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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2003 - September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted under GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005
Reference Type:
publication
Title:
Developmental toxicity studies in Crl:CD (SD) rats following inhalation exposure to trichloroethylene and perchloroethylene.
Author:
Carney EW, Thorsrud BA, Dugard PH, Zablotny CL
Year:
2006
Bibliographic source:
Birth Defects Res B Dev Reprod Toxicol. 2006 Oct;77(5):405-12.

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): perchloroethylene (Tetrachloroethylene)- Analytical purity: > 99%- Source: obtained from INEOS Chlor Ltd, Cheshire, UK- Substance type: colourless liquid- Lot/batch No.: 204-825-9- Storage condition of test material: ambient temperature in the dark
Specific details on test material used for the study:
Supplied by INEOC Chlor Ltd. Purity > 99%.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River (UK) Ltd- Age at study initiation: 10-11 weeks- Weight at study initiation: 221-314 g- Housing: barriered rodent facility. Individually housed in solid bottom polypropylene cages fitted with wire mesh tops. Nesting material was provided in each cage. For the daily exposures, the animals were transferred to exposure chambers where they were housed in individual animal exposure cages of the suspended basket type, constructed of stainless steel mesh. - Diet: Standard rodent diet ad libitum. Each batch of diet was analysed routinely by the supplier. - Water: ad libitum- Acclimation period: 3-5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 19-23 (actual recorded 20-24°C)- Humidity (%):40-70 (actual recorded 31-57%)- Photoperiod (hrs dark / hrs light): 12 / 12Temperature and relative humidity were maintained within the designated laboratory specified ranges, except for slight, transient excursions. However, these deviations were not considered to have adversely affected the scientific integrity of the study.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Animals were whole body exposed in 0.75-cubic-meter exposure chambers. Chamber airflow was maintained at approximately 150 L/min. The control animals received air alone. The animals were exposed at approximately the same time each day and a separate exposure chamber was used for each group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of perchloroethylene were measured multiple times each exposure day using GC analysis.
Details on mating procedure:
Virgin female rats were mated overnight at the supplier with male rats (one female to one male). The day on which evidence of positive copulation was established was documented as GD 0.
Duration of treatment / exposure:
gestation day 6-19
Frequency of treatment:
6 hours/day, 7 days/week
Duration of test:
20 days
No. of animals per sex per dose:
22 females
Control animals:
yes
Details on study design:
Dose selection rationale: There have been previous developmental toxicity studies of rats exposed to perchloroethylene via the inhalation route. All studies referred to below involved whole body exposure which matches the design of the current study.Schwetz et al (1975) exposed Sprague-Dawley rats on gestation days 6 - 15 to a perchloroethylene dose level of 300 ppm. A slight bodyweight effect was evidence of maternal toxicity. Schwetz et al also reported a reduction in fetal weight and a possible slight increase in resorptions. No effects on the dam or offspring were reported by Beliles et al (1980) and Hardin et al (1981) in a study that employed Sprague-Dawley rats and exposure to 500 ppm from day 1 - 19 of gestation. In a study with a developmental neurotoxicity focus, Nelson et al (1980) observed frank maternal toxicity at 900 ppm and no effects at 100 ppm in Sprague-Dawley rats exposed for either gestation days 7 - 13 or 14 - 21. The results of a multigeneration study in AP, Wistar-derived, rats, and its preliminary studies, yield the following information for perchloroethylene exposure during pregnancy: In both preliminary studies (Tinston 1992 and undated) maternal effects were seen during a pregnancy-only exposure regime at a dose of 750 ppm in the form of reduced food consumption and bodyweight gain. In the first preliminary study (Tinston undated) possible small effects on food consumption and weight gain were seen at 500 ppm. In the main study, substantial effects were seen on litters at a dose of 1,000 ppm which diminished to a slight effect on fetal growth with no evidence of maternal toxicity at 300 ppm. The data indicated that the onset of maternal toxicity may be as low as a dose level of 300 ppm of perchloroethylene, although no effects were seen in one study that employed 500 ppm. Clear maternal toxicity was evident at 750 ppm and higher dose levels. The limit dose defined in the EPA guidelines for developmental toxicity studies by the inhalation route is an atmosphere of 2mg/l (equal to a level of 290 ppm) perchloroethylene. This limit dose could have been selected for this study, but it was decided that the evidence showed that pregnant rats could tolerate a higher level. Accordingly, a dose level of 600 ppm was selected as the top dose since it is likely to cause detectable maternal toxicity. The mid (250 ppm) and low (75 ppm) doses were then selected to give an appropriate spread of dose levels and a probable overall no observed effect level at the lowest dose.Analytical concentration: The analysed mean chamber concentrations for Group 3 (249 ppm) and Group 4 (600 ppm) were in good agreement with target values 250 and 600 ppm respectively. The analysed mean chamber concentration for Group 2 (65 ppm) was lower than target (75 ppm). On Day 1 a 10-fold error led to the Group 2 concentration being only 9.6 ppm, resulting in a group mean concentration 13% of the target. This only affected the first batch of animals exposed. The overall study mean concentration for the other batches of animals exposed at the lowest concentration was similar but below target (approximately 10% lower). This was considered not to affect the integrity of the study.

