Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (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 male and female fertility study; no restrictions, fully adequate for assessment
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Principles of method if other than guideline:
Male and female fertility study
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: > 99.9%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Saint-Aubin-Les Elbeuf, France
- Age at study initiation: 6 weeks (weanling)
- Fasting period before study: during 6 hr exposure
- Housing: 4 to a cage (males) and 5 to a cage (females), in wire mesh stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks (weanling), 3 weeks (young adult)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-25
- Humidity (%): 45-70

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material was led from cylinders via a pressure reducer, stainless steel tubing and three mass flow controllers to the mixing device of the inhalation chamber, where it was diluted with filtered air from the air conditioning system (filter efficiency: 80-90% for particles having a diameter of 0.2-0.4 µm) to obtain the desired concentration.
Details on mating procedure:
Male fertility evaluation
After 15 weeks of exposure each male animal of the subset “weanlings” was mated with two untreated, young adult nulligravida-females of the same strain and obtained from the same breeder in week 11, being four weeks prior to the start of the mating period. Mating took place overnights inside the inhalation chamber. During the nightly mating periods each male was randomly caged with two females until pregnancy occurred or two weeks had elapsed. Vaginal smears were taken each morning to determine whether mating had occurred. The day on which a copulation plug was found or sperm was present was considered day 0 of pregnancy. Females of which copulation was established were moved to individual cages. During the day the males stayed in the respective inhalation chambers for the normal exposure until successful mating with two different females had been achieved or two weeks had elapsed. After the mating period the males were killed. The pregnant females were killed on day 15 of gestation and the uterus and ovaries were removed.

Female fertility evaluation
After 15 weeks of exposure each female animal of the subset “weanlings” was mated with one untreated, young adult male of the same strain and obtained from the same breeder in week II, being four weeks prior to the start of the mating period. Mating took place overnights outside the inhalation chamber. During nightly mating periods each female was randomly caged with one male until pregnancy occurred or two weeks had elapsed. Vaginal smears were taken each morning to determine whether mating had occurred. The day on which a copulation plug was found or sperm was present in the smear, was considered day 0 of pregnancy. Females of which copulation was established were moved to individual cages. During the day the females were returned to the respective inhalation chambers for the normal exposure until successful mating and 5 days of pregnancy had been recorded or two weeks had elapsed. The females were then removed from the exposure chambers and housed individually. On day 15 of gestation the pregnant females were killed and the uterus and ovaries were removed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test atmospheres were performed by a total carbon analyzer, type Intersmat GC 120, fitted with a flame ionisation detector. Calibration of the total carbon analyzer was performed four times, once just prior to start the exposure, once in week 4 and 8, and once after the exposure period. The calibration was performed by using a calibration gas, prepared by mixing known volumes of VF2 and air by means of calibrated mass flow controllers. The mass flow controller for the VF2 flow was calibrated with a soap bubble meter and that for the air with a wet test meter. Next the detector response of the total carbon analyzer was determined at at least 6 concentration levels, covering the measuring range needed.
The total carbon analyzer was connected with a micro-computer interfaced serial data aquisition system, which monitored and controlled the exposure levels of the test material. Samples of the test atmospheres were taken from one location.
Duration of treatment / exposure:
15 weeks prior to mating, 2 weeks during mating, first 5 days of pregnancy (pretreated females only)
Frequency of treatment:
- prior to mating: 6 hours/day, 5 days per week;
- during mating: 6 hours/day, 7 days per week;
- after mating: 6 hours/day, 5 days per week (pretreated females only)
Details on study schedule:
See 'Details on mating procedure'
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 1000 and 7000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
251, 997, 6998 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent no treatment
Details on study design:
In order to make a decision as to which subset (weanling or young adult) should be used for the fertility study, an additional period of at least one week after the 13 week exposure in the concurrent repeated dose toxicity study was necessary to collect the relevant results of autopsy findings and organ weights of animals of both subsets.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

WATER CONSUMPTION: No

FERTILITY EVALUATION AND REPRODUCTIVE PERFORMANCE
The following observations in treated animals and in untreated males and females mated with these animals were recorded:
- mating index (nr of females mated/nr of females placed with males)
- male fertility index (nr of males that became sire/nr of males placed with females)
- female fertility index (nr of females pregnant/nr of females placed with males}
- fecundity index (nr of pregnancies/nr of matings)
- pre-coital time (time between the moment of placing males with females and successful mating)

In both treated and untreated females the following reproduction parameters and litter data were also be recorded:
- nr of corpora lutea of pregnancy
- nr of implantation sites
- nr of early and late resorptions.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
No data
Postmortem examinations (parental animals):
After the mating period each male of the subgroups was killed and selected to a thorough autopsy. The following organs were weighed:
testis, epididymis, seminal vesicles, prostate.

In both treated and untreated females, killed on day 15 of pregnancy, ovary weights were recorded.

Postmortem examinations (offspring):
No data
Statistics:
Body weights and organ weights were determined by means of electronic scales on line with a computer. Calculations of the mean values and standard errors were also done by computer. Body weights of the parent rats were subjected to analysis of variance.
The number of females showing evidence of mating, the number of pregnant females and the number of females bearing live foetuses were evaluated by the Chi-square test.
The differences between the median pre-coital times were evaluated by the Mann-Whitney U-test while the mean pre-coital time, including only females that showed evidence of mating was evaluated by the Student t-test.
Statistical analysis of differences in organ weights, litter data and pre-implantation loss was carried out by applying analysis of variance, followed by Dunnett's Multiple Comparison test.
Percentage pre-implantation loss (PRIL) was calculated for each litter by the formula:
PRIL = ((number of corpora lutea – number of implantation sites)/ number of corpora lutea) x 100 %
Reproductive indices:
Treated females
Mating indices were 100 in all exposed groups and 95 in the control group
Fertility indices were 90 (in the low and high dose group) and 95 (in the mid dose group)

Treated males
Mating indices vary from 92.5 in the controls to 100 in both higher concentration groups
Fertility indices were well within the range as observed in historical control series (the presence of implantation sites was slightly lower in the control and low dose group than in the two higher dose groups)
Offspring viability indices:
No data

Results and discussion

Results: P0 (first parental generation)

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
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
During the study, no deaths occurred and no signs of ill-health were observed in any of the groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of untreated females determined on day 15 of pregnancy were similar in all groups.
The mean body weights of males that were exposed during a 15-week premating period and during the successive mating period and that were killed at the end of the mating period did not show any differences between any of the groups.
The mean body weights of females exposed to VF2 during a 15-week premating period, during the successive mating period and during the first five days of pregnancy and that were killed on day 15 of pregnancy were similar in all groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
From the 20 males placed with 40 untreated females in each group, 37 to 40 females showed evidence of mating. Through this the mating indices varied from 92.5 in the controls to 100 in both higher concentration groups.
Although the mean pre-coital time seemed to be slightly longer in the two higher concentration groups when compared with the controls, no significant differences were observed. Moreover, the median pre-coital time was similar in all groups.
The number of females that actually became pregnant, as was demonstrated by the presence of implantation sites observed at autopsy, was slightly lower in the control- and low-concentration group than in the two higher concentration groups. However, the female fertility indices of all groups were well within the range as observed in historical control series. Apart from one male in the control- and one male in the low-concentration group, all treated males became sire. Male reproductive performance was slightly better in the two higher concentration groups than in the control- and low-concentration group as was demonstrated by a somewhat higher number of treated males that became sire of two litters instead of one litter in these groups.

From the 20 females, previously exposed to VF2 during a 15-week premating period, and placed with 20 untreated males in each group, only one control female did not show evidence of mating. Through this, mating indices were 100 in all exposed groups and 95 in the control group.
Both mean and median pre-coital time did not reveal any changes that could be related to the treatment.
The number of treated females that actually became pregnant was 18 or 19 in each group, revealing female fertility indices that ranged from 90.0 (in both the high- and low-concentration group) to 95.0 (in the mid-concentration group). Since the animals were mated on a “1 male to 1 female”-basis, the number of males that became sire and the male fertility index were similar to the number of pregnant females and the female fertility index.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Mean absolute and relative weights of all reproductive organs of males that were exposed to VF2 for 17 weeks and that were mated with untreated females, were similar in all groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At autopsy of the untreated female parent rats mated with treated males, no abnormalities were observed.
No significant differences between the different groups were observed in the mean number of corpora lutea and the mean number of implantation sites. The pre-implantation loss, reflecting not only the loss of zygotes in the pre-implantation stage, but also the number of ova not being fertilized, was even lower (though not significantly) in any of the groups of females, mated with VF2-treated males than in the controls.
The mean number of early and late resorptions per litter was also comparable in all groups. No differences between groups were observed in mean ovary weights.

At autopsy of the treated female parent rats, mated with untreated males, no abnormalities were observed that could be related to treatment. One animal of the low-concentration group showed some minor haemorrhagic encrustations around both eyes. The mean number of corpora lutea and the mean number of implantation sites were comparable in all groups. Although pre-implantation loss values varied between 4.4 in the control- and 12.5 in the mid-concentration group, all values were well within the range as observed in historical control series. Moreover, there was no concentration-response relationship.
The mean number of early and late resorptions per litter was comparable in all groups. Mean ovary weights were also similar in all groups.

Effect levels (P0)

Dose descriptor:
NOAEC
Remarks:
general toxicity and fertility
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion