Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

one-generation reproductive toxicity
based on test guideline (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study, conducted in accordance with the sponsor protocol, but taking into account OECD Guideines 414 and 415, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
equivalent or similar to
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
equivalent or similar to
other: OECD Guideline 414
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): 1,1,1,3,3-pentafluorobutane, HFA 365mfc
- Molecular formula (if other than submission substance): C4H5F5
- Molecular weight (if other than submission substance): 148 g/mol
- Substance type: organic
- Physical state: volatile colourless liquid
- Analytical purity: ≥99.5%
- Lot/batch No.: CV 009400
- Expiration date of the lot/batch: May 2000
- Stability under test conditions: stable under normal using conditions
- Storage condition of test material: container tightly closed in a cool, well-ventilated place, kept away from direct sunlight and other sources of heat or ignition

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males 5-6 weeks; females 10-11 weeks
- Housing: 4/sex in suspended stainless steel group cages (45 x 32 x 18 cm)
- Diet (e.g. ad libitum): commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3), ad libitum, except during the periods of exposure
- Water (e.g. ad libitum): tap water in polypropylene bottles, cleaned weakly, ad libitum, except during the periods of exposure
- Acclimation period: 2 days carantine, 6 days acclimatization

- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
unchanged (no vehicle)
Details on exposure:
- Exposure apparatus: nose-only inhalation unit
- Method of holding animals in test chamber: plastic animal holders (Battelle)
- Source and rate of air: vapours of the test material were generated by passing mass flow controlled amounts of compressed air through stainless steel evaporators in which mass flow controlled amounts of test material were injected. The amounts of air passed through were about 17.5, 17.5 and 27.0 L/min for the low, mid and high concentration test atmospheres, respectively.
- Air flow rate: ca. 100 L/min
- Air change rate:
- Treatment of exhaust air:

- Brief description of analytical method used: IR analysis at wavelength ca. 3.5 μm.

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: at the most 1 week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration of the test material in the test atmosphere of each unit was determined by IR analysis (Polytron IR ex, Dräger, Germany) at a wavelength of ca. 3.5 μm.
Duration of treatment / exposure:
Males were exposed 10 weeks and females 2 weeks prior to mating. Mated females were exposed until gestation day 15.
Frequency of treatment:
During premating: 5 d/week, 6 h/day
During mating: 6 h/day
Mated females, males and non-mated females: 6h/day
Details on study schedule:
Males were killed after the mating period and macroscopically examined. The dams were killed on gestation day 21 and macroscopically examined.
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 5000, 1500 and 30000 ppm
nominal conc.
Doses / Concentrations:
4967(22), 14905(61) and 29970 (121) ppm for males; 4971 (20), 14903 (60) and 30000 (85) ppm for females
analytical conc.
No. of animals per sex per dose:
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: the concentration levels were selected in consultation with the sponsor and were the same as used in a sub-chronic inhalatory study with 1,1,1,3,3-pentafluorobutane (TNO study no. 2187, TNO report no. V99.1121)
- Rationale for animal assignment (if not random): proportionately by body weight by computer randomization


Parental animals: Observations and examinations:
- Time schedule: daily in the morning. A group wise observation was also made during each exposure day. On exposure days, all animals were checked again in the afternoon for dead and moribund animals. During remating, animals were only checked once on Saturdays and Sundays.

- Time schedule for examinations: body weights of male rats were recorded shortly before the start of the study for randomization and weekly during the study until sacrifice. Body weights of females were recorded shortly before the start of the administration of the test substance for randomization and weekly during the premating, mating and gestation period. Non-mated females were weighed weekly until sacrifice. All animals were weighed on the day of sacrifice.

- The quantity of food consumed was measured by weighing the feeders weekly during the premating period for all animals. Food consumption of mated females was recorded weekly during gestation.


Sperm parameters (parental animals):
Parameters examined in males:
testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility,
Litter observations:
All fetuses of each litter were examined carefully for anomalies. The sex of the fetuses was determined and the anogenital distance of the fetuses was measured. Half of the fetuses was examined for soft tissue anomalies. The other half was eviscerated and examined for skeletal and carilage abnormalities. The fetuses of all groups were examined for visceral, skeletal and cartilage abnormalities.

Postmortem examinations (parental animals):
- Male animals: all male rats were examined grossly for gross abnormalities.
- Maternal animals: Mated females were killed on gestation day 21 and examined for gross abnormalities. The uteri (including the fetuses), ovaries and placentas were examined for the following parameters: number of corpora lutea, number of implanation sites, number of early and late resorptions, number of live and dead fetuses, sex of the fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities; weight of ovaries, weight of uterus, containing placentas and fetuses, weight of uterus, empty, weight of fetuses, weight of the placentas, gross evalution of the placentas

Clinical findings were evaluated by Fisher's exact probability test. Body weight, body weight gain, organ weights and food consumption were subjected to one-way analysis of variance followed by Dunnett's multiple comprison tests. Fisher's exact probability test was used to evaluate the number of mated and pregnant females, females with live fetuses and number of males that became sire. The number of corpora lutea, implantations, live and dead fetuses and early and late resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test. Fisher's exact probability test was used to evaluated the mortality data, data of the pathology of the parent females and the fetopathological screening. Sperm parameters were evaluated by ANOVA followed by Dunnett's multiple comparison test.
Reproductive indices:
For each group the following data/indices were presented: number of males placed with females, number of males mated with females, number of successul copulations, number of males that became sires, pre-coital time (calculated as time between the start of mating and successful copulation), mating index = (number of females mated/number of femaes placed with males) x 100; male fertility index = (number of males that became sire/ number of males placed with females) x 100; female fertility index = (number of pregnant females/number of females placed with males) x 100; female fecundity index = (number of pregnant females/number of females mated) x 100; pre-implantation loss = [(number of corpora lutea - number of implantation sites)/number of corpora lutea] x 100; post-implantation loss = [number of implantation sites - number of live embryos)/number of implantation sites] x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
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:
no effects observed
Gross pathological findings:
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:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): one female animal of the high-concentration group died by accident during the premating period. All other animals survived until scheduled sacrifice. Daily clinical observations during the premating, mating and gestation period did no reveal any remarkable findings in the animal's appearance, general condition or behaviour amongst the exposed and control groups.