Examinations

Maternal examinations:
Maternal necropsies were performed on GD 20. Dams were evaluated for clinical signs, body weight and feed consumption. On the scheduled day of euthanasia each surviving female underwent a gross necropsy.Clinical observationsAnimals were inspected visually at least once a day during the acclimatisation period and at least twice daily during the treatment period for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health of the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition. Throughout the treatment period, detailed observations were recorded daily at the following times in relation to exposure: Pre-exposure observation.During exposure: restricted to gross changes on a group basis, of animals that can be seen. Within one hour of return of animal to home cage. Body weightThe weight of each adult was recorded on arrival (Day 1 or 3 depending on batch) and on Days 3, 6, 9, 13, 17 and 20 after mating.Food consumptionThe weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 3-5, 6-8, 9-12, 13-16 and 17-19 after mating inclusive. NecropsyAnimals were killed on Day 20 after mating. Animals were killed by carbon dioxide asphyxiation. Fetuses were killed by chilling on a cold plate (approximately 0°C). The sequence in which the animals were killed after completion of the study was selected to allow satisfactory inter-group comparison.Macroscopic pathology All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral midline incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
On the scheduled day of euthanasia each surviving female underwent a gross necropsy. The gravid uterus was weighed prior to dissection; this weight included the weight of the ovaries. For each animal, the number of corpora lutea in each ovary and the number of implantation sites, the number and distribution of resorption sites (classified as early or late) and live and dead fetuses were recorded for each uterine horn. For apparently non-pregnant animals, the number of uterine implantation sites was checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, E, 1964)). The ovaries and pituitary for non-pregnant animals were retained in 10% neutral buffered formalin. Placenta weights were measured.
Fetal examinations:
All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded.Approximately half of the fetuses in each litter were subject to gross internal examination of the viscera of the neck, thorax and abdominal cavities. These fetuses were then eviscerated and their skeletons were fixed in Industrial Methylated Spirit, prior to processing and staining with Alizarin Red S and Alcian Blue. The remaining fetuses were fixed whole in Bouin’s fluid.Free-hand serial sections were prepared from the Bouin’s fixed fetuses and were examined under the microscope for visceral abnormalities. Fetuses stained with Alizarin Red S and Alcian Blue were assessed for skeletal and cartilaginous development and abnormalities. The criteria for evaluation of cartilaginous and osseous development are tabulated on an individual basis and as a group incidence of fetuses/litters affected, within the following categories:- skeletal and cartilage minor abnormalities- rib, vertebral and cartilage configuration- incomplete ossification - a lesser degree of ossification of individual bones in a fetus, seen as a reduction in Alizarin stained area or density within the outlines of a structure- precocious ossification - a greater degree of ossification than expected for Day 20.The fetuses were stained with Alcian blue for visualisation of cartilaginous structures and abnormalities recorded.
