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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:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and reported study conducted according to a relevant guideline and under GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: Flux Oil (MCS 2170/Therminol@ 59 heat transfer fluid)
Chemical Name: Mixture of alkyl substituted aromatics
Lot Number: NBP 2933500 t
EHL Code Number: T850028
Appearance: Light yellow liquid
Source: Monsanto Chemical Company.
Stated Stability: No special precautions
Stated Purity: Distilled

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Species and- Strain: Sprague-Dawley rat (Crl : CD@ (SD) BR)
Source: Charles River Breeding Laboratories, Inc.,
Kingston, NY
Approximate Age and Weight at Initiation of Mating:
Females - 12 weeks, > 210 g
Males - 11 weeks, > 299 g
Number on Study : Twenty-f ive mated females /group.

Identification: Animals were individually identified using metal ear tags containing an animal quarantine number. An animal study number was assigned to each mated female when selected for study.

Housing: Animals were individually housed in susp,ended wire-bottom, stainless steel cages. All wire-inhalation cages were used during exposure. Environmental conditions were maintained at approximately 72 +/-2 deg. F and 40-60% relative humidity, with 12 hours of artificial illumination daily.
Food and Water: Purina Certified Rodent Chow@ 5002 and tap water were available ad libitum, except during exposure.
Pretest examination and Acclimation: Upon receipt, animals were retained in quarantine for 10 days in order to determine their health status. Inspections were performed daily in an effort to identify unhealthy or diseased animals. Only healthy animals were released for use on Study.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Exposure System: The exposure system consisted of 10 m3 glass and stainless steel New York University-style inhalation chambers and an atmospheric generation system. Test material atmospheres were generated as aerosols by metering test material through Laskin-style nebulizers. A particle-size separator was employed to prevent most of the large, non-respirable aerosol particles from entering the exposure chambers. Airflow in the
chambers was nominally maintained at 1700 liters per minute.


Unifomrmity of distribution of the test material in the exposure chambers was determined once for each expostire level either during the study period or just prior to the study period. Particle-size analysis was performed gravimetrically using an Andersen Cascade impactor for the mid and high exposure levels. These analyses were performed four times during the first exposure week and weekly therafter .The mass median aerodynamic
diameter and geometric standard deviation and'percentage of particles less than 10 microns were calculated for each analysis. Chamber airflow, temperature, and humidity were monitored continuously, with specific values recorded every 30 minutes per chamber per day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Test Atmospheres: Exposure chamber environment was monitored (for test'material concentrations at least three times per day in all chambers except the control. The control chamber was checked for the presence of test material twice during the study. Samples of test atmosphere for analysis were drawn through and collected in impingers containing, iso-octane. The amount of test material in each sample was analytically determined using gas chromatography. Because the test material was a mixture of components the most prominent peak in the chromatogram (m-ethyl-diphenylethage) was used to quantitate the overall relative amount of test material. Nominal concentration measurements were performed daily for each exposure level.
Details on mating procedure:
Unfit animals were eliminated prior to cohabitation. Selected females and males were cohoused 1:1 for mating. The occurrence of copulation was determined by inspection for copulatory plugs each day following cohabitation. The day of which a plug was detected was designated gestation day 0.
Duration of treatment / exposure:
The test material was administered by inhalation, six hours per day on days 6-15 of gestation.
Frequency of treatment:
daily from GD6 - 15
Duration of test:
Mating, then Exposure from GD6 through to 21
No. of animals per sex per dose:
25 females per dose group
Control animals:
yes, sham-exposed
Details on study design:
Test Groups and Dose Levels: Three treatment groups, each consisting of 25 mated females, were exposed to target flux oil concentrations omf 20, 100 and 400 mg/m3. These exposure levels are also expressed in this report as 0.02, 0.1 and 0.4 mg/l. A concurrent control group of equal size received house-conditioned air only.

Dose levels were se1ected from results of a prior 30 day inhalation study (EHL 84105).

Dosing Regimen: The test material was administered by inhalation, six hours per day on days 6-15 of gestation.

Assignment to Test Groups: Mated females were xandomly assigned to treatment and control groups using a block design based on equivalent gestation day 0 body weights.

Examinations

Maternal examinations:
Mated females underwent a detailed physical 'examination on gestation day 0 and daily on days 6-21. Examinations were performed after exposure on treatment days. In addition, observations were made during exposure for treatment-related signs of toxicity. Only those animals in view in the chambers underwent exposure observations. Clinical signs were not recorded during exposure on exposure days 14 and 18-20.

Body Weight: Individual female body weights were recorded on gestation days 0, 6, 10,'13, 16, and 21. Individual body weight change was determined for the corresponding intervals.

Sacrifice: Surviving dams were sacrificed on, gestation day 21 by C02 inhalation.

Dams underwent a gross necropsy and abnormal maternal findings were recorded.

Early Deliveties: Dams which were observed to deliver prior to the nominal gestation day 21 were sacrificed as soon as possible and underwent a gross necropsy. Abnormal maternal findings and the number of ilmplantations i n each uterine horn were recorded.
Ovaries and uterine content:
Immediately following' sacrifice the uterus was opened and the fetuses removed. The number and relative placement of live fetuses, dead fetuses, early resorptions, and late resorptions were recorded. The number of corpora lutea per ovary was also determined.
Fetal examinations:
All live fetuses from dams which survived to 'scheduled sacrifice were subjected to a gross external examination, sexed, weighed, and tagged for identification. Approximately one-half of each litter was placed in Bouin's fixative for subsequent serial sectioning and visceral examination. The remaining fetuses were eviscerated and fixed in alcohol for subsequent staining with Alizarin Red and skeletal examination.

Abnormal findings were classified as malformations or variations. Fetuses from Dams which delivered were subjected to a gross external examination if there was little or no evidence of fetal autolysis or cannibalism. These fetuses were then processed and examined for visceral or. skeletal malformations.
Statistics:
Data. were analyzed statistically to aid in interpretation of results. Differences between the treatment groups and the control group were analyzed using one-gided comparisons and a critical level of 0.05, except two-sided comparisons were used for comparison of fetal sex distribution.
Maternal body weight, maternal body weight change, and fetal body weight were compared using Dunnett's test.
Pre- and postimplantation loss and the number of liv;e fetuses, dead fetuses, early resorptions, late resorptions, implantations, corpora lutea and fetal sex distribution were compared using the Mann-Whitney U test.

Pregnancy rate and the number of litters with malformed fetuses or variatons were compared using Fisher's Exact test