Mean body weights were statistically significantly decreased from the 2nd and 7th week of the premating period until the end of this period for males of the hgh- and mid-concentration group, respectively. Mean body weight change was also statistically lower for several weeks in both the mid and high-concentration group compared to the control group. No statistically significant differences in mean body weights and mean body weight change were found for females during the premating period. During the gestation period a statistically significant decrease for maternal body weight change was found for the high-concentration group during the first week of gestation.
During the 2nd week of the premating period the mean food consumption of males was significantly decreased for the high-concentration group. Mean maternal food consumption was only decreased during the 1st wek of gestation in the high-concentration group. The terminal body weight of males was statistically significantly lower for the mid- and high-concentration group.

Sperm parameters recorded were motility, count and morphology of epididymal sperm and count of testicular sperm. No significant differences were observed for any sperm parameter.

At gestation day 21, 25, 24, 22 and 23 rats in the control, low-, mid- and high-concentration group, respectively, were pregnant. There were no statistically significant differences between the control group and the exposed groups concerning the number of corpora lutea, implantation sites, live and dead fetuses concerning the number of corpora lutea, implantation sites, live and dead fetuses, early and late resorptions nor concerning the pre- and post implantation loss. No statistically significant differences ingravid and empty uterus weight, ovary weight, carcass weight and net weight change of females were observed between the control group and exposed groups.

Effect levels (P0)

Dose descriptor:
Effect level:
>= 30 000 ppm
Basis for effect level:
other: No effects on fertility and development observed at the highest dose tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Six small fetuses, 1 large fetus and 1 fetus with flexed limbs were observed. No statisticall significant differences were calcualted for the individual findings nor for the findings per litter.

The mean weight of all viable fetuses was statistically significantly decreased in the hihg-concentration group compared to the control group. this was also true when taking male and female fetuses apart. However, in the high-concentration group more fetuses were born compared to the control group (9.96 vs. 9.48 fetuses per dam). Multiplying the quotient 9.96/9.48 with 4.4982 g (fetal weight of high-concentration group) gives 4.7260 g, compared to 4.7306 g for the fetal weight of the control group. Therefore, this effect regarding the mean weight of the fetuses is not a concentration effect, but follows from the non-statistically significant higher number of fetuses per litter in the high-concentration group. No statistically significant differences in mean anogenital distance and the mean placenta weight were observed.


No visceral malformations were seen in any of the groups. Visceral anomalies were limited to 1 fetus in the mid-concentration groups with small kidneys and several fetuses with a dilated urinary bladder in all groups. No statistically significant differences were found between the various concentration groups regarding visceral anomalies. Low and non-statistically significant incidences of haemorrhagic areas in liver, increased renal pelvic cavitation, bent uterus and subcutaneous haemorrhagic areas were found among all concentration groups. Lower incidences of pericard filled with haemorrhagic fluid were observed in all concentration groups in comparison to the control group. In the low-concentration group a statistically significant lower fetal incidence was found compared to the control group. On litter incidence no statistically significant difference existed.
No statistically significant differences were observed aong the control, low-, mid- and high-concentration groups in skeletal malformations and skeletal anomalies. Skeletal variations observed were irregularly ossified parietalis, inter parietalis, supra occipitalis, hyoid and one or three or more ribs, accessory lumbar ribs and one or two or more sternebrae which had an irregular shape. These skeletal variations are normal for rats and none of them was statistically significant, except the latter. The incidence of fetuses with two or more sternebrae with an irrefular shape was statistically signicant in the mid-concentration group, both on individual (4.9%) and on litter basis (23%). In the control group the incidence was 0%. However, in the high-concentration group no statistically significant increase was observed. moreover, in recent studies performed in the same institute, the incidence of irregular shape of two or more stenebrae in the control group ranges 4.1-16% and 24-52% based on fetus and litter incidence, respectively. Therefore the finding is not considered to be related to the exposure to the test substance.
Several statistically significant difference in ossification were observed between the control and the exposed groups. In the low-concentration group the frontalis, parietalis and the caudal arches showed statistically significantly advanced ossification, whereas the frontal distal phalanges showed a retardedossification in comparison to the cotrol group. In the mid-concentration group the ossification of the frontalis and caudal arches (also on litter basis) was more pronounced compared to the control group. In the high-concentration group retarded ossification was observed in the supra occipitalis (also on litter basis), rpoximal phalanges of front and hind limbs and metatarsales (also on litter basis)k whereas more pronounced ossification was observed in the sternebrae and caudal arches compared to the control group. Effects on ossification were not consistent. Moreover, in the high-concentration group the retarded ossification was most probably due to the lower fetal weight and higher number of fetuses compared to the control group. Furthermore, no other statistically significant differences were observed in the number of fetuses showing variations in skeletal ossification.
the incidence of fetuses showing a hole in the xyphoid process was significantly decreased in the hihg-concentration group. This finding is considered to be a fortuitous finding and not related to exposure. No other statistically significant difference in cartilage findings were observed in the number of fetuses showing cartilage findings.



Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion