trans-dichloroethylene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: U. S. Environmental Protection Agency. Toxic Substances Control Act Test Guidelines: Final Rules. Federal Register 50: 39426-39428 and 39433-39434 (1985).

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

trans-dichloroethylene

EC Number:

205-860-2

EC Name:

trans-dichloroethylene

Cas Number:

156-60-5

Molecular formula:

C2H2Cl2

IUPAC Name:

(1E)-1,2-dichloroethene

Details on test material:

- Purity: 99.64%

Test animals

Species:

rat

Strain:

other: Crl:CD®BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Female - 63 days old; Male - 84 days old
- Weight at study initiation: Female -175.6 to 220.5 g; Male - 307.9 to 376.5 g
- Housing: individually housed in suspended, stainless steel, wire-mesh cages
- Diet: Purina® Certified Rodent Chow® #5002, ad libitum
- Water: Wilmington Suburban Water Corporation, ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): targeted between 21 and 25°C
- Humidity (%): between 40 and 60%
- Photoperiod: 12 hrs dark /12 hrs light

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

other: conditioned house air.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 150 liter stainless steel and glass exposure chambers
- Method of holding animals in test chamber: Rats were individually housed during exposures in stainless- steel cage modules that were placed within the chamber
- Source and rate of air: Filtered, conditioned dilution air
- System of generating vapour: Test atmospheres were generated by vapourization. The liquid test material was placed in gas washing bottles within water baths. The water baths were regulated to a temperature of 22-26°C to facilitate evaporation of the test material. it was vapourized into a nitrogen stream, and swept into 3-neck, glass mixing flasks. Filtered, conditioned dilution air was added to the mixing flasks at approximately 30 L/min to sweep the vapours into the 150 liter stainless steel and glass exposure chambers.
- Temperature, humidity, pressure in air chamber: temperatures, based on 2 to 3 daily measurements per chamber, ranged from 22-30, 23-33, 23-31, and 22-34°C for the control, low, intermediate, and high concentration, respectively; mean percent relative humidity, based on a single measurement per chamber, was 53±9, 52±7, 52±7, and 55±11 for the control, low, intermediate, and high concentration,-respectively; pressure not reported
- Air flow rate: approximately 30 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of test substance in each test chamber was determined at approximately 30-minute intervals by gas chromatography. Gas samples were obtained from within each test chamber through Teflon® sample lines. Gas samples were analysed with a Hewlett Packard Model 5890A gas chromatograph equipped with a gas sample loop, a Hewlett Packard Model 3393A integrator and a flame ionization detector. Replicate gas samples were analysed at an oven temperature of 40°C on a 5 m x 0.53 mm fused silica megabore column coated with HP-1 (methyl silicone gum) to a film thickness of 2.65 µm. Chamber concentrations were determined by comparing the detector response of chamber samples with test substance standards. Standards were prepared prior to each exposure by diluting known amounts of liquid test substance (99.64% pure) in known volumes of air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method of Analysis: gas chromatograph, see above

The mean daily chamber concentrations were within ± 5% of the desired concentrations throughout the test period.

Details on mating procedure:

- Impregnation procedure: Females were cohabited with males (1:1) until copulation was confirmed by the presence of a copulation plug in the vagina or on the cageboard. Checks for copulation plugs were made each morning; the day copulation was confirmed was designated as Day 1 of gestation (Day 1G).

