Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / 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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene
EC Number:
200-815-3
EC Name:
Ethylene
Cas Number:
74-85-1
Molecular formula:
C2H4
IUPAC Name:
ethylene
Details on test material:
- Name of test material (as cited in study report): Compressed ethylene (technical grade)
- Supplied by: Linde Gas UK Ltd., Stoke on Trent, UK.
- Analytical purity: 99.94%
- Lot/batch No.: 50ST6299541
- Storage condition of test material: Steel container, outside at ambient temperature

Test animals

Species:
rat
Strain:
other: Crl: CD BR
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

Administration / exposure

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.
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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 1000, 5000 ppm
Basis:
other: target concentration
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

Examinations

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.
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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
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.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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.
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.



Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
5 000 ppm
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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.