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Effects on fertility

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Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
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other: GLP compliant, guideline study, available as unpublished report, fully adequate for assessment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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
the deviations were considered not to have influenced the outcome of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, F.R.G
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: 278.8 - 321.6 g (mean 299.4g) for males and 191.5 - 219.0 g (mean 204.0 g) for females
- Fasting period before study: None
- Housing: 4 per sex/cage prior to exposure, and individually after mating, in suspended stainless steel cages.
- Diet: Stock diet ad libitum except for about 30 minutes prior to, and during exposure.
- Water: ad libitum except for about 30 minutes prior to, and during exposure.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Humidity: 37-80% (with occasional higher values after cleaning)
- Air changes: 10 per hr
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: From: 30 January 1992 To: 15 March 1992
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: Modified H 1000 multitiered inhalation chambers manufactured by Hazleton Systems Inc., USA. Stainless steel, with glass doors, with a capacity of approximately 2.2 m3
- Method of holding animals in test chamber: Individually in wire mesh stainless steel cages
- Each chamber was fitted with a micromanometer which showed the negative pressure inside ( 1-4 mm water column).
- To generate the test atmospheres, the test material was passed from the cylinder via a pressure reducer, stainless steel tubing and two calibrated massflow controllers and rotameters to the inlet of the inhalation chambers, where they were diluted with filtered air from the air conditioning system to the desired concentrations. The atmospheres were directed downward to the location of the animals.
- At the bottom of the chamber the test atmosphere was exhausted.
- Control rats were exposed to filtered air only.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere samples were taken sequentially from each of the chambers (control chamber included). They were drawn through heated sampling lines and were passed via a controlled valves system (Kuax-Control, Kuhnke 61.000) to the total carbon analyser (Ratfisch RS 55, FRG). The response of the analyser was recorded by a Kipp analogue recorder.
- The concentration was determined approximately twice each hour in each test atmosphere. Samples were taken from the chambers at a location close to the cage units in which the animals were housed.
- During preliminary experiments, it was checked whether a uniform distribution was obtained by measuring the concentration at several locations.
- The response of the total carbon analyser was recorded in scale units and converted into concentration values (ppm) based on the response of the calibration mixtures.
- The nominal concentration was determined using the mean amount of test material used per hour divided by the mean hourly volume of air passed through the exposure chamber (control chamber excluded).
Details on mating procedure:
- M/F ratio per cage: 1:1 (from about 3.30 p.m. until 8.30 a.m)
- If no sperm was detected the female was caged again with the same male the next afternoon
- Length of cohabitation: until mating occurred or 1 week had elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Males and females, not showing evidence of copulation were housed individually after the mating period had elapsed and were exposed until necropsy at the end of the study.
- 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 actual concentrations (± standard deviation) of butene-2 in the test atmospheres were 2476 (68) and 5009 (88) ppm, or 5.7 and 11.5 g/m3, for the low- and high-concentration respectively.
Duration of treatment / exposure:
Exposure: daily during the premating period (2 weeks), through mating (1 week) and gestation (up to and including day 19)
Study duration was 39-46 days for males and up to day 19 of gestation for females
Frequency of treatment:
6 hrs/day
Details on study schedule:
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.
Remarks:
Doses / Concentrations:
0, 2500 or 5000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 2476 or 5009 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
5.7 and 11.5 g/m3
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, sham-exposed
Details on study design:
none
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: As necessary, to appraise physical condition.

BODY WEIGHT: Yes
- Time schedule for examinations: Male rats were weighed weekly. Female rats were weighed weekly during the premating period, on days 0, 7, 14 and 21 of pregnancy and on day 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption was measured weekly (the second week during premating over a 6-day period) during the premating period in both males and females. Food consumption of parent females was also recorded weekly during pregnancy and through day 1-4 of lactation. Food consumption of parent males and not-mated females was recorded weekly after the mating period until scheduled sacrifice.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy
- Anaesthetic used for blood collection: Yes; ether anaesthesia
- Animals fasted: No data
- How many animals: All F0 animals
- Parameters checked: haemoglobin, packed cell volume, red blood cell count, red blood cell distribution width (RDW-SD), reticulocyte count, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy
- Animals fasted: No data
- How many animals: All F0 animals
- Parameters checked: alanine amino transferase (ALAT)/glutamic-pyruvic transaminase (GPT), albumin, aspartate amino transferase (ASAT), bilirubin total, calcium (Ca), chloride (Cl), creatinine, y-glutamyl transferase (yGT), inorganic phosphate, potassium (K), sodium (Na), total protein, urea.
Oestrous cyclicity (parental animals):
not examined.
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
- The total litter size and numbers of each sex as well as number of stillbirths and grossly malformed pups were recorded on days 1 and 4 post partum.
- The litters were weighed on days 1 and 4

GROSS EXAMINATION OF DEAD PUPS:
- yes. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. Pups sacrificed on day 4 were also examined macroscopically.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- All male and female parent rats were killed by exsanguination from the abdominal aorta under ether anaesthesia. A complete gross examination was performed.
- Pregnancy was demonstrated on the basis of implantation sites observed at autopsy of the female rats.

