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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
Study period:
Jan - May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research, Kalamazoo, MI
- Age at study initiation: 5-9 months on gestation day 0
- Weight at study initiation: 2.9 - 4.5 kg on gestation day 0
- Housing: Individual in suspended stainless steel cages elevated above ground corncob bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light):12

IN-LIFE DATES: From: Jan 2008 To: June 2008

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass whole body exposure chambers (1500 to 2000 liters)
- Method of holding animals in test chamber: Individual cages
- Source and rate of air: HEPA and charcoal filtered
- System of generating test substance atmosphere: Metering of gas from headspace of storage cylinder controlled by appropriate valves and flow meters
- Temperature, humidity, in air chamber: 65.3°F to 66.1°F (18.5°C to 19.0°C), mean daily relative humidity ranged from 50.0% to 58.5% during the study
- Air change rate: At least 10/hour

TEST ATMOSPHERE
- Brief description of analytical method used: Samples taken at least hourly for analysis by gas chromatography
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography
Details on mating procedure:
- Impregnation procedure: Purchased timed pregnant
Duration of treatment / exposure:
Gestation days 6-28
Frequency of treatment:
6 hours a day
Doses / concentrationsopen allclose all
Dose / conc.:
4 000 ppm
Remarks:
Group 2: Low dose.
Dose / conc.:
5 500 ppm
Remarks:
Group 3: Mid dose.
Dose / conc.:
7 500 ppm
Remarks:
Group 4: High dose.
No. of animals per sex per dose:
24
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on rabbit teratogenicity range finding study which indicated significant maternal mortality at 10000 ppm and 50000 ppm.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: Daily

FOOD CONSUMPTION:
- Food consumption for each animal was determined and mean daily diet consumption was calculated as g food/kg body weight/day.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # 29
- Organs examined: Heart, lungs, liver, kidney, brain

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: GD 0, 4, 6-29 (daily)

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # 29
- Organs examined: Uterus, liver, kidney, brain, heart, lungs, all gross lesions
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
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
Statistics:
Two tailed for a minimum significance of 5% and 1% with means and standard deviations presented. All statistical tests were done by computer with appropriate programming. Data obtained from nongravid animals were excluded from statistical analyses. Due to the different rounding conventions inherent in the types of software used, the means, standard deviations and standard errors on the summary and individual tables may differ by ±1 in the last significant figure. Where applicable, the litter was used as the experimental unit.

Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal and combined) and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-exposed groups to the control group.
Historical control data:
Included in report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Some of the animals which were found dead or killed in extremis (2 of 4 at 5500 ppm and and 3 of 7 at 7500 ppm) also exhibited labored and/or decreased respiration and/or hypoactivity prior to death or euthanasia.
No test substance related clinical findings were noted in females that survived to scheduled necropsy.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
In total, 4 and 7 females in the 5500 and 7500 ppm groups, respectively, were found dead or euthanized in extremis. All other females survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weight gain was noted in the 7500 ppm group during gestation days 12-20 with corresponding occasional reductions in mean daily food consumption. Because several females died or were euthanized prior to the scheduled necropsy, mean net body weight and net body weight change in this group were not significantly different from the control group. Mean body weights in the 7500 ppm group were similar to the control group throughout the study. A slightly lower mean body weight gain and a mean body weight loss were observed in the 5500 ppm group during gestation days 12-20 and 20-29, resulting in a lower mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated. A large mean net body weight loss was observed in this group.
Correspondingly lower mean food consumption was observed in this group during gestation days 20-29. However, mean body weights throughout the study and mean net body weight in the 5500 ppm group were similar to the control group. Slightly lower mean body weight gains were observed in the 4000 ppm group during gestation days 12-20 and 20-29, resulting in a lower mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated.
However, because there were no test substance-related effects on mean food consumption, mean net body weight or net body weight change, and there were no clinical or macroscopic findings indicative of systemic toxicity observed in this group, the lower mean body weight gain noted in the 4000 ppm group was not considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
See 'body weight and weight changes' field.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Mean gravid uterine weights were unaffected by exposure to the test substance at all concentrations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All females found dead or euthanized in extremis were gravid with normally developing implantations or fetuses in utero.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination revealed these deaths were of undetermined causation (4/5 at 5500 ppm, 6/10 at 7500 ppm), due to subacute inflammation of the heart (1/5 at 5500 ppm and 3/10 at 7500 ppm) or a result of edema/hemorrhage of the lung (1/10 at 7500 ppm). Test substance exposure was also associated with subacute inflammation in the heart in the 2500 (n=8), 4000 (n=12), 5500 (n=10), and 7500 (n=15) ppm groups, coagulation necrosis of the heart in the 7500 (n=1) ppm group, and renal tubular necrosis in the 5500 n=3) and 7500 (n=1) ppm groups. At 5500 ppm, renal necrosis was noted in 2 animals that died or were euthanized. Although not dose related, these changes were considered adverse.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
One and 3 females in the 5500 and 7500 ppm groups, respectively, aborted on gestation day 26, 28 or 29. The abortions in the 5500 and 7500 ppm groups were attributed to the test substance; no test substance-related macroscopic findings were observed in these females.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 5500 ppm had a late resorption and one female at 7500 ppm had an early resorption in utero.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
One female in the 7500 ppm group delivered on gestation day 29. The premature delivery in the 7500 ppm groups were attributed to the test substance; no test substance-related macroscopic findings were observed in these females.
Other effects:
no effects observed
Description (incidence and severity):
Corpea lutea were unaffected by exposure to the test substance at all concentrations.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEC
Remarks:
General toxicity
Effect level:
> 2 500 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
mortality
Key result
Dose descriptor:
LOAEC
Remarks:
Maternal developmental toxicity
Effect level:
> 2 500 ppm
Based on:
test mat.
Basis for effect level:
effects on pregnancy duration
number of abortions

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal/pup weight by sex and with sexes combined were not affected by test substance exposure at any concentration.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio were not affected by test substance exposure at any concentration.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean number and percent of live offspring were not affected by test substance exposure at any concentration.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related developmental variations were observed at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test substance-related developmental variations were observed at any exposure level.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related visceral malformations of the heart and/or great vessels were observed at frequencies above the WIL historical control values in the maternally toxic 5500 (2 fetus total; 2 litters) and 7500 (3 fetus total; 2 litters) ppm groups. These findings, were not statistically different from the concurrent study control group, consisted of bulbous aorta, stenotic pulmonary arch, interventricular septal defect (absent septa), absent tricuspid valve and/or interrupted aortic arch. No test substance-related developmental variations were observed at any exposure level.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
750 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: cardiovascular
Description (incidence and severity):
Exposure related visceral malformations in the heart and/or great vessels were observed in 2 fetus at 5500 and 3 fetus at 7500 ppm groups. These effects were not statistically significant and occurred at exposure levels that resulted in maternal lethality.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1.  Animals found dead, euthanized in extermis, aborted or delivered prior to scheduled necropsy

Dose (ppm) 0 2500 4000 5500 7500
found dead 0 0 0 3 (GD13, 14,27) 6 (GD12(1),14(3),18(1),29(1))
euthanized in extremis 0 0 0 1 (GD28) 1 (GD16)
Aborted 0 0 0 1 (GD28) 3 (GD26(2), 29(1))
Delivered  0 0 0 0 1 (GD29)

Attachment with body weight changes and food comsumption are included in the background information section below

Table 2. Incidence of selected histopathologic findicngs and cause of death in the rabbit

Exposure Level (ppm):
   
0
2500
4000
5500
7500
 
Sample size
24
24
24
24
24
 
# animals adverse effects
0
8
12
14
22

 
Heart

 

 

 

 

 
 
  Inflammation, subacute
0
8
12
10*
15*
 
      Minimal
-
3
1
2
2
 
      Mild
-
2
2
5
6
 
      Moderate
-
3
8
3
6
 
      Severe
-
0
1
0
1
 

 

 

 

 

 

 
 
Number of Deaths
0
0
0
5
10
 
Cause of Death

 

 

 

 

 
 
  Undetermined
-
-
-
4
6
 
  Heart Inflammation
-
-
-
1
3
 
  Lung hemorrhage and
            edema
-
-
-
0
1
 

Applicant's summary and conclusion

Conclusions:
Based on mortality, moribundity, abortions, premature delivery, lower mean body weight gain, mean body weight loss and/or lower food consumption observed at 5500 and 7500 ppm (25630 and 34950 mg/m3), an exposure concentration of 4000 ppm (18650 mg/m3) was considered to be the no-observed-adverse-effect level (NOAEC) for maternal lethality. Non statistically significant test substance-related visceral malformations in the heart and/or great vessels were observed in the 5500 (2 fetus) and 7500 ppm (3 fetus) groups only in the presence of maternal toxicity (mortality). Therefore, an exposure concentration of 7500 ppm was considered to be the NOAEL for embryo/fetal development when pregnant New Zealand White rabbits were exposed to the test substance via whole-body inhalation. These conclusions are drawn from the underlying data from this study which meets the requirements cited in OECD 414.

Histological evaluation of selected tissues (brain, heart, liver, lungs, kidneys) failed to reveal the cause of death in the majority of animals. However, cardiac inflammation were observed at 2500 ppm (11620 mg/m3) and higher. The significance of these findings is not understood.

Executive summary:

Administration of filtered air or test substance atmospheres to pregnant female New Zealand White [Hra:(NZW)SPF] rabbits, approximately 5.5 to 6 months of age, as 6-hour, daily whole-body exposures during the period of major organogenesis (gestation days 6 through 28) at concentrations of 2500, 4000, 5500, and 7500 ppm was associated with early death in the 5500 (n=5) and 7500 (n=10) ppm groups, and these deaths were of undetermined causation (4/5 at 5500 ppm, 6/10 at 7500 ppm), due to subacute inflammation of the heart (1/5 at 5500 ppm and 3/10 at 7500 ppm) or a result of edema/hemorrhage of the lung (1/10 at 7500 ppm). Test substance exposure was also associated with subacute inflammation in the heart in the 2500 (n=8), 4000 (n=12), 5500 (n=10), and 7500 (n=15) ppm groups, coagulation necrosis of the heart in the 7500 (n=1) ppm group, and renal tubular necrosis in the 5500 n=3) and 7500 (n=1) ppm groups. At 5500 ppm, renal necrosis was noted in 2 animals that died or were euthanized. Although not dose related, these changes were considered adverse.