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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although OECD guideline 416 was not available at the time of the study, this study follows an appropriate number of recommandations (despite the lack of data on reproductive organs, oestrous cycle and sperm parameters). The study showed a high variability for some of the endpoints, but these were identified by the authors of the study and additional investigations were included in the study to address these, e.g. additional generations and cross-fostered litters.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
no data available on oestrous cycle and on sperm parameters; no data on reproductive organs (weight, macroscopic and microscopic examinations)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): vinylidene chloride (1,1-dichloroethylene)
- Substance type: production grade
- Physical state: liquid
- Other: distilled prior to use in order to remove the inhibitor monomethyl ether of hydroquinone (MEHQ)
- Analytical purity: 99.5 % minimum

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Spartan Research Animals, Inc., Haslett, MI
- Age at study initiation: 6-7 weeks
- Housing: housed separately by sex, except during mating periods in suspended wire-bottom cages (2 per cage)
- Diet: laboratory chow (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
- Rate of preparation of diet (frequency): daily
- Nominal concentration in vehicle: 0, 50, 100 or 200 mg/L
Details on mating procedure:
For initial mating F0:
- Length of cohabitation: 15 days
After weaning the F1a litters, F0 rats were remated to produce the F1b litters (because of low fertility rate including the controls), M/F ratio = 1
- Proof of pregnancy: presence of a vaginal plug
After successful mating each pregnant female was caged in individual cage containing ground corn cob litter for nesting

For F1 and F2 generations:
one female was placed with a male for two 6-day mating periods (different male for each period) separated by a 6-day resting period

For F2 and F3 generations, the mating period was shortened to 6-day period because F0 rats did not mate after 6 days of cohabitation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the water solutions were conducted 35 times during the test period. The mean (± SD) concentration of vinylidene chloride in the drinking water, determined by gas chromatographic analyses, was 68 ± 21, 99 ± 22 and 206 ± 33 ppm for the respective nominal concentrations of 50, 100 and 200 mg/L.
The concentration of vinylidene chloride present in water decreased approximately 10% in a 24 hour period.
Duration of treatment / exposure:
100 days for the F0 rats producing F1a litters
100 days + 10 days after the last F1a weaning for the F0 rats producing F1b litters
110 days for the F1b and F2 rats producing F2 and F3a, respectively
100 days + 10 days after the last F3a weaning for the F2 rats producing F2b litters
100 days + 10 days after the last F3a weaning + 10 days after the last F3b weaning for the F2 rats producing F3c litters
Frequency of treatment:
ad libitum
Details on study schedule:
The F0 rats were mated at approximately 20-21 weeks to produce the F1a litters. 10 days after the last litter was weaned, F0 rats were remated to produce the F1b litters.
The rats serving as parents for the F2 and F3 generation were randomly selected with the aid of a random number table from F1b and F2 litters, respectively.
The F1b and F2 rats were mated at approximately 110 days of age to produce the F2 and F3a litters, respectively. The F2 rats were remated to produce the F3b and F3c litters, 10 days after the last F3a and F3b litters were weaned, respectively.

cross fostered litters:
Pups from eight dams ingesting 200 ppm VDC in the drinking water from the tab litters were randomly reduced to 8 per litter on the day of birth and raised by dams ingesting only water. Pups from eight dams ingesting water from the F3b litters were randomly reduced to eight per litter on the day of birth
and raised by dams ingesting 200 ppm VDC.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/L
Basis:
nominal in water
corresponding to 7 and 9 mg/kg/day bw for males and females respectively (Quast 1983)
Remarks:
Doses / Concentrations:
100 mg/L
Basis:
nominal in water
corresponding to 10 and 14 mg/kg bw/day for males and females respectively (Quast 1983)
Remarks:
Doses / Concentrations:
200 mg/L
Basis:
nominal in water
corresponding to 20 and 30 mg/kg bw/day for males and females respectively (Quast 1983)
No. of animals per sex per dose:
F0 rats :
control group : 15 males / 30 females
50, 100, 200 mg/L groups : 10 males / 20 females

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the maximum concentration of 1,1-dichloroethene given in the drinking water (200 mg/L) was the upper limit of solubility of the compound in water
- Animal selection: Pups from 8 dams of the 200 mg/L group from the F3b litters were randomly reduced to 8 per litter on the day of birth and raised by dams ingesting only water. Pups from 8 dams of the control group from the F3b litters were randomly reduced to 8 per litter on the day of birth and raised by dams ingesting 200 mg/L of 1,1-dichloroethene.

- Rationale for animal assignment (if not random): gross fostered litters
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Body weight and water consumption were recorded at least weekly until mating
After mating, the females were observed daily for signs of normal or abnormal parturition.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
Date of parturition (Day 0), number of live and dead newborn, number of live pups on days 1, 7, 14, and 21, litter weights on days 1, 7, and 14, and individual weanling weights on day 21.
On days 1, 7, 14 and 21, each pup was examined for external anomalies, signs of toxicity and sex
Postmortem examinations (parental animals):
Blood urea nitrogen levels and serum glutamic pyruvic transaminase and alkaline phosphatase activities from 10 F2 rats/sex/dose level
Weights of brain, heart, liver, kidneys and testes/ovaries from at least 10 males and 20 females of F1 and F2 generation at each dose
Paraffin-embedded sections of liver and kidneys from at least 10 F1 and F2 rats/sex/dose level stained with hematoxylin and eosin for light microscopy observations. All grossly visible masses of the F2 rats histologically examined after similar preparation.
Postmortem examinations (offspring):
Weanlings not selected as future parents :
Body weights, liver and kidney weights from 2 to 7 21-day old pups/sex/concentration level from the F1b, F2 and F3b litters
Paraffin-embedded sections of liver and kidneys stained with hematoxylin and eosin for histological examination
Statistics:
Survival indices were analyzed by Wilcoxon test
Fertility index, sex ratio and histopathologic data analyzed by Fisher Exact Probability Test
Neonatal and maternal body weights, food and water consumption, number of days between observing a vaginal plug and parturition and number of days between first cohabitation and parturition were analyzed by an analysis of variance and the means were compared with control values by Dunnett's test.
Reproductive indices:
Fertility Index
Sex ratio
Number of days between observing a vaginal plug and parturition
Number of days between first cohabitation and parturition
Offspring viability indices:
Average number of pups per litter at birth
Postnatal Survival Index
Postnatal body weight

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

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 fertility of the F0 rats producing the F1a litters was unusually low in the control rats and in 100 and 200 mg/L groups. The F0 adults were remated according to a modified mating regimen, 1:1 instead of 1:2, after which the fertility in the 200 mg/L group was lower than the other groups but higher than the first time. Since, this lower fertility was not observed anymore for the 200 mg/l group later in the study, it was considered as a isolated, non-representative finding. When the F2 rats were mated for the F3c litter, a decreased fertility was observed in the control and 100 mg/L groups probably due due to the age of the rats: 276-298 days) (Table 1)

No significant effect on the mean number of days from copulation to delivery in any generation and at any dose level.

A significant increase in the average number of days from cohabitation to delivery was observed in the second mating of the F0 rats in the 100 mg/L group but not in the 200 mg/L group (mean (± SD): 26±2, 33±9, 26±2, in the control, 100 mg/L and 200 mg/L group, respectively). Due to the absence of a dose response, this finding was considered not to be causally linked to vinylidene chloride.


Gross examination at necropsy of the F1 and F3 adults revealed no alterations considered to be related to the treatment.
F2 males in the 200 mg/L group had an increased incidence and severity of chronic renal disease and a loss of body fat (but chronic renal disease occurs spontaneously in this species). Gross examination of the liver of F2 females of 100 and 200 mg/L groups revealed a pale appearance, small pale foci and an accentuated lobular pattern.
Light microscopic examination of the liver and kidneys revealed an accentuated hepatic lobular pattern and a minimal degree of hepatocellular fatty change in the F1 rats of the 100 and 200 mg/L groups. Similar hepatocellular fatty changes were observed in all groups of F2 rats ingesting vinylidene chloride. The hepatocellular fatty changes as observed in the 50 mg/l groups in absence of concurrent effects were judged not sufficient to consider the dose as "adverse".
Elevated relative liver weights of the female F2 rats of the 200 mg/L group were observed as well as an increased serum glutamic pyruvic transaminase activity (25 % above control mean) but no change in the blood urea nitrogen levels and in alkaline phosphatase activity.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
9 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: systemic toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
100 mg/L drinking water
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

No significant change in the average number of pups per litter and in the percentage of pups alive at birth was observed.

The postnatal survival index for the F2 litters in 50, 100 and 200 mg/L groups was significantly decreased (most likely due to the larger litter size at birth). The postnatal survival index for the F3a litters was significantly decreased in a dose-dependent manner but not for the F3b and F3c litters (Table 2). In order to investigate possible causes for the decreased postnatal survival index which appeared in a non-consistent manner, cross-fostered litters were also investigated. The postnatal survival index for the two sets of cross fostered litters (0 and 200 ppm VDC in the drinking water) were comparable to each other and the f3b control litters.


Significant decreases in postnatal body weight were observed in the F2 litters in the 50 and 100 mg/L groups (but most likely due to the increased number of pups/litter at birth) but not in the 200 mg/L group. The average body weight of the F3a pups of the 200 mg/L group was occasionally significantly decreased from that of the control group but not of the F3b and F3c litters.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/L drinking water
Sex:
male/female
Basis for effect level:
other: no effect on reproductive parameters were observed

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
200 mg/L drinking water
Sex:
male/female
Basis for effect level:
other: no developmental effects were observed, the fertility index when producing the f3c generation was overall lower, which was probably due to the age of the breeding rats.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

TABLE 1 : Fertility Index (FI) of Rats Ingesting 1,1 -dichloroethene (VDC)

mg/L VDC in the drinking water

0

50

100

200

Generation

FIa

%

FI

%

FI

%

FI

%

F0 adults:

   F1a litters

17/30

57

19/20b

95

13/20

65

9/20

45

   F1b litters

23/30

77

15/20

75

16/20

80

11/20

55

F1 adults:

   F2 litters

27/30

90

14/20

70

16/20

80

20/20

100

F2 adults:

   F3a litters

29/30

97

19/20

95

16/20

80

19/20

95

   F3b litters

32/36

89

22/26

85

19/23

83

21/24

88

   F3c litters

20/36

56

16/20

80

9/23

39

17/24

71

 

aFertility Index (FI) = number of females delivering a litter/number of females placed with a male.

bSignificantly different from the control value by Fisher's exact probability test, p<0.05.

TABLE 2: Postnatal Survival Index (PSI) Among Litters of Rats Ingesting 1,1 -dichloroethene (VDC)

mg/L VDC in the Drinking Water

0

50

100

200

Generation

PSIa

%

PSI

%

PSI

%

PSI

%

F1a

153/178

86

143/168

85

115/126

91

81/102

79

F1b

155/190

82

112/140

80

101/140

72

65/87

75

F2

234/252

93

121/149

81b

148/192

77b

169/206

82b

F3a

215/296

73

95/211

45b

74/192

38b

60/215

28b

F3b

214/306

70

183/260

70

167/207

81

114/148

77

F3c

193/205

94

145/171

85

86/91

94

117/152

77

aPostnatal Survival Index (PSI) is the number of liveborn pups surviving to 21 days of age/number of liveborn pups.

bSignificantly different from the control value by the Wilcoxon test as modified by Haseman and Hoel,p<0.05.

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive toxicity in this study is 200 mg/L (equivalent to 30 mg/kg) for exposure to 1,1 -dichloroethene in drinking-water
Executive summary:

Three generations of Sprague-Dawley rats were exposed to drinking-water containing nominal 1,1 -dichloroethene concentrations of 0, 50, 100, or 200 mg/L. After 100 days of exposure, the rats were mated. There were no biologically significant changes in fertility index, average number of pups per litter, average body weight of pups, or pup survival at any exposure. Light microscopic examination of the liver and kidneys revealed an accentuated hepatic lobular pattern and a minimal degree of hepatocellular fatty change in the F1 rats of the 100 and 200 mg/L groups. Similar hepatocellular fatty changes were observed in all groups of F2 rats ingesting vinylidene chloride. The hepatocellular fatty changes as observed in the 50 mg/l groups in absence of concurrent effects were judged not sufficient to consider the dose as a NOAEL. Exposure to 1,1-dichloroethene in drinking-water at concentrations causing mild, dose-related changes in the liver did not affect the reproductive capacity of rats through three generations, which produced six sets of litters. The NOAEL for reproductive toxicity in this study is 200 mg/L, corresponding to an average exposure of 30 mg/kg/d.