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Effects on fertility

Description of key information

The effects of tetrachloroethylene on fertility and general reproductive performance have been thoroughly investigated in a well-conducted two-generation study in rats exposed to 0, 100, 300 and 1000 ppm (0, 690, 2070 and 6900 mg/m3). Observed effects on reproduction (reductions in litter size and pup survival at 1000 ppm, and pup body weight at 1000 and 300 ppm) are considered likely to be the non-specific consequences of maternal toxicity. No adverse effects on fertility or mating parameters were apparent, even at 1000 ppm. 

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1992 - June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
The study design was based on the EPA TSCA guidelines for a two-generation study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Sub-strain: Alpk:APfSD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Specific Pathogen Free (SPF) colony maintained at the Barriered Animal Breeding Unit (BABU) at Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK. - Age at study initiation: 21-22 days old- Housing: stainless steel long-term exposure chambers of approximately 3.4m3 capacity. Rats were housed by sex in litters on arrival and the cages were fitted with solid cage floors until randomisation. After randomisation (F0 generation) or selection (F1 generation) the animals were housed two per cage by sex. During the pairing period one male was housed with one female.- Diet: ad libitum, (CT1 diet supplied by Special Diets Services Limited), except during exposure when all food and water were removed - Water: ad libitum, except during exposure when all food and water were removed- Acclimation period: approximately one week ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24- Humidity (%): 40-60- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Atmospheres were generated by evaporating liquid tetrachloroethylene in a heat exchanger warmed to approximately 60 degrees Celsius. The tetrachloroethylene was metered into the haet exchanger using a peristaltic pump. Clean dry air was passed through the generation equipment and the vapour/air mixture was then passed into the exposure chamber. The air flow through each chamber was approximately 700 l/min.
Details on mating procedure:
BREEDING PROGRAMME:In each generation, the females were mated with males of the same group.After the pre-mating period, one F0 female was continuously housed with one male from the same group i.e. from the adjacent cage on the same level in the chamber for a maximum pairing period of 21 days. Initial pairing was done in the afternoon. Vaginal smears were taken each morning and examined to determine when mating had occurred. The presence of blood in a vaginal smear, bodyweight gain and abdominal enlargement were also used as evidence of mating, if necessary and the duration of pregnancy estimated where possible. A male showing evidence of mating with a female was separated from the female immediately and individually housed in a separate chamber. The females remained in the same position within the chamber until day 20 of gestation.Any female which failed to show positive indication of mating after a 21 day mating period, together with any female which did not show the expected weight gain to day 15 of gestation was remated where necessary with a different male from the same treatment group after a rest period of at least three days (so that the paternity of any ensuing litter could be unequivocally determined). The males used in remating were ones which had shown positive indication of mating with at least one female.Those females which appeared to be pregnant but failed to litter were killed on day 25 (approximately) of supposed gestation and the uterus examined for the presence of implantation sites.All F1A litters were weaned at day 29 post partum and from the F1 litters a further 24 males and 24 females per group were randomly selected to become the parents for the next generation (litters derived from rematings were included in this process). The parentage of the selected animals was recorded. These animals were maintained for a pre-mating period of at least 11 weeks prior to a 21 day mating period for the F2A litter.The breeding programme was continued until the F2A litters had been weaned. Mating for the F2B and F2C litters was as described for the parental animals except that the maximum mating period was 14 days for the F2B litter and 10 days for the F2C litter. During the course of the study brother/sister matings were avoided for all litters.Three different types of F2 litters were produced. For the F2A litters, the dams and pups were treated in a similar manner to the F1 litters (i.e. exposed during lactation from day 7 to day 29 post-partum), except that the dams and the offsprings in the 1000 ppm group were not exposed during lactation. This change was the result of effects (sedation of the dams with consequent neglect of their litters) apparent at 1000 ppm in the first generation. The F2B litters were derived from matings that followed at least 2 weeks daily exposure to tetrachloroethylene (0, 300 or 1000 ppm only). Exposure of the dams then continued on days 1 to 20 of gestation, but there was no exposure of the dams and their litters during lactation. Finally, the F2C litters were produced by mating males in the control and 1000 ppm groups with unexposed females.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The atmospheres were sampled using an automatic air sampling system and analysed automatically using a gas chromatograph equipped with a gas sampling valve and flame ionisation detector. Each test atmosphere (including control and room air) was analysed at least every two hours during each exposure period, with a small number of exceptions. The analysis system was calibrated using an appropriate range of freshly prepared standards prior to the study, daily during the first week of exposures and at regular intervals thereafter.
Duration of treatment / exposure:
11 weeks before mating; daily during the mating period of up to 21 days and continued at 7 days/week until sacrifice for the males and until day 20 of gestation for the females. The dams and the first generation (F1) litters were then removed from exposure until day 6 after birth. Exposure recommenced on day 7 after birth until selection of the weanlings to be second generation (F2) parents on about day 29 after their birth. The selected weanlings were then exposed at least a further 11 weeks before mating.
Frequency of treatment:
6 hours/day, 5 days/week for 11 weeks before mating; daily during the mating period of up to 21 days and continued at 7 days/week until sacrifice for the males and until day 20 of gestation for the females.The dams and the first generation (F1) litters were then removed from exposure until day 6 after birth. When exposure recommenced on day 7 after birth, the animals (the dams with their litters) were exposed 7 days/week until selection of the weanlings to be second generation (F2) parents on about day 29 after their birth. The selected weanlings were then exposed 5 days/week for at least a further 11 weeks before mating.
Details on study schedule:
- F1 parental animals not mated until 11 weeks after selected from the F1 litters.- Selection of parents from F1 generation when pups were 29 days of age.
Dose / conc.:
690 mg/m³ air (nominal)
Dose / conc.:
2 070 mg/m³ air (nominal)
Dose / conc.:
6 900 mg/m³ air (nominal)
No. of animals per sex per dose:
Groups of 24 male and 24 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
Atmosphere analytical results (presented overall results, analysed in two chambers for target concentration 100, 300, and 1000 ppm):Generation 1 (mean and sd): 104 +/-5 and 102 +/-5298 +/-12 and 303 +/-14991 +/-51 and 1005 +/-51Generation 2 (mean and sd): 100 +/-5 and 100 +/-5303 +/-12 and 300 +/-141002 +/-40 and 1000 +/-33
Positive control:
not applicable
Parental animals: Observations and examinations:
The investigations carried out on parents included clinical observations, body weight, food consumption, fertility (indicated by the success of the matings), length of gestation, precoital interval, organ (testes, liver and kidney) weights, necropsy and histopathology of certain tissues. CAGE SIDE OBSERVATIONS: Yes Prior to the start of the study and daily observation during the study. DETAILED CLINICAL OBSERVATIONS: Yes once weekly during the pre-mating periods a detailed examination of each rat was made. Subsequent clinical observations were recorded for those weeks when the animals were weighed, the occasions depending on the stage of gestation and lactation. In addition, animals were observed at regular intervals during each exposure. Any abnormalities or the observation of no abnormality detected were recorded. Any rats requiring euthanasia were killed and subjected to a post mortem examination. Any rats found dead were subjected to a post mortem examination as soon as possible after death.BODY WEIGHT: YesThe bodyweights of all rats were recorded at weekly intervals throughout the pre-mating periods. The initial weights for the F0 parents were recorded immediately before the first exposure and the initial weights for the F1 parents were recorded at selection.After the pre-mating period the males were weighed approximately every four weeks until they were killed prior to post mortem examination. All females were weighed on the first day of the mating period. Subsequently, the females were weighed on presumed days 1, 8, 15 and 22 of gestation (day 1 being the day on which a sperm-positive vaginal smear was seen) and on days 1, 5, 11, 16, 22 and 29 post partum (F1A and F2A litters) or days 1 and 5 post partum (F2B litter). Dams were not weighed during lactation for the F2C litter. If there was no evidence of successful mating they were weighed at weekly intervals during the mating period as an aid to detection of pregnancy. All rats were weighed at termination.FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption for each cage of rats was recorded throughout the pre-mating periods and calculated on a weekly basis. Food consumption was recorded for F1 rats from the time the first animal selected was placed in its cage.REPRODUCTIVE PERFORMANCE- Fertility: established by the success of each mating. The criterion for a successful mating was the production of a viable litter ie a litter in which at least one pup was found alive at day 1.- Lenqth of gestation: measured in days from the date of the positive smear to date of birth (but only in females fulfilling the criterion above ie production of a viable litter).- Pre-coital interval: days between the date of pairing and the date of the positive smear was measured.
Oestrous cyclicity (parental animals):
not investigated
Sperm parameters (parental animals):
not investigated
Litter observations:
PARAMETERS EXAMINEDInvestigations of the offspring included clinical condition, percentage live births, survival rates, body weights and (for selected pups only) organ weights, macroscopic and histological examinations.A count of all live and dead pups was made within 24 hours of parturition (day 1) and thereafter at days 5, 11, 16, 22 and 29 post partum for the F1A and F2A litters, and day 5 post partum for the F28 and F2C litters. The sexes of the pups were also recorded at these times. Any clinical abnormalities seen in the pups were recorded. Individual pup bodyweights were recorded within 24 hours of birth (day 1) and at days 5, 11, 15, 22 and 29 post partum for the F1A and F2A litters, and day 5 post partum for the F2B and F2C litters. Since pups were not individually identified, data were recorded by sex and litter.The pups selected to be the parents of the next generation were weighed on day 29 post partum to give the initial F1 parent bodyweight values. Subsequently they were weighed at weekly intervals for the duration of the pre-mating period.GROSS EXAMINATION OF DEAD PUPS:Litters were examined for dead or moribund pups at least once daily and any such pups were subjected to a macroscopic post mortem examination. For pups killed or found dead up to and including 18 days of age, abnormalities were recorded and the pups were discarded. Pups over 18 days of age were subjected to a full post mortem examination.
Postmortem examinations (parental animals):
All rats surviving to scheduled termination and those requiring euthanasia for humane reasons were killed by exsanguination. These, and any rat found dead, were necropsied and the following tissues removed and submitted for possible histological examination:Cervix, epididymis, kidney, liver, mammary gland (adult females only}, ovary, pituitary gland, prostate gland, seminal vesicle including coagulating gland, testis, uterus, vagina and macroscopically abnormal tissues.Initially, histological examination was limited to kidney and liver from the controls and 1000ppm groups, cervix, epididymis, mammary gland, ovary,prostate gland, seminal vesicle, testis, uterus and vagina from suspected infertile animals in all groups. Subsequently, kidney from the F0 males and females in the 100 and 300ppm groups, kidney from the F1 males and females in the 300ppm group and liver from the F1 males in the 300ppm group were also examined histologically. In addition, testis from the fertile F1 males in the control and 1000ppm groups were also examined histologically and the testis from the infertile F1 males were re-examined at this time to ensure consistency. All remaining tissues were stored.Testes (left and right separately), kidneys (left and right separately) and liver were weighed from the adult animals terminated as scheduled.
Postmortem examinations (offspring):
All F1A and F2A pups surviving to scheduled termination (except those F1A pups selected to be F1 parents) were killed by the same method as used for the adults on approximately day 29 post partum. Approximately five males and five females per group from the F1A litters and approximately ten males and ten females per group from the F2A litters were selected randomly for a full post mortem examination in which the tissues from the same list as for the parents, but excluding mammary gland, were taken. The pups were selected from those with no clinical abnormalities with the proviso that no more than one pup of each sex from a litter received a full post mortem examination. After selection of pups for full post mortem examination, any pups showing clinical abnormalities were subjected to a macroscopic post mortem examination in which abnormalities only were taken. In addition, two male and two female clinically normal pups were selected at random from each litter, where possible, and given a macroscopic post mortem examination.Testes (left and right separately), kidneys (left and right separately) and liver were weighed from the pups subjected to a full post mortem examination. In addition, these organs were also weighed from approximately 5 F1A pups per sex per group selected at random from the pups given a macroscopic post mortem examination, so that organs from approximately 10 male and 10 female pups per group from each generation were weighed.
Statistics:
- Analysis of (co)variance: Bodyweights, food consumption, parental organ weights, initial (day 1) pregnancy and lactation bodyweights, litter size, mean gestation length, mean pre-coital interval, initial (day 1) mean pup weight and total litter weight, mean pup organ weights, final mean pup bodyweight. - Fisher's Exact test: proportion of fertile animals, the proportion of whole litter losses, the proportion of litters with gestation length <22, 22 and >22 days, and the proportion of litters with pre-coital interval 1, 2, 3, 4 and >4. The proportion of pups born live, the proportion of pups surviving, the proportion of litters with all pups born live and the proportion of litters with all pups surviving. - Analysis of variance following the double arcsine transformation of Freeman and Tukey (1950): Percentages live born pups and pup survival.
Reproductive indices:
The fertility of each male and female was established by the success of each mating. The criterion for a successful mating was the production of a viable litter i.e. a litter in which at least one pup was found alive at day 1. The method of calculation is shown in Appendix H of the report. There were no effects on the lenght of gestation or male and female fertility in the parental animals.
Offspring viability indices:
Litters were examined for dead or moribund pups at least once daily and any such pups were subjected to a macroscopic post mortem examination. For each litter, the percentage of pups live born and the percentage of pups suviving to day 5 were calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs consistent with depression of the central nervous system such as decreased activity and reduced response to sound were seen during exposure to 1000 ppm for the first 2 weeks of exposure. These signs were not present approximately 30 minutes after the end of each exposure.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was low and not related to exposure to tetrachloroethylene.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights in the 1000 ppm males were slightly lower than controls during the first few weeks of the pre-mating phase, but from week 6 onwards body weights were similar to controls. The largest diffference was seen in week 2 where the difference from controls was approximately 5% when adjusted for intiial weight. A slimilar pattern was seen in the 1000 ppm females, but the reduction was marginal. There no adverse effects on body weights at 100 or 300 ppm in either sex during the pre-mating phase.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 ppm, the food consumption was slightly lower than controls in both sexes during week 1 of the pre-mating phase.
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:
effects observed, treatment-related
Description (incidence and severity):
Testes, kidney and liver were weighed from adult animals. Kidney weighs (absolute and adjusted for body weight) were statiscallly significantly higher than controls for males in the 1000 ppm group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence of findings in the F0 adults at termination was low and unrelateted to treatment. No macroscopic findings to account for suspected fertility were detected in any animal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes related to treatment with tetrachloroethylene were observed in the kidney. In animals killed at terminated, there was a slightly increased incidence of mininmal chronic progressive glomeruloenphorpathy and an increased pleomorphism within proximal tubular nuclei in all males exposed to 1000 ppm. In females in the same group there was a slight decrease in incidence and a more pronounced decrease in severity of intratubular microlithiasis with increaed nuclear pleomorphism in 50% of the rat.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
The few deaths that occurred among parents during the study were not related to exposure to tetrachloroethylene. Clinical signs of toxicity to the parents, present in the 1000 ppm dose group, were consistent with depression of the CNS such as reduced activity and reduced response to sound. Other clinical signs of toxicity included breathing irregularities, salivation, hair loss, pale appearance, hunched posture, piloerection and tip-toe gait. Hair loss, pale appearance, hunched posture, piloerection and increased breathing rate were also observed at 300 ppm. The animals had recovered from these effects by 30 minutes after the end of the exposure and there were no clinical findings at 100 ppm. Reductions in parental body weight gains, apparent only at 1000 ppm, were generally small (up to 5% of the controls) and often transient (during the first few weeks of the pre-mating phase). Macroscopic findings in parents were generally unremarkable, although 3 F0 females (1 at 100 ppm, 2 at 1000 ppm) were removed from the study intercurrently due to dystocia. For the F0 parents, statistically significant changes in organ weights were largely confined to increased absolute and relative values for kidney (by 13%) and liver (by 9%) in the males exposed at 1000 ppm, with no treatment-related effect being found for testes. Treatment-related histopathological changes were confined to the kidneys of animals exposed to 1000 ppm tetrachloroethylene and were generally minor in nature (slightly increased incidence of minimal chronic progressive glomerulonephropathy and increased nuclear pleomorphism within the proximal tubules).There were no treatment-related effects of tetrachloroethylene on reproductive performance, as judged by male or female fertility, pre-coital interval or length of gestation. However, at 1000 ppm there were statistically significant decreases in the proportions of F1 pups born live (206/222, 91%) and litters with all pups born live (12/21) compared with the control group (224/226, 99% and 21/23 respectively).
Key result
Dose descriptor:
NOAEL
Remarks:
effects on fertility
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility and mating performance were apparent in this study, even not at 1000 ppm (6900 mg/m3) (highest dose tested)
Key result
Dose descriptor:
NOAEL
Remarks:
paternal toxicity
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs consistent with depression of the central nervous system such as decreased activity and reduced response to sound were seen during exposure to 1000 ppm for the first 2 weeks of exposure. These signs were not present approximately 30 minutes after the end of each exposure.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was low and not related to exposure to perchloroethylene.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Initial body weights of all groups in the F1 generation were lower than controls for both sexes, the reduction being most marked at 1000 ppm where the difference from controls was 26 and 24% for males and females respectively. There was evidence for some subsequent divergence from controls in the 1000 ppm males even after adjustment for initial body weight whilst growth of the 1000 ppm females was similar to controls. Subsequent growth in the 100 and 300 ppm male and female groups was similar to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the 1000 ppm group of the F1 generation was lower than controls in week 1 of the pre-mating phase in both sexes, but recovery to controls levels was evident by week 6 in males and week 3 in females. Subsequent food consumption was generally similar to controls in males and higher than controls in females. At 300 ppm slightly lower food consumption was seen duriing week 1 in females and weeks 1 and 2 in females, but subsequent values were similar or higher than controls. There were no adverse effects on food consumption in the 100 ppm group.
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:
effects observed, treatment-related
Description (incidence and severity):
Absolute kidney and liver weights for males in the 1000 ppm group were slightly higher than the controls although the differences were not statistically significant. When kidney and liver weights were adjusted for body weight the increases (14% for the liver and 11% for the kidney) were statiistically significant. There were no dose-related changes in males at 100 or at 300 pmm females at any exposure level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
In animals killed at termination, a mass was observed in the kidney of one male exposed to 300 ppmm and cysts were present in the kidney of one male exposed to 300 ppm and in two males exposed to 1000 ppm. The incidence of all other macroscopic findings was low and unrelated to treatment with perchloroethylene.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes similar to those seen in the F0 generation were observed in the F1 adults except that a slightly increased incidence of chronic progressive glomerulonephropathy was observed in females exposed to 1000 ppm and in most of the males in the control and 1000 ppm groups reflecting the greater age of the animals in the second generation. Increaed nuclear pleomorphism within proximal kidney tubules was confined to males at 1000 ppm in this generation. The mass observed in one male exposed to 300 ppm was a mesenchymal tumour (nephroblastoma). This was an isolated incidence which was considered to be unrelated to treatment. Minimal or slight focal unilateral tubular degeneration of the testis was observed in two control males and two males in the 1000 ppm group. This is a spontaneous lesion and unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
F1 parents: pre-coital interval, lenght of gestation and male and female fertility of the treated groups were similar to the controls for all litters.The incidence of pups born live and the incidence of litters wtih all pups born live in the 1000 ppm group was lower than controls for the A and B litters although this was only statistically significant for the incidence of pups born live. There were no statistically significant changes at 300 ppm for the B litter or at 1000 ppm for the C litter.