ORGAN WEIGHTS: Yes
- The following organs were weighed: testes, epididymides, liver, kidneys, thymus, lungs with trachea and larynx.

HISTOPATHOLOGY: Yes
- The following organs were examined microscopically from control and 5000 ppm animals: testes, epididymides, liver, kidneys, thymus, lungs with trachea and larynx, ovaries, uterus, seminal vesicles, adrenals, brain, heart, spleen, nose and any abnormal tissues and organs.
Postmortem examinations (offspring):
SACRIFICE
- All offspring were killed at 4 days of age.

GROSS NECROPSY
- Macroscopic abnormalities were recorded.
Statistics:
Parental clinical findings, mating and litter data, and pathological changes were evaluated by Fisher's exact probability test; bodyweight and food consumption by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. Duration of gestation, litter size implantations and pre- and post-implantation loss were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test, and pup bodyweights by analysis of variance followed by Dunnett's multiple comparison tests.
Reproductive indices:
The following indices were calculated for each group: mating index, female fertility index, fecundity index and gestation index.
Offspring viability indices:
The following indices were calculated for each group: live birth index, viability index (days 1-4) and sex ratio.
Clinical signs:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the males were comparable in all groups. Some slight differences in body weight change were observed. Fourteen days and 7 and 14 days after the start of exposure to 2500 and 5000 ppm respectively, female rats showed statistically significantly decreased mean body weights compared with controls. No effects on body weights of females were observed during gestation. On day 1 of lactation the mean body weight of the females of the 5000 ppm group was statistically significantly decreased. No effects on body weight change of the females was observed during the study.

FOOD CONSUMPTION
There was a statistically significantly decreased food intake of the females of the 5000 ppm group during the first week of the premating period only.
Dose descriptor:
NOAEC
Effect level:
5 000 other: ppm (11474 mg/m3, 11.5 mg/L) nominal.
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
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
BODY WEIGHT (OFFSPRING)
Mean body weight of the pups was slightly but not statistically significantly lower in the 2500 and 5000 ppm group, (possibly due to the higher number of pups in these groups).
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
5 000 other: ppm (11474 mg/m3, 11.5 mg/L) nominal.
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
Reproductive effects observed:
not specified

In the original report a NOAEL for toxicity was set at 2,500 ppm based on the slight effects on body weights in males and females, and possibly food consumption in females. A re-analysis of the data by RIVM (The Netherlands) concluded that as these effects were not dose-related and not consistently present during the study, the NOAEL for butene-2 should be ≥5000 ppm.

 

The data in question is shown in Table 1.

 

  • The changes in the body weight gain of the males was not considered to be adverse. It was only observed in week 1 and in week 4, when only the low dose was decreased, not showing a dose-response relationship. The change of 12 to 20 g, compared with the body weight of about 300 g is small. Furthermore, this effect was not observed in week 2 and 3, and no significant effects were shown on the body weight of the males.
  • In females, the effect on food consumption was not accompanied by an effect on body weight.
  • The effect on body weights of females was also not consistent through the whole study and was small (at the most about 5%).

 

Table 1. Treatment-related effects on body weight, body weight gain and food consumption

 

 

0

2500

ppm

5000

ppm

MALES

 

 

 

 

Week 1

Body weight

317.9± 4.36

313.9± 2.91

314.4± 3.63

Weeks 0-1

Body weight change

21.78 ±1.56

 

12.66±1.20

(58.1%)**

13.48± 1.54

(61.9%)**

Week 5

Body weight

369.6± 6.70

361.8± 4.14

367.0± 4.59

Weeks 4-5

Body weight change

10.87±1.38

5.84±1.74

(53.7%)*

11.60±1.20

(106.7%)

FEMALES

 

 

 

 

Week 0

Body weight

205.33± 2.10

203.60± 1.55

203.15± 1.91

Week 1

Body weight

214.25± 2.23

210.83± 1.55

(98.4%)

207.79± 1.13

(97.0%)*

Weeks 0-1

Body weight change

8.93± 1.21

7.23± 1.43

4.64± 1.49

Week 2

Body weight

220.28± 2.13

212.78± 2.05

(96.6%)*

213.19± 1.42

(96.8%)*

Weeks 1-2

Body weight change

6.0± 1.46

1.94± 1.79

5.40± 1.00

Body weight

Lactation Day 1

243.13±2.00

238.42±3.44

(98.1%)

230.61±2.67

(94.9%)*

Weeks 0-1

Food consumption

70.42±1.40

68.76±1.52

(97.6%)

65.28±0.96

(92.7%)*

Anova + Dunnet test; *= p, 0.05, **=p< 0.001

Conclusions:
No effects on reproduction or pup development were seen when male and female rats were exposed to 2-butene at concentrations of 2500 or 5000 ppm (approximately 5737 or 5737mg/m3) for two weeks prior to breeding, during breeding and until day 19 of gestation.
Executive summary:

Male and female rats were exposed to 2-butene at target concentrations of 2500 or 5000 ppm (approximately 5750 or 11500 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. Some decreases in body weight were observed in females at 2500 or 5000 ppm during the mating period but no other treatment-related changes were observed. There was no evidence of significant 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. Microscopic evaluation of gonadal function in parental males revealed no difference between treated and control groups. The NOAEC for reproductive toxicity was 2500 ppm (5700 mg/m3) based on the decreased body weights. 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 5000 ppm (11,500 mg/m3), the highest concentration tested.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 700 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate for assessment
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity data are available for one category stream member, the C2-C4 alkanes and propene.