Statistics:
The litter was used as the statistical unit for analysis for litter/fetal data. Continuous data were tested for homogeneity of variance using Bartlett's test at alpha = 0.01 (Bartlett, 1937). The raw, log-transformed and square root-transformed data were tested. Based on the results of Bartlett's test, data were analyzed using either parametric or nonparametric tests. The study used the Williams test (Williams, 1972) or Shirley's test (Shirley, 1977) if the dose-response was monotonic. If not monotonic, the Dunnett's test or Steel's test were used. In the study, if 75% of the data (across all groups) were the same value, then a frequency analysis was performed. Treatment groups were compared using a Mantel test for a trend in proportions (Mantel, 1963) and also pairwise Fisher's Exact tests (Fisher, 1973) were used for each dose group against the control. Skeletal variants were analyzed by a generalized mixed linear model with a logit link function and used litter as a random effect (Lipsitz et aI., 1991). Each treated group was compared to the control group using a Wald chi-square test. In both studies, 0.05 was considered the nominal alpha level for statistical significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the course of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in mean gestation bodyweight for the test article-treated dams (groups 2-4) when compared with the Control group. Group mean bodyweight gain in the Intermediate and High dose females (249 and 600 ppm) was slightly and transiently lower than the concurrent control group during the first 3 days of exposure (Days 6 to 8 of gestation), with the difference from controls at 600 ppm being statistically significant. From Day 9 of gestation the pattern of bodyweight gain in these treated groups was similar to that of the concurrent control group. Overall bodyweight gain during days 6 to 20 of gestation was similar to controls. There were considered to be no treatment-related effects on bodyweight change amongst females receiving 65 ppm. Bodyweight change on Day 20, adjusted for gravid uterine weight, showed very little variation between the groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Amongst animals exposed to 600 ppm, group mean food consumption was marginally yet statistically significantly lower than the controls during the first 3 days of treatment. There were no treatment-related effects on group mean food consumption in animals exposed to 65 or 249 ppm throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic changes detected at post mortem examination of any female on Day 20 of pregnancy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was considered to be no treatment related effect in the group mean number of implantations. Although there were slight intergroup differences in the mean number of corpora lutea, these events were established before treatment commenced on Day 6 of gestation.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The resorption rate was low in all groups, such that there were no treatment-related effects on litter size or group mean sex ratio.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
All rats survived to scheduled maternal necropsy. The study revealed no clinical observations that could be attributed to treatment. Maternal responses to tetrachloroethylene were limited to slight, but statistically significant reductions in body weight gain and feed consumption during the first 3 days of exposure to 600 ppm, resulting in a maternal NOEC of 250 ppm. A total of 2, 0, 5 and 1 rats were found to be non-pregnant in the control, 65-, 250-, and 600-ppm groups, respectively. No treatment related effects were noted in the mean number of implantations. Although there were slight intergroup differences in the mean number of corpora lutea, these events were established before treatment commenced on gestation day 6 and do not reflect a treatment related effect.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEC
Effect level:
250 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEC
Effect level:
250 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight but statistically identified decrease in mean gravid uterine weight (9.4%) was observed in females exposed to 600 ppm. This correlated with a slight decrease in mean litter weight and statistically significant decreases in mean fetal bodyweight (3.39g) and placental weight (0.48g) at this dose level. The mean fetal and placental weights were found to be outside the historical control range of 3.56 – 3.96 and 0.54 – 0.62g respectively. These observations indicate a possible relationship to treatment at the 600 ppm exposure level. Although decreases in mean fetal bodyweight and placental weight were reported at a dose level of 249 ppm, they were not considered to be toxicologically meaningful because the mean fetal weight was within the historical range and the litter and gravid uterine weights were comparable to those in the control group. Fetal, placental and litter weights at 65 ppm were similar to control values.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean fetal and placental weights were found to be outside the historical control ranges of 3.56-3.96 and 0.54-0.62 g, respectively. Therefore, the changes at 600 ppm were interpreted to be treatment related. Decreases in mean fetal body weight (4.3%) and placental weight (12.3%) were statistically identified at a dose level of 250 ppm. While the non statistically significant changes at 250 ppm were judged to be treatment related, they were considered of minimal toxicological significance based on the fact that mean fetal weight was within the historical control range, and there was no apparent impact on gravid uterine weight. Fetal, placental, and litter weights at 65 ppm were similar to control values.