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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, near-guideline study. Published in peer reviewed literature. No restrictions, fully adequate for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
US-NTP Continuous Breeding Protocol. Comprises 4 phases through dose range-finding phase, F0 cohabitation and lactation phase, a F0 crossover mating trial and a F1 fertility assessment phase.
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): 4-vinylcyclohexene
- Substance type: a dimer of 1,3-butadiene
- Physical state: clear volatile liquid
- Analytical purity: >99%
- Lot/batch No.: A061181/B03

Test animals

Species:
mouse
Strain:
other: CD-1 (ICR)BR outbred Swiss albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh, NC, USA.
- Age: approximately 9 weeks old upon arrival
- Housing: solid bottom polycarbonate cages with stainless steel wire lids.
- Diet: pelleted food NIH-07 (Zeigler Brothers, Gardners PA, USA) ad libitum
- Water: deionized and filtered water ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 72±0.5°F
- Humidity: 53±6%
- Air changes (per hr): Not reported
- Photoperiod: 10 hrs dark / 14 hrs light

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 weeks
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of each dosing formulation analysed at approximately 1-month intervals for concentration of 4-vinylcyclohexene by gas chromatography. Concentrations were acceptable, between 89 and 117% of nominal.
Duration of treatment / exposure:
See below
Frequency of treatment:
See below
Details on study schedule:
See below
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 250 or 500 mg/kg/day (F0), 0 and 500 mg/kg/day (F1)
Basis:
other: nominal in corn oil
No. of animals per sex per dose:
40/sex controls, 25/sex 100, 250 and 500 mg/kg test groups (F0)
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes (no details reported)

BODY WEIGHT: Yes
- Time schedule: during weeks 1, 2, 5, 9, 13 and 18 (F0 females).

FOOD CONSUMPTION: Yes
- Time schedule: during weeks 1, 2, 5, 9, 13 and 18 (F0 females), during weeks 1 (breeding), 2, 3, and 4 (F1 male and female).

WATER CONSUMPTION: Yes
- Time schedule: during weeks 1, 2, 5, 9, 13 and 18 (F0 females), during weeks 1 (breeding), 2, 3, and 4 (F1 male and female).
Oestrous cyclicity (parental animals):
After delivery of the litters, vaginal smears were prepared from F1 females for 12 days.
Sperm parameters (parental animals):
Parameters examined in F1 male parental generations:
sperm motility, concentration, morphology and homogenization-resistant spermatid concentration
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
Parent (F0) cohabitation parameters included: date of delivery of each litter, number, sex, weight of pups per litter, number of litters per pair, and PND 0 dam body weight. On PND 0, 4, 7, 14, and 21, surviving pups were counted, sexed, and weighed for all dams delivering a litter after week 16.

F1 generation cohabitation parameters included: date of delivery of each litter, number, sex, weight of pups per litter, number of litters per pair, and PND 0 dam body weight.
Postmortem examinations (parental animals):
At study end, F1 parents were subject to necropsy and body weight, kidney/adrenal weights, liver, testis, prostate, seminal vesicle (+ coagulating gland), ovary/oviduct and uterus weights collected. The ovaries were processed for microscopic assessment.
Statistics:
Data were analysed using Williams' modification of Dunn's or Shirley's nonparametric multiple comparison procedures.
Reproductive indices:
Mating index = Number of females vaginal plug positive / number cohabitated
Fertility index = Number of females delivering a litter / number cohabitated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Although there were some mortalities (due to gavage-related injuries or indeterminate causes) none were considered to be treatment-related. There were still 36, 19, 19 and 16 breeding pairs available for the 0, 100, 250, and 500 mg/kg/day (F0) groups and 20 and 19 breeding pairs available for the 0 and 500 mg/kg/day (F1) groups.

BODY WEIGHT: (PARENTAL ANIMALS): F0 females at 500 mg/kg/day had an 8% reduction in body weight compared to controls during week 18 (data not reported). For F1 animals, there were significantly reduced body weights (7-8%) at 500 mg/kg/day during the F1 breeding phase (PND 77±10 up to and including PND 117±10). At delivery of F2 pups, 500 mg/kg/day dam weights were 7% lower than controls.

FOOD CONSUMPTION (PARENTAL ANIMALS): No effect on F0 parents. For F1 increased relative food consumption in 500 mg/kg/day males during weeks 2 and 4 (19% and 12% respectively) and in 500 mg/kg/day females during weeks 3 and 4 (11% and 29% respectively).

WATER CONSUMPTION: Cohabited pair relative water consumption for F0 was transiently increased at 100 mg/kg/day (7% at 5 weeks of exposure) and 500 mg/kg/day (27% and 17% at 5 and 9 weeks of exposure respectively). F1 relative water consumption was increased by 9% in 500 mg/kg/day females during week 3.

FERTILITY AND REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
F0 parents: 4-vinylcyclohexene at all treatment doses had no effect on reproductive competence including initial fertility, litters per pair, live pups per litter, total pups born alive, proportion of pups born alive, and sex ratio of pups.
F1 parents: 4-vinylcyclohexene at all treatment doses had no effect on reproductive competence including mating index, fertility index, gestation length, live F2 pups per litter, total number of F2 pups born alive, total number of F2 male pups per litter, or live F2 pup weight.

REPRODUCTIVE FUNCTION: F1 SPERM MEASURES (PARENTAL ANIMALS): 4-vinylcyclohexene had no effect on epididymal sperm concentration or morphology, but did cause a statistically significant increase in sperm motility and a statistically significant decrease (16%) in spermatid concentration in the right testis homogenates. No histopathological lesions were noted for the testis.