 

Members and constituents of the Other Petroleum Gases category are flammable gases at room temperature and therefore exposure via the dermal or oral routes is unlikely and the requirement to test is waived in accordance with REACH Annex XI.

 

Liquefied Petroleum Gas

HLS (2009) exposed groups of rats to target concentrations of 0; 1,000; 5,000; or 10,000 ppm liquefied petroleum gas (propane and propylene 93.513%) for 6 hours per day, 5 days per week, for 13 weeks. No treatment-related effect on estrous cycle in females or sperm count, motility, or morphology in males was observed at any exposure concentration. The experimentally defined NOAEC is 10,000 ppm.

 

Methane CAS Number 74-82-8

No reproductive toxicity data are available specifically for methane.

 

Ethane CAS Number 74-84-0

HLS (2010) report an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen in rats. No effects on mating, fertility, or gestation indices or reproductive performance were observed in a 6-week study in which 0, 1,600, 5,000, or 16,000 ppm ethane was administered to male and female rats by inhalation. The experimentally defined NOAEC is 16,000 ppm (19678 mg/m3).

 

Propane CAS Number 74-98-6

In male and female rats exposed to 0, 1,200, 4,000, or 12,000 ppm propane by inhalation for 6 weeks (OECD Guideline 422), no effects on mating, fertility, or gestation indices or reproductive performance were observed. The experimentally defined NOAEC is 12,000 ppm (21641 mg/m3) (HLS 2009).

 

Isobutane CAS Number 75-28-5

There were no effects on mating, gestation indices or pup endpoints (survival, body weight and development up to postnatal day 4) when isobutane was tested in an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen (HLS, 2010). Rats were exposed by inhalation for up to 6 weeks to 0, 900, 3,000, or 9,000 ppm isobutane. The NOAEC was 3000 ppm (7131 mg/m3), based on equivocal effects at 9000 ppm (21,394 mg/m3), on both fertility and post-implantation loss.

Nine out of 12 female rats exposed to 9000 ppm isobutane became pregnant following successful mating, a difference that was not significantly different from the controls (75% of females became pregnant compared with 100% of controls), and of the 9000 ppm exposed rats that became pregnant a statistically significant increase in post-implantation losses was recorded (1.8 per litter compared to 0.8 in controls). A detailed review of the study report supports the possibility that the lower pregnancy rate may have been a chance occurrence on the basis that the group size was small (12 animals per group) and the percentage of females becoming pregnant was near historical levels (75% compared with a historic range of 87.5-100% with a mean of 93.7% in studies conducted between 2001 and 2002). The mean number of corpora lutea, implantation sites, pre-implantation losses, live pups per litter, pup survival to post-natal day 4, and pup sex ratio were not significantly different, all further evidence that a real effect on fertility is questionable. The limitations of this study should be taken into account when considering the potential hazard posed by isobutane. The weight of evidence from the other C1- C4 petroleum gases, where no effects on fertility or reproduction were seen, also supports the likely lack of effect of isobutane.

 

Butane CAS Number 106-97-8

HLS (2008) report an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen in rats. No effects on mating, fertility, or gestation indices or reproductive performance were observed in a 6-week study in which 0, 900, 3,000, or 9,000 ppm butane was administered to male and female rats by inhalation. The experimentally defined NOAEC is 9,000 ppm (21394 mg/m3).

 

Propene CAS Number 115-07-1

Overall, there is a considerable amount of data from well conducted and reported guideline studies with adequate and reliable coverage of key parameters for the assessment of the reproductive toxicity potential of propene. A weight of evidence evaluation for propene and its proximate metabolite, propene oxide indicate that there is no evidence for adverse reproductive effects (including fertility). Findings include; no effects in reproductive tissues in repeated dose studies of up to 2 years on propene (NTP, 1985).

Butene isomers (butenes)

A weight of evidence evaluation for butene isomers indicates that they have no effect on fertility. No reproductive toxicity was seen in OECD Guideline 422 (reproduction screening) studies on rats via inhalation exposure for 1-butene (Huntingdon, 2003) or 2-butene (TNO 1992b). There was no evidence of systemic toxicity for 1-butene in the parents. Slight reductions in maternal body weight occurred with 2-butene but these were inconsistent and no other treatment-related changes occurred. There were no effects on mating behaviour, fertility and gestation indices, the number of implantation sites 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 NOAECs for reproductive toxicity were 8000 ppm (18,359 mg/m3) for 1-butene and 5000 ppm (11,474 mg/m3) for 2-butene, the highest concentrations tested. In addition, no effects on male and female reproductive parameters in rats and mice were observed in 14 week inhalation exposure studies of 2-methylpropene. These repeat dosing studies included parameters such as sperm analysis, estrus cycle analysis and histopathology (although mating was not carried out). NOAECs of 8000 ppm (18,359 mg/m3) for both rat and mouse studies were established (NTP 1998).