Clinical signs of toxicity to the parents, present in the 1000 ppm dose group, were consistent with depression of the CNS such as reduced activity and reduced response to sound. Other clinical signs of toxicity included breathing irregularities, salivation, hair loss, pale appearance, hunched posture, piloerection and tip-toe gait. Hair loss, pale appearance, hunched posture, piloerection and increased breathing rate were also observed at 300 ppm. The animals had recovered from these effects by 30 minutes after the end of the exposure and there were no clinical findings at 100 ppm. When exposure of the dams and litters in the 1000 ppm group was resumed on day 6 post partum in the F1, sedation of the dams with consequent neglect of their litters was evident. In the F1 generation, initial (at 29 days post-partum) body weights of all F1 parent groups exposed to tetrachloroethylene were statistically significantly lower than controls for both sexes, with reductions of 7-8%, 9-11% and 24-26% at 100, 300 and 1000 ppm respectively. Reductions in parental food consumption, apparent prevalently at 1000 ppm, were also generally small and transient (during week 1 of the pre-mating phase). For the F1 parents, relative (but not absolute) liver and kidney weights were increased in males at 1000 ppm (by 14% for the liver and by 11% for the kidney) and statistically significant decreases in absolute testes weight were found at 300 (by 6%) and 1000 ppm (by 16%). Macroscopic findings in parents were generally unremarkable, although 4 F1 females (2 at 300 ppm, 2 at 1000 ppm) were removed from the study intercurrently due to dystocia. Also, cysts were present in the kidney in 1 male exposed at 300 ppm and 2 males at 1000 ppm. Treatment-related histopathological changes were confined to the kidneys of animals exposed to 1000 ppm tetrachloroethylene and were generally minor in nature (slightly increased incidence of minimal chronic progressive glomerulonephropathy and increased nuclear pleomorphism within the proximal tubules).There were no treatment-related effects of tetrachloroethylene on reproductive performance, as judged by male or female fertility, pre-coital interval or length of gestation, for either of the parental generations. However, at 1000 ppm there were statistically significant decreases in the proportions of F2 pups born live of the A and B litters only. For F2A, the proportion of pups born live was 165/191, 86% (compared with 264/274, 96% for the controls) and for F2B, the values were 126/158, 80% and 220/233, 94% respectively. Additionally, total litter size at birth was also reduced (by about 30% compared with controls). The lack of any effect in the F2C litter (201/209 at 1000 ppm compared to 218/220 for the controls) indicated that these changes were unlikely to be mediated through the males.
Key result
Dose descriptor:
NOAEC
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Reduced pup survival at 1000 ppm, which may have been maternally mediated
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
On day 6 post partum (when the dams and their litters resumed exposure) and on each subsequent day of exposure the pups in the 1000 ppm group showed signs of sedation. When the pups were examined after exposure they also showed signs of hypothermia. There were no significant clinical findings in the 100 or 300 ppm groups.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
F1A litters: reduced pup survival was seen in the 1000 ppm group compared with controls and was statistically significant for days 5-22 post partum. This reduction was evident for both the incidence of pups surviving and for the incidence of litters with all pups surviving. There were no effects at 100 or 300 ppm.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup body weights in the 1000 ppm group were approximately 10% lower than controls at birth and, following re-exposure on day 6 post partum, there was a further divergence from controls such that day 29 boy weights were approximately 20% below controls after adjutment for initial body weight.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The reductions seen in kidneys, liver and testes were all reflections of differences in final body weight since there were no statistically signifcant changes after adjustment for body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence of all macroscopic findings in pups dying or killed intercurrently over 18 days of age and those killed at termination was low and unrelated to treatment with perchloroethylene.
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Statistically significant effects among F1 offspring during the lactation period were largely limited to the 1000 ppm group; pup survival was decreased (for the F1 litters survival was only about 50% compared with 90% among controls) and pup weights were reduced, by about 10% at birth and about 20% at weaning relative to the controls. On day 6 (when exposure to tetrachloroethylene was resumed) and subsequently, the pups in the 1000 ppm group showed signs of sedation, as well as increased incidence of small appearance associated with the poor survival and growth. At 300 ppm pup bodyweights were also reduced, but to a lesser extent (up to 11%) than observed at 1000 ppm, and only at weaning. There were no treatment-related, toxicologically significant effects on relative organ weight, macroscopic or microscopic appearance in the F1 offspring.
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 ppm
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Increased incidences of whole litter loss and statistically significant reductions in litter size were noted in the 1000 ppm group. For example, the mean F2A litter size was 8.7 (standard deviation 3.8) on day 1 compared with the control value of 11.6 (3.2). On day 5, the values were 5.5 (4.3) and 10.6 (3.7) respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no exposure of the 1000 ppm F2A and F2B litters during lactation. Pup weights were reduced by about 10% at birth and about 20% on day 5, but started to recover after day 5. On day 29 they were actually slightly higher than in the control group. Decreased (by 7-8%) pup weights were also occasionally (on days 5 and 11) found for the 300 ppm F2A females and for the 300 ppm F2B males. For the F2C litters, weights, litter sizes and total litter weights were similar for the 2 groups.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related, toxicologically significant effects on relative organ weights in the F2 offspring.
Description (incidence and severity):
There were no treatment-related, toxicologically significant effects on macroscopic appearance in the F2 offspring.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related, toxicologically significant effects on microscopic appearance in the F2 offspring.
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Effects on pup survival and body weights were found at 1000 ppm for the F2A and F2B as for the F1 litters. Increased incidences of whole litter loss and statistically significant reductions in litter size were also noted. For example, the mean F2A litter size was 8.7 (standard deviation 3.8) on day 1 compared with the control value of 11.6 (3.2). On day 5, the values were 5.5 (4.3) and 10.6 (3.7) respectively. Unlike with the F1 litters, however, there was no exposure of the 1000 ppm F2A and F2B litters during lactation and pup weights, which were reduced by about 10% at birth and about 20% on day 5, started to recover after day 5; on day 29 they were actually slightly higher than in the control group. Decreased (by 7-8%) pup weights were also occasionally (on days 5 and 11) found for the 300 ppm F2A females and for the 300 ppm F2B males. For the F2C litters, weights, litter sizes and total litter weights were similar for the 2 groups. Thus the changes seen in the other litters are unlikely to be male-mediated. There were no treatment-related, toxicologically significant effects on relative organ weight, macroscopic or microscopic appearance in the F2 offspring.
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
300 ppm (nominal)
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 ppm
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 ppm (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
The effects of tetrachloroethylene on fertility and general reproductive performance have been thoroughly investigated in a well-conducted two-generation study in rats exposed to 0, 100, 300 and 1000 ppm (0, 690, 2070 and 6900 mg/m3). Reduction in parental boy weight gain was observed at 1000 ppm during the pre-pairing period and lactation in both generations and during pregnancy in the second generation. A similar but less marked effect was also seen in the 300 ppm group. Observed effects on reproduction (reductions in litter size and pup survival at 1000 ppm, and pup body weight at 1000 and 300 ppm) are considered likely to be the non-specific consequences of maternal toxicity. No adverse effects on fertility or mating parameters were apparent, even at 1000 ppm. The NOEL for reproductive effects in this study was 300 ppm.
Executive summary:

Groups of 24 male and 24 female weanling Wistar rats (F0) were exposed whole-body to tetrachloroethylene vapour (purity 99.9%; 0, 100, 300 or 1000 ppm, 0, 690, 2070 or 6900 mg/m3) for 6 hours/day, 5 days/week for 11 weeks before mating. Exposure became daily during the mating period of up to 21 days and continued at 7 days/week until sacrifice for the males and until day 20 of gestation for the females. The dams and the first generation (F1) litters were then removed from exposure until day 6 after birth. When exposure recommenced on day 7 after birth, the animals (the dams with their litters) were exposed 7 days/week until selection of the weanlings to be second generation (F2) parents on about day 29 after their birth. The selected weanlings were then exposed 5 days/week for at least a further 11 weeks before mating.

Three different types of F2 litters were produced. For the F2A litters, the dams and pups were treated in a similar manner to the F1 litters (i.e. exposed during lactation from day 7 to day 29 post-partum), except that the dams and the offsprings in the 1000 ppm group were not exposed during lactation. This change was the result of effects (sedation of the dams with consequent neglect of their litters) apparent at 1000 ppm in the first generation. The F2B litters were derived from matings that followed at least 2 weeks daily exposure to tetrachloroethylene (0, 300 or 1000 ppm only). Exposure of the dams then continued on days 1 to 20 of gestation, but there was no exposure of the dams and their litters during lactation. Finally, the F2C litters were produced by mating males in the control and 1000 ppm groups with unexposed females.

The investigations carried out on parents included clinical observations, body weight, fertility (indicated by the success of the matings), length of gestation, precoital interval, organ (testes, liver and kidney) weights, necropsy and histopathology of certain tissues. The range of tissues examined varied between groups, but generally included liver, kidney and, usually only in animals suspected of being infertile, a range of reproductive tissues such as cervix, epididymis, ovary, prostate gland, testis, uterus and seminal vesicle. Investigations of the offspring included clinical condition, percentage live births, survival rates, body weights and (for selected pups only) organ weights, macroscopic and histological examinations.

The few deaths that occurred among parents during the study were not related to exposure to tetrachloroethylene. Clinical signs of toxicity to the parents, present in both generations of the 1000 ppm dose group, were consistent with depression of the CNS such as reduced activity and reduced response to sound. Other clinical signs of toxicity included breathing irregularities, salivation, hair loss, pale appearance, hunched posture, piloerection and tip-toe gait. Hair loss, pale appearance, hunched posture, piloerection and increased breathing rate were also observed at 300 ppm. The animals had recovered from these effects by 30 minutes after the end of the exposure and there were no clinical findings at 100 ppm. When exposure of the dams and litters in the 1000 ppm group was resumed on day 6 post partum in the F1, sedation of the dams with consequent neglect of their litters was evident. Reductions in parental body weight gains, apparent only at 1000 ppm, were generally small (up to 5% of the controls) and often transient (during the first few weeks of the pre-mating phase). However, initial (at 29 days post-partum) body weights of all F1 parent groups exposed to tetrachloroethylene were statistically significantly lower than controls for both sexes, with reductions of 7-8%, 9-11% and 24-26% at 100, 300 and 1000 ppm respectively. Reductions in parental food consumption, apparent prevalently at 1000 ppm, were also generally small and transient (during week 1 of the pre-mating phase).

For the F0 parents, statistically significant changes in organ weights were largely confined to increased absolute and relative values for kidney (by 13%) and liver (by 9%) in the males exposed at 1000 ppm, with no treatment-related effect being found for testes. For the F1 parents, relative (but not absolute) liver and kidney weights were again increased in males at 1000 ppm (by 14% for the liver and by 11% for the kidney) and statistically significant decreases in absolute testes weight were found at 300 (by 6%) and 1000 ppm (by 16%). Macroscopic findings in parents were generally unremarkable, although 3 F0 females (1 at 100 ppm, 2 at 1000 ppm) and 4 F1 females (2 at 300 ppm, 2 at 1000 ppm) were removed from the study intercurrently due to dystocia. Also, cysts were present in the kidney in 1 male exposed at 300 ppm and 2 males at 1000 ppm. Treatment-related histopathological changes were confined to the kidneys of animals exposed to 1000 ppm tetrachloroethylene and were generally minor in nature (slightly increased incidence of minimal chronic progressive glomerulonephropathy and increased nuclear pleomorphism within the proximal tubules).

There were no treatment-related effects of tetrachloroethylene on reproductive performance, as judged by male or female fertility, precoital interval or length of gestation, for either of the parental generations. However, at 1000 ppm there were statistically significant decreases in the proportions of F1 pups born live (206/222, 91%) and litters with all pups born live (12/21) compared with the control group (224/226, 99% and 21/23 respectively). Similar effects were also apparent at 1000 ppm for F2 pups of the A and B litters only. For F2A, the proportion of pups born live was 165/191, 86% (compared with 264/274, 96% for the controls) and for F2B, the values were 126/158, 80% and 220/233, 94% respectively. Additionally, total litter size at birth was also reduced (by about 30% compared with controls). The lack of any effect in the F2C litter (201/209 at 1000 ppm compared to 218/220 for the controls) indicated that these changes were unlikely to be mediated through the males.

Statistically significant effects among F1 offspring during the lactation period were largely limited to the 1000 ppm group; pup survival was decreased (for the F1 litters survival was only about 50% compared with 90% among controls) and pup weights were reduced, by about 10% at birth and about 20% at weaning relative to the controls. On day 6 (when exposure to tetrachloroethylene was resumed) and subsequently, the pups in the 1000 ppm group showed signs of sedation, as well as increased incidence of small appearance associated with the poor survival and growth. At 300 ppm pup bodyweights were also reduced, but to a lesser extent (up to 11%) than observed at 1000 ppm, and only at weaning.

Somewhat similar effects on pup survival and body weights were found at 1000 ppm for the F2A and F2B as for the F1 litters. Increased incidences of whole litter loss and statistically significant reductions in litter size were also noted. For example, the mean F2A litter size was 8.7 (standard deviation 3.8) on day 1 compared with the control value of 11.6 (3.2). On day 5, the values were 5.5 (4.3) and 10.6 (3.7) respectively. Unlike with the F1 litters, however, there was no exposure of the 1000 ppm F2A and F2B litters during lactation and pup weights, which were reduced by about 10% at birth and about 20% on day 5, started to recover after day 5; on day 29 they were actually slightly higher than in the control group. Decreased (by 7-8%) pup weights were also occasionally (on days 5 and 11) found for the 300 ppm F2A females and for the 300 ppm F2B males. For the F2C litters, weights, litter sizes and total litter weights were similar for the 2 groups. Thus the changes seen in the other litters are unlikely to be male-mediated. There were no treatment-related, toxicologically significant effects on relative organ weight, macroscopic or microscopic appearance in the F1 or the F2 offspring.

Overall, this study showed that tetrachloroethylene exposure at 1000 ppm (6900 mg/m3) elicited adverse effects on reproduction, observed as reductions in litter size, pup survival and pup body weight. However, since severe maternal toxicity (CNS depression, reduced activity, breathing irregularities, salivation, hair loss, pale appearance, hunched posture, piloerection, tip-toe gait and sedation with consequent neglect of their litters) was observed at this concentration level, the observed developmental effects are considered likely to be a secondary non-specific consequence of maternal toxicity. Exposure at 300 ppm (2070 mg/m3) produced a reduction in pup body weight at weaning in the F1 pups (up to 11%) that was also occasionally seen in the F2 pups (by 7-8%). This marginal reduction in pup body weight was observed in the presence of some maternal toxicity (hair loss, pale appearance, hunched posture, piloerection and increased breathing rate). At 100 ppm, there was a 7-8% reduction in offspring body weight in the F1 only. However, as this was an isolated finding occurring at 29 days post-partum only and was not observed either at birth or during lactation, or at weaning, or later in life at subsequent weeks during the pre-mating phase, or in the F2 offspring at birth, during lactation, or at weaning, it is likely to be an incidental finding of no toxicological significance. No effects on fertility and mating performance were apparent in this study, even at 1000 ppm (6900 mg/m3).

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
6 900 mg/m³
Species:
rat
Quality of whole database:
OECD guideline study under GLP, available as unpublished report, fully adequate for assessment
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No firm conclusions can be made from human data regarding fertility and reproductive performance. The effects of tetrachloroethylene on fertility and general reproductive performance have been thoroughly investigated in a well-conducted two-generation study (Tinston, 1995) in rats exposed to 0, 100, 300 and 1000 ppm (0, 690, 2070 and 6900 mg/m3). Observed effects on reproduction (reductions in litter size and pup survival at 1000 ppm, and pup body weight at 1000 and 300 ppm) are considered likely to be the non-specific consequences of maternal toxicity. No adverse effects on fertility or mating parameters were apparent, even at 1000 ppm. Therefore tetrachloroethylene is not considered a fertility and reproductive perfomance toxicant. From the available data, there are no indications that dams are more sensitive regarding systemic effects compared to animals exposed in the repeated dose toxicity studies.


