Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August 2007 - 30 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Colorless clear liquid
- Purity: 99.9%
- Lot/batch No.: 3SMZD
- Stability under test conditions: Stable

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (Atsugi Farm)
- Age at study initiation: 9 weeks-old
- Weight at study initiation: Male: 317-361 g, Female: 199-251 g
- Fasting period before study: none
- Housing: Males and females were housed in stainless-steel wire-meshed cages except for gestation and lactation periods. During gestation and lactation periods, females were housed in polycarbonate cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week including quarantine period
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8-23.6°C
- Humidity (%): 49.8-61.8%
- Air changes (per hr): 6-20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared daily before administration Using purified water as a vehicle, solution with a concentration of 30 mg/mL was prepared first. Then, the
solution was diluted with vehicle and prepared as solutions with concentrations of 1 and 6 mg/L.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage:1:1
- Length of cohabitation: Length of cohabitation: 14 days (maximum)
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Male: 42 days including 14-days pre-mating period and mating period
Female: 42-52 days including 14 days pre-mating, mating and gestation periods, and days until lactation day 4.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 60, 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Administration groups:
Control and high dose groups: 7 males and 12 females per dose
Low and mid dose groups: 12 per sex per dose
Recovery groups:
Control and high dose groups: 5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In 14-days preliminary study, a dose of 500 mg/kg/day tested was proved to be excess because of poor general conditions for the test animals. As a consequence, the top dose in the
main study was set down to 300 mg/kg/day. The mid and low doses were set as 60 and 10 mg/kg/day, respectively.
- Rationale for selecting satellite groups: Satellite groups for recovery study were set for control and high dose groups.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times (before and just after administration and about 3 hrs after administration) per day during administration period. Once daily during period other than administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: (Males) Day 1, 8, 15. 22, 29, 36, 42 and 43. (Females) Pre-mating Day 0, 7 and 17, once a week during mating period, gestation day (GD) 0, 7, 14 and 20, and lactation day 0 and 4. (Males and females, recovery groups) Day 50 and 56.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: (Males) Day 43; (Females) Lactation day 5; (Males and females, recovery) Day 57
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, 18-23 hrs from the day before.
- How many animals: 5 per sex per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the same schedule as haematology
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the day before start of administration, once a week during administration until Week-6.
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other: reactivity to stimuli
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: all per litte
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
For parametric data, homogeneity of variance was examined by Bartlett method. When the variance was equal, ANOVA was used. While variance was not equal or non-parametric data was applied, Kruskal-Wallis
method was used. When significant difference was recognized among the groups, either Dunnett method or Dunnett multiple comparison test was used. For qualitative data, Dunnett multiple comparison test,
Wilcoxon rank sum test, Fischer's exact probability, Student or Aspin-Welch's t-test were used as necessary.
Indices:
Implantation index (%) = (Number of implantation sites/number of corpora lutea)×100
Delivery index (%) = (Number of pups delivered/number of implantation sites)×100
Live birth index (%) = (Number of live pups on day 0/number of pups delivered)×100
Viability index (%) = (Number of live pups on day 4/number of live pups on day 0)×100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
See 7.5.1 Repeated dose toxicity oral
300 mg/kg: death (female), clinical signs (depression of central nervous system, anesthetic action)(both sexes), decrease in body weight
(both sexes), irritating changes in gastrointestinal tract.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In offspring at 300 mg/kg, among fetuses or dead offsprings of 3 dams including the dead dam during the gestation period, generalized edema and protruding tongue were observed in 20 fetuses/offsprings and in 22 fetuses/offsprings, respectively.
In this group, increased pleural fluid was observed in 3 offsprings.
Blackish and/or dark reddish abnormal contents in the intestine were observed in 6 offsprings.
Since it is reported that ethanol similar to a monohydric alcohol (C3) of PHF affected the offspring’s intestines by embryonic exposure, this change was suspected to be test substance effects.
At 300 mg/kg, in addition to total litter loss in 3 dams including the above mentioned dystosia, the number of dead offsprings increased until 4 days after birth.
Consequently, the number of live offspring and viability index on day 4 decreased.
For these changes, the anesthetic action of the test substance was likely to work on the maternal behavior.
Moreover, although the body weight at birth was comparable to that of the control group in both sexes, the variability index and body weight on day 4 decreased.
There were no significant differences between the test substance-treated groups and control group in number of days until copulation, incidence of females without the estrous cycle, fertility index, implantation index, numbers of corpora lutea and implantation, gestation index, and number of offspring.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1 Delivery and litter data in rats treated orally with 2-Propanol, 1,1,1,3,3,3-hexafluoro- the combined repeated dose and reproductive/developmental toxicity screening test

Dose level (mg/kg) Administration period
0 10 60 300
Number of females examined 12 12 11 10

