Polyhaloalkene

Developmental toxicity / teratogenicity

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

Test material information

Constituent 1

Reference substance name:

754-12-1

Cas Number:

754-12-1

IUPAC Name:

754-12-1

Constituent 2

Reference substance name:

-

EC Number:

468-710-7

EC Name:

-

Cas Number:

754-12-1

Molecular formula:

C3H2F4

IUPAC Name:

2,3,3,3-tetrafluoroprop-1-ene

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 / concentrations

Doses / concentrations 1

Dose / conc.:

2 500 ppm

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

4 000 ppm

Remarks:

Group 3 and 5. Phase I. Two test substances manufactured by different supplier

Doses / concentrations 3

Dose / conc.:

5 500 ppm

Remarks:

Group 3 and 5. Phase II. Two test substances manufactured by different supplier

Doses / concentrations 4

Dose / conc.:

7 500 ppm

Remarks:

Group 4

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):

No test substance-related clinical findings were noted females that survived to the scheduled necropsy at the daily examinations, during mid-point of exposure or 1 hour following exposure at any exposure concentration. Findings noted in the test substance-treated groups, including hair loss on various body surfaces, occurred infrequently or, at similar frequencies in the control group, and/or were observed prior to the exposure period or in a manner that was not dose-related.

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.
On the day of or prior to death or euthanasia, 1 and 3 females in the 5500 and 7500 ppm groups, respectively, had labored and/or decreased respiration, 2 females in the 5500 ppm group were hypoactive, 1 female in the 7500 ppm group had clear material around the nose and 1 female in the 7500 ppm group had red material on the left hindlimb. In addition, the female in the 5500 ppm group had red material on the urogenital area on the day prior to euthanasia and 1 occurrence of labored respiration several days prior to death. Aside from a single occurrence of decreased defecation in 1 female in the 7500 ppm group, no other noteworthy clinical findings were observed during the exposure period for females that were found dead or euthanized in extremis. The only noteworthy finding for the aborted females was 1 or 11 occurrences of decreased defecation noted in 3 females in the 7500 ppm group. The abortions and premature delivery were attributed to the test substance. All other females survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean maternal body weight gain in the 7500 ppm group was similar to the control group during gestation days 6-9 and 9-12. A significantly (p<0.01) lower mean body weight gain was observed in this group during gestation days 12-20 relative to the control group. Mean body weight gain in the 7500 ppm group was similar to the control group during gestation days 20-29, presumably because the most sensitive animals had died. As a result of the lower mean body weight gain during gestation days 12-20, mean body weight gain in the 7500 ppm group was slightly lower (not statistically significant) when the entire exposure period (gestation days 6-29) was evaluated. The decrements in mean body weight gain in this group were not of sufficient magnitude to result in lower mean body weights. Mean net body weight and gravid uterine weight in this group were similar to the control group.
Mean body weight gains in the 5500 ppm group were similar to the control group during gestation days 6-9 and 9-12. A slightly lower mean body weight gain was observed in this group during gestation days 12-20 (significant, p<0.05, during gestation days 12-13 only), and a significant (p<0.01) mean body weight loss was noted during gestation days 20-29. The decrements in mean body weight gain in the 5500 ppm group resulted in a significantly (p<0.01) lower mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated. A significant (p<0.01) mean net body weight loss (174.8 g) was also observed in this group when compared to the control group. Although there was no apparent exposure-related trend, the effects on mean body weights during the last week of exposure and mean net body weight loss were considered test substance-related because the most sensitive population in the 7500 ppm group had been eliminated by this point. However, the mean body weight loss and lower mean body weight gain in the 5500 ppm group were not of sufficient magnitude to result in lower mean body weights or a lower mean net body weight. Mean gravid uterine weight in this group was similar to the control group value.
Mean body weight gain in the 4000 ppm group was similar to the control group during gestation days 6-9 and 9-12. Slightly (not statistically significant) lower mean body weight gains were noted in this group during gestation days 12-20 and 20-29, resulting in a significantly lower (p<0.05) mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated. Although the slight reduction in mean body weight gain in the 4000 ppm group was considered test substance-related, mean body weights in this group were similar to the control group throughout the exposure period, mean net body weight was similar to the control group value, and only a slight (not statistically significant) mean net body weight loss was noted. Therefore, the slightly lower mean body weight gain in the 4000 ppm group was not considered adverse.
Mean body weights, body weight gains, net body weight, net body weight gain and gravid uterine weight in the 2500 ppm group were unaffected by exposure to the test substance. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, in 7500 ppm group was generally similar to the control group throughout the study. However, significantly (p<0.05 or p<0.01) lower mean food consumption was observed in this group relative to the control group during several daily intervals (gestation days 13-14 [g/kg/day only], 18-19, 22-23 and 23-24).
Mean food consumption in the 5500 ppm group was similar to the control group during gestation days 6-9, 9-12 and 12-20, with the following exception. Significantly (p<0.05 or p<0.01) lower mean food consumption was noted compared to the control group during gestation days 18-19. During gestation days 20-29, food consumption in the 5500 ppm group was significantly (p<0.01) lower compared to the control group; the lower food consumption corresponded to a mean body weight loss noted during this same period.
Mean food consumption in the 2500 and 4000 ppm groups was similar to that in the control group throughout the exposure period. No statistically significant differences were noted.

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:

effects observed, treatment-related

Description (incidence and severity):

Four and 7 females in the 5500 and 7500 ppm groups, respectively, were found dead or euthanized in extremis during the study. Dark red areas or dark red discoloration in the lungs were noted in 3 and 2 females in these respective groups, and 1 female each in the 5500 and 7500 ppm groups had dark red areas in the thymus gland. In addition to these findings, 1 female in the 5500 ppm group had green contents in the uterus and 1 female in the 7500 ppm group had dark red areas in the spleen. Another female in the 7500 ppm group had brown discoloration in the lungs, red fluid contents and white material in the thoracic cavity, a small lobe of the liver and dark red areas in the kidneys.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was subacute inflammation in the heart of animals exposed to ≥ 2500 ppm with increased incidence and severity with increasing dose. Subacute inflammation was not present in control group animals. The inflammation was characterized by a mixture of infiltration of lymphocytes and heterophils, degeneration of cardiac myofibers, expansion of endomyseal basophilic ground substance, and fibrosis. The lesion was multifocal throughout the left and right ventricles, and occasionally present in papillary muscles and atrial walls.
One 7500 ppm group female had moderate locally extensive necrosis of a papillary muscle of the heart, which was characterized by coagulation necrosis without inflammation.
The kidney had multifocal discrete foci of necrosis of the cortical renal tubules in the 5500 and 7500 ppm dose groups. The incidence of this change did not follow a dose response profile. Necrotic tubules had intact basement membrane lined by remnants of eosinophilic cytoplasm within the lumen. There was no remaining cellular architecture. Rarely, there were pyknotic nuclei and/or cellular debris. The affected tubules were interpreted to be proximal convoluted tubules, but in the absence of recognizable architecture, this identification of tubule type is not definitive. Tubules lying adjacent to necrotic tubules consisted mostly of distal tubules, and were intact and normal. Necrosis of kidney tubules appeared similar to the necrosis of the kidney tubules in early death animals, and this change was not present in control group animals.
There were no test substance-related findings in the lungs. There were a variety of findings including congestion, alveolar hemorrhage, and subacute inflammation in all treatment groups, however, these findings did not show a clear dose response profile and were also present in control animals. Subacute inflammation was characterized by multifocal discrete foci with intra-alveolar neutrophils and alveolar macrophages, and thickened hypercellular alveolar walls with occasional prominence of alveolar epithelial cells. The slight increase in incidence and/or severity of these changes in treated animals when compared to the control group represented normal biologic variability, and the changes were considered incidental.
Collections of pigmented and non-pigmented alveolar macrophages were present in the control and all test substance-exposed groups, and there was no clear test substance or dose-related response profile for this particular finding. There was an increase in severity and incidence of these macrophages in the 2500 and 4000 ppm groups only. The finding was characterized by individualized and well-demarcated clusters of densely packed macrophages, ranging in numbers from 15-50, residing within distended alveoli. In many of the macrophages, there was golden brown pigment typical of hemosiderin.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

See Table 2 in 'Any other information on results incl. tables'.

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 4 females in the 5500 and 7500 ppm groups, respectively, aborted or delivered during the study. The female in the 5500 ppm group aborted 2 viable and 5 dead fetuses with no apparent malformations and 1 viable and 2 dead fetuses that were partially cannibalized; 2 viable fetuses were noted in utero. This female also had dark red areas in the lungs. Three females in the 7500 ppm group aborted late resorptions or 1 dead fetus; 2 late resorptions in the same litter had open eyelids. The female that aborted 1 dead fetus also had 9 viable fetuses in utero. A swollen spleen and mottled liver were observed for another of the females in this group that aborted.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

All of the females that were found dead or euthanized in extremis were gravid with normally developing implantations or fetuses in utero. In addition, 1 female in the 5500 ppm group had a late resorption, and 1 female in the 7500 ppm had an early resorption in utero.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

One female in the 7500 ppm group delivered on gestation day 29 and had dark red areas in the lungs. Other macroscopic findings (accessory spleen, small gallbladder or cystic oviducts) noted in females that were found dead, euthanized in extremis, aborted or delivered are common in this species and strain.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

All other females survived to the scheduled necropsy. At the scheduled necropsy on gestation day 29, no test substance-related internal findings were observed at any exposure level. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

Intrauterine growth and survival were unaffected by test substance administration at exposure concentrations of 2500, 4000, 5500 and 7500 ppm. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights and fetal sex ratios. No statistically significant differences from the concurrent control group were noted, and values were within the ranges of the historical control data for definitive studies. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of preimplantation loss were similar across all groups.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEC

Remarks:

General toxicity

Effect level:

< 2 500 ppm

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEC

Remarks:

Maternal developmental toxicity

Effect level:

4 000 ppm

Based on:

test mat.

Basis for effect level:

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

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 206(24), 191(23), 206(24), 153(18) and 132(14) in the control, 2500, 4000, 5500 and 7500 ppm groups, respectively.

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):

External malformations were observed in 1(1), 2(1) and 1(1) fetuses (litters) in control, 2500 and 7500 ppm groups, respectively. Fetus no. 53771-01 in the 7500 ppm group had carpal flexure (bilateral) and hyperflexion of the hindpaw (bilateral); no skeletal origin as apparent for these malformations. Fetus nos. 53745-03 and 53745-10 in the 2500 ppm group had microphthalmia (unilateral). Skeletally, the orbit was smaller than normal for fetus no. 53745-10; however, there was no apparent skeletal origin for microphthalmia in the other fetus. These findings were not considered test substance-related because they occurred infrequently, not in an exposure related manner, the mean litter proportions were not statistically significantly different from the concurrent control group and/or the values were within the ranges of the historical control data. In the control group, fetus no. 54153-08 had a short tail that consisted of only 12 caudal vertebrae present and misshapen and fused caudal vertebrae.
No external developmental variations were noted at any exposure level. Four fetuses in 1 litter in the 2500 ppm group had white areas in the skin (dorsal neck or entire external surface). This finding was not classified as a malformation or developmental variation and was not included in any tabulation.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were observed in 3(2), 4(4), 0(0), 3(2) and 1(1) fetuses (litters) in
the control, 2500, 4000, 5500 and 7500 ppm groups, respectively. Vertebral anomalies
with or without associated rib anomalies were noted in 1(1), 2(2), 0(0), 2(1) and 1(1) fetuses (litters) in the same respective groups. For 1 fetus in each in the 2500, 5500 and 7500 ppm groups, this finding consisted of an extra arch, rib and/or half of centrum, forked and/or fused ribs (5500 and 7500 ppm groups), malpositioned arches and halves of centra (2500 and 5500 ppm groups) and costal cartilage from the extra rib associated with the sternum (2500 and 7500 ppm groups) resulting in malpositioning of subsequent costal cartilages (2500 ppm group). The fetus in the 7500 ppm group also had a costal cartilage anomaly consisting of costal cartilages that fused, then bifurcated prior to associating with the sternum normally. One fetus each in the control and 5500 ppm group had vertebral anomalies that consisted of an absent half of a centrum and malpositioned arches and halves of centra; the 5500 ppm group fetus also had a malproportioned arch.
One fetus in the 2500 ppm group had a vertebral anomaly consisting of a malproportioned centrum attached to an adjacent centrum, malpositioned arches and halves of centra and fused ribs. Also in the 2500 ppm group, 1 fetus had a vertebral centra anomaly consisting of centra or halves of centra that were absent, attached to an adjacent centrum or malpositioned and another fetus had a severely malaligned sternebra resulting in fused sternebrae. Two (2) and 1(1) fetuses (litters) in the control and 5500 ppm groups, respectively, had rib anomalies consisting of bifurcated and/or fused ribs and/or extra rib, costal cartilage from extra rib or anterior fork of bifurcated ribs that associated with the sternum and/or malpositioned costal cartilage. Because skeletal malformations occurred similarly in the control group, there was no exposure-response relationship, the mean litter proportions were not statistically significantly different from the concurrent control group and/or the values were within the ranges of the historical control data, the malformations observed in the test substance-exposed groups were considered spontaneous.
Skeletal developmental variations observed in all groups, including the control group, consisted of 13th rudimentary rib(s), 13th full rib(s), sternebra(e) nos. 5 and/or 6 unossified, bent hyoid arch(es) and 27 presacral vertebrae. Other skeletal developmental variations occurred similarly in the control group, were not observed in an exposure-related manner, the mean litter proportions were not statistically significantly different from the concurrent control group and/or the values were within the range of the historical control data. No relationship to the test substance was evident.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Soft tissue malformations were observed in 2(2), 0(0), 2(1), 5(4) and 3(2) fetuses (litters)
in the control, 2500, 4000, 5500 and 7500 ppm groups, respectively. In the 7500 ppm group, fetus nos. 54108-08, 54108-10 and 54135-08 had a bulbous aorta (ascending and/or arch), stenotic pulmonary trunk and an absent interventricular septum; fetus nos. 54108-08 and 54135-08 also had an absent tricuspid valve. Fetus no. 54145-01 in the 5500 ppm group also had a bulbous aorta (ascending and arch), a stenotic pulmonary trunk and an absent interventricular septum. Fetus no. 54127-05 in the same group had an interrupted aortic arch (the right and left subclavian and carotid arteries arose from the ascending aorta, with no brachiocephalic trunk). The mean litter proportions of these findings were not statistically significant compared to the concurrent control group; however, the values for bulbous aorta (0.8% and 2.4% per litter) and stenotic pulmonary trunk (0.8% and 2.4% per litter) in the 5500 and 7500 ppm groups, respectively, exceeded the maximum mean values in the historical control data (0.5% per litter for both findings). Absent tricuspid valve and interrupted aortic arch have not been observed in the historical control data. Based on the mean litter proportion and the similarity of the multiple findings (cardiovascular), the heart and great vessel anomalies in the 5500 and 7500 ppm groups were attributed to the test substance. Fetus no. 54170-01 in the 5500 ppm group had a diaphragmatic hernia (a portion of the liver protruded into the thoracic cavity through an opening in the diaphragm). Lobular agenesis of the lungs (absent right accessory lobe) was noted in 1(1), 2(1) and 2(1) fetuses (litters) in the control, 4000 and 5500 ppm groups, respectively. These malformations were not considered test substance-related because there was no exposure- response relationship and/or the finding was observed similarly in the control group. A malpositioned kidney (located more posterior than normal) was observed in fetus no. 53735-02 in the control group. No other visceral malformations were noted.
Soft tissue developmental variations noted in all groups, including the control group, consisted of retrocaval ureter, extra papillary muscle in the heart, major blood vessel variations (the left carotid artery arose from the brachiocephalic trunk or retroesophageal right subclavian artery) and accessory spleen. Other visceral developmental variations occurred infrequently, similarly in the control group and/or not in an exposure-related manner. No relationship to the test substance was evident.
Several visceral findings were not classified as either a malformation or developmental variation and were not included in any tabulation. Other findings were not attributed to the test substance because they occurred infrequently, were noted similarly in the control group or did not occur in an exposure- related manner.
See Table 3 in 'Any other information on results incl. tables'.

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

4 000 ppm

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:

yes

Lowest effective dose / conc.:

5 500 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

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 findings and cause of death in the rabbit

			Females
Exposure Level (ppm):	0	2500	4000	5500	7500
Heart ^a	24	24	24	24	24
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
Kidneys ^a	24	24	24	24	24
Necrosis	0	0	0	3	1
Mild	-           -           -			2	0
Moderate	-           -           -			1	0
Severe	-           -           -			0	1
Lungs ^a	24	24	24	24	24
Congestion	0	2	5	3	3
Mild	-	1	1	2	2
Moderate	-	1	4	1	1
Hemorrhage	2	6	3	3	1
Minimal	2	2	0	1	0
Mild	0	4	3	1	0
Moderate	0	0	0	1	1
Inflammation	4	8	8	3	4
Minimal	4	5	6	3	2
Mild	0	3	2	0	2
Alveolar macrophages ^b	2	10	7	2	3
Minimal	1	6	6	2	3
Mild	1	4	0	0	0
Moderate	0	0	1	0	0
Cause of Death	0	0	0	5	10
Undetermined	-	-	-	4	6
Heart Inflammation        -		-	-	1	3
Lung hemorrhage and   -		-	-	0	1

            edema 

^a = Number of tissues examined from each group.

^b = Included the incidence of pigmented and non-pigmented macrophages

Table 3. Visceral Findings Not Classified As Malformations Or Developmental Variations

	Number of Fetuses Affected
Finding	0 ppm	2500 ppm	4000 ppm	5500 ppm	7500 ppm
Renal papilla not fully developed (Woo and Hoar grade 1)	1	1	-	-	-
Cyst(s)	1a	1b	-	2c	-
Thymus gland - dark red areas or dark red discoloration	-	3	-	-	-
Distended stomach	-	1	-	-	-
Thoracic cavity - dark red contents	1	1	-	-	-
Thoracic cavity - fluid contents or fluid-filled	-	3	-	1d	1d
Abdominal cavity - fluid contents or fluid-filled	-	4	-	2d	1d
- = Not observed. ^a = On gallbladder. ^b = On diaphragm. ^c = On oviduct(s). ^d = One fetus with heart and great vessel malformations.

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, mortality, moribundity, abortions, premature delivery, lower mean body weight gain, mean body weight loss and/or lower food consumption were observed at 5500 and 7500 ppm and test substance-related, adverse microscopic findings were noted in all exposure groups that consisted of subacute inflammation of the heart at ≥ 2500 ppm, coagulation necrosis of the heart at 7500 ppm, and renal tubular necrosis at ≥ 5500 ppm. Therefore, an exposure concentration of < 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on the abortions observed at 5500 and 7500 ppm, the maternal developmental NOAEL has been set at 4000 ppm. Because test substance-related visceral malformations in the heart and/or great vessels were observed in the 5500 and 7500 ppm groups in the presence of maternal toxicity, an exposure concentration of 4000 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.

