Hydrocarbons, C4, 1,3-butadiene-free

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant (minor exception detailed below), guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Justification for type of information:

Justification for Category/Read-across approach:
See justification document for category approach and read-across to individual UVCB constituents in section 13.2.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Deviations:

no

GLP compliance:

yes

Remarks:

GLP deviation: no lot number available of test material

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isobutane

EC Number:

200-857-2

EC Name:

Isobutane

Cas Number:

75-28-5

Molecular formula:

C4H10

IUPAC Name:

isobutane

Details on test material:

- Name of test material (as cited in study report): isobutane
- Supplier: MG Industries, 3 Great Valley Parkway, Malvern, Pennsylvania 19355, USA
- Substance type: Industrial gas
- Physical state: colourless gas
- Analytical purity: 99.0% per supplier
- Lot/batch No.: Not available
- Stability under test conditions: 99.00% before study, 99.53% after study
- Storage condition of test material: Ambient

Test animals

Species:

rat

Strain:

other: Sprague-Dawley CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Species: Albino rats (Outbred) VAF/Plus®, Sprague-Dawley derived (CD®), Crl:CD®(SD)IGS BR
- Source: Charles River Laboratories, Raleigh, North Carolina 27610, USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Males mean 277 g (range 229-301 g); females mean 194 g (range 171-216 g)
- Fasting period before study: None
- Housing: Individually in stainless steel suspended cages with wire mesh floors and fronts (except for mating period when 1 male and 1 female were housed together)
- Diet: Certified Rodent diet No 5002 (PMI Nutrition International, St Louis, Missouri, USA) ad libitum
- Water: Municipal water ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20- 22°C
- Humidity: 42-63%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 15 December 2003 To: 6 February 2004

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L glass and stainless steel whole-body exposure chamber
- Method of holding animals in test chamber: housed individually, the placement of animals in the chamber was rotated daily to ensure uniform exposure
- System of generating particulates/aerosols: the test substance was delivered from a single cylinder, through a regulator and two backpressure gauges via a flowmeter into the exposure chambers
- Time to T99: 23 minutes maximum
- Airflow rate: 204 Lpm
- Temperature and humidity in chamber: 21-25°C, 52-59%
- Oxygen level: at least 19%
- Air flow rate: minimum flow rate of 200 L/minute
- Air change rate: final airflow set to provide at least one air change in 5 mins (12 air changes/hour)
- Method of particle size determination: yes; weekly
- Treatment of exhaust air: filtered through a system which consisted of a coarse filter, a HEPA filter and an activated charcoal bed

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared spectrophotometer (IR) 4 times per chamber per day
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of mating was seen, or for two consecutive 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 (how): individually in plastic "shoebox" cages with bedding
- After the mating period was over, females without evidence of copulation were removed from the mating cages, housed individually and monitored for visible signs of pregnancy with corresponding bodyweight gain.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure levels were determined using an infrared spectrophotometer 4 times/chamber/day. The test substance was evenly distributed within each chamber. The mean (± SD) analytical concentrations were 0 ± 0, 930.0 ± 27.8, 3122 ± 83 and 9148 ± 201 ppm

Duration of treatment / exposure:

Males for 2 weeks prior to mating and for an additional 28 days (minimum) after mating.
Females for 2 weeks prior to mating and gestation days 0-19.

Frequency of treatment:

6 hours/day, 7 days/week

Details on study schedule:

Females without evidence of mating that appeared to be pregnant were killed on an estimated gestation day 19.
Females that littered and their offspring were killed on post partum day 4.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Basis: target concentration

Doses / concentrations 2

Dose / conc.:

900 ppm

Remarks:

Basis: target concentration

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

Basis: target concentration

Doses / concentrations 4

Dose / conc.:

9 000 ppm

Remarks:

Basis: target concentration

Doses / concentrations 5

Dose / conc.:

0 ppm (analytical)

Doses / concentrations 6

Dose / conc.:

930 ppm (analytical)

Remarks:

930 ± 27.8 ppm
Basis: analytical conc.

Doses / concentrations 7

Dose / conc.:

3 122 ppm (analytical)

Remarks:

3122 ± 83 ppm
Basis: analytical conc.

Doses / concentrations 8

Dose / conc.:

9 148 ppm (analytical)

Remarks:

9148 ± 201 ppm
Basis: analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality and clinical condition)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: - Time schedule: Male rats were examined prior to randomisation and once weekly throughout the study. Female rats were examined prior to randomisation and once weekly throughout the premating period and on gestation days 0, 7, 14, 20 and lactation days 0 (except if parturition was not completed on the same day), 1 and 4.

BODY WEIGHT: Yes
- Time schedule for examinations: Male rats were weighed at randomisation and then weekly throughout the study. Females were weighed at randomisation, on the first day of exposure and twice weekly until evidence of copulation was observed, on gestation days 0, 7, 14 and 20, and on lactation days 1 and 4. Females were not fasted prior to recording terminal bodyweights.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Once weekly throughout the premating period. For pregnant or confirmed mated females, food consumption was recorded on gestation days 0-7, 7-14, 14-20 and on lactation days 1-4.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

During the microscopic examination of the testes, special emphasis was placed on the stages of spermatogenesis and the histopathology of interstitial testicular cell structure.

Litter observations:

Not applicable

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (after a minimum of 28 days post-mating).
- Maternal animals: All surviving animals (28 days post-mating).

GROSS NECROPSY: Yes (all animals).
- Tissues examined: adrenal glands, bone (sternum/femur), bone marrow, brain (medulla/pons, cerebrum and cerebellum), epididymides, heart, kidneys, caecum, colon, rectum, larynx, liver, lungs (with mainstem bronchi), lymph nodes (mesenteric and mediastinal), mammary glands (with adjacent skin), nasopharynx, ovaries (with oviducts), prostate, seminal vesicles, duodenum, ileum, jejunum, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid with parathyroids, tibial nerve, trachea, urinary bladder, uterus with vagina and all macroscopic lesions and tissue masses.

ORGAN WEIGHTS: Yes. Adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus and uterus with vagina.

HISTOPATHOLOGY: Yes (male and female main study only).
- tissues examined: adrenal glands, bone (sternum/femur), brain (cerebellum, cerebrum and cerebellum), epididymes, heart, kidneys, large intestine (caecum, colon and rectum), liver, lungs (with mainstream bronchi), lymph node (mesenteric), lymph node (mediastinal), mammary glands (with adjacent skin), ovaries (with oviducts), prostate, seminal vesicles, small intestine (duodenum, ileum and jejunum), spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroids with parathyroids, tibial nerve, trachea, urinary bladder, uterus with vagina, all macroscopic lesions and tissue masses.

Postmortem examinations (offspring):

Macroscopic postmortem examinations (external only) were performed on all surviving F1 pups on lactation day 4.

Statistics:

Group mean values of parameters for all the exposure groups were compared to the control group mean values at each time interval, using appropriate statistical methods.

Reproductive indices:

Male and female mating indices, pregnancy rates, male and female fertility indices, gestation indices and the incidence of dams with no viable pups, were analysed statistically.

Offspring viability indices:

Live birth index, litter survival and mean pup survival indices (days 0 and 4) were analysed statistically.

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

Reproductive function / performance (P0)

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS).
- There was a low incidence of transient red nasal discharge between test days 8 and 29 in all groups including control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Almost all mated females were found pregnant and delivered live pups; one each in the 900 and 3000 ppm groups and three in the 9000 ppm group (25%) were not pregnant. None of the differences were statistically significant. The mating index for the male rats was compared to control. Mating and gestation indices for females were comparable to control. Almost all females mated at the first opportunity. There were no treatment-related differences in the other reproductive parameters up to time of parturition, including percentange of females completing delivery and duration of gestation.
- There was a dose-related increase in post-implantation loss (as defined by the difference between the number of live pups born and the number of implantations, including any stillborn pups) in 9000 ppm females. Otherwise, there were no treatment-related differences in other parturition parameters including pre-implantation loss, the total number of pups delivered, the number of pups dying, the viability (day 4 survival) index, the pup sex ratio and the number of live pups per litter, compared to control. There were no microscopic findings considered to be treatment-related.

Effect levels (P0)

1

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

9 000 other: ppm (21,394 mg/m3)

Sex:

male/female

Basis for effect level:

other: no treatment-related effects at the highest concentration tested

2

Dose descriptor:

NOAEC

Remarks:

Fertility

Effect level:

3 000 other: ppm (7131 mg/m3)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEC

Remarks:

Reproductive endpoints

Effect level:

3 000 other: ppm (7131 mg/m3)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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

Histopathological findings:

not examined

Details on results (F1)

There were no effects on numbers of live or dead pups, pup abnormalities, sex or weight.

Effect levels (F1)

Dose descriptor:

NOAEC

Remarks:

developmental effects

Generation:

F1

Effect level:

9 000 other: ppm (21,394 mg/m3)

Sex:

male/female

Basis for effect level:

other: no treatment-related effects on survival, body weight and development up to postnatal day 4 at the highest concentration tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Delivery and litter data for rats exposed to isobutane in this study

	control	900 ppm	3000 ppm	9000 ppm
No. Corpora lutea	16.3	15.1	14.9	16.4
No. implantation sites	14.6	14.1	13.9	15.1
Pre-implantation loss (no. corpora lutea minus no. implantation sites)	1.8	1.0	1.0	1.3
No. pups per litter	13.8	13.5	13.2	13.4
Post-implantation loss (no. implantation sites minus no. pups per litter)	0.8	0.6	0.7	1.8*

* Statistically significant (P<0.05)

Applicant's summary and conclusion

Conclusions:

Isobutane was tested in an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen. There was no systemic toxicity, no effects on mating, gestation indices and no effects on pup endpoints. The NOAEC for these endpoints was 9,000 ppm (21,394 mg/m3). Equivocal effects at 9000 ppm on both fertility and increased post-implantation loss led to the NOAEC for fertility and reproductive endpoints being concluded to be 3000 ppm (7131 mg/m3).

Executive summary:

Rats were exposed to isobutane by inhalation (900, 3000 and 9000 ppm) in an OECD Guideline 422 combined repeated-exposure toxicity study with the reproduction/developmental screening test. Males and females were exposed for 6 hours/day, 7 days/week for 2 weeks prior to mating. Main study females were evaluated for subchronic effects and were exposed for 28 days. A satellite group of females was evaluated for reproductive effects only - exposed for at least two weeks prior to mating initiation, then during mating and gestation (days 0-19). Main study male rats were exposed during the mating and post-mating periods until euthanized for a minimum exposure of 28 days.

Exposure of male and female rats for 4-6 weeks resulted in no general systemic or neurotoxic effects. A NOAEC of 9000 ppm (21,394 mg/m3) was determined for all general systemic/neurotoxic endpoints in this study. The NOAEC for pup endpoints was also 9000 ppm (21,394 mg/m3) based on no effects on survival, body weight and development up to postnatal day 4.

Almost all mated female animals were found pregnant and delivered live pups. In the 9000 ppm group, 25% of the mated females did not become pregnant, and this reduction in the male and fertility indices (75%) was considered to be exposure-related even though it was not statistically significant. The NOAEC for fertility was therefore concluded to be 3000 ppm (7131 mg/m³). This effect is considered to be equivocal - small group sizes (12) were employed in the study and the differences reported between the control and high dose-exposed animals were small. There was also no evidence of a gradation of response, which might have been expected, where some females were not pregnant and others had a reduced number of implantations if this effect had been treatment related. The lower pregnancy rate may therefore have been a chance occurrence. Mating indices for the male rats treated with the isobutane were comparable to controls. Mating and gestation indices for the female rats treated with isobutane were also comparable to controls. 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, compared to controls. The 75% of females in this study becoming pregnant was also near historical control levels (87.5-100% with a mean of 93.7% in studies conducted between 2001 and 2002).

An exposure-related increase in post-implantation loss was noted for the 9000 ppm group and the NOAEC for reproductive endpoints was also concluded to be 3000 ppm (7131 mg/m³). This effect is also considered to be equivocal. There were no exposure-related differences in any of the parturition parameters including pre-implantation loss, the total number of pups delivered, the number of pups dying, the viability (4 day survival) index, the pup sex ratio and the number of live pups/litter, when compared to control. There were no exposure-related differences in body weights, weight gains or macroscopic differences in the pups at post-natal day 4.The mean number of corpora lutea were similar in both groups whilst the control group was less successful in achieving implantation and therefore had a higher pre-implantation loss than the test group. Both groups give birth to a similar number of pups per litter and therefore the treated group had a higher post-implantation loss than the control group. As both groups had the same potential number of implantations and both gave birth to a similar number of pups there is no indication of any adverse effect of isobutane on embryo/foetal survival. The apparent effect on post-implantation loss is therefore a numerical difference in this calculated parameter whilst the measured parameters showed no effect.