Tetrachloroethylene

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4700 (Reproduction and Fertility Effects)

Test guideline 2

Qualifier:

equivalent or similar to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tetrachloroethylene

EC Number:

204-825-9

EC Name:

Tetrachloroethylene

Cas Number:

127-18-4

Molecular formula:

C2Cl4

IUPAC Name:

tetrachloroethene

Details on test material:

Tetrachloroethylene was supplied by ICI Chemicals & Polymers Ltd., Runcorn, Cheshire, UK as a colourless liquid. A single batch (PD299) of perchloroethylene with a purity of 99.9% w/w was used throughout the study and was assigned the CTL reference number Y00207/005.A bulk supply of the test substance was stored in drums under external ambient conditions. Appropriate aliquots were taken from the drums, transferred to the Inhalation Unit and stored at room temperature in ventilated cupboards until used for atmosphere generation. Re-analysis of the test substance following termination of the study demonstrated satisfactory stability.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Sub-strain: Alpk:APfSD

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

690 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

2 070 mg/m³ air (nominal)

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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).

Effect levels (P0)

1

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)

2

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'

3

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)

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Details on results (P1)

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.

Effect levels (P1)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 ppm

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

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.

Effect levels (F2)

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

300 ppm (nominal)

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 ppm

Overall reproductive toxicity

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

Applicant's summary and conclusion

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).