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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Principles of method if other than guideline:
Deviations from OECD guideline 414:

1. The type of environmental enrichment given to the animals was not documented prior to the week of 16 January 2012.
2. Mechanical difficulties caused the daily mean chamber concentration value (262 ppm) to be below the acceptable range of 300 ppm +/- 10%.
3. A paper jam occured on 29 January 2012 causing improper sample collection for chambers one and three. Manual samples were taken and replaced the incorrect data.
4. The carcass and fetuses in group 1 had been placed in Bouin's solution rather than 100% ethyl alcohol. A skeletal examination was therefore not possible for this carcass.

These deviations did not negatively impact the quality or integrity of the data nor the outcome of the study.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
na

Test animals

Species:
rat
Strain:
other: Sprague Dawley [Crl:CD(SD)]
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, N.C.
- Age at study initiation:79 days
- Sex: female
- Fasting period before study: No Data
- Housing: Individually in stainless steel wire-mesh cages. Rats were paired for mating in the home cage of the male.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 degrees C +/- 3 degrees C
- Humidity (%): 50% +/- 20%
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 dark/ 12 light
IN-LIFE DATES:
From: 3 January 2012 To: 13 March 2012

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 cubic meter stainless-steel and glass exposure chambers. (Four chambers one for each exposure concentration and one for the control group exposures)
- Method of holding animals in test chamber: No data
- Source and rate of air: 12 to 15 air changes per hour
- System of generating particulates/aerosols: Vapors were generated using a glass-bead column-type vaporization system
- Temperature, humidity, pressure in air chamber: 18 to 26 degrees C, 30 to 70% humidity, under slight negative pressure
- Air change rate: 12 to 15 air changes per hour
- Method of particle size determination: Casella Microdust 880 nm Aerosol Monitoring System was used for particle evaluation
- Treatment of exhaust air: No data
TEST ATMOSPHERE
- Brief description of analytical method used: Exposure concentrations were calculated for each test substance chamber from the total amount of the substance consumed during the exposure and the total volume of air passed through the chamber during exposure. Analyzed exposure concentrations were also determined at approximately 45-minute intervals using a gas chromatograph (GC) under the control of the WINH system
- Samples taken from breathing zone: yes
USE OF RESTRAINERS FOR PREVENTING INGESTION: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC) under the control of the WINH system
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: Until evidence of mating
- Age at mating of the mated animals in the study: Approximately 14 weeks
- Further matings after two unsuccessful attempts: No data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
6 hours daily, from gestation days 6 through 19
Frequency of treatment:
Daily from gestation days 6 through 19
Duration of test:
Exposures occured from gestation days 6 through 19
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 1000, and 3000 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
3 groups of 25 bred females (25 per exposure group) and 25 bred control females exposed to filtered air
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were determined based on a previous 13 week inhalation study
- Rationale for animal assignment (if not random): Random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily for mortality and moribundity
- Cage side observations checked in table were included
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: "Appropriate intervals"
BODY WEIGHT: Yes
- Time schedule for examinations: "Appropriate intervals"
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Uteri, placentae, ovaries, fetuses
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
- Head examinations: Yes: all per litter
Statistics:
All analyses will be two-tailed for significance levels of 5% and 1%. All statistical tests will be performed using a computer with appropriate programming as referenced below. Data from nongravid females will be excluded from calculation of means and from comparative statistics. The litter, rather than the fetus, will be considered as the experimental unit.

Maternal in-life data:
Continuous data variables [mean body weights (absolute and net), body weight gains (absolute and net) and food consumption of each interval] will be
subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup difference. If the results of the ANOVA are significant (p<0.05), Dunnett's test (Dunnett, 1964) will be applied to the data.

Laparophysterectomy data:
The group mean numbers of corpora lutea, implantation sites, viable fetuses, maternal gravid uterine weights and mean fetal weight (separately by sex, and
combined) will be subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) and Dunnett's test (1964) as described
above. The mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and
post-implantation loss and the fetal sex distribution) will be subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to
determine intergroup difference. If the results of the ANOVA are significant (p<0.05), the Dunn’s Test (Dunn, 1964) will be applied to the data.

Fetal Morphology data:
The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each
particular external, visceral and skeletal malformation or variation will be tabulated. The mean litter proportions of fetal malformations and
developmental variations will be subjected to the Kruskal-Wallis nonparametric ANOVA test (1952) followed by the Dunn’s Test (1964)
Indices:
Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss.
Historical control data:
Indicental fiindings in treatment and controls groups were within the laboratory's historical control ranges.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations noted at any exposure level.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 300, 1000, and 3000 ppm groups were unaffected by test substance exposure. The only significant (p<0.01 or p<0.05) differences from the control group were increased mean body weight gains in the 1000 ppm group on gestation days 16-18 and in the 3000 ppm group on gestation days 14-15 and 16-17. These changes in mean body weight gains did not affect mean body weights in this group or the overall mean body weight gain during gestation days 6-20, and were therefore not considered to be related to test substance exposure.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Description (incidence and severity):
Maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 300, 1000, and 3000 ppm groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
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:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Gravid uterine weights were unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 300, 1000, and 3000 ppm. Macroscopic findings observed in the test substance-treated groups occurred infrequently and in a manner that
was not exposure-related. All females were gravid with the exception of 1 female each in the 1000 and 3000 ppm groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 300, 1000, and 3000 ppm.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre- and post-implantation loss were not impacted by treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter size was not impacted by treatment.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early and late resorptions were not impacted by treatment.
Dead fetuses:
no effects observed
Description (incidence and severity):
Litter survival was similar between control and treatment groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Pregnancy duration was not impacted by treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility was not impacted by treatment.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEC
Effect level:
39.5 mg/L air
Based on:
test mat.
Basis for effect level:
other: na
Remarks on result:
not determinable because of methodological limitations

