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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid procedures and according to GLP guidelines. All parameters described are closely related or comparable to testing guideline methods.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3404 HE Zeist, the Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charlese River Deutschland, Sulzfeld, Germany
- Age at study initiation: females were 11 weeks old at mating; males were 13 weeks old at mating
- Weight at study initiation: Female weights ranged from 196.90 to 201.16 g on gestation day 0.
- Fasting period before study: None
- Housing: The rats were housed in macrolon cages with a bedding of wood shavings and strips of paper (Enviro-dri) as environmental enrichment. During the quarantine and acclimatisation period, the males were housed individually and the females were housed in groups of 4. For mating, one male and two females were housed together in type 3 macrolon cages. Mated females were housed individually in macrolon cages which were placed in a separate cage rack. The location of the mated females in the new cage racks were determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by the animal number (within each lot the mated females was housed in the order of animal number).
During exposure periods, the rats were individually housed in the exposure unit.
- Diet: The rats received a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England). Feed was provided ad libitum from the arrival of the rats until the end of the study, except during exposure.
- Water: The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Drinking water was provided ad libitum from the arrival of the rats until the end of the study, except during exposure.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- 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
Vehicle:
air
Details on exposure:
EEPOSURE EQUIPMENT:
Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections and each rodent tube section accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The location of the rats was changed weekly by rotating the animals 5 places clockwise and moving the animals from the upper section to the middle section, the animals from the middle section to the lower section and the animals from the lower section to the upper section. The units were illuminated externally by normal laboratory TL-lighting.

GENERATION OF TEST ATMOSPHERE:
- The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, a flow of cooled liquid test material controlled by a peristaltic pump was allowed to evaporate in a flow of humidified air (mass flow controlled). The air was supplemented with mass flow controlled oxygen for the mid and high concentrations to ensure a sufficiently high and equal oxygen concentration. Total test atmosphere flow was between 54 and 55 L/min for all groups except on one day, where it was between 29 and 30 L/min. At all times the flow was more than 1 L/min/animal with regard to the amount of animals present. The exposure unit for control animals was supplied with a mass flow controlled stream of humidified compressed air only.
- The measured concentrations were used in a PI feedback system to control the peristaltic pumps. The feed back system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the set point.
- The settings of the mass flow controllers were checked each morning at the start of the generation and subsequently at regular intervals during exposure (approximately bi-hourly, i.e. three times a day).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ACTUAL CONCENTRATIONS:
- The actual concentration of the test material in the atmospheres was measured by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and were passed to total carbon analyzers. The response of the analyzers was recorded on a pc every minute using a CAN transmitter.
- The daily mean response was calculated by averaging values read every minute.
- The total carbon analyzers were calibrated by sampling from 3 concentrations (in duplicate) in a range including the target concentration. The concentrations of the test material used to calibrate the total carbon analyzer were prepared in a sample bag by injecting known amounts (by mass) of the (cooled) test material.
Details on mating procedure:
At the start of mating, 2 nulliparous females were caged with each male for mating until a sperm positive vaginal smear was detected. Every consecutive morning vaginal examinations were made to ascertain copulation by detection of sperm cells in a vaginal smear. Upon evidence of copulation, positive females were housed individually. The day a sperm-positive smear is detected was considered as gestation day 0. The mated females were distributed over the 4 experimental groups in such a way that the animals from the same day of pregnancy were, as far as possible, equally distributed over all groups. Females mated by the same male were placed in different groups.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
From gestation day 6 up to and including gestation day 19.
Duration of test:
In-life termination of female rats occurred on gestation day 21.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 4000, 10000 and 15000 ppm
Basis:
other: Target concentrations
Remarks:
Doses / Concentrations:
0, 3988, 9981 and 14906 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
24 pregnant female rats
Control animals:
yes
Details on study design:
- Dose selection rationale: The concentration levels were selected in consultation with the sponsor and were the same as used in the sub-chronic (90-day) inhalation toxicity study in rats (TNO Triskelion study V8964).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily and, if necessary, the animal was handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

CLINICAL OBSERVATIONS DURING EXPOSURE:
- Time schedule: A group-wise observation was made halfway through each exposure day.

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation days 0, 3, 6, 9, 12, 15, 19 and 21.

FOOD CONSUMPTION: Yes
- The food consumed for each mated female was measured over the periods: gestation days 0-3, 3-6, 6-9, 9-12, 12-15, 15-19 and 19-21.
- The results were expressed in g per animal per day and g per kg body weight per day.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri (including the fetuses), ovaries and placentas of all females.
Ovaries and uterine content:
The uteri (including the fetuses), ovaries and placentas of all females killed on gestation day 21 were examined for the following parameters: number of corpora lutea, number of implantation 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 empty uterus, weight of live fetuses, weight of the placentas of live fetuses, gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: [all]
- Soft tissue examinations: Yes [half per litter ]
- Skeletal examinations: Yes [half per litter ]
Statistics:
The resulting data were analyzed using the methods mentioned below. As a level of significance was considered: p< 0.05.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live fetuses.
- Number of corpora lutea, implantation sites, 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.
- Mortality data, data of the pathology of parent females and the fetopathological screening were evaluated by the Fisher’s exact probability test.
Indices:
As far as applicable for each group the following indices were recorded:
- 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 fetuses)/number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- Clinical signs and mortalities: Daily clinical observations during gestation did not reveal any remarkable findings in animals’ appearance, general condition or behaviour among the test and control groups. None of the animals died during the study.

- Body weight and body weight change: No statistically significant differences in body weight and body weight change were observed between the females of the test groups and the control group.

- Food consumption: Food consumption (both g/kg body weight/day and g/animal/day) of the test groups did not differ from the control group.

- Reproduction and litter data: In each group, 24 females were mated. A total of 18, 17, 20 and 18 females of the control, low-, mid-, and high-concentration test substance groups, respectively, appeared to be pregnant. One female of the control group had no viable fetuses including a late resorption. No statistically significant differences were observed in the gestation index and female fecundity index among the groups. No statistically significant differences were observed in the number of corpora lutea, implantation sites, pre- and post-implantation loss, live and dead fetuses, resorptions and sex ratio between test groups and control group.

- Macroscopic findings in dams at necropsy: No statistically significant differences were observed in the incidence of parental necropsy observations among the groups. The findings observed were incidental and not related to treatment.

- Organ weights and net weight change: The mean weight of the gravid uterus, carcass, net weight change from day 0, empty uterus and ovaries of the groups exposed to the test substance did not differ from the control group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
15 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
10 000 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: A statistically significant increase was observed in the incidence of a dilated urinary bladder in fetuses of the high-concentration (15,000 ppm) group.

Details on embryotoxic / teratogenic effects:
- Fetal external observations: There were no treatment-related statistically significant differences in fetal external observations. One female of the low-concentration test substance group showed a fetus with a soft skull, malformed ears and subcutaneous oedema. Because no similar effect was observed in the mid- and high-dose groups, these findings were considered as incidental and not related to treatment. At skeletal examination of the one female fetus of the low-concentration test substance group, only incomplete ossification of the frontal, parietal, interparietal and supra occipital bones was observed. Other fetal external observations that occurred at low incidences in all groups included small fetuses and small subcutaneous haemorrhages/petechiae on the skin. These incidental observations are common for this strain and not related to treatment.

- Findings of the placenta: In one female of the control group, a large placenta and an amnion sac filled with red fluid were observed with a late resorption. This female had no viable fetuses. No treatment-related findings of the placenta were observed.

- Fetal and placental weight: No statistically significant differences were observed on placenta and fetal weight among the groups.

- Visceral malformations: No visceral malformations were observed.