Bonferroni's inequality was used to assess the significance of comparing multiple treatment groups with a control group when the Mann-Whitney U test or Fisher's exact test indicated apparent statistical significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No females died spontaneously in any of the exposure groups or the control group. Most pregnant females in the high exposure group exhibited wet fur and staining/encrustation (principally of the head, neck and forelimbs), urineary staining and piloerection. A few pregnant females in the high exposure group also exhibited hunched posture/head tilt and single pregant female incidencess of ptosis and red vaginal discharge were observed. Fur staining/encrustation was also observed in the mid and low exposure groups, but the incidence was much lower than in the high exposure group. One pregnant female exhibited piloerection in the mid exposrue group. Alopecia was observed at a low incidence in all of the exposure groups and in the control group. No remarkable clinical signs were recorded during exposure. Most females did not exhibit any remarkable postmortem findings and there did not appear to be any treatment related findings with the possible exception of reduced body fat recorded for two females in the high exposure group.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No females died spontaneously in any of the exposure groups or the control group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The only statistically significant effects on mean body weight were observed in the high exposure groups on gestation days 10, 13, and 16. Mean body weight of high exposure females was a little over 8% lower than control group mena body weight on gestation day 16. Statistically significant differences from mean control group values in body weight change were observed for both the mid and high exposure groups. In the high exposure group body weight loss was observed for gestation day 6-10 and reduced body weight gain was observed for gestation days 10-13 and for the overall period of gestation days 6-21. Reduced body weight gain was observed for mid exposure group females for gestation days 6-10 and 10-13.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistically significant differences between the control group values and any of the exposure groups were observed for reproductive parameters including numbers of live fetuses, dead fetuses, early or late resorptions, preimplantation loss and post implantation loss. The data indicate that there were no treatment related effects on reproductive parameters.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No statistically significant differences between the control group values and any of the exposure groups were observed for reproductive parameters including numbers of live fetuses, dead fetuses, early or late resorptions, preimplantation loss and post implantation loss. The data indicate that there were no treatment related effects on reproductive parameters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No statistically significant differences between the control group values and any of the exposure groups were observed for reproductive parameters including numbers of live fetuses, dead fetuses, early or late resorptions, preimplantation loss and post implantation loss. The data indicate that there were no treatment related effects on reproductive parameters.
Dead fetuses:
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): not specified
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs of wet, matted or stained- fur, piloerection and urinary staining were observed in most females of the high exposure group and hunched posture/head tilt was observed in several females. High exposure level females also exhibited lower mean body weights on gestation days 10, 13 and 16 and body weight loss (gestation days 6-10) and reduced weight gain (gestation days 10-13) compared to control values. Some toxicity was also observed at the mid exposure level. A few females exhibited fur staining or encrustation and piloerection was observed in one female. Reduced body weight gain was observed for mid exposure females for gestation days 6-10 and 10-13. The body weight effects were most severe during the initial exposure period (gestation days 6-10) and weight gains after completion of exposures (gestation days 16-21) were comparable to control values. No treatment related toxic effects were observed in low dose level females. Most females did not exhibit remarkable postmortem findings and, except for reduced abdominal fat observed in two high exposure level females, there were no findings judged to be treatment related.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
20 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
100 mg/m³ air
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between any of the exposure groups and control group values for fetal body weights or fetal sex distribution. Although high exposure group male and female mean fetal weights were lower than corresponding values for the control group and the other exposure groups, the lack of statistical significance indicates that this should not be ascribed as a treatment related effect.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between any of the exposure groups and control group values for fetal body weights or fetal sex distribution. Although high exposure group male and female mean fetal weights were lower than corresponding values for the control group and the other exposure groups, the lack of statistical significance indicates that this should not be ascribed as a treatment related effect.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
An increase in the total number of litters with malformations was observed for the high exposure group compared to the control group. The increase was not statistically significant at the 5% level after application of the Bonferroni inequality for comparison of multiple treatment with controls. Most of this was due to a number of litters with fetuses exhibiting bent ribs. Other malformations in the high exposure group were restricted in distribution to single fetuses with four of the eleven malformation findings occuring in a single fetus and two other malformation findings (sternoschesis and anus absent/tail short) each occurring in a single fetus. The only malformation observed in the mid exposure group was a single fetus with microphthalmia and the only malformations observed in the low dose group were two fetuses in two litters with bent ribs.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A variation finding of reduced ossification of inner ear bones was elevated on a fetal basis in all exposure groups compared to the control group incidence. However, the elevations in incidence were not statistically significant at the 5% level with the Bonferroni inequality correction and were not significant in terms of nmbers of litters affected for any of the exposure groups. Although there were elevations in some types of other variation findings in the high exposure group liters (reduced skull ossification, sternebrae #5 and /or 6 unossified, cervical #7 rib, and 13 full pairs of rudimentary ribs), none of these findings were statistically significant compared to the control group on either a litter or fetal incidence basis.
Visceral malformations:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
An increase above control in the number of bent ribs observed inhigh dose fetuses. (7 fetuses in 5 litters, compared to 1 fetus in the control.). Although the increased incidence of bent ribs in the high,exposure group may have been treatment related, the incidence of this finding alone was not statistically significant when compared to the control group incidence. Other malformation findings in the high exposure level group were restricted to single incidences in three fetuses (one with multiple head malformations). Because of the restricted distribution of these findings they are not '
considered to be definitely treatment related. One malformation (microphthalmia) was observed in one fetus in-the mid exposure group and two fetuses with bent ribs were observed in the low exposure group.

Effect levels (fetuses)

Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study identified an increased maternal toxicity in the mid and high dose groups and some evidence of malformations but only in conjunction of maternal toxicity. It is therefore possible that these effects may be as a result of the general toxicity observed in the Das rather than specific teratogneicity or developmental toxicity. With the exception of some small changes in malformation incidence, there was no evidence of embryotoxicity or fetotoxicity, that would lead to a conclusion that this substance is developmentally toxic.
Executive summary:

Mated female Sprague-Dawley rats, in groups of 25, were exposed by inhalalation to target flux oil concentrations of 20, 100 and 400 mg/m3 for six hours per day on gestation days 6-15. A concurrent control group was exposed to house-conditioned air only. Dams were sacrificed on gestation day 21, implantations were described, and fetuses were removed from the uterus. Fetuses were weighed, underwent a gross external examination and were processed for subsequent visceral or skeletal examination. Abnormal fetal findings were classified as malformations or variations.

Study mean exposure concentrations and standard deviations determined by analytical methods were 19 ± 2, 110 ± 30 and 410 ± 54 mg/m3 for the target levels of 20, 100 and 400mg/m3, respectively. Distribution of test material within the exposure chambers was found to be reasonably uniform and most (>80%) of the aerosol particles in the mid and high exposure level groups were less than 10 urn in size. No significant decomposition of the test material was observed during the study.

There were no spontaneous deaths in any of the exposure groups or -the control group. Maternal toxicity was observed in the high exposure group. Clinical signs of wet, matted or stained- fur, epiloerection and urinary staining were observed in most females of the high exposure group and hunched posture/head tilt was observed in several females. High exposure level females also exhibited lower mean body weights on gestation days 10, 13 and 16 and body weight loss (gestation days 6-10) and reduced weight gain (gestation days 10-13) compared to control values. Some toxicity was also observed at the mid exposure level. A few females exhibited fur staining or encrustation and piloerection was observed in one female. Reduced body weight gain was observed for mid exposure females for gestation days 6-10 and 10-13. The body weight effects were most severe during the initial exposure period (gestation days 6-10) and weight gains after completion of exposures (gestation days 16-21) were comparable to control values. No treatment related toxic effects were observed in low dose level females. Most females did not exhibit remarkable postmortem findings and, except for reduced abdominal fat observed in two high exposure level females, there were no findings judged to be treatment related.

The total number of litters with malformations was elevated in the high exposure level group. The difference in total litters formations was significant at the 5% level without, but not with, the Bonferroni inequality correction for comparison of multiple treatment groups with a control group. The increase was primarily because of findings of bent ribs in 7 fetuses in 5 litters in the high exposure group. This finding was also observed in one fetus in the control group. Although the increased incidence of bent ribs in the high,exposure group may have been treatment related, the incidence of this finding alone was not statistically significant when compared to the control group incidence. Other malformation findings in the high exposure level group were restricted to single incidences in three fetuses (one with multiple head malformations). Because of the restricted distribution of these findings they are not considered to be definitely treatment related. One malformation (microphthalmia) was observed in one fetus in the mid exposure group and two fetuses with bent ribs were observed in the low exposure group.

There were no clear, treatment-related effects on reported variations in any of the exposure level groups. Although increases were observed in the fetal incidence of reduced inner ear ossification of all exposure groups compared to the control group the increases were not judged to be treatment related because of lack of a dose response pattern, lack of significant effects on litter incidence, lack of fetal weight effects, and lack of statistical significance with the Bonferroni inequality correction. Although increases in certain other variation findings were observed in the high exposure group, none of these increases was statistically different from control values on either a litter or fetal "incidence" basis. These increases in, variation findings in the high exposure group are not judged to be clear indications of treatment related effects. The incidences of variation findings other than reduced ear ossification in the -low and mid exposure level groups were comparable to control incidence levels and did not indicate treatment related effects.

In, conclusion, maternal toxicity was observed at exposure levels of 400 and 100 mg/m3 but not at 20 mg/m3. No treatment related embryotoxicity or fetotoxicity was observed at any of the exposure levels. An increase in litters with malformations and litters with bent ribs was observed at the 400 mg/rn3 level; These observations are considered t o be possible indications of a treatment related effect , although it is also possible that the effects are not treatment related. Moreover, significant maternal toxicity was evident in the high exposure level females and the effects observed may have been secondary to maternal toxicity. No treatment related effects on malformations or variations were observed at the 100 or 20 mg/m3 exposure levels. Therefore, these effects may be as a result of the general toxicity observed in the Dams rather than specific teratogenicity or developmental toxicity. With the exception of some small changes in malformation incidence, there was no evidence of embryotoxicity or fetotoxicity, that would lead to a conclusion that this substance is developmentally toxic.