Duration of treatment / exposure:

Gestation Days 7 to 16

Frequency of treatment:

6 hours daily

Duration of test:

16 days

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A pilot study was conducted to determine the "maximum tolerated dose" in pregnant rats exposed for six hours daily on days 7-16 of gestation. Exposure levels for the pilot were 0, 6000, 9000 and 12000 ppm and were based on limited exposure data for nonpregnant rats at 5000 and 10000 ppm. At the highest exposure level, animals were narcotized but recovered rapidly when compound administration ceased; some tolerance to the compound was evident as exposures progressed. Some animals, of those that could be observed during exposure, showed signs of incoordination at 6000 and 9000 ppm. Statistically significant weight losses occurred during the first two days of exposures at the two highest levels and feed consumption was depressed. All animals exposed to the test substance showed clinical signs of ocular irritation, not unexpected given the irritating nature of the compound. No effects on pregnancy or on in utero survival were seen, but fetal weights were depressed at the highest level. No external alterations were detected. Based on these data, the high and intermediate dose levels were selected. A level ten times the threshold limit value was chosen for the low exposure group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded on the day after arrival and before mating; observations for morbidity and mortality were made daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: individual clinical signs were recorded each morning on Days 1-22G and each afternoon on Days 7-16G (the dosing period)

BODY WEIGHT: Yes
- Time schedule for examinations: Females selected for the study were weighed on Days 1, 7-17 and 22G.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 22
- Organs examined: liver, gravid uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Live fetuses were weighed, sexed and examined for external alterations. For each litter, the maximum stunted weight (MSW) was calculated by subtracting the lightest weight from the total weight, dividing by the remaining number of fetuses and multiplying by 0.666. A fetus weighing the same or less than the MSW was considered stunted; its weight was omitted when the mean litter weight was calculated. The first live fetus and thereafter every other fetus in each litter were decapitated and examined for visceral alterations and the sex verified. The heads were fixed in Bouin's fluid and examined. All stunted and externally malformed fetuses were also examined for visceral alterations; a decision to do a head examination was made on an individual basis.

The remaining fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. All fetuses were fixed in 70% ethanol, eviscerated (if not done earlier during visceral examination), macerated in 1% aqueous potassium hydroxide solution, stained with alizarin red and examined for skeletal alterations.

Statistics:

See Statistics Table below

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was evidenced by significant effects on body weight and feed consumption at 12000 ppm and by a significant decrease in feed consumption at 6000 and 2000 ppm. The decrease in feed consumption at 6000 ppm only translated into an observed effect on body weight change on gestation days 11-13. At 2000 ppm, the effect on feed consumption, seen only on Days 13-15G, was minimal and not accompanied by a significant body weight change. Although a significant trend was noted in the incidence of females with clinical findings on days 7-16G, this was the result of ocular irritation in most of the animals. This finding was not unexpected in view of the irritating nature of the compound and the route of exposure. The significant trend and increase at 6000 and 12000 ppm for total and early resorptions did not appear to be biologically significant. The significance stems from the relatively low control value (0.3 mean resorptions per litter). The values observed in the low, intermediate and high concentration groups are all within the limits of the historical control data from studies conducted within the past two years at the testing facility (i.e., 1.0, 0.8, 1.5, 0.6, and 0.9 mean resorptions per litter).

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

2 000 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Significant embryo-fetal toxicity was evident among fetuses from dams which were exposed to 12000 ppm. The toxicity was expressed as a decrease in the mean weight of female fetuses. Although a slight increase in hydrocephaly occurred in fetuses in the high exposure group the increase was not significant and occurred at a maternally toxic level. A significant increase at all three concentrations was seen in the mean percent of fetuses affected per litter with developmental variations. This was due to an increase in the number with visceral variations at the low and intermediate dose, as well as a significant increase in skeletal variations in the high concentration group. In both cases the results do not show a dose-response relationship and therefore, are not believed to be compound-related. This conclusion is supported by the fact that the overall percent of fetuses per litter with variations was not significant and variations due to retarded development, usually a more sensitive indicator of fetal toxicity, were reduced in the experimental groups.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