Number of females with live pups

12 12 11 9
Gestation index (%) a) 100.0 100.0 100.0 90.0
Gestation length (days)   22.3 ± 0.5  22.6 ± 0.5  22.7 ± 0.5  23.4 ± 0.7**
Number of corpora lutea 16.3 ± 2.3  17.5 ± 1.8  16.9 ± 2.8  17.7 ± 2.5
Number of implantation sites 15.2 ± 2.0  16.4 ± 1.8  15.3 ± 3.4  16.2 ± 2.4
Implantation index (%) b) 93.03 ± 4.60  93.93 ± 5.44  89.18 ± 9.10  91.78 ± 4.35
Delivery index (%) c) 95.68 ± 4.75  92.38 ± 4.38  95.40 ± 6.44  80.64 ± 18.94
Number of pups delivered 14.5 ± 2.0  15.2 ± 1.9  14.5 ± 3.4  13.3 ± 4.5
Number of live pups on day 0 14.5 ± 2.0  15.1 ± 1.9  14.3 ± 3.4  10.4 ± 4.9
Number of live pups on day 4 14.3 ± 2.1  14.9 ± 1.6  14.2 ± 3.4  5.2 ± 5.3**
Live birth index (%) d) 100.00 ± 0.00  99.44 ± 1.93  98.10 ± 3.30  70.20 ± 33.60**
Viability index on day 4 (%) e) 98.23 ± 3.28  99.07 ± 2.18  99.44 ± 1.87  39.71 ± 40.52**
Sex ratio of total number of offspring at birth (M/Total) 0.53 (92/174)  0.51 (92/182)  0.46 (73/160)  0.53 (62/117)
Sex ratio of total number of live offspring at birth (M/Total) 0.53 (92/174)  0.51 (92/181)  0.45 (71/157)  0.57 (54/94)
Sex ratio of total number of live offspring on day 4 (M/Total) 0.53 (91/171)  0.51 (91/179)  0.45 (70/156)  0.55 (26/47)
Sex ratio of total number of offspring at birth (M/Total, litter) 0.531 ± 0.098  0.497 ± 0.124  0.464 ± 0.110  0.497 ± 0.208
Sex ratio of total number of live offspring at birth (M/Total, litter ) 0.531 ± 0.098  0.499 ± 0.122  0.458 ± 0.105  0.577 ± 0.145
Sex ratio of total number of live offspring on day 4 (M/Total, litter ) 0.533 ± 0.097  0.501 ± 0.123  0.455 ± 0.103  0.650 ± 0.208
Body weight of pups (g)
on day 0 male 6.7 ± 0.4  6.9 ± 0.6  7.1 ± 0.7  6.3 ± 0.4
on day 0 female 6.3 ± 0.4  6.5 ± 0.6  6.7 ± 0.9  5.8 ± 0.3
on day 4 male 10.4 ± 0.9  10.7 ± 1.0  11.1 ± 2.2  7.1 ± 2.3*
on day 4 female 9.9 ± 1.0  10.2 ± 1.0  10.5 ± 2.2  7.4 ± 1.5*

a) Gestation index (%) = (Number of females with live pups/number of pregnant females)×100

b) Implantation index (%) = (Number of implantation sites/number of corpora lutea)×100

c) Delivery index (%) = (Number of pups delivered/number of implantation sites)×100

d) Live birth index (%) = (Number of live pups on day 0/number of pups delivered)×100

e) Viability index (%) = (Number of live pups on day 4/number of live pups on day 0)×100

Values are expressed as Mean±S.D.

Significantly different from 0 mg/kg group; * p<0.05, **p<0.01

Applicant's summary and conclusion

Conclusions:
As for the reproductive and developmental toxicity, the NOEL and NOAEL for parental animals and offspring were judged to be 60 mg/kg/day, since prolonged estrous cycle and gestation length, decreased copulation index and delivery index in dams as well as generalized edema and protruding tongue, a change in the color of the gastrointestinal contents, decreased number of live offspring, body weight, and viability index in offspring were noted in the 300 mg/kg group.
Executive summary:

In accordance with the OECD guideline on combined repeated dose and reproductive/developmental toxicity screening test, 2-propanol, 1,1,1,3,3,3-hexafluoro- (PHF) was studied for oral toxicity in rats at doses of 0, 10, 60, and 300 mg/kg/day.

At 300 mg/kg, the mean estrous cycle was prolonged to 6 days in 4 dams and the mean estrous cycle in this group was significantly prolonged. The copulation index was 83.3% (10/12 animals) at 300 mg/kg while other groups were all 100 % (12/12 animals). During the gestation period, none of the dams completed the delivery on day 22 of gestation and one of them showed dystosia as it delivered on day 25 of gestation. The gestation length was prolonged in this group. These changes were suspected to be caused by anesthetic action of the test substance. In numbers of corpora lutea, implantations, and offspring, although they were comparable to the control values, number of live offspring and delivery index decreased.

In offspring at 300 mg/kg, among fetuses or dead offsprings of 3 dams including the dead dam during the gestation period, generalized edema and protruding tongue were observed in 20 fetuses/offsprings and in 22 fetuses/offsprings, respectively. In this group, increased pleural fluid was observed in 3 offsprings. Blackish and/or dark reddish abnormal contents in the intestine were observed in 6 offsprings. Since it is reported that ethanol similar to a monohydric alcohol (C3) of PHF affected the offspring’s intestines by embryonic exposure, this change was suspected to be test substance effects. At 300 mg/kg, in addition to total litter loss in 3 dams including the above mentioned dystosia, the number of dead offsprings increased until 4 days after birth. Consequently, the number of live offspring and viability index on day 4 decreased. For these changes, the anesthetic action of the test substance was likely to work on the maternal behavior. Moreover, although the body weight at birth was comparable to that of the control group in both sexes, the variability index and body weight on day 4 decreased.

There were no significant differences between the test substance-treated groups and control group in number of days until copulation, incidence of females without the estrous cycle, fertility index, implantation index, numbers of corpora lutea and implantation, gestation index, and number of offspring.