Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): vinyl acetate
- Storage condition of test material: In stainless steel drums at ambient temperature
- Physical state: Clear, flammable liquid
- Source: Society of Plastics Industry Inc., New York, USA
- The target specification for each batch of VA was to contain less than 50 ppm acetaldehyde and 1 ppm or less of hydroquinone
- Each batch of vinyl acetate was analyzed for water, acetaldehyde and acetic acid by the supplier before shipping, by HLE on arrival, prior to returning the unused material, and again by the supplier after receipt of the unused sample

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate, Kent, UK
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: no data
- Housing: During the premating period the F0 rats were housed 6 per cage in stainless-steel mesh cages suspended over cardboard-lined trays; rats in the F1 generation were housed 5 per cage. During gestation and lactation, female rats were housed in polypropylene cages with autoclaved sawdust bedding.
- Diet: SQC Rat and Mouse Breeder Diet No. 3, expanded (Special Diets Services, Witham, UK) ad libitum
- Water: Drinking water or drinking water formulations were available water ad libitum
- After approximately 24 hr control and test formulations were discarded and the bottles were refilled with fresh material
- Animal husbandry of the F1 generation was similar to that of the F0 generation
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Environmentally controlled
- Photoperiod: 12 hrs dark / 12 hrs light)

IN-LIFE DATES: From: no data

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Solutions containing 0, 200, 1000 or 5000 ppm vinyl acetate (v/v) in untreated drinking water) were prepared daily. The dosing solutions at each level were formulated by an extra 5% to correct for the loss of vinyl acetate by hydrolysis over the 24-h period.
Details on mating procedure:
- M/F ratio per cage: 1:2
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility (F1 only), for a maximum period of 5 days.
- During gestation and lactation, female rats were housed individually in polypropylene cages with autoclaved sawdust bedding.
- Any other deviations from standard protocol: Cross-mating of F1 rats: Approximately half of the male rats continued to be dosed with vinyl acetate for production of a second set of litters (F2b). All high-dose (5000 ppm) females were paired with the remaining control group males and all control group females were paired with the remaining high-dose males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Drinking water formulations were analyzed to verify the concentration of vinyl acetate. Duplicate analyses were done daily for the first 9 days of dosing, in weeks 5, 6, and 9, and at approximately 4-week intervals throughout the remainder of the study.
Duration of treatment / exposure:
Through 2 generations
Frequency of treatment:
Continuous
Details on study schedule:
- F0 rats were mated to produce F1 offspring after 70 days treatment.
- F0 males continued on treatment for 4 weeks after mating, then killed. Pregnant F0 females allowed to deliver F1 pups (day delivery complete = Day 1 of lactation. Any female that did not deliver was presumed not pregnant.
- Litter size reduced to a total of 10 pups of equal sex, when possible, on Day 7 of lactation.
- One rat/sex from 25 litters randomly selected/group to continue the reproduction study and become F1 parents of F2 offspring.
- One pup/sex from 10 litters randomly selected for a full necropsy.

- F1 parents mated to produce F2 offspring after 70 days treatment, sibling pairings avoided. The initial male replaced by a proven male from the same group if mating did not occur within 10 days. Cohabitation with the second male continued for a maximum of 5 days.
- F1 females allowed to deliver and rear their offspring to weaning, at which time the pups were killed.
- Approximately half of the F1 males had been killed when the decision was made to conduct a cross-mating of the F1 rats; the remaining male rats were continued on VA for production of a second set of litters (F2b).

F1 cross-mating: All 5000 ppm females were paired with the remaining control group males and all control group females were paired with the remaining high-dose males. In general, two females were paired with one male for up to 10 days. Non-productive rats were paired with those of known fertility. Water bottles removed during cohabitation to avoid exposure of control animals to treated water and vice versa. Mated females killed and necropsied on Day 13 of gestation to determine the pregnancy status and the number of implantations
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
nominal in water
Dose / conc.:
200 ppm
Remarks:
nominal in water
Dose / conc.:
1 000 ppm
Remarks:
nominal in water
Dose / conc.:
5 000 ppm
Remarks:
nominal in water
Dose / conc.:
0 mg/kg bw/day
Remarks:
other: Equivalents based on a dose conversion from Health Canada 1994
Dose / conc.:
28 mg/kg bw/day
Remarks:
other: Equivalents based on a dose conversion from Health Canada 1994
Dose / conc.:
140 mg/kg bw/day
Remarks:
other: Equivalents based on a dose conversion from Health Canada 1994
Dose / conc.:
700 mg/kg bw/day
Remarks:
other: Equivalents based on a dose conversion from Health Canada 1994
No. of animals per sex per dose:
18 males and 36 females
Control animals:
yes, concurrent vehicle
Details on study design:
The cross-mating trial of control and 5000 ppm male and female rats was conducted in the F1 generation to investigate the slightly decreased litter production in the high-dose group.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the males were recorded weekly. Body weights of the females were recorded weekly until mating was confirmed and then on Days 0, 6, 12, 15 and 20 of gestation. During lactation, body weights were recorded on Days I, 7, 14 and 21 except for the second mating of the F1 females when body weights were only recorded up to Day 12 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured daily throughout the premating periods by dividing the total amount of water consumed by the number of animals in the cage. Individual water consumption was also measured daily for mated females during gestation and lactation, but not during the mating periods or for weaned litters awaiting randomization.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 7 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
- In the F1 and F2 generations, developmental parameters (pinna unfolding, generalized hair and fur growth, tooth eruption, and eye opening) and functional tests (grip strength, pupillary reflex, visual placing response, and auditory response) were performed on pups from 10 randomly selected litters in each group on Day 21 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
- yes (no further details)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 and approximately half the F1 males: 4 weeks after mating phase completed. Remaining F1 males: after completion of cross-mating phase
- Maternal animals: Females not recorded as having mated were euthanized 13 days after the end of the mating period and the uterine contents were examined.
Mated females in the cross-mating phase were killed and necropsied on day 13 of gestation to determine pregnancy status and the number of implantations.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera: Yes
- The following organs were removed and preserved in 10% neutral-buffered formalin (except for the testes, which were stored in Bouin's fixative): cervix, coagulating gland, epididymides, lung, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, and vagina.
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring not selected as parental animals: One pup/sex from 10 litters randomly selected for full necropsy
- F2 offspring: Killed at weaning
- 10 male and 10 female pups from each group of the F1 and F2 litters underwent full necropsy. The following organs (at least) were removed and preserved in 10% neutral-buffered formalin (except for the testes, which were stored in Bouin's fixative): cervix, coagulating gland, epididymides, lung, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, and vagina.
Statistics:
Continuous or semi-continuous responses: analysis of variance (Snedecor and Cochran, 1967), followed by Student’s t-tests for normally distributed errors and Kruskal-Wallis test and Wilcoxon rank sum test for non-normally distributed errors (Siegel, 1956).

Mating, fertility, fecundity, and gestation indices: Fisher's exact test.

Pup numbers: Fisher's two-sum randomization (permutation) test with a Monte-Carlo simulation for computation of significance levels (Bradley, 1968; Van Julsingha, 1967).

The litter was the experimental unit and a square root transformation was used for weight and litter size adjustment. The level of significance for all determinations was set at 5%.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the F0 and F1 male rats and in the F1 female rats in the 5000 ppm group tended to be slightly lower than those of the control group. Body weight gain was significantly decreased during lactation of the F0 females at 5000 ppm and in the F1 females at 1000 and 5000 ppm.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food intake of animals exposed at 5,000 ppm v/v vinyl acetate was mar-ginally lower than that of the control animals during the exposure period. For the remainder of the study, the mean food intake of this group was similar to that of the control group. The mean food intake of animals exposed to 200 and 1,000 ppm v/v was similar to that of the control animals throughout the study
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The amount of VA consumed per kg of body weight decreased over the course of the premating period which is consistent with the decline in water consumption rela-tive to body weight that occurred over this time period in control and treated rats.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The number of litters produced in the F1 generation in the 5000 ppm group was slightly lower than that of the control group and was attributed to lower fertility. Few-er pups were produced when control females were mated with the 5000 ppm males, however, the decrease was due to poor mating performance rather than decreased fertility. No decrease was apparent when the 5000 ppm females were mated with the control group males.
However, VA did not produce a consistent effect on reproduction at any of the dose levels tested, although several changes in reproductive end points were observed at 5000 ppm. The changes in fertility parameters included a decrease in fertility of the F1 generation, and a decrease in F1 male mating performance in a cross-mating trial. In addition, the fertility index in a subset of F0 rats mated for the chronic study was lower than that for control rats. The inconsistency of the response across gen-erations and the small magnitude of the changes do not allow a definitive conclu-sion of compound-related effects to be made. No effects were also observed during histopathological examinations of gonads and accessory sex organs in any of the treated groups. Moreover, these effects were only observed in doses that induced some parental toxicity (decreased body weight gain and water consumption) and were within the historical control data of the laboratory. Based on these considera-tions, a conservative evaluation of the data leads to the conclusion that the no-observed adverse fertility effect level is 1000 ppm.

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- No significant effects on body weight in males or females during the premating period, although body weights of 5000 ppm F0 and F1 males rats tended to be lower than controls, possibly due to decreased water consumption. Body weight gain was significantly lower in the F0 (5000 ppm) and F1 (1000 and 5000 ppm) females during lactation. Given the increased nutritional demands placed on the dam during lactation, weight gain during this period could have been more readily affected by decreased water consumption.