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major abnormalities was low and showed no relationship to treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of malformations was low and showed no relationship to treatment with PERC, nor were there any treatment related effects on the incidence cartilage variations. Among animals exposed to 600 ppm, there was a marginal, non-statistically significant increase in the incidence of incomplete ossification of thoracic vertebral centra (bipartite ossified/asymmetrically ossified/dumb bell ossified), which was outside the historical control range, appeared to correlate with reduced fetal body weights, and was therefore considered to be a treatment related effect. The incidence of incomplete ossification of the 5th and/or 6th sternebrae in the high-dose group (54% of fetuses, 95% affected litters) was slightly higher than that of the control group (44% of fetuses, 85% affected litters). However, the incidence of this variation was well within the historical control range (48-77% of fetuses) and, thus, not considered treatment-related. In all treatment groups, there was a higher incidence of fetuses with unossified hyoid, in comparison with the control. However, in the absence of any dose-response relationship, this finding was considered coincidental and unrelated to treatment. There were no other noteworthy skeletal variations.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects in the incidence of minor visceral abnormalities.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
The resorption rate was low in all groups, such that there were no treatment-related effects on litter size or group mean sex ratio. A slight, but statistically significant, decrease in mean gravid uterine weight (9.4%) was observed in females exposed to 600 ppm. This correlated with statistically significant reductions in mean fetal body weight (9.4%) and placental weight (15.8%). The mean fetal and placental weights were found to be outside the historical control ranges of 3.56-3.96 and 0.54-0.62 g, respectively. Therefore, the changes at 600 ppm were interpreted to be treatment related.Decreases in mean fetal body weight (4.3%) and placental weight (12.3%) were statistically identified at a dose level of 250 ppm. While the non statistically significant changes at 250 ppm were judged to be treatment related, they were considered of minimal toxicological significance based on the fact that mean fetal weight was within the historical control range, and there was no apparent impact on gravid uterine weight. Fetal, placental, and litter weights at 65 ppm were similar to control values.The incidence of malformations was low and showed no relationship to treatment with PERC, nor were there any treatment related effects on the incidence of visceral and cartilage variations. Among animals exposed to 600 ppm, there was a marginal, non-statistically significant increase in the incidence of incomplete ossification of thoracic vertebral centra (bipartite ossified/asymmetrically ossified/dumb bell ossified), which was outside the historical control range, appeared to correlate with reduced fetal body weights, and was therefore considered to be a treatment related effect. The incidence of incomplete ossification of the 5th and/or 6th sternebrae in the high-dose group (54% of fetuses, 95% affected litters) was slightly higher than that of the control group (44% of fetuses, 85% affected litters). However, the incidence of this variation was well within the historical control range (48-77% of fetuses) and, thus, not considered treatment-related. In all treatment groups, there was a higher incidence of fetuses with unossified hyoid, in comparison with the control. However, in the absence of any dose-response relationship, this finding was considered coincidental and unrelated to treatment. There were no other noteworthy skeletal variations.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Maternal and fetal toxicities were assessed following the inhalation administration of perchloroethylene to pregnant rats on days 6 – 19 after mating. Exposures to atmospheres of 0, 65, 249 and 600 ppm were for 6 hours per day. Based on a small, transient adaptive effect on maternal bodyweight gain and food consumption, the NOAEL for maternal toxicity is considered to be 600 ppm. Fetal effects seen at the dose level of 600 ppm were decreased mean gravid uterine, litter, fetal, and placental weights and an associated reduction in ossification of thoracic vertebral centra (classified as a variation). No toxicologically meaningful effects were seen at a dose level of 249 ppm which is therefore the NOAEL for fetal toxicity. No compound-related effects of any kind were observed at 65 ppm, and no teratogenic or other severe effects were apparent at dose levels up to and including 600 ppm.
Executive summary:

The effect of perchloroethylene on embryo-fetal survival and development in Crl:CD® (SD) IGS BR rats was assessed following inhalation administration during the organogenesis phase of gestation. Three groups of 22 female rats were exposed to perchloroethylene for 6 hours a day by whole body exposure at target concentrations of 75, 250 or 600 ppm from Days 6 to 19 after mating. A similarly constituted control group was exposed to air alone throughout the same period. Animals were killed on day 20 after mating for reproductive assessment and fetal examination. Based on a small, transient effect on maternal bodyweight gain and food consumption, the NOAEL for maternal toxicity is considered to be 600 ppm. Fetal effects seen a the dose level of 600 ppm were decreased mean gravid uterine, litter, fetal and placental weights and an associated reduction in ossification of thoracic vertebral centra (classed as a variant). No toxicologically meaningful effects were seen at a dose level of 249 ppm which is therefore the NOAEL for fetal toxicity. No compound-related effects of any kind were observed at 65 ppm, and no teratogenic or other severe effects were apparent at dose levels up to and including 600 ppm