6 000 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

REPRODUCTIVE OUTCOME		ppm
		0	2000	6000	12000
No. Copulated		24	24	24	24
No. Pregnant		22	24	24	24
No. Pregnant		0	0	0	0
No. With Total Resorptions		0	0	0	0
No. of Litters		22	24	24	23 a
Means Per Litter Live Fetuses:	Total	15.3	15.0	15.3	14.3
	Males	7.1	7.0	7.3	6.3
	Females	8.2	8.0	8.0	8.1
Dead Fetuses		0.0	0.0	0.0	0.0
Resorptions:	Total †	0.3	0.6	0.8 *	1.1 *
	Early †	0.3	0.6	0.8 *	1.1 *
	Late	0.0	0.0	0.0	0.0
Nidations		15.6	15.6.	16.1	15.5
Mean Corpora Lutea		16.7	16.6	16.8	16.3
Mean Fetal Weight b	Total †	4.97	5.13	5.01	4.76 *
	Males	5.05	5.27	5.19	4.96
	Females†	4.88	5.00	4.86	4.59 *
No. of Stunted Fetuses		0	0	1	0
a One animal delivered on study day 18 with term fetuses; removed from study.
b Grams. Weights of stunted fetuses were excluded from calculations of means.
† Significant trend detected (Jonckheere's test).
* Significantly different from controls (Mann-Whitney U test);  p < 0.05

Applicant's summary and conclusion

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
NOAEC Maternal toxicity = 2000 ppm
NOAEC Embryo-fetal toxicity = 6000 ppm

Executive summary:

The test item was administered to rats by inhalation six hours daily on Days 7-16 of gestation at exposure levels of 0, 2000, 6000, or 12000 ppm.

 

Overt maternal toxicity was expressed at levels of 6000 ppm and above. During the first two days of dosing, the group receiving 6000 ppm showed a slight weight gain suppression and the group receiving 12000 ppm lost weight. For the entire dosing period, a significantly reduced weight gain was only seen at the 12000 ppm level. Feed consumption, on the other hand, was significantly reduced at 6000 and 12000 ppm for the entire dosing period. At 2000 ppm marginal maternal toxicity was evident as a significant decrease in feed consumption only during days 13-15 of gestation. This change, however, was not accompanied by any significant changes in body weight. No significant differences in body weight change or feed consumption were noted for the pre-dosing or post-dosing periods. Except for ocular irritation in animals at all exposure levels, the only compound-related clinical or postmortem findings were an increase in alopecia, salivation and lethargy during the exposure period at 12000 ppm. The ocular effects, as well as the alopecia and salivation were not unexpected in view of the irritating nature of the compound and the route of exposure. In addition, lethargy was not surprising given the test items narcotic effect which was observed during the exposure periods at 12000 ppm.

 

A significant increase in the mean number of resorptions per litter was seen in females at the 6000 and 12000 ppm levels. However, these values are within the historical ranges for studies conducted at the test facility over the past year. No differences were seen in pregnancy rate, fetuses per litter, number of stunted fetuses or in corpora lutea counts.

 

Embryo-fetal toxicity was expressed as reductions in female and combined fetal weights at 12000 ppm. A significant increase at all three concentrations, accompanied by a significant trend, was seen in the mean percent of fetuses affected per litter with developmental variations. This was due to an increase in the number with visceral variations at the low and intermediate concentrations, as well as a significant increase in skeletal variations in the high concentration group. These increases are not believed to be compound related since there was neither a dose-response relationship observed in the visceral or skeletal variations per litter nor in the overall percent of fetuses per litter with developmental variations. The mean percent of fetuses per litter with variations due to retarded development was decreased at all concentrations compared to controls and the overall percent of fetuses per litter with variations was not different from controls. In addition, no significant differences were detected for the mean percent of fetuses per litter with malformations.

 

In this study, overt maternal and fetal toxicity were demonstrated at concentration levels of 6000 ppm and above and 12000 ppm, respectively. At 2000 ppm marginal maternal toxicity was evident as a decrease in feed consumption only during days 13-15 of gestation. Therefore, the no-observable adverse- effect level (NOAEC) approached 2000 ppm for the dam and was 6000 ppm for the conceptus.