Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The developmental toxicity study was performed to comply with product stewardship as well as other global regulatory requirements.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The deviations observed are considered not to have affected the validity and integrity of the study. See any other information on material and methods for details
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The deviations observed are considered not to have affected the validity and integrity of the study. See any other information on material and methods for details.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Purity: >99.9%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at arrival: Males were approximately 10-11 weeks, females were approximately 9-10 weeks
- Weight at study initiation: GD 0 body weights ranged from 183.8-225.3 grams at grouping
- Housing: During the premating period the rats were housed in groups of four per cage, separated by sex. Cages with male animals were distributed between the cages holding females in the same rack. For mating one male and two females were housed together. Mated females were housed individually in macrolon cages. During the daily exposure periods the rats were individually housed in the exposure unit. Immediately after each exposure, the animals were returned to their home cages. When not exposed animals were housed in macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment.
- Diet: ad libitum, except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: At least 5 days prior to the start of the mating

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45-65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton Systems, Inc., Aberdeen, MD, USA
- Method of holding animals in test chamber: During exposure, animals were housed individually in Type II Macrolon cages. Each whole body chamber could accommodate 60 cages. The places that were not filled with animal cages, were filled with plastic blocks to reduce the chamber volume and thereby the amount of test material required. Animals were rotated at least every week with respect to their position in the exposure chamber.
- Source and rate of air: 10 times/hour
- Temperature, humidity, pressure in air chamber: 22 ± 3ºC, 30 and 70%
- Air flow rate: The average total air flows (± standard deviation) were 262 (± 2), 267 (± 3), 243 (± 2), 244 (± 7) and 258 (± 3) L/min for the control, low-, mid-, high-, and top concentration group, respectively.

Generation of the test atmosphere:
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmospheres were generated by mixing a mass flow controlled stream of gaseous test material with a controlled flow of clean air, available as a laboratory provided source of (non-pressurized) filtered air (HEPA filter). The readings of the mass flow controllers were checked at regular intervals (approximately each hour). The resulting test atmosphere was directed to the inlets at the top of the exposure chamber; the atmosphere was exhausted at the bottom. A heating jacket was used to compensate the heat of evaporation of the liquefied gas from the cylinder containing the test material. The flow of test atmosphere was controlled using a constant volume controller and was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor (type CTV110-AOD150). The air flow was continuously measured and recorded on a PC every minute using a CAN transmitter. The animals were placed in the exposure chamber prior to the start of the test atmosphere generation. Test atmosphere generation was stopped six hours after the start of generation. The animals were removed from the exposure chamber after the concentration had dropped below a level of 1% of the target concentration. The exposure unit for the control animals was supplied with a controlled flow filtered air only. Prior to the first exposure of the animals, homogeneous distribution of the test substance in the exposure chambers was confirmed by analysis of samples taken taken at five different locations in each exposure chamber (deviation of individual values from the mean of all five samples should not exceed 10%; the actual maximum deviation was 2.6% for the top concentration group).

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test substance in the test atmospheres was measured by total carbon analysis. Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line. The response of the analysers was recorded on a PC every minute using a CAN transmitter. The responses of the analysers were converted to concentrations by means of calibration graphs (the formula’s used to convert responses into concentrations are given below). For each exposure day, the mean concentration was calculated from the values determined every minute in the period 30 minutes after the start until the end of the generation of the test atmosphere
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall average actual concentrations (± standard deviation) of the test material in the test atmospheres as determined by total carbon analysis were 984 (± 31), 4992 (± 115), 7535 (± 205) and 14783 (± 897) ppm for the low-, mid-, high-, and top concentration, respectively. These concentrations were close to the respective target concentrations of 1000, 5000, 7500 and 15000 ppm.
Details on mating procedure:
One hundred and forty untreated females were mated with 70 untreated males (two females were caged with one male) in order to obtain 24 mated females per group. Animals were caged together until mating occurred. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0 (GD 0). Upon evidence of copulation the females were caged individually. Females mated by the same male were placed in different groups. The remaining females were discarded after mating without further examination
Duration of treatment / exposure:
Beginning on gestation day (GD) 6 through GD 19
Frequency of treatment:
Daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
5 000 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
15 000 ppm
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours (pre-dosing on exposure days), animals were also observed about halfway through the 6-hour exposure period and also thoroughly checked again after exposure.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days (GD) 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes; Gestation days (GD) 0-6, 6-9, 9-12, 12-15, 15-18 and 18-21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Whole body

Other: The following maternal tissues were preserved after dissection: Adrenals, pharynx, aorta, parotid salivary glands, axillary lymph nodes, pituitary, brain (brain stem, cerebrum and cerebellum), rectum, caecum, skeletal muscle (thigh), colon, skin (flank), eyes (with optic nerve), small intestines (duodenum, ileum, jejunum), exorbital lachrymal glands, spinal cord (three levels), femur with joint, spleen, heart, sternum with bone marrow, kidneys, stomach (glandular, non-glandular), liver, thymus, lungs/trachea/larynx, thyroid, mammary glands, tongue, mandibular (cervical) lymph nodes, tracheobronchial (mediastinal) lymph nodes, nasal passages (with teeth), ureter, peripheral sciatic nerve, urinary bladder, oesophagus, uterus (with cervix), olfactory bulb, ovaries, pancreas, parathyroids, and all relevant gross lesions. These organ were fixed in a neutral, aqueous phosphate buffered 4% solution of formaldehyde and were not examined microscopically.
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: Yes
- Number of late resorptions: Yes
- Other: For each female with visible malformed fetuses and fetuses with external abnormalities, the number of live and dead fetuses, early and late resorptions were recorded. Gross evaluation of placentas, and abnormal tissues or organs in dams were examined.
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter
Statistics:
For continuous data, the Shapiro Wilks data for normality was run. If the data were normally distributed Levene’s test was run. If the continuous data were not normally distributed, logarithmic transformation of the values was performed. Levene’s test was followed by ANOVA or Kruskal-Wallis. If ANOVA was significant, Dunnett or Tukey Kramer were run. If Kruskal Wallis was significant, Dunn’s or Wilcoxon’s test was run. For dichotomous data, Chi-Square Standard or Exact followed by Fisher Exact or Cochran-Armitage was run. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 or p<0.01. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 or p<0.01.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pre-dosing signs: All rats survived until scheduled necropsy on GD 21. The few clinical signs noted were related to the skin (encrustations and sparsely haired area) represented normal background findings in of rats of this strain and age. Therefore, these findings were not ascribed to the exposure to the test material.
Signs during exposure: Restlessness was observed in animals of the top concentration group during group-wise observation about halfway through exposure. No further clinical abnormalities were noted during exposure.
Post-dosing signs: There were no treatment-related clinical abnormalities directly after exposure.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences in body weights among the groups were observed. In the top concentration group body weight gain was statistically significantly lower during the complete gestation period and a loss was observed during GD6-9 after the start of exposure on GD6. The slightly lower body weight gain in the top concentration level was considered to be related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the top concentration group a statistically significant decreased food consumption was observed from GD6-9 and from GD9-12. This was considered to be related to the start of exposure and recovered thereafter. In the low, mid and high concentration groups food consumption was comparable to the control group. A transient lower food consumption in the top concentration group from GD6-12 was considered to be related to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant changes in the mean weight of the full uterus (containing placentas and foetuses), mean empty uterus weight or in mean ovary weight. The net weight change in carcass weight (from GD 0) was statistically significantly lower in the low concentration group and the top concentration group as compared to controls. In absence of a dose-response relationship, the effect in the low dose was not considered treatment-related. Taking into account the observed effects on body weight gain and food consumption in the top concentration group, a relation to treatment was considered.
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
On dead foetus was observed in the top concentration group, no dead foetus were observed in the other groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
7 500 ppm
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean placenta weight was statistically significantly reduced in the top concentration groups in both male and female fetuses. Foetus weight was also statistically significantly reduced in both male and female foetuses in the top concentration group as compared to the control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was comparable in all groups (expressed as the percentage of male littermates, 48.3%, 46.5%, 48.9%, 52.8% and 46.8% for the control, low, mid, high and top concentration groups, respectively
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One foetus in the low concentration group showed protrusion of the tongue at external observations. Upon visceral examination the nasal septum was not fused with the palate in this animal. A single foetus in the high concentration group showed a bent limb. Variations were related to general observations (subcutaneous haemorrhage, small size, distended abdomen and hyperflexion of a limb) and occurred in single incidences. Based on the incidences in single foetuses, these observations were not treatment related.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Two foetuses from one litter in the top concentration group were lost during processing (coloring) for skeletal examinations. This was related to the very small size of the fetuses (foetus weight were 2.989 and 3.282 grams, respectively), which caused the skeleton to fall apart. One animal in the low concentration group showed multiple skeletal malformations, including three or more fused ribs and a fused thoracic arch. No other malformations were observed. Variations observed were mainly related to the ossification of the bones. In the top concentration group a statistically significant increase in the number of litters comprising fetuses showing incomplete ossification of one or two bodies was observed and also an increase in the percentage fetuses per litter that showed incomplete ossification of the metacarpals in the forelimbs. In addition, although not statistically significant, the number of fetuses showing incomplete ossification or no ossification of the digits in the fore- and hindlimbs, metatarsals in the hind limb, and incomplete ossification of the sternebrae were increased in the top concentration group. These findings are indicative for a retardation in ossification and are considered related to treatment. Based on the incidence and distribution of the skeletal observations no treatment related effects were observed in the low, mid and high concentration groups. In the top-concentration group a reduced ossification was observed.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One foetus in the control group showed situs inversus and one foetus in the low concentration group showed agenesis of the eyes. No further malformations were observed. Variations that were observed included variations in the lens and retina of the eye and ureters, dilatation of the ventricles in the brain, dilatation of the renal pelvis, distension of the urinary bladder and general observations (fluid filled abdomen and small fetus size). Based on the above incidences and distributions of the observations, it was concluded that visceral examination did not reveal any exposure-related effects.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
7 500 ppm
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: forelimb
skeletal: sternum
skeletal: hindlimb

