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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:
other: Similar to guideline study; GLP

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Developmental toxicity study in Sprague-Dawley rats by whole-body exposure to N,N-diethylethanolamine vapor.
Author:
Leung, H-W, Murphy, SR
Year:
1998
Bibliographic source:
J. Appl. Toxicol. 18: 191-196.
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The objective of this study was to determine the potential of MRD-87-086 to induce embryo- and/or fetotoxicity, or fetal structural and/or other abnormalities when administered to the rat during pregnancy by the inhalation route.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N,N-diethylethanolamine (MRD-87-086), diethylaminoethanol DEAE-40
- Physical state: colorless liquid
- Analytical purity: >99.9 %
- Storage condition of test material: Room temperature, nitrogen blanketed

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Breeding Labs, Inc. Kingston Facility, Stoneridge, N.Y.- Age at study initiation: MALES: 83 days; FEMALES: 91 days- Weight at study initiation: MALES: Approximately 350 grams - 468 grams; FEMALES: Approximately 230 grams - 320 grams- Fasting period before study: none- Diet: Purina Certified Rodent Chow (5002 Meal), ad libitum- Water: Automatic watering system, ad libitum- Acclimation period: 14 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24- Humidity (%): 40-70- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The developmental toxicity of the test substance was evaluated in groups of 25 pregnant rats exposed daily from day 6 to day 15 of gestation. The test material was administered via the breathing air.The experimental and control animals were placed into appropriate-inhalation chambers which were operated under dynamic conditions. The exposure period was 6 hours per day plus time for equilibration.The chambers used for exposure were stainless steel and glass and had a total volume of approximately 1,000 liters. They were operated at a flow rate (approximately 12-15 air changes/ hour) sufficient to ensure timely equilibration. The flow of air through each chamber was monitored continuously using a calibrated flow meter and recorded approximately every 30 minutes. All chambers were maintained at a slight negative pressure. 
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A nominal exposure concentration was calculated. The weight of test material used was evaluated and divided by the total volume of air passing through the chamber to give the nominal concentration.Analytical concentrations were determined by an online GC. They were measured hourly. Samples were drawn from 4 points around the horizontal center plane of each of the exposure chambers to determine homogeneity of the test material distribution.
Details on mating procedure:
After approximately 3:00 p.m., on the initial scheduled mating day, females were placed in males’ cages in a 1:1 (male:female) ratio. Males and females were paired based on sequential PIN number. A suitable number of animals were co-housed in an attempt to produce a suitable number of mated animals to accommodate lab scheduling. Mating was confirmed on the following morning by observation of a copulatory plug in the female’s vagina or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was the female's Day OG (Gestation). After confirmation of mating, each mated female was returned to its own cage. New females then were placed in the males’ cages until the required number of mated females was obtained by continuous cohabitation. Mated females were assigned to dose groups in the order of mating. Accordingly, the first confirmed mated female was assigned to Group 1, the next to Group 2, and so on until all mated animals for a given day had been assigned to dose groups. On subsequent days, the next group in sequence was filled by the first confirmed mated female on that day, and so on. Assignments were made until all confirmed mated females were assigned to groups, or until as many were assigned as could be examined on Day 21G. Upon completion of the mating period, all males and unassigned females were transferred to the stock colony maintained by the Developmental Toxicology Group.
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
until day 21 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.160, 0.320 and 0.486 mg/l (160, 320, and 486 mg/m3)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
33, 66, 100 ppm
Basis:
other: The concentrations were selected based on results from a range finding study using 8 pregnant females/group exposed to 10, 50, 100, 150, 200 ppm of DEAE on GD 6-15.
No. of animals per sex per dose:
100 sexually mature virgin females and 42 sexually mature virgin males were used in total.Each test group comprised 25 pregnant females.
Control animals:
yes, concurrent no treatment

