Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
No other data
Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Justification for type of information:
N/A
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
N/A
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: Crl: CD BR
Details on species / strain selection:
not specified
Sex:
male/female
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
Type of inhalation exposure (if applicable):
head only
Vehicle:
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.
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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The range of mean daily concentrations of ethylene within the chambers were 187-243, 966-1082 and 4961-5171 ppm which were considered satisfactory.
Duration of treatment / exposure:
~ 28 days (6 hr/day) 2 weeks prior to mating, during mating, until the day prior to necropsy for the males (Day 28) or until Day 20 of gestation for the females. 
Frequency of treatment:
6 hrs daily
Details on study schedule:
not specified
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
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).
Positive control:
None
Parental animals: Observations and 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.

Oestrous cyclicity (parental animals):
The stage of oestrus cycle was recorded when positive evidence of mating was seen.
Sperm parameters (parental animals):
N/A
Litter observations:
STANDARDISATION OF LITTERS - no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, clinical condition, individual pup weights

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
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 10% neutral buffered formalin. Histopathological examination of the ovaries, testes and epididymides of the control and high dose group were conducted using light microscopy.
Postmortem examinations (offspring):
F1 pups were examined externally only.
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.
Reproductive 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 were calculated.
Offspring viability indices:
N/A
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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 preventing normal grooming behaviours.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In females (early gestation, days 0-7) from the low dose group only, a significant increase in body weight was observed as compared to controls. Group Mean Body weight: 282 and 287g for controls and 200 ppm respectively. Treatment was not seen to adversely affect body weight gain of males or females pre-mating, during gestation or lactation at any of the doses.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of treated males and females during pre-mating (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. Group Mean Food intake: 34 and 41 g/animal/day for controls and 5000 ppm respectively.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
N/A
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
N/A
Reproductive performance:
no effects observed
Description (incidence and severity):
N/A
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): 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 preventing normal grooming behaviours. 

BODY WEIGHT (PARENTAL ANIMALS): In females (early gestation, days 0-7) from the low dose group only, a significant increase in body weight was observed as compared to controls. Group Mean Body weight: 282 and 287g for controls and 200 ppm respectively. Treatment was not seen to adversely affect body weight gain of males or females pre-mating, during gestation or lactation at any of the doses.  

FOOD CONSUMPTION (PARENTAL ANIMALS): Food intake of treated males and females during pre-mating (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. Group Mean Food intake: 34 and 41 g/animal/day for controls and 5000 ppm respectively.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no ethylene-induced effects on fertility or fecundity. All females became pregnant. No adverse outcome on pregnancy was observed and no adverse effect on litter size was seen.

PATHOLOGY: Necropsy findings did not suggest toxicity due to ethylene treatment. Histopathology finding in the testis, epididymis and ovary of the control and high dose were similar to what could be expected in normal control rats. One high dose male, showed a minor unilateral tubular atrophy in the testis which is occasionally seen in the background pathology of F344 rats. Proportions of tubules at specific points in the cycle were similar between control and treatment groups. In addition, cell association in the testis and sperm maturation were considered to be within the normal range.
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no evidence of toxicity or adverse effects on male or female reproductive performance, fertility, pregnancy, maternal or suckling behaviour at highest dose concentration. Equivalent to 5737 mg/m3.
Clinical signs:
not examined
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
not examined
Description (incidence):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
not examined
Description (incidence and severity):
N/A
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on results:
N/A
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
N/A
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
N/A
Reproductive performance:
not examined
Description (incidence and severity):
N/A
N/A
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
no mortality observed
Description (incidence and severity):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
In females (early gestation, days 0-7) from the low dose group only, a significant increase in body weight was observed as compared to controls. Group Mean Body weight: 282 and 287g for controls and 200 ppm respectively. Treatment was not seen to adversely affect body weight gain of males or females pre-mating, during gestation or lactation at any of the doses.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Food intake of treated males and females during pre-mating (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. Group Mean Food intake: 34 and 41 g/animal/day for controls and 5000 ppm respectively.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
not examined
Description (incidence and severity):
N/A
Nipple retention in male pups:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
no effects observed
Description (incidence and severity):
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.
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no evidence of adverse effects on the offspring to day 4 post partum at highest dose concentration. Equivalent dose 5737 mg/m3.
Clinical signs:
not examined
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
not examined
Description (incidence and severity):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
not examined
Description (incidence and severity):
N/A
Nipple retention in male pups:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Reproductive effects observed:
not specified

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.

 

Target ethylene concentration (ppm)

0

200

1000

5000

Mean Pup Weights (g)

Male and female combined - 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

 

Conclusions:
Head-only administration of ethylene at nominal concentrations of 200; 1,000 or 5,000 ppm was without evidence of toxicity or adverse effects on male and female reproductive performance, fertility, pregnancy, maternal, and suckling behaviour of the offspring. The highest dose of 5000 ppm (5737 mg/m3) is concluded to be a NOAEC for reproduction/development in rats.
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; 1,000; or 5,000 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. Ovaries, testes and epididymides of the control and high dose animals were subject to a histopathological examination.

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 male and female reproductive performance, fertility, pregnancy, maternal, and suckling behaviour of the offspring from conception to day 4 post-partum.

The highest dose of 5000 ppm ( 5737 mg/m3) is concluded to be a no adverse effect concentration (NOAEC) for the reproduction/development screening test in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no 2-generation reproduction study available for ethylene, however, data are available from an OECD 421 reproductive/developmental screening study, two 90-day studies (one to modern guidelines) (Dow, 2010 and Rhudy 1978) and the 2-year repeat dose study (Hamm et al, 1984).

In addition, data on C3 alkene homologues provide sufficient additional information for evaluation. These data are summarised below, and justification to support their use for the purposes of read-across is attached at the end of this CSR and appended to Section 13 of the IUCLID dossier.

An extended one-generation reproductive toxicity study is proposed for ethylene. This will form part of a read-across approach to addressing this endpoint in both ethylene and the read-across target propene.

 

Information on ethylene

Human data

There are no available data concerning the effects of ethylene on reproduction in humans.

Non-human data

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. 

A modern guideline 13-wk inhalation toxicity study was conducted in rats at target exposure concentrations of 300, 1000, 3000 and 10,000 ppm (344.2, 1147, 3442 and 11,473 mg/m3) (Dow, 2010). Consistent with an earlier 90 day study on ethylene (Rhudy, 1978) at the same dose concentrations and a 2-year lifetime study (Hamm, 1984) at target concentrations of 300, 1000 and 3000ppm, no gross or microscopic lesions of the reproductive organs were observed (including prostate/testes/seminal vesicles and ovaries/oviducts/uterus/vagina/mammary glands/fallopian tubes).

 

Metabolism to ethylene oxide

Approximately 15% of inhaled ethylene is absorbed, a significant proportion of that absorbed is eliminated unchanged in exhaled air; while some systemically available ethylene is metabolised to ethylene oxide, it is estimated that only approximately 3% of atmospheric ethylene is converted to ethylene oxide by metabolism ( Csanády et al., 2000). Neither ethylene nor closely-related substances are mutagenic, carcinogenic, or reproductive toxicants. Therefore, although ethylene oxide is classified for carcinogenicity and genotoxicity and affects fertility and development in animal studies, the metabolism of ethylene to ethylene oxide does not impact the toxicity of ethylene.

 

 

Summary & Conclusions

 

Overall, there is sufficient information available from well conducted and reported guideline studies with adequate and reliable coverage of key parameters for the assessment of the reproductive toxicity potential of ethylene. On the basis of this information, there is no evidence to indicate potential for adverse reproductive effects. These data include

•          A reproduction/developmental toxicity screening study done according to OECD 421 (Corning Hazleton, 1997). No adverse effects of ethylene were reported. Whilst this screening test is limited in detecting effects on all aspects of reproduction and development the negative result does provide some reassurance for lack of effect.

•          No gross or microscopic lesions of the reproductive organs were observed (including prostate/testes/seminal vesicles and ovaries/oviducts/uterus/vagina/mammary glands/fallopian tubes) in either of the 90 day studies (Dow, 2010; Rhudy, 1978) or the 2 year study (Hamm, 1984).

•          A comprehensive database indicates that ethylene is not genotoxic (in contrast, direct administration of the genotoxic material ethylene oxide has been linked directly to its reproductive toxicity).

•          A supporting database on alkene homologue propene (C3) which includes lack of findings in reproductive tissues (histopathological examination of the gonads) in repeated dose studies up to 2 years and lack of prenatal developmental effects.

 

Short description of key information:
A weight of evidence from a reproductive toxicity study, reproductive tissues in repeat dosing studies on ethylene, and data from alkene homologues from guideline tests at the highest concentrations that can be safely tested for these substances, indicates no evidence of reproductive toxicity.

Further testing is required to address data requirements under REACH. An OECD 443 in rats is therefore proposed, with ethylene as the testing material.

Effects on developmental toxicity

Description of key information

The weight of evidence from studies on ethylene and alkene homologues indicates no evidence of developmental toxicity from guideline studies conducted at the highest concentrations that may be safely tested.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
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
Justification for type of information:
N/A
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
not specified
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
Type of inhalation exposure (if applicable):
head only
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

Dose / conc.:
0 ppm (nominal)
Remarks:
air
Dose / conc.:
200 ppm (nominal)
Remarks:
187-243 ppm
Dose / conc.:
1 000 ppm (nominal)
Remarks:
966-1082 ppm
Dose / conc.:
5 000 ppm (nominal)
Remarks:
4966-5171 ppm
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
Blood sampling:
N/A
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.
Historical control data:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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 consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
N/A
Gross pathological findings:
not specified
Description (incidence and severity):
N/A
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on results:
N/A
Number of abortions:
no effects observed
Description (incidence and severity):
There were no ethylene-induced effects on fertility or fecundity. All females became pregnant.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Total litter losses by resorption:
not specified
Description (incidence and severity):
N/A
Early or late resorptions:
not specified
Description (incidence and severity):
N/A
Dead fetuses:
not specified
Description (incidence and severity):
N/A
Changes in pregnancy duration:
not specified
Description (incidence and severity):
N/A
Changes in number of pregnant:
not specified
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other:
Remarks:
5737 mg/m3 air
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of pups alive on days 1 and 4 in all treatment groups was no different from control
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The % of male pups (sex ratio) in all treatment groups on day 1 and 4 was similar to control.
Changes in litter size and weights:
not specified
Description (incidence and severity):
N/A
Anogenital distance of all rodent fetuses:
not specified
Description (incidence and severity):
N/A
Changes in postnatal survival:
not specified
Description (incidence and severity):
N/A
External malformations:
not specified
Description (incidence and severity):
N/A
Skeletal malformations:
not specified
Description (incidence and severity):
N/A
Visceral malformations:
not specified
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Developmental Toxicity
Remarks on result:
other:
Remarks:
5737 mg/m3 air
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:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Human data

There are no available data concerning the effects of ethylene on reproduction in humans.

Non-human data

Limited developmental toxicity studies on ethylene are available other than an OECD guideline 421 reproduction/developmental toxicity screening study (Corning Hazleton, 1997). No adverse effects of ethylene were seen in this study. Head-only exposure to ethylene gas at target concentrations of up to 5737 mg/m3 did not affect pregnancy, or the number, growth or survival of the offspring to postnatal day 4.

In addition, data on propene provide sufficient additional information for evaluation. These data are summarised below, and justification to support the use of these data for the purposes of read-across is attached to the end of this CSR and appended to Section 13 of the IUCLID dossier.

A prenatal developmental toxicity study is proposed for ethylene in a second species. Combined with the OECD 414 in rats using propene, this will form part of a read-across approach to addressing this endpoint in both substances.

 

Information on alkene homologue – propene

The developmental toxicity of propene has been reported in a guideline (OECD 414) rat study (BASF, 2002).

Time-mated female Wistar rats were exposed to propene gas at 0, 200; 1,000 and 10,000 ppm (0, 340, 1,720 and 17,200 mg/m3) for 6 hours per day on day 6 through day 19 post coitum (14 exposures). On day 20 post coitum, all animals were sacrificed and assessed by gross pathology. For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead foetuses) were determined. The foetuses were removed, sexed, weighed and further investigated for any external findings. Thereafter, half of the foetuses of each litter were examined for soft tissue findings and the remaining foetuses for skeletal (including cartilage) findings.

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).

  

Summary & Conclusion

Only limited developmental toxicity studies on ethylene are available other than a reproduction/developmental toxicity screening test (Corning Hazleton, 1997); no adverse effects of ethylene were reported. Whilst this screening test is limited in detecting effects on all aspects of reproduction and development the negative result does provide some reassurance for lack of effect. The study is supported by a guideline prenatal developmental toxicity study on propene (BASF, 2002). No developmental effects were induced in rats exposed to propene up the 10,000 ppm (17,200 mg/m3), half the lower explosive limit.

Further testing is required to address data requirements. An OECD 414 in rabbits is therefore proposed, with ethylene as the testing material.

 

Justification for classification or non-classification

There is adequate information available from which to assess the potential of ethylene to induce reproductive or developmental effects and to conclude that classification under CLP is not warranted.

Additional information