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:
Based on the effects observed on body weight gain during gestation and a transient effect on food consumption in the top-concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was placed at the high concentration (7500 ppm). Based on the effects observed on foetal weight and placental weight, accompanied by a retardation in ossification observed in the foetuses in the top-concentration group, the No Adverse Effect Concentration (NOAEC) for developmental toxicity was placed at the high concentration (7500 ppm).
NOAEC (maternal and developmental): 7500 ppm
Executive summary:

The objective of this study was to provide data on the possible effects of the test substance in pregnant female rats, and on the development of the embryo and foetus. The test material was administered by inhalation (whole body exposure) to groups of 24 mated females from gestation day (GD) 6 up to and including GD 19. The target concentrations in the air were 1000, 5000, 7500 and 15000 ppm. Animals of a control group were exposed to clean air only. At Caesarean section on GD 21, dams and foetuses were examined macroscopically, and foetuses, placentas and maternal reproductive organs were weighed. Half of the foetuses of each litter were subjected to visceral examination, the other half to skeletal examination. The overall average actual concentrations of the test material in the test atmospheres as determined by total carbon analysis were 984, 4992, 7535 and 14783 ppm for the low-, mid-, high-, and top concentration, respectively and close to the respective target concentrations of 1000, 5000, 7500 and 15000 ppm.

 

Maternal data:

There was no mortality, and daily observations revealed no treatment-related predosing or post-dosing clinical signs.

Maternal body weights and food intake were not affected by the exposure in the low, mid and high concentration group. In the top-concentration group a lower body weight gain during gestation was observed and a reduced food intake after the start of exposure from gestation day (GD) 6-12 was noted.

Macroscopic examination of the dams did not reveal exposure-related changes.

There were no effects on the mean number of corpora lutea and pregnancy status.

There were no changes in the weights of the uterus (containing placentas and foetuses), the empty uterus and the ovaries.

Based on these findings, the no-observed-adverse-effect concentration (NOAEC) for maternal toxicity was placed at the high-dose concentration (7500 ppm).

 

Foetal examination:

There were no exposure-related effects on the number and distribution of implantation sites, early and late resorptions and live and dead foetuses. No effects were observed on post-implantation loss.

Mean foetus weight and mean placenta weight were decreased in the top concentration group for both male and female foetuses.

External and visceral examination of foetuses did not reveal any treatment related effects.

Skeletal examination showed reduced ossification in the foetuses in the top concentration group, which was indicative for growth retardation and considered to be related to the lower foetus weight in this group.

Based on the effects observed on body weight gain during gestation and a transient effect on food consumption in the top-concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was placed at the high concentration (7500 ppm). Based on the effects observed on foetal weight and placental weight, accompanied by a retardation in ossification observed in the foetuses in the top-concentration group, the No Adverse Effect Concentration (NOAEC) for developmental toxicity was placed at the high concentration (7500 ppm).