Examinations

Maternal examinations:
Animals were examined for viability at least twice daily during the treatment period and at least once daily at other times during the study. Body weights were determined for each animal prior to selection, weekly during the mating period, and for each mated female assigned to a study group, on Days 0, 6, 9, 12, 15, 18, and 21G.Food consumption was measured for mated females and was calculated based on feed jar weights measured on Days 0, 6, 9, 12, 15, 18, and 21G.A clinical examination was given to each female prior to selection and daily thereafter.
Ovaries and uterine content:
Surviving mated females were euthanized by carbon dioxide asphyxiation and exsanguination on Day 21G. All females assigned to groups were examined by a cursory necropsy performed by appropriately trained personnel. Uterine weight with ovaries attached was recorded at the time of necropsy. Uterine contents were examined and the numbers and locations of implantation sites, early and late resorptions, live, dead, and externally malformed fetuses were recorded. Corpora lutea were counted and recorded. All live fetuses were euthanized by intramuscular injection into the tongue of sodium pentobarbital prior to internal examination. Abnormal tissues from mated adult females were saved in 10 % neutral buffered formalin at the discretion of the Study Director or his designee for possible histological evaluation.
Fetal examinations:
Each live fetus was weighed and examined externally for gross malformations, including cleft palate. Fetal sex was determined by external examination and internally confirmed on those fetuses receiving visceral examination. The viscera of approximately one-half of the fetuses of each litter were examined by fresh dissection. After visceral examination, these fetuses were decapitated and heads were preserved in Bouin's solution for at least two weeks. Free-hand razor blade sections of the Bouin's-fixed fetal heads were examined for the presence of abnormalities. The remaining live fetuses were eviscerated after being euthanized, processed for skeletal staining with Alizarin red, and examined for the presence of malformations and ossification variations. Representative malformations were photographed at the discretion of the Study Director.
Statistics:
Mean body weights and body weight changes of dams, food consumption, number of corpora lutea/dam, number of implantations/dam, number of resorptions/dam, number of live fetuses/litter, number of live male, live female, dead, and malformed fetuses/litter, number of affected fetuses (resorbed, dead, malformed), number of offspring/litter, number of fetuses with developmental variations/litter.Fetal weight was analyzed by standard nested analysis of covariance with fetuses nested within dams and with dams nested within doses, and litter size as the covariant. All tests were reported at the 5 or 1% level of significance. The following were calculated/analyzed for statistical significance: incidences of individual and total malformations, of individual and total variations, number of litters in groups with >= 1 resorption, number of litters in groups with >= 1 dead fetus, number of litters in groups with >= 1 malformed fetus, number of litters in groups with >= 1 affected (resorption, dead or malformed) conceptus, number of litters in groups >= 1 fetus with developmental variations. A standard chi-square analysis was performed to determine if the proportion of incidences differed between the groups tested. Each treatment group was then compared to each control group using a 2 x 2 Fisher Exact test. Armitage's test for linear trend in the dosage groups was performed..
Indices:
Live fetuses as % of implants, dead fetuses as % of implants, and resorptions as % of implants, were calculated for each litter. Percentages were transformed by Cochran's transformation. The raw and the transformed percentages were tested for statistical significance using Bartlett's test of homogeneity of variance to determine if the groups had equivalent variances at the 5 % or 1 % level of significance (If the variances were equivalent, the hypothesis that there was no difference in response between the groups was tested using a standard one-way analysis of variance. If the ANOVA was significant, Dunnett's test was performed to determine which treated groups differed from the control. If the variances were not equivalent, then a Kruskal-Wallis (non-parametric) test was performed to determine if the treatment effects were equivalent. If there was a difference, a rank sum comparison was used to determine which treatment groups differed from the control. Jonckheere's test for ordered response was also performed.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yesDetails on maternal toxic effects:CLINICAL IN-LIFE OBSERVATIONSDry rales was observed during Days 11-21G in up to nine animals in the high dose group and was considered to be related to the known irritant nature of the test material on the respiratory tract. Single or low incidences of alopecia, scabs, sores, ocular discharge, nasal discharge, and maloccluded incisors were observed intermittently throughout the groups. These abnormalities were considered incidental and unrelated to treatment. All animals were pregnant and survived to scheduled cesarean sections on Day 21G, although one animal in the mid dose group delivered offspring prior to terminal euthanasia. BODY WEIGHTSThere were no statistically significant differences in mean gestation body weights between treated and control groups during the study, except for a 6 % lower weight in high dose animals on Day 15G. This slight difference between control and high dose dams on Day 15G was not considered biologically significant. However, statistically significant body weight gain suppression was observed during the study in the high dose group compared with controls. Mean gestation body weight changes in the mid and low dose groups were generally comparable with controls during the study, although statistically significant body weight gain suppression in mid-dose animals was observed during the Day 12-15G interval. The statistically significant increase in body weight gain observed in the low dose group compared with controls during the Day 9-12G interval was considered spurious and unrelated to treatment with the test material in the absence of similar increases at other intervals. FOOD CONSUMPTIONStatistically significant and dose-related decreases in mean maternal food consumption were observed in the high dose group at the majority of food consumption intervals when compared with controls, including both the treatment period (Day 6-15G) and the entire study period (Day 0-21G), although the latter decrease was slight (9 % ). The mid dose group was observed with statistically significant decreases in mean food consumption during the Day 12-15G interval, as well as during the overall treatment period when compared with those of controls, although the latter decrease was slight (8 % ). MATERNAL NECROPSY OBSERVATIONSIncidental findings were observed at necropsy in control and treated groups which were considered unrelated to treatment with respect to their incidence and/or severity. These findings consisted of single or low incidences of alopecia, scab(s), or maloccluded incisors. UTERINE IMPLANTATION DATAThere were no meaningful differences between treated and control animals for any uterine implantation parameter. A statistically significant decrease in mean resorptions was observed in the high dose group when compared with controls. This finding was considered spurious and unrelated to treatment and would not be considered an adverse effect in any event. No other statistically significant differences were observed between uterine implantation data of control and treated groups when evaluated by pairwise comparison. Mean uterine implantation data do not include total variations or malformations of delivered pups.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
0.16 mg/L air
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:FETAL BODY WEIGHTSMean fetal body weights for treated groups were comparable to controls. Mean body weights did not include weights of delivered pups.FETAL VARIATIONS AND MALFORMATIONSNo meaningful differences in the incidences of fetal variations or malformations were observed in treated groups when compared with controls on either a per fetus or per litter basis. Visceral variations were observed in all groups and included dilated renal pelvis and distended and/or convoluted ureters. Skeletal variations were observed throughout the groups and consisted primarily of hypoplastic or unossified elements of the skull, sternebrae, vertebrae, ribs, or extremities. These findings are common in gestation Day 21 rat fetuses and their incidences or severities were not considered treatment-related. However, a dose-related decrease was observed in the incidence of hypoplastic bones of the forepaw on a per fetus basis and was statistically significant in the high dose group when compared with controls after analysis by pairwise comparison. These findings were not considered adverse effects. A dose-related increase in the incidence of advanced ossification of the bones of the hindpaw on a per fetus basis was observed in treated groups when compared with controls. However, no statistically significant differences were observed on either a per fetus or per litter basis and this finding was not considered to be biologically significant. The majority of malformations were observed in single instances in the control group and included agnathia, microphthalmia and microtia in one fetus, folded retina, cyst dorsal to rhombencephalon and malformed mandible. One fetus in the low dose group was observed with a cyst external to turbinate bones, another low dose fetus was observed with extremely dilated lateral ventricles/thin cortex, and one fetus in the high dose group was observed with agenesis of the sternebrae. All observed malformations were considered spurious and unrelated to treatment, with the test material.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
0.486 mg/L air
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

The average analytical concentrations were 33, 66, 100 ppm.

These concentrations are comparable to ca. 38, 76 and 116 mg/kg bw per day doses assuming 100% lung deposition and absorption.

 The maternal NOAEL (No Observable Adverse Effect Level) was established as 33 ppm of the test material, MRD-87-086, due to body weight gain suppression and decreased food consumption observed in the mid and high dose groups. The developmental NOAEL was established as greater than 100 ppm based on the absence of adverse effects on fetal parameters. The test material was not teratogenic or embryo- or fetotoxic when administered under the conditions of this study.

Applicant's summary and conclusion