- Visceral anomalies: A statistically significant increase was observed in the incidence of a dilated urinary bladder in fetuses of the high-concentration test substance group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses of the test substance. Therefore this finding is considered to be a treatment-related effect. One fetus of the mid-concentration group showed diverticulum of the intestines. This finding occurred in the mid-concentration group only; therefore, this finding was considered not to be a treatment-related effect. No other statistically significant effects in the incidence of visceral anomalies were observed among the groups.

- Visceral variations: Visceral variations in all groups included haemorrhagic areas in the oral cavity, haemorrhagic areas in the nasal cavity, folded retinas, not well-defined soft lenses, pericard and stomach filled with haemorrhagic fluid, and kinked and bent ureters. The incidences of folded retina and stomach containing haemorrhagic fluid were statistically significantly decreased in the high-concentration test substance group and the low-concentration group, respectively. Overall, the total incidence of fetal visceral variations was decreased in the low- and high-concentration groups compared to the control group.

- Skeletal malformations: No skeletal malformations were observed.

- Skeletal anomalies: No skeletal anomalies were observed.

- Skeletal variations: Skeletal variations were seen in parietal and interparietal bones (supernumerary), supraoccipital bones (holes), ribs (accessory lumbar ribs), and sternebrae (irregular ossification of one or irregular shape of one or more) and caudal bodies (one or two supernumerary). No statistically significant differences were observed in skeletal variations among the groups.

- Skeletal retardations: A few statistically significant differences were observed in the incidences of fetuses and/or litters showing skeletal retardations after treatment with the test substance: (1) Decreased fetal incidence of three or more incompletely ossified caudal arches in the low- and mid-concentration groups. (2) Decreased fetal incidence of 1-2 incompletely ossified metacarpals in the low-concentration group. The observed statistically significant differences in retardations of fetal skeletons were incidental, inconsistent and/or not dose-related. This kind of variation in skeletal ossification is considered as normal developmental variability. No other indications of developmental toxicity were observed. Therefore, these variations are considered to be not treatment-related.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the increased incidence of dilated urinary bladders in fetuses of the high-dose group (15000 ppm or 79650 mg/m3) was considered to be a treatment-related effect. Therefore, the NOEL for developmental toxicity was determined to be 10000 ppm (53100 mg/m3). Maternal toxicity was not observed up to and including the highest dose tested. Therefore, the NOEL for maternal toxicity was determined to be 15000 ppm.
Executive summary:

In a developmental toxicity study performed according to OECD 414 and GLP, 24 pregnant Wistar rats per dose group were exposed by nose-only inhalation from Gestation Day (GD) 6 to GD 19. The animals were exposed every day to clean air (air control) or to the test substance at exposure concentrations of 4,000, 10,000 or 15,000 ppm for 6 hours per day. Inhalation of the test substance did not induce any relevant changes in the condition or macroscopic findings of the dams. No treatment-related effects were observed on body weight, body weight gain and food consumption of the females exposed to the test substance. Gestation index, female fecundity index, the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead fetuses, resorptions and sex ratio after exposure to the test substance did not differ from none-treated controls. Parental necropsy did not reveal any differences in reproductive organ weights and macroscopic findings. Fetal external- and placental observations and weights did not reveal any treatment-related effects. A statistically significant increase in the incidence of dilated urinary bladders was observed in fetuses of the high-dose group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses. Therefore this finding is considered to be a treatment-related effect. No treatment-related effects were observed on visceral malformations and variations, and on skeletal malformations, anomalies, variations, and retardations. In conclusion, the increased incidence of dilated urinary bladders in fetuses of the high-dose group was considered to be a treatment-related effect. Therefore, under the conditions of this study, inhalation of the test substance at concentrations up to 10,000 ppm during gestation of females was tolerated without obvious signs of developmental toxicity. Under the conditions of this study, inhalation of the test substance at concentrations up to 15,000 ppm during gestation of females was tolerated without obvious signs of maternal toxicity.