WATER CONSUMPTION
- Water consumption decreased in the 5000 ppm F0 and F1 male and female rats and in the 1000 ppm F1 female rats during the first week of dosing and persisted throughout the study (probably due to the palatability of vinyl acetate). The magnitude of the effect on water consumption (> 20% reduction at 5000 ppm) suggests that concentrations used were probably the maximum allowable concentrations for a drinking water study.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Vinyl acetate intake at the start of the premating period, for drinking water concentrations of 200, 1000 and 5000 ppm, was 29, 145 and 650 and 38, 177 and 763 mg/kg/d, in F0 males and females respectively. At the end of the premating period, vinyl acetate intake was 16, 76 and 320 and 25, 117 and 431 mg/kg/d, in F0 males and females respectively.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- F0 generation, no significant differences in mating, fertility, or gestation indices, no effect on gestation length.
- F1 generation, no effect on mating or gestation indices. Although not statistically significant, the F1 fertility index of the 5000 ppm group was lower than that for the control group (approximately 79 versus 96% for control). No effect on F1 gestation length.
- In the cross-mating trial, vinyl acetate had no apparent effect on the 5000 ppm female rats that were mated with the control males; the mating and fertility indices were approximately 96%. Fewer pregnancies occurred when the 5000 ppm male rats were mated with the females from the control group, resulting in a mating index of 76%. The fertility index was not affected (100%).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: approximately 100 mg/kg/d. At 5000 ppm, reduced weight gain of F1 pups to weaning. No similar effect in F2 pups.
Remarks on result:
other: Generation: 2 generations (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

BODY WEIGHT (OFFSPRING)
- With the exception of decreases in the weights of the F1 pups at 5000 ppm during the lactation period, there were no effects of treatment on any of the parameters associated with the litters. Similar weight effects were not observed in the F2 pups at 5000 ppm; the decreased weight gain in the F1 pups was considered secondary to the lower weight gain observed in the dams during this period.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Summary of mean water consumption (g)

(Table based on Mebus et al, 1995, Fundamental and Applied Toxicology 24, 206-216, Tables 4, 5, 6 and 7)

Parameter/sex

Dose level of vinyl acetate (ppm)

0

200

1000

5000

F0 males (premating, wks 1-10)

275.1

279.0

258.3

218.4**

F1 males (premating, wks 1-10)

267.6

252.8

249.4

207.6**

F0 females (premating, wks 1-10)

223.7

217.8

216.1

162.8**

F1 females (premating, wks 1-10)

219.9

215.6

183.0**

163.5**

F0 females (gestation, overall)

54

54

51

38**

F0 females (lactation, overall)

95

93

89

72**

F1 females (gestation, overall)

55

56

49**

38**

F1 females (lactation, overall)

96

89

83**

76**

*significantly different from control (p=0.05) **significantly different from control (p=0.01)

 

Summary of female body weight changes (g)

(Table based on Mebus et al, 1995, Fundamental and Applied Toxicology 24, 206-216, Table 3)

Parameter

Dose level of vinyl acetate (ppm)

0

200

1000

5000

F0 females: gestation (days 0-20)

118

112

111

108

F0 females: lactation (days 1-21)

53

46

45

32**

F1 females: gestation (days 0-20)

123

115

115

117

F1 females: lactation (days 1-21)

46

44

29**

29*

*significantly different from control (p=0.05) **significantly different from control (p=0.01)

 

Reproductive indices of rats

(Table based on Mebus et al, 1995, Fundamental and Applied Toxicology 24, 206-216, Table 12)

Parameter

Dose level of vinyl acetate (ppm)

0

200

1000

5000

Mating index (%)

 

 

 

 

F0

94.9

97.1

97.2

97.2

F1

100.0

100.0

100.0

100.0

Cross-mating

76.0

-

-

95.8

Fertility index (%)

 

 

 

 

F0

88.2

88.2

94.3

91.4

F1

96.0

96.0

96.0

79.2

Cross-mating

100.0

-

-

95.7

 

Applicant's summary and conclusion

Conclusions:
In a two generation reproduction study in rats with vinyl acetate in drinking water, the no-observed adverse effect level for reproductive effects was 1000 ppm, equivalent to approximately 100 mg/kg/day.
Executive summary:

In a two-generation reproduction study in rats, at concentrations of 0, 200, 1000 and 5000 ppm vinyl acetate in drinking water, there was no consistent effect on reproduction at any of the dose levels tested, although several changes in reproductive end points were observed at 5000 ppm. The changes in reproductive parameters included a decrease in F1 pup weight (Day 21), a decrease in fertility in the F1 generation and a decrease in F1 male mating performance in a cross-mating trial. In addition, the fertility index in a subset of F0 rats mated for a chronic study was lower than that for control rats. The inconsistency of the responses across generations and the small magnitude of the changes do not allow a definitive conclusion of compound-related effects to be made.

Some minor and slight effects on male fertility, not forming a specific pattern, were observed in the high dose group receiving 5000 ppm orally in drinking water, which also induced slight parental toxicity. No effects were observed on any parameters of female fertility. Based on these data it can be concluded that VA shows no specifically toxic effects on fertility. A conservative NOAEL of 1000 ppm (equivalent aproximately 100 mg/kg/day) in drinking water could be derived for male fertility, and 5000 ppm for female fertility.

   

Limitations of the study: Study not formally following OECD Guideline 416, but most relevant fertility parameters were addressed. No investigation on sperm parameters were performed. No data on GLP conformity.