Ethylene

A reproduction/developmental toxicity screening test (OECD test guideline 421) has been conducted on ethylene (Corning Hazleton, 1997). This test was designed to provide initial information on the possible effects of ethylene on reproduction and/or development when inhaled by rats (head only exposure) at concentrations of 0, 200, 1000 or 5000 ppm (corresponding to 0, 230, 1147 or 5737 mg/m3). The exposure concentrations were calculated to give approximately 80, 400 or 2000 mg/kg bw/day respectively.

Parent males and females (10 per sex/group) were exposed to ethylene for 6 hours/day and for 14 days prior to the start of a mating period; exposure of the males continued through to termination i.e. for a minimum of 28 days and exposure of the females, until day 20 of gestation. In the post-exposure period, the females were allowed to litter and then terminated on day 4 post partum. The ovaries, testes and epididymides of the control and 5000 ppm parent animals were examined histologically. 

No adverse effects of ethylene were seen in this study. Therefore, head-only exposure to ethylene gas at target concentrations of up to 5737 mg/m3 did not impair reproductive performance of the parent animals, their fertility or pregnancy, or the number, growth or survival of the offspring to postnatal day 4. 

C-4 rich stream:

Male and female rats were exposed to C4 Crude Butadiene (CAS no. 68476-52-8) by inhalation at levels of 0, 2, 10, or 20 mg/L (6h/day, 7 d/week) for two weeks prior to breeding, during breeding (up to two weeks), and continuing through day 19 of gestation. Pups were maintained until postnatal day 4. There were no treatment-related adverse effects on any measures of reproductive function. Based on these data, the NOAEC for reproductive toxicity was 20 mg/L (20,000 mg/m3), the highest concentration tested (Dow, 2001).

 

Summary

No quantitative data were located on the effects on fertility and reproductive parameters of Other Petroleum Gases in humans. There are no 2-generation reproduction studies available but there is sufficient weight of evidence from the component substances to conclude that further testing is scientifically unjustified (Annex XI adaptation). Inhalation exposure is the most relevant route, and a GLP-compliant guideline study is available on one category stream, the major components being propane and propylene (93.513%). In a 90 day study on liquefied petroleum gas, parameters such as sperm analysis, oestrus cycle analysis and histopathology were included (although mating was not carried out); there were no effects with a no observed adverse effect level (NOAEC) of 10,000 ppm, the maximum dose level tested.

GLP-compliant guideline studies (OECD 422) are available in animals for C2 – C4 alkanes up to 6 weeks in duration that indicate members of this category have low potential for reproductive toxicity (including effects on fertility). No biologically significant treatment-related reproductive toxicity or effects on reproductive endpoints in repeat dosing studies were observed in rats after inhalational exposure to butane, isobutane, propane or ethane. The NOAEC for fertility is 3000 ppm (7131 mg/m³) based on the study on isobutane where equivocal effects on fertility occurred at 9000 ppm (21,394 mg/m3). The limitations of this study, together with the weight of evidence from the other C2 – C4 petroleum gases support an absence of hazard for effects on fertility.

 

A more extensive database exists for main component propene which similarly supports a conclusion of low potential for reproductive toxicity/fertility effects.

Benzene

In a one-generation reproduction toxicity study, there were no significant effects at any dose tested (up to 300 ppm) on maternal body weight and body weight gain and on fertility/reproductive endpoints as measured by percentage of pregnant animals, mean gestational length, number of litters, litter size, and viability of the pups and the weanlings. The only statistically significant postnatal developmental effects for offspring were lower female pup body weight on day 21 and lower liver weight at the 300 ppm exposure level. These differences were small (10 and 14%, respectively) and considered not to be adverse. An exposure concentration of 300 ppm (960 mg/m3) is a NOAEC for fertility/reproductive toxicity in adults and postnatal developmental effects for offspring.

1,3-Butadiene

Data from dominant lethal assays indicate that it has an adverse effect on germ cells in male mice but not rats. The results from long term toxicity and carcinogenicity studies indicate that the ovary and testes are target organs for 1,3-butadiene toxicity in mice. The sensitivity of the mouse ovary is thought to be due to the high levels of butadiene diepoxide present in 1,3-butadiene-exposed mice. It is not known whether ovarian atrophy in 1,3-butadiene-exposed mice affects reproductive function. However, the rat is considered to be more relevant to humans, and ovarian atrophy is not observed in rats exposed to 1,3-butadiene up to 8,000 ppm (Owen 1987). The more recent study of WIL (2003) supports the conclusion that 1,3-butadiene does not affect fertility in rats and the NOAEC for fertility is 6000 ppm (13,276 mg/m3) based on this key study.


Short description of key information:
The weight of evidence from studies on one category stream member and main components C2-C4 alkanes and propene indicates no evidence of reproductive toxicity.

Justification for selection of Effect on fertility via oral route:
These streams are gases at room temperature, hence fertility testing via the oral route is not technically feasible.