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weight was not impacted by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Liver litter size was not impacted by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not impacted by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size and body weight were not impacted by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Postnatal survival was not impacted by treatment.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external malformations were noted at any exposure level. External malformations were noted for 1(1), 6(2), 1(1), and 0(0) in the control, 300, 1000, and 3000 ppm groups, respectively. In the 1000 ppm group, fetus no. 37780-16 was noted with anophthalmia (left). This finding had no skeletal origin. This malformation was not considered to be related to test substance exposure, as it occurred in a single fetus, similarly in a control group fetus (no. 37783-09), and in a non-exposure related manner. In the 300 ppm group, 5 fetuses from dam no. 37825 (nos. 1, 5, 8, 16, and 18) were noted with syndactyly (digits fused; phalanges fused) and polydactyly (extra digits;no skeletal origin). Additionally in the 300 ppm group, fetus no. 37772-09 was noted with ectrodactyly (absent digit no. 2) and gastroschisis (portion of the liver, stomach, and several loops of intestine protruded through an opening in the ventral midline). Skeletally, ectrodactyly consisted of absent proximal, medial, and distal phalanges. These malformations observed in the 300 ppm group were not considered to be related to test substance exposure, as the malformations occurred in only 2 litters, and were not observed in an exposure-dependent manner.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related skeletal malformations were noted at any exposure level. Skeletal malformations were observed in 0(0), 1(1), 1(1), and 0(0) fetuses in the control, 300, 1000, and 3000 ppm groups. In the 1000 ppm group, fetus no. 37780-16, which was also noted with anophthalmia (see Section 6.7.1), was noted with a vertebral centra anomaly that consisted of halves of centra absent or malpositioned. In the 300 ppm group, fetus no. 37772-09 was noted with a vertebral anomaly with an associated rib anomaly that consisted of absent, fused, malpositioned, and/or bifurcated ribs, arches, and/or costal cartilage. This fetus was also noted with ectrodactyly and gastroschisis . These malformations seen in the 300 and 1000 ppm groups were not considered to be related to test substance exposure, as they occurred in single fetuses and in a manner that was not exposure-related. No test substance-related developmental skeletal variations were noted at any dosage level. Skeletal developmental variations observed in all groups, including the control group, consisted of sternebra(e) malaligned (slight or moderate), 14th rudimentary rib(s), sternebra(e) no. 5 and/or no. 6 unossified, and cervical centrum no. 1 ossified. Additional skeletal developmental variations noted in this study were not observed in an exposure-related manner, occurred at similar frequencies in the control group or in single animals, and/or the mean litter proportions were not statistically significantly different from the control group or were within the ranges of the WIL historical control data, and therefore are not considered to be test substance-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No visceral malformations were noted at any exposure level.
Visceral developmental variations consisted of distended ureter(s) in 1 fetus each in the 300 and 1000 ppm groups and major blood vessel variation (right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]) in a single fetus in the 3000 ppm group. These visceral developmental variations were noted in single fetuses, did not occur in an exposure-related manner, were not statistically significantly different from the concurrent control group when evaluated on a proportional basis, and/or the mean litter proportions were within the ranges of the WIL historical control data, and therefore were not considered to be related to test substance-exposure.
Renal papilla(e) that were not fully developed (Woo and Hoar Grade 1) was observed in 3(1) and 1(1) fetuses (litters) in the 300 and 1000 ppm groups, respectively. This finding was not classified as either a malformation or developmental variation and was not considered to be test substance-related because it occurred infrequently and in a manner that was not exposure-related.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
39.5 mg/L air
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable because of methodological limitations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the study the no-observed-adverse-effect-concentration (NOAEC) for maternal toxicity and embryo/fetal development when the test article was administered via whole body inhalation to bred Crl:CD(SD) rats was 3056 ppm (39.5 mg/L).
Executive summary:

A developmental toxicity study was conducted on the test article by exposing bred Sprague Dawley Crl:CD(SD) rats to 300, 1000, and 3000 ppm in a whole body inhalation chamber.   The study was conducted according to OECD 414 in compliance with OECD GLP.  The rats were exposed to the test article for 6 hours daily from gestation days 6 through 19. A concurrent control group composed of 25 bred females was exposed to filtered air on a comparable regimen. 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 20, a laparohysterectomy was performed on each 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 also recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external visceral and skeletal malformations and developmental variations. All females survived to necropsy on gestation day 20. Analytically-measured test atomospheres were 0 (control), 305 ppm (3.9 mg/L), 1006 ppm (13 mg/L) and 3056 ppm (39.5 mg/L).  There were no test substance-related clinical observations noted at any exposure level. Additionally, there were no test substance-related maternal macroscopic findings noted at the scheduled necropsy. Intrauterine growth and survival were unaffected by the test substance exposure at all exposure levels tested. There were no test substance-related external, visceral, or skeletal malformations or developmental variations observed at any dosage level. Based on the results of the study the no-observed-adverse-effect-concentration (NOAEC) for maternal toxicity and embryo/fetal development when the test article was administered via whole body inhalation to bred Crl:CD(SD) rats was 3056 ppm (39.5 mg/L).