F1 VAGINAL CYTOLOGY (OESTRUS CYCLICITY): 4-vinylcyclohexene had no effect on normal cyclic patterns of vaginal cytology or mean cycle length following approximately 95 days of exposure to 500 mg/kg bw/day.

F1 ORGAN WEIGHTS (PARENTAL ANIMALS): Statistically significant increase in liver weights in F1 males (55.59±1.2 for controls vs. 60.46±1.37 for treated) and in F1 females (57.52±1.18 for controls vs. 62.08±1.28 for treated) at necropsy compared to controls. All other organ weights assessed were considered normal.

F1 SPERM ANALYSIS: No effect on epididymal sperm concentration or morphology, but did cause a statistically significant increase in sperm motility and a statistically significant decrease (16%) in spermatid concentration in the right testis homogenates. No histopathological lesions were noted for the testis.

F1 SECTIONED OVARY RESULTS: At 500 mg/kg bw/day for approximately 95 days there was a statistically significant reduction in the number of primordial oocytes/follicles by 33%, the number of growing follicles by 55%, and the number of antral follicles by 33%.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects on reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related

Details on results (F1)

Body weights of Male and Female F1 pups born after the end of F0 cohabitation (Week 16)on postnatal days (PND) 0, 7, 21, 77, and 117 were slightly reduced when compared to controls but only the reductions observed at weeks 77 and 117 were identified as statistically significant.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Generation:
F1
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no effects on reproductive performance
Dose descriptor:
LOAEC
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: clear ovarian toxicity was apparent in F1 females as evidenced by significant, marked decrements in numbers of primordial oocytes, growing follicles and antral follicles
Dose descriptor:
LOAEC
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: slight, statistically significant reductions in sperm motility in F1 males (concentration and morphology unaffected)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Parental Effects

Parameter

Dose (mg/kg/day)

0

100

250

500

No. fertile/No. cohabitated

36/36

19/19

19/19

16/16

Litters per pair

4.8

4.7

4.8

4.6

Live pups per litter

12.2

13.5

12.5

11.5

Pups born alive (%)

97

99

99

99

Live males per litter

49

48

48

49

Live pup weight (g)

1.64

1.58

1.58

1.58*

Adjusted live pup weight (g)

1.63

1.61

1.58

1.55

* statistically significant

 

 

F1 body weights (g)

PND

Dose (mg/kg/day)

males

females

0

100

250

500

0

100

250

500

0

1.75

1.69

1.75

1.63

1.66

1.59

1.64

1.59

7

4.38

4.35

1.75

4.23

4.27

4.35

4.22

4.08

21

11.07

10.98

10.79

10.94

10.3

10.93

10.2

10.56

77

34.07

 

 

31.51*

28.4

 

 

26.20*

117

35.24

 

 

32.79*

30.6

 

 

28.00*

* statistically significant

 

 

F2 Fertility and reproductive performance

Parameter

Dose (mg/kg/day)

0

500

Mating index

16/20

18/20

Fertility index

19/20

19/20

Number of days to litter

18.7

19.2

Live F2 pups per litter

11.6

10.6

F2 pups born alive

99

98

F2 male pups born alive

41

39

Live F2 pup weight

1.52

1.47

 

 

Sperm analysis

Parameter

Dose (mg/kg/day)

0 (n=20)

500 (n=19)

Epididymal sperm concentration 

988

876

Epididymal sperm motility

68.9

85.5*

Epididymal sperm morphology

2.4

2.9

Testicular sperm concentration

13.6

11.3*

* statistically significant

 

 

F1 sectioned ovary results

Follicular stage

Dose (mg/kg/day)

0 (n=20)

500 (n=19)

Primordial oocytes/follicles

208.9

140.6*

Growing follicles

51.2

23.2*

Antral follicles

7.4

4.95*

* statistically significant

 

 

Applicant's summary and conclusion

Conclusions:
4-vinylcyclohexene administered at 500 mg/kg bw/day was clearly toxic to ovarian follicles in female offspring and produced a slight but statistically significant effect on spermatogenesis in male offspring, but did not adversely affect reproductive performance in either the F0 or F1 generations. The NOEL for reproductive performance was 500 mg/kg/day.
Executive summary:

F0male and female CD-1 mice were administered 4-VCH by oral gavage at doses of 0, 100, 250 or 500mg/kg body weight/day for 16 weeks prior to conception of an F1breeding generation. Direct dosing (0 or 500 mg/kg body weight/day, by gavage) of 21-day old weaning F1adults commenced 7-8 weeks prior to conception of an F2generation, therefore, adults were exposed to 4-VCH before and during mating, throughout pregnancy and lactation, with continuous exposure of the foetuses and pups occurring secondary to maternal treatment (i.e. occurring in utero or via milk, respectively).

4-VCH, at doses up to 500 mg/kg body weight/day, which resulted in generalised toxicity, was clearly toxic to ovarian follicles in female offspring (significant reduction in primordial oocytes, growing follicles and antral follicles) and produced a slight but statistically significant effect on sperm motility in F1male offspring (concentration and morphology unaffected). However there were no effects on reproductive performance of the F0or F1generations, including mating and fertility indices, live litter size, sex ratio and pup survival to post-natal day 4.

NOAEL reproductive performance/foetotoxicity/teratogenicity = 500 mg/kg/day

LOAEL gonadal toxicity (F1 males and females) = 500 mg/kg