Justification for selection of Effect on fertility via inhalation route:
Results from an OECD 422 study on iso-butane provide a NOAEC of 7131 mg/m3 for possible effects on fertility, based on an equivocal effects at 21,394 mg/m3; this is considered a conservative conclusion as the effects seen were relatively minor in nature and not statistically significant. Supporting information regarding an absence of a potential to adversely affect fertility is provided by screening studies on ethane, propane and butane.

Justification for selection of Effect on fertility via dermal route:
These streams are gases at room temperature, hence fertility testing via the dermal route is not technically feasible.

Effects on developmental toxicity

Description of key information

The weight of evidence from studies on liquefied petroleum gas, main components C1-C4 alkanes and propene indicates no evidence of development toxicity. However, category stream members may contain carbon monoxide which could trigger classification for developmental effects.

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, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl: CD BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 272.8-354.7 g (males), 194.9-248.7 g (females)
- Fasting period before study: No
- Housing: stainless steel wire-mesh suspended cages. From day 20 of gestation onwards in solid floor polypropylene cages with wire mesh stainless steel lids
- Diet: SQC Rat and Mouse Breeder Diet No 3, Expanded, Special Diet Services Ltd., Witham, UK ad libitum ( except during exposure)
- Water: mains drinking water ad libitum
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: 15 per hr
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: From: 20 December 1995 To: 3 February 1996
Route of administration:
inhalation: gas
Vehicle:
other: air
Details on exposure:
Animals were exposed (head-only) in rodent restraint tubes secured to each of the two sides of the chamber through ports let into the chamber walls. Ethylene was ducted past the noses of the animals in a regulated flow and extracted from the chamber via ports at each end. The high dose concentration was generated through a miniature flowmeter and prepared in excess quantity of that needed for the high dose exposure. Balance of the flow served as a feedstock for the second stage of dilution for the low and intermediate doses.
Temperature and relative humidity in the exposure chamber was recorded 2 times/hour throughout the exposure period and monitored continuously using a digital thermometer and a paper hygrometer.
Chamber airflow was also monitored continuously with recordings twice per hour.
The air flow was maintained at a rate sufficient to provide the normal concentration of oxygen to the animals. The concentration of ethylene in each chamber was determined approximately 2 times per hour with a Miran 1A infrared spectrophotometer which was calibrated and the analytical concentration during each exposure was interpolated from the standard curve.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The ranges of mean daily concentrations of ethylene within the chambers were 187-243, 966-1082 and 4961-5171 ppm which were considered satisfactory.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: when mating confirmed or 10 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
6 hours/day, 7 days/week, 2 weeks prior to mating, during pairing, until the day prior to necropsy for the males (Day 28) or Day 20 of gestation for the females.
Frequency of treatment:
6 hrs daily
Duration of test:
Approximately 28 days

No. of animals per sex per dose:
10/sex/group

Control animals:
other: yes, air exposed
Details on study design:
Dose selection rationale: Following review of data from previous toxicity studies and based on levels that are safe in practice (high dose) and anticipated human exposure (low dose).

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Daily cage-side observations included mortality (twice daily), clinical reactions, abnormal behaviour, and availability of food and water. Post dosing observations were initially recorded up to 2 hours after the end of exposure; however, as no adverse clinical signs were seen on the first two days, the observations were conducted 0.5 and 1 hour after the end of exposure for the remainder of the week and immediately and 0.5 hours after the end of exposure for the remainder of the treatment period.

BODY WEIGHT: Yes
- Body weights were measured prior to the first treatment and weekly thereafter. Mated females were weighed on days 0, 7, 14, and 20 of gestation and on days 1 and 4 post-partum.

FOOD CONSUMPTION
Per cage of animals, food consumption was determined weekly during the pre-mating period. For mated females, food intake was recorded for days 0-4, 4-7, 7-10, 10-14, 14-17, and 17-20 of gestation and on days 1-4 post-partum.

LITTER DATA: Yes
For each female that littered the following were recorded: date of mating, date of parturition, duration of gestation, any abnormal behaviour. The following data were recorded for each litter: pup numbers (live and dead), number and sex of live pups recorded daily and reported for days 1 and 4 post-partum, clinical condition of pups on days 1-4 post-partum, individual pup weights on days 1 and 4, necropsy findings of dead pups.

POST MORTEM EXAMINATIONS:
At necropsy, all parental animals were subjected to macroscopic examination for structural or pathological changes. For each parental female, the numbers of corpora lutea and implantation sites were recorded. Blood samples were taken from all parental animals. 20 tissues were fixed and retained in formalin. Histopathological examination of the ovaries, testes and epididymides of the control and high dose group were conducted using light microscopy.
Ovaries and uterine content:
The ovaries and uteri were examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: No
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Body weights, body weight gains, and food intake were analyzed by analysis of variance. To test for equality of variances between groups, Levene's test was performed. Dunnett's test was used to compare each group and control. Regression analysis was performed for dose-response. Non-parametric analysis was used to evaluate the number of implantation sites, number of pups born, % male pups on days 1 and 4, post-implantation survival index, mean pup weights on days 1 and 4 and % weight change on days 1 and 4. This included Kruskall-Wallis analysis together with the protected Wilcoxon Rank Sum test for each treated group against control. The Terpstra-Jonckheere test for dose response was also performed. The Cochran-Armitage test for dose-response between groups and Fisher-Irwin tests for pairwise comparisons between control and treated groups were performed where data included a high proportion of tied values. This included mating index, mean duration of gestation, pup deaths on days 0-1, and 1-4, live birth index and viability index. Group values for fertility, fecundity, number of females with live pups at day 4, and gestation index were identical and therefore were not analyzed.
Indices:
Reproductive function was evaluated by calculation of mating index, female and male fecundity index, and female and male fertility index. In addition, the gestation index, post implantation survival index, live birth index, viability index and % male pups was calculated.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
5 737 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Basis for effect level:
other: Developmental Toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
5 737 mg/m³ air
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

 Fertility:

No deaths were observed that were attributable to treatment. Clinical observations did not indicate an adverse effect of treatment. Observations included minor changes such as swollen ears, damaged or missing tip of tail or ear, sores and lesions chromodacryorrhoea, hair loss, etc. Of note, several females in all groups during the latter half of the gestation period showed a red discharge from the vulva which was attributed to the restraint method which prevented normal grooming behaviours. No adverse outcome on pregnancy was observed.

In females (early gestation, days 0-7) from the low dose group, a significant increase in body weight was observed as compared to controls. Treatment was not seen to adversely affect body weight gain of males or females during premating, or females during gestation or lactation at any of the doses. Food intake of treated males and females during pre-pairing (males and females) as well as gestation and lactation (females only) was unaffected. For females treated with the high dose, food intake was shown to be significantly increased for days 1-4 of lactation.

There were no ethylene-induced effects on fertility or fecundity. All females became pregnant.

Mean number of corpora lutea and implantation sites per dam was seen to be slightly higher than controls in all treatment groups, although this effect was not statistically significant.

The post-implantation survival index was slightly lower in the treated groups; however no adverse effect on litter size was seen.

Developmental Parameters:

Litter parameters were unaffected by ethylene treatment. The number of pups alive on days 1 and 4 in all treatment groups was no different from control. The % of male pups (sex ratio) in all treatment groups on day 1 and 4 was similar to control. The live birth index and viability index were both unaffected by ethylene treatment.

Mean pup weights were seen to be higher than controls in all treated groups at days 1 and 4 post-partum, although this increase was not statistically significant.

Mean pup weights (g)

male and female combined

ethylene concentration (ppm)

0

200

1000

5000

Day 1

6.0

6.4

6.3

6.5

Day 4

8.6

9.1

8.9

9.2

% Weight change Day 1-4

43.3

42.2

41.3

41.5

Necropsy findings did not suggest toxicity due to ethylene treatment. Clinical observations on day 1 post-partum included findings attached umbilical cord, haemorrhagic; nose, hindpaw, abdomen, back, and mouth, pups which were cold and unfed, etc. These findings were distributed equally among all experimental groups including control. At necropsy, on post-partum day 4, findings including pups without milk in the stomach, microphthalmia and damage of a tail tip. Again, these findings were not attributed to ethylene treatment.

Conclusions:
Head-only exposure of rats to ethylene at concentrations of 0, 200, 1000, or 5000 ppm did not induce effects on reproductive performance, fertility, or pregnancy. The NOAEC in this study was 5000 ppm (5737 mg/m3).
Executive summary:

The potential effects of ethylene inhalation on rat reproduction and on growth and development of the offspring was studied in a combined reproduction/development toxicity screening test, conducted according to GLP. Four groups of rats (10 females and 10 males per group) were dosed by head only inhalation for 6 hours daily with air only (control); 200, 1000 or 5000 ppm of ethylene (corresponding to 0, 230, 1147 or 5737 mg/m3). Ethylene was administered to parent animals for two weeks prior to mating, during the mating period and until the day prior to necropsy of the males (minimum 28 days) or until day 20 of gestation for the females. The females were allowed to litter and rear their offspring to day 4 post-partum, when they and their offspring were killed. Morbidity, mortality, clinical condition, weight and food intake were observed throughout the study, and mating was carefully observed. For each female, litter data and also observations for each offspring were recorded. At termination of the study, all animals were subject to macroscopic examination for structural or pathological changes.

There were no deaths attributable to the test article, and body weight gain was not adversely affected during the pre-mating, gestation or lactation periods. The treatment had no effect on fertility or fecundity and all females became pregnant. Litter size, sex ratio, mean pup weight and pup growth and clinical condition were not adversely affected by treatment. Necropsy revealed no macroscopic finding suggestive of toxicity due to test substance administration. There was no evidence of any toxic effect on the testis due to test substance administration and there were no other microscopic findings suggestive of toxicity due to the exposure.

In conclusion, head-only administration of ethylene at nominal concentrations of 200, 1000 or 5000 ppm was without evidence of toxicity or adverse effects on growth and development of the offspring from conception to day 4 post-partum.

The highest dose of 5000 ppm (5736 mg/m3) is concluded to be a no adverse effect concentration (NOAEC) for the reproduction/development screening test in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
5 737 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate for assessment
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Human data

Two cases are reported of butane exposure in pregnant women, one accidentally exposed in pregnancy week 27, the other intentionally as a suicide attempt in week 30 (Health Council of the Netherlands, 2004).The first woman gave birth to a child with hydranencephaly, while the second woman gave birth to a child that died after 11 hours with severe encephalomalacia and hypoplastic kidneys. In both cases, the brain effects were not considered to be caused by butane but by intrauterine anoxia. In neither of these cases were estimations of the concentrations inhaled made, also, as the history prior to the exposures is unknown, the relationship of the developmental outcomes to butane exposure this study is highly uncertain.

 

Non human data

Liquefied Petroleum Gas

Liquified petroleum gas (propane and propylene 93.513%) was tested in an OECD Guideline 414 Prenatal Developmental Toxicity Study (HLS 2010). Exposure of pregnant rats to target concentrations of 1000, 5000 or 10,000 ppm liquified petroleum gas by whole-body inhalation on gestation days 6 -19 resulted in no effects of exposure. Therefore, a no observed adverse effect concentration (NOAEC) for maternal toxicity and developmental toxicity of 10,000 ppm was indicated.

 

Methane CAS Number 74-82-8

No developmental toxicity data are available specifically for methane.

 

Ethane CAS Number 74-84-0

HLS (2010) report an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen in rats. There were no effects on offspring survival (to postnatal day 4), pup body weight, or macroscopic post mortem evaluations in a 6 week study to GLP in which rats were exposed by inhalation prior to mating, during mating, and after mating. The experimentally defined NOAEC is 16,000 ppm (19678 mg/m3), the maximum dose tested.

 

Propane CAS Number 74-98-6

In male and female rats exposed to 0, 1,200, 4,000, or 12,000 ppm propane by inhalation for 6 weeks (OECD Guideline 422), prior to mating, during mating, and after mating, no effects on offspring survival (to post natal day 4), pup body weight, or macroscopic post mortem evaluations were observed. The experimentally defined NOAEC is 12,000 ppm (21641 mg/m3) (HLS 2009).

 

Isobutane CAS Number 75-28-5

HLS (2010) report the findings of an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen in rats exposed up to 6 weeks to 0, 900, 3,000, or 9,000 ppm isobutane by inhalation prior to mating, during mating, and after mating. There were no treatment-related differences in offspring survival (to post natal day 4), pup body weight, or macroscopic post-mortem evaluations. The NOAEC for pup endpoints is 9000 ppm (21394 mg/m3).

 

Butane CAS Number 106-97-8

HLS (2008) report an OECD Guideline 422 combined repeated-exposure toxicity, reproduction and neurotoxicity screen in rats. No effects on offspring survival (to post natal day 4), pup body weight, or macroscopic post mortem evaluations were observed in a 6-week study in which 0, 900, 3,000, or 9,000 ppm butane was administered to male and female rats by inhalation. The experimentally defined NOAEC is 9,000 ppm (21394 mg/m3).

 

Propene CAS Number 115-07-1

No developmental toxicity was reported when propene was tested in a rat developmental toxicity study (OECD Guideline 414) by inhalational exposure (BASF, 2002). No maternal toxicity was expressed at any concentration up to 10,000 ppm (17,200 mg/m3). There were no treatment-related influences on the gestational parameters and no signs of prenatal developmental toxicity, in particular no indications of teratogenicity. The NOAEC for prenatal developmental and maternal toxicity from inhalation exposure to propene is 10,000 ppm (17,200 m/m3), half the lower explosive limit.

  

Butene isomers (butenes)

The butenes are not toxic to development. 2-Methylpropene has been tested in a key rat developmental toxicity study (OECD Guideline 414) by inhalational exposure (CTL 2002). There were no effect on the females during gestation, no effects on the number, growth or survival of the foetuses in utero and no effects on foetal development (determined by visceral and skeletal analysis). A NOAEC of 8000 ppm (18,359 mg/m3) (the highest concentration tested) was established for maternal toxicity and foetal toxicity (CTL 2002). 2-Butene and 1-butene also had no effect on developmental toxicity when tested during the reproductive toxicity element of OECD Guideline 422 studies by inhalation exposure. There were no effects on pup body weight gain or observed during macroscopic examination of pups at post mortem (Huntingdon 2003, TNO 1992b). The NOAEC of 8000 ppm (18,359 mg/m3) for developmental toxicity is based on the NOAEC for 2-methylpropene in the developmental toxicity study (CTL 2002).

C-4 rich streams

Male and female rats were exposed to C4 Crude Butadiene (CAS no. 68476-52-8) by inhalation at levels of 0, 2, 10, or 20 mg/L (6h/day, 7 d/week) for two weeks prior to breeding, during breeding (up to two weeks), and through day 19 of gestation. Pups were killed on postnatal day 4. There was no treatment-related maternal toxicity or effects on developmental toxicity or teratogenicity. Based on these data, the NOAEC for reproductive toxicity was 20 mg/L (20,000 mg/m3), the highest concentration tested (Dow, 2001).

 

Summary

No quantitative data were located on the effects on developmental parameters of petroleum gases in humans. Limited human data demonstrated birth defects in 2 pregnant women were associated with intrauterine anoxia rather than butane exposure, also as the history prior to the exposures is unknown, the relationship of the developmental outcomes to butane exposure this study is highly uncertain.

In animals, a prenatal developmental toxicity study is available on liquified petroleum gas (major components propane and propylene) by whole-body inhalation; a no observed adverse effect concentration (NOAEC) for maternal toxicity and developmental toxicity of 10,000 ppm was indicated. Current GLP-compliant guideline studies (OECD 422) are also available for C2-C4 alkanes up to 6 weeks in duration; there were no developmental malformations or evidence of foetal toxicity. Also data on the component substance propene further indicate that members of this category have low potential for developmental toxicity. Based on these data, no labelling is warranted.

However, some streams in this category may contain carbon monoxide and data on this material indicates evidence of developmental toxicity:

 

Benzene

The pre-natal developmental toxicity (or teratogenic) potential of benzene inhaled at doses ranging from 1 to 500 ppm by pregnant Sprague-Dawley rats on gestation days 6-15 has been investigated in two studies (Coate et al, 1984; Kuna and Kapp, 1981). No maternal toxicity was observed at 100ppm (Coate et al, 1984) but maternal body weight and bodyweight gain were decreased at 50 and 500 ppm (Kuna and Kapp, 1981). Reduced foetal weight was seen at 100 ppm (Coate et al, 1984) and at 50 and 500 ppm (Kuna and Kapp). Reduced crown rump lengths and associated delay in ossification of extremities and sternebrae were seen in the same foetuses.

Overall, the NOAEC for teratogenicity in the rat was 500 ppm (1600 mg/m3), the NOAEC for maternal and developmental toxicity was 10 ppm (32 mg/m3).

The effect of inhaled benzene on pre-natal developmental toxicity/teratogenicity was also assessed in CF-1 mice and New Zealand white rabbits.There were no significant effects on maternal clinical condition, body weight or body weight gain. Benzene exposure did not significantly affect the incidence of pregnancies or the average number of live foetuses or resorptions per litter in either species. Mean foetal body weight, but not crown-rump length, was decreased significantly in benzene exposed mice but there were no effects in rabbits. No teratogenic malformations were observed in either species. However, increases in the occurrence of several minor skeletal variants (including delayed ossification of sternebrae, skull bones and of unfused occipital bones of the skull) were reported in offspring of benzene-exposed mice.

The NOAEC for maternal toxicity was 500 ppm (1600 mg/m3) in rabbits and mice. 500 ppm (1600 mg/m3) was the NOAEC for pre-natal developmental toxicity in rabbits and the LOAEC for pre-natal developmental toxicity in mice.

1,3-Butadiene

Developmental toxicity occurred in rats and mice, in the presence of maternal toxicity, manifested as reduced weight gain or increased developmental variations (HLE 1982, Hackett 1987a&b). There was no evidence of developmental toxicity in the absence of maternal toxicity. The available evidence indicates that 1,3-butadiene has low potential for developmental toxicity in humans. The NOAEC for developmental toxicity is 40 ppm (88 mg/m3), based on the key study of Hackett (1987b) in mice.

Carbon Monoxide CAS number 630-08-0

(Classification: CLP Category 1A, H360D)

The World Health Organisation published an extensive review of carbon monoxide in 1999 (WHO, 1999, updated 2004). In the human body, it reacts readily with haemoglobin to form carboxyhaemoglobin (COHb). Its toxic effects on humans are due to hypoxia, which becomes evident in organs and tissues with high oxygen consumption such as the brain, the heart, exercising skeletal muscle and the developing foetus. Carbon monoxide readily diffuses crosses the placenta and binds to foetal haemoglobin with a higher affinity than for maternal haemoglobin. Furthermore, the gas is cleared from foetal blood slower than from maternal blood, leading to the accumulation of carbon monoxide which, at steady state, may be up to 10 – 15 % higher than maternal concentrations. WHO state that maternal carbon monoxide exposures of 170–230mg/m3 (150–200 ppm), leading to approximately 15–25% carboxyhaemoglobin, can produce reductions in birth weight, cardiomegaly, delays in behavioural development and disruption in cognitive function.

 

Reference

World Health Organisation, 1999

Environmental Health Criteria 213 (Carbon Monoxide, second edition)

1999, updated 2004


Justification for selection of Effect on developmental toxicity: via oral route:
These streams are gases at room temperature, hence developmental toxicity testing via the oral route is not technically feasible.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Results from an OECD 414 developmental toxicity study on liquified petroleum gas (comprising propane and propylene, 93.513%) provide no evidence of foetal toxicity at exposures up to10,000 ppm LPG (equivalent to approx. 17,200 mg/m3 propene).

Justification for selection of Effect on developmental toxicity: via dermal route:
These streams are gases at room temperature, hence developmental toxicity testing via the dermal route is not technically feasible.

Toxicity to reproduction: other studies

Additional information

No other studies are available

Justification for classification or non-classification

The weight of evidence from studies on one stream member, the main components C1-C4 alkanes and propene, indicates there is adequate information available from which to assess the potential of Other Petroleum Gases to induce reproductive or developmental effects and to conclude that classification is not warranted. However category streams containing ≥ 0.3% carbon monoxide should be classified Cat 1A H360D. 

Additional information