Short description of key information:
No effects on reproductive function were observed at the highest dose tested (1000 ppm; 6900 mg/m3) in a good quality two-generation study in rats. It is concluded that tetrachloroethylene is not toxic to reproduction.

Justification for selection of Effect on fertility via oral route:
A multi-generation study is available via the inhalation route (most likely route of exposure). See discussion below.

Justification for selection of Effect on fertility via inhalation route:
There is one multi-generation study available

Justification for selection of Effect on fertility via dermal route:
A multi-generation study is available via the inhalation route (most likely route of exposure). See discussion below.

Effects on developmental toxicity

Description of key information
In humans, there is no clear evidence that exposure to tetrachloroethylene results in an increased risk of developmental toxicity including spontaneous abortion.
From a well-performed prenatal developmental toxicity study, a NOAEL of 250 ppm for developmental toxicity was derived (maternal NOAEL: 250 ppm). At 600 ppm, reduced gravid uterus, placental and foetal body weights, and decreased ossification of thoracic vertebral centra were observed in the presence of maternal toxicity. In a two-generation reproduction at maternal toxic concentrations also developmental effects were observed.
Link to relevant study records
Reference
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
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
Specific details on test material used for the study:
Supplied by INEOC Chlor Ltd. Purity > 99%.
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.
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.
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.
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
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.
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
Key result
Abnormalities:
no effects observed
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.
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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

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
1 725 mg/m³
Species:
rat
Quality of whole database:
Guideline GLP study, no restrictions, adequate for assessment
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Although there is no convincing evidence that tetrachloroethylene causes developmental toxicity in humans due to a number of limitations in the studies conducted, concern has been raised regarding the risk of spontaneous abortion, particularly, in dry-cleaning workers. An investigation conducted in the form of a retrospective epidemiological study of a cohort of dry-cleaners has shown no significant difference in the risk of spontaneous abortion between laundry workers and dry-cleaning workers (Doyle et al., 1997). However, in this study small, but statistically significant increases were observed in so-called dry-cleaning ‘operators’ compared with ‘non-operators’. Overall, due to the lack of specific exposure measurements for tetrachloroethylene, it was impossible to conclude with any confidence that this increase was due to exposure to tetrachloroethylene, and hence, the significance of this finding is unclear. Moreover, no consideration was given to any different distribution between operators and non-operators of many important confounding factors such as bending down and heavy lifting. Previous evidence for a positive association with tetrachloroethylene exposure is derived mainly from two case-control studies (Windham et al., 1991; Kyyronen et al., 1989), both of which involve small numbers and may be subject to various criticisms, including the fact that they failed to take into account known work-related risk factors for spontaneous abortion such as strenuous work, prolonged standing, bending down, shift-work and high workload.

In animals, several studies designed specifically to investigate the developmental toxicity of tetrachloroethylene by the inhalation route have been conduced in rats, mice and rabbits at concentrations ranging from 65 to 1000 ppm. Individually, most of these studies have weaknesses, notably the use of a single exposure level. However, a modern standard inhalation developmental toxicity study in the rat, using concentrations of 65, 250 and 600 ppm (509, 1695 and 4068 mg/m3) is available (Carney et al., 2006). Furthermore, a modern inhalation 2-generation study also in rats, using concentrations of 100, 300 and 1000 ppm is available (Tinston, 1995).

Regarding the developmental study, 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 NOAEL of 250 ppm. Developmental effects at 600 ppm consisted of reduced gravid uterus, placental and foetal body weights, and decreased ossification of thoracic vertebral centra. At 250 ppm very slight decreases in foetal body weight and placental weight were observed, but the differences were small, not statistically significant, and within historical control ranges. These effects were considered too small to have any adverse impact on development.There were no maternal and developmental effects at 65 ppm.

In the two-generation reproduction at maternal toxic concentrations, developmental effects also were observed.

There is no evidence from the available studies that inhalation exposure to tetrachloroethylene can cause foetal malformations.

The NOAELs for developmental and maternal toxicity are not lower (more critical) compared to the repeated dose N(L)OAELs.


Justification for selection of Effect on developmental toxicity: via oral route:
A more standard inhalation development toxicity study is available (most likely route of exposure). See discussion below.

Justification for selection of Effect on developmental toxicity: via inhalation route:
In animals, several studies designed specifically to investigate the developmental toxicity of tetrachloroethylene by the inhalation route have been conduced in rats, mice and rabbits at concentrations ranging from 65 to 1000 ppm. Individually, most of these studies have weaknesses, notably the use of a single exposure level. However, a modern standard inhalation developmental toxicity study in the rat, using concentrations of 65, 250 and 600 ppm (509, 1695 and 4068 mg/m3) is available (Carney et al., 2006).

Justification for selection of Effect on developmental toxicity: via dermal route:
A more standard inhalation development toxicity study is available (most likely route of exposure). See discussion below.

Justification for classification or non-classification

Based the available data regarding effects on fertility and developmental toxicity, classification is not warranted according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.