Executive summary:

The objectives of the study were to determine the potential of the test article to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. Five groups of time-mated female New Zealand White rabbits (12/group per phase; approximately 5.5-6 months of age at initiation of exposure) were exposed to either clean filtered air or vapor atmospheres of the test substance for approximately 6 hours daily in whole-body inhalation chambers during gestation days 6-28. Because of the limited amount of space available in the exposure chambers, each group was divided into 2 phases, with the following exception. The 4000 and 5500 ppm test substance concentrations were administered only during Phase I and Phase II, respectively. Target test substance concentrations were 0, 2500, 4000, 7500 and 4000 parts per million (ppm) for each phase, with the following exception. Because no toxicity was noted at 4000 ppm in Phase I, the sponsor elected to increase the mid-exposure concentration to 5500 ppm for Phase II. In order to determine if there were any differences in the test substance manufactured by a different supplier, the 4000 and 5500 ppm groups were divided into 2 subgroups each, exposed to the test substance supplied by either the sponsor or the other manufacturer. Overall mean measured exposure concentrations were 2504, 3982, 7512 and 4013 ppm for the 2500, 4000, 7500 and 4000 ppm groups, respectively, in Phase I and 2479, 5408, 7441 and 5479 ppm for the 2500, 5500, 7500 and 5500 ppm groups, respectively, in Phase II.
All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 29, a laparohysterectomy was performed on each surviving female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. In addition, microscopic examination of the brain, heart, kidneys, liver, and lungs was performed for all females. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.
Four and 7 females in the 5500 and 7500 ppm groups, respectively, were found dead or euthanized in extremis. Labored and/or decreased respiration and/or hypoactivity were observed prior to death or euthanasia for 2 and 3 females in the same respective groups. One and 3 females in the 5500 and 7500 ppm groups, respectively, aborted on gestation day 26, 28 or 29, and 1 female in the 7500 ppm group delivered on gestation day 29; these deaths were of undetermined causation, due to subacute inflammation of the heart, or a result of edema/hemorrhage of the lung. The mortality, moribundity, abortions and premature delivery in the 5500 and 7500 ppm groups were attributed to the test substance; no test substance-related macroscopic findings were observed in these females. All other females survived to the scheduled necropsy.
No test substance-related clinical findings were noted at the daily examinations or at mid-point of or 1 hour following exposure at any concentration.
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 the most sensitive 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. Mean gravid uterine weights were unaffected by exposure to the test substance at all concentrations.
Intrauterine growth and survival were not affected by test substance exposure at any concentration. However, test substance-related (not statistically significant) visceral malformations of the heart and/or great vessels were observed at frequencies above the historical control values in the maternally toxic 5500 and 7500 ppm groups. These findings, 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.
The histopathological evaluation and interpretation of these data were completed after issuance of the final report. Microscopic examination revealed subacute inflammation in the heart in the 2500, 4000, 5500 and 7500 ppm groups, coagulation necrosis of the heart in the 7500 ppm group, and renal tubular necrosis in the 5500 and 7500 ppm groups. All of these changes were considered related to test substance administration and considered adverse.
Based on the results of this study, mortality, moribundity, abortions, premature delivery, lower mean body weight gain, mean body weight loss and/or lower food consumption were observed at 5500 and 7500 ppm and test substance-related, adverse microscopic findings were noted in all exposure groups that consisted of subacute inflammation of the heart at ≥ 2500 ppm, coagulation necrosis of the heart at 7500 ppm, and renal tubular necrosis at ≥ 5500 ppm. Therefore, an exposure concentration of < 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Because test substance-related visceral malformations in the heart and/or great vessels were observed in the 5500 and 7500 ppm groups in the presence of maternal toxicity, an exposure concentration of 4000 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.