingston, New York, USA.
- Age at study initiation: c.a. 8 weeks
- Weight at study initiation: Males 200-300g, females 150-250 g
- Housing: Individually in stainless steel, wire mesh cages within a 1.0 m3 glass and stainless steel whole body exposure chamber except during the mating period when one male and one female were housed together in a cage of suitable size.
- Diet: Certified Rodent Diet No. 5002 (PMI Nutrition International, St. Louis, MO, USA) ad libitum except during exposure
- Water: Mains water ad libitum except during exposure.
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 36-82%
- Air changes: Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1 July 2002 To: 25 August 2002

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The whole-body exposure chambers each had a volume of approximately 1000 L. The chambers were operated at a minimum flow rate of 200 L/minute. The final airflow was set to provide at least one air change in 5.0 minutes (12 air changes/hour) and a T99 equilibrium time of at most 23 minutes At the end of the 6-hour exposure, all animals remained in the chambers for a minimum of 30 minutes. During this time, the chambers were operated at approximately the same flow rate using clean air only. The chambers were exhausted through the in housed filtering system, which consisted of a coarse filter, a HEPA filter and activated charcoal.
For the treated groups, the test article was delivered from the cylinder via a regulator with a backpressure gauge via 1/4" tubing through a flowmeter via 1/4" tubing. The outlet of the flowmeter was regulated by a built-in metering valve. The test article laden airstream was directed into the turret of a 1.0 m3 glass and stainless steel exposure chamber via 1/4" tubing, where it was diluted with room air as it was drawn into the chamber. For controls, room air was drawn into the turret of the 1.0 m3 glass and stainless steel exposure chamber.



TEST ATMOSPHERE
During each exposure, measurements of airborne concentrations were performed in the animals' breathing zone at least 4 times using an appropriate sampling procedure and on-line GC analytical method. During each week of exposure, particle size determinations were performed using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean (± standard deviation) analytical (GC) concentrations for the control and the exposure groups were as follows: 0 ± 0, 524 ± 40, 2062 ± 126 and 8271 ± 683 ppm. The analytically measured exposure levels of the airborne test article were reasonably close to the targeted exposure levels.

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female
- Length of cohabitation: until evidence of mating seen or for 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged Individually

Duration of treatment / exposure:

Two weeks prior to breeding, during breeding, and continuing through day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until lactation day 4.

Frequency of treatment:

6 hrs/day, 7 days/week

Duration of test:

After an initial exposure period of 2 weeks, males and females were mated for 1 week. Females were allowed to litter and to rear their progeny until day 4 of lactation; after which the parent animals and the pups were killed

Dose / conc.:

500 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

Dose / conc.:

8 000 ppm (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

none

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Females weekly during pre-mating and on gestations days 0, 7, 14, 20 and lactation days 0, 1 and 4.

BODY WEIGHT: Yes
- Time schedule: Females day treatment initiated, twice/week until copulation confirmed, gestations days 0, 7, 14 and 20, lactation days 1 and 4. Females without evidence of mating were weighed weekly. All weighed at termination.

FOOD CONSUMPTION: Yes
- Time schedule: Females weekly during pre-mating period, gestation days 0-7, 7-14, 14-20 and lactation days 1-4.

SACRIFICE
- Maternal animals: Mated animals on day 5 post partum, animals when evidence of mating was detected but failed to deliver 26 days after evidence of mating, animals with no evidence of mating 26 days after completion of mating period.

Ovaries and uterine content:

GROSS NECROPSY
- Maternal animals: Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Number of implantation sites and corpora lutea were assessed.

ORGAN WEIGHTS
Maternal animals: ovaries with oviducts, uterus with vagina, adrenals, brain with brainstem, heart, kidneys, liver, thymus, spleen, lungs.

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined as soon as possible after parturition and twice/day thereafter: number of live and dead pups and sex of pups, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities and presence/absence of milk in stomach

Postmortem examinations (Offspring)
External macroscopic postmortem examinations performed on all F1 pups found dead during lactation and at termination on lactation day 4. No microscopic examination of pups.

Statistics:

Mean values of all exposure groups were compared to the mean value for the control group at each time interval. For all parameters except for organ weights, the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance was used. If significant differences among the means were indicated, additional testing was performed using Dunnett's t-test to determine which means were significantly different from the control. Organ weight data was analyzed only by parametric methods. Bartlett's test was performed to determine if groups had equal variances. The standard
one-way analysis of variance (ANOVA) using the F ratio to assess significance was used. If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett's t-test for homogeneous data, or Cochran and Cox's modified t-test for non-homogeneous data. All t-tests were conducted at the 5% and 1% significance levels. For incidence data, a Fisher Exact Test with Bonferonni correction was performed to identify differences between the control and treatment groups.

Reproductive indices
Gonadal function, mating behaviour, implantation, and general fertility were evaluated.

Offspring viability indices
Litter size, pup survival, sex, body weight, and the presence of gross external malformations were assessed in the offspring.

Indices:

none

Historical control data:

not cited

Details on maternal toxic effects:

Maternal toxic effects: no effects

Details on maternal toxic effects: no effects

Key result

Dose descriptor:

NOAEC

Effect level:

8 000 other: ppm (18,359 mg/m3, 18.4 mg/L) nominal

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects: no effects

Key result

Dose descriptor:

NOAEC

Effect level:

8 000 other: ppm (18,359 mg/m3, 18.4 mg/L) nominal

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

BODY WEIGHT AND WEIGHT GAIN (PARENTS)

No effects

BODY WEIGHT (OFFSPRING)

No effects

Conclusions:

No effects on or pup development were seen when male and female rats were exposed to 1-butene at concentrations of 500, 2000, or 8000 ppm for two weeks prior to breeding, during breeding and until day 19 of gestation. The NOAEC for reproductive and developmental toxicity was 8000 ppm (18,359 mg/m3), the highest concentration tested.

Executive summary:

Male and female rats were exposed to 1-butene at target concentrations of 500, 2000, 8000 ppm (1147, 4589, 18,359 mg/m3) for two weeks prior to breeding, during breeding and until day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until post-natal day 4. There was no evidence of significant systemic toxicity in the parents. There were no treatment-related effects on the development of pups. There were no effects on body weight gain or observed during macroscopic examination of pups at post mortem.

Under the test conditions, the NOAEC for reproductive and developmental toxicity was 8000 ppm (18,359 mg/m3), the highest concentration tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

But-1-ene

EC Number:

203-449-2

EC Name:

But-1-ene

Cas Number:

106-98-9

Molecular formula:

C4H8

IUPAC Name:

but-1-ene

Test material form:

gas

Details on test material:

- Name of test material (as cited in study report): 1-butene
- Analytical purity: ≥99%

Test animals

Species:

rat

Strain:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Kingston, New York, USA.
- Age at study initiation: c.a. 8 weeks
- Weight at study initiation: Males 200-300g, females 150-250 g
- Housing: Individually in stainless steel, wire mesh cages within a 1.0 m3 glass and stainless steel whole body exposure chamber except during the mating period when one male and one female were housed together in a cage of suitable size.
- Diet: Certified Rodent Diet No. 5002 (PMI Nutrition International, St. Louis, MO, USA) ad libitum except during exposure
- Water: Mains water ad libitum except during exposure.
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 36-82%
- Air changes: Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1 July 2002 To: 25 August 2002

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Remarks on MMAD:

MMAD: 0.822 ± 0.1, 0.893 ± 0.2, 0.7986 ± 0.0343 and 3.6503 ± 7.0 for 0, 500, 2000 and 8000 ppm respectively
GSD: 2.019 ± 0.4, 1.869 ± 0.3, 1.893 ± 0.3 and 2.181 ± 0.8 for 0, 500, 2000 and 8000 ppm respectively

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The whole-body exposure chambers each had a volume of approximately 1000 L. The chambers were operated at a minimum flow rate of 200 L/minute. The final airflow was set to provide at least one air change in 5.0 minutes (12 air changes/hour) and a T99 equilibrium time of at most 23 minutes At the end of the 6-hour exposure, all animals remained in the chambers for a minimum of 30 minutes. During this time, the chambers were operated at approximately the same flow rate using clean air only. The chambers were exhausted through the in housed filtering system, which consisted of a coarse filter, a HEPA filter and activated charcoal.
For the treated groups, the test article was delivered from the cylinder via a regulator with a backpressure gauge via 1/4" tubing through a flowmeter via 1/4" tubing. The outlet of the flowmeter was regulated by a built-in metering valve. The test article laden airstream was directed into the turret of a 1.0 m3 glass and stainless steel exposure chamber via 1/4" tubing, where it was diluted with room air as it was drawn into the chamber. For controls, room air was drawn into the turret of the 1.0 m3 glass and stainless steel exposure chamber.



TEST ATMOSPHERE
During each exposure, measurements of airborne concentrations were performed in the animals' breathing zone at least 4 times using an appropriate sampling procedure and on-line GC analytical method. During each week of exposure, particle size determinations were performed using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present.

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female
- Length of cohabitation: until evidence of mating seen or for 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean (± standard deviation) analytical (GC) concentrations for the control and the exposure groups were as follows: 0 ± 0, 524 ± 40, 2062 ± 126 and 8271 ± 683 ppm. The analytically measured exposure levels of the airborne test article were reasonably close to the targeted exposure levels.

Duration of treatment / exposure:

Two weeks prior to breeding, during breeding, and continuing through day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until lactation day 4.

Frequency of treatment:

6 hours/day, 7 days/week

Details on study schedule:

see below

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

analytical: 0 ± 0 ppm

Doses / concentrations 2

Dose / conc.:

500 ppm (nominal)

Remarks:

analytical: 524 ± 40 ppm

Doses / concentrations 3

Dose / conc.:

2 000 ppm (nominal)

Remarks:

analytical: 2062 ± 126 ppm

Doses / concentrations 4

Dose / conc.:

8 000 ppm (nominal)

Remarks:

analytical: 8271 ± 683 ppm

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Females weekly during pre-mating and on gestations days 0, 7, 14, 20 and lactation days 0, 1 and 4.

BODY WEIGHT: Yes
- Time schedule: Females day treatment initiated, twice/week until copulation confirmed, gestations days 0, 7, 14 and 20, lactation days 1 and 4. Females without evidence of mating were weighed weekly. All weighed at termination.

FOOD CONSUMPTION: Yes
- Time schedule: Females weekly during pre-mating period, gestation days 0-7, 7-14, 14-20 and lactation days 1-4.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

not determined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined as soon as possible after parturition and twice/day thereafter: number of live and dead pups and sex of pups, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities and presence/absence of milk in stomach

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: Mated animals on day 5 post partum, animals when evidence of mating was detected but failed to deliver 26 days after evidence of mating, animals with no evidence of mating 26 days after completion of mating period.

GROSS NECROPSY
- Maternal animals: Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Number of implantation sites and corpora lutea were assessed.

ORGAN WEIGHTS
Maternal animals: ovaries with oviducts, uterus with vagina, adrenals, brain with brainstem, heart, kidneys, liver, thymus, spleen, lungs.

MICROSCOPIC EXAMINATION: No

Postmortem examinations (offspring):

External macroscopic postmortem examinations performed on all F1 pups found dead during lactation and at termination on lactation day 4. No microscopic examination of pups.

Statistics:

Mean values of all exposure groups were compared to the mean value for the control group at each time interval. For all parameters except for organ weights, the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance was used. If significant differences among the means were indicated, additional testing was performed using Dunnett's t-test to determine which means were significantly different from the control. Organ weight data was analyzed only by parametric methods. Bartlett's test was performed to determine if groups had equal variances. The standard
one-way analysis of variance (ANOVA) using the F ratio to assess significance was used. If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett's t-test for homogeneous data, or Cochran and Cox's modified t-test for non-homogeneous data. All t-tests were conducted at the 5% and 1% significance levels. For incidence data, a Fisher Exact Test with Bonferonni correction was performed to identify differences between the control and treatment groups.

Reproductive indices:

Gonadal function, mating behaviour, implantation, and general fertility were evaluated.

Offspring viability indices:

Litter size, pup survival, sex, body weight, and the presence of gross external malformations were assessed in the offspring.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

see below

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

8 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects on dams at the highest concentration tested, no effects on reproductive performance or fertility

Remarks on result:

other: (18359 mg/m3, 18.4 mg/m3) nominal

Results: F1 generation

General toxicity (F1)

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:

no effects observed

Details on results (F1)

see below

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

8 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects on pups at the highest concentration tested, no effects on pup viability, body weight or development

Remarks on result:

other: (18359 mg/m3, 18.4 mg/L) nominal

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

All mated female animals (except one animal in the 2000 ppm group) were found pregnant and delivered live pups. Mating indices for the male rats treated with the test article were comparable to controls. Mating, fertility and gestation indices for the female rats treated with the test article were comparable to controls. Most of the females mated at the first opportunity. There were also no treatment-related differences in the other reproductive parameters up to the time of parturition including the percent of females completing delivery and the duration of gestation, when compared to controls. There were no treatment related differences in all parturition parameters including the total number of pups delivered, the number of pups dying, the viability (4 day survival) index, the number of implantation sites and corpora lutea per dam, the pup sex ratio and the number of live pups/litter, when compared to controls.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, repeated inhalation exposure of 1-butene to female Sprague Dawley rats at levels of 0, 500, 2000, or 8000 ppm produced no evidence of adverse effects on any measures of reproductive function. Therefore, the NOAEC was 8000 ppm (18359 mg/m3).

Executive summary:

In a Combined Repeated dose toxicity study with Reproductive/Developmental toxicity screening Test performed according to the OECD Guideline No. 422 and in compliance with GLP, male and female rats were exposed by inhalation (whole body) to 1-butene at target concentrations of 500, 2000, 8000 ppm (1147, 4589, 18359 mg/m3) for two weeks prior to breeding, during breeding and until day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until post-natal day 4.

There was no evidence of systemic toxicity in the parents.

There were no effects on mating behaviour, fertility and gestation indices, the number of implantation sites and corpora lutea per dam, numbers of pups delivered, viability of pups at and after birth and the pup sex ratio when compared to the control group.

Based on these data, the NOAEC for reproductive toxicity was the highest concentration tested 8000 ppm (18359 mg/m3).

There were no treatment-related effects on the development of pups. There were no effects on body weight gain or observed during macroscopic examination of pups at post mortem.

Based on these data, the NOAEC for developmental toxicity was also the highest concentration tested 8000 ppm (18359 mg/m3).