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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study which meets basic scientific principles, comparable to guideline

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Developmental toxicity of diethanolamine applied cutaneously to CD rats and New Zealand White rabbits
Author:
Marty MS, et al.
Year:
1999
Bibliographic source:
Regul. Toxicol. Pharmacol., 30, 169-181
Report Date:
1999
Reference Type:
study report
Title:
Unnamed
Year:
1992
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992
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:
Prenatal developmental toxicity study with dermal application in rats
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethanolamine
- Analytical purity: >99.4%
- Supplier: Union Carbide Corp., North Seadrift, TX, USA

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Portage, MI
- Weight at study initiation: 209-251g
- Housing: individual
- Diet Ground certified rodent diet (RMH 3200, Agway. Inc. Waverly, NY) ad libitum
- Water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-21
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
- Area of exposure: clipped back skin
- % coverage: 100
- Type of wrap if used: steril gauze and further occluded with polyvenyl film attached to a specially designed Lycra-Spandex jacket  with Velcro closures
- Time intervals for shavings or clipplings: no data


REMOVAL OF TEST SUBSTANCE
- Washing (if done): warm water
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 ml
- Constant volume or concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
gas chromatography
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
gestation day 6 -15
Frequency of treatment:
6 h/day
Duration of test:
gestation day 0-21
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 380 (500), 1500 mg/kg bw/day
Basis:
other: target dose, remark: due to a preparation error the mid dose had to be reduced to 380 mg/kg bw/day
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection was based on the results of developmental toxicity range finding studies using dermally applied DEA

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes (skin)
- Time schedule: twice daily during the dosing period, once daily during the post-treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15, 18

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: 3-day intervals from GD 0-21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: liver, kidney

BLOOD:
- Prior to sacrifice
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 (variation/malformation) / half per litter (thoracic, abdominal visceral abnormalities)
- Soft tissue examinations: No data
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Continuous variables were compared for homogeneity of variance using  Levene's test for equal variances (Levene, 1960). A parametric or nonparametric analysis of variance (ANOVA) was performed , f parametric ANOVA analyses were significant, pooled T-tests were used for pairwise comparisons. If results from a nonparametric ANOVA were significant, separate variance T tests for pairwise comparisons were performed. Data  from nongravid females and females delivering early were not included in  the statistical analyses. Nonparametric data were statistically evaluated  using the Kruskal-Wallis test, followed by the Mann Whitney U test. Incidence data were compared using the Fisher's exact test (Sokal and  Rohlf, 1969), with the exception of frequency data for fetal malformations and variations, statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). For all statistical tests, the critical level of significance was set a priori at a = 0.05 (two-tailed).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Body weight data: Dams at 1500 mg/kg bw showed significant declines in body weight gains at the end of the dosing period and during the post-dosing period (GD  15-21). This corresponded with a 4.5% decrease in body weight, when corrected for gravid uterine weight. Despite significant differences in  body weight gain at discrete time points, there was no significant difference in body weight gains across dose groups over the entire  duration of the study (GD 0-21). At 150 and 380 (500) mg/kg/day, there  were no treatment-related declines in maternal weight gain at any time  interval. 
- Food consumption: no effect 
- Clinical signs: At 380 (500) and 1500 mg/kg bw signs of skin irritation, which were dose-dependent in incidence and severity). At the high dose, this  condition persisted into the post-treatment period. No significant effects on skin irritation were observed at 150 mg/kg bw. No skin irritation was observed in control animals.

EXAMINATION OF THE DAMS AT TERMINATION:
- Reproduction data of dams:  DEA administration had no effect on pregnancy rate, corpora lutea number, implantation number, litter size, resorption rate, number of dead  fetuses, fetal body weight, fetal sex ratio, or gravid uterine weight at  any of the dose levels tested.
- Hematology:  DEA slightly decreased red blood cell parameters, including hematocrit, MCV, MCH, hemoglobin concentration, and erythrocyte number at all  concentrations. The highest dose level of DEA produced increased numbers of leukocytes and lymphocytes, but decreased platelet numbers. Changes in the profile of red cell morphology (poikilocytosis, anisocytosis, polychromasia) were observed in treated animals from all dose groups.  - Organ weights: Dose dependent increases in both absolute and relative kidney weights at  the 380 (500) and 1500 mg/kg/day dose levels. No significant changes in absolute or relative liver weights were observed.

Effect levels (maternal animals)

Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effect on the overall incidence of external, visceral, or skeletal  malformations or variations observed in rat fetuses. Although there was no difference in the overall incidence, litters from the 1500 mg/kg bw  group had significantly increased incidences of six specific skeletal  alterations. These alterations consisted primarily of delays in ossification. An elevated incidence of "all proximal hindlimb phalanges unossified corresponded with significant reciprocal reductions in litter frequency for "some proximal hindlimb phalanges poorly ossified." There  was also an increase in the litter incidence of "some forelimb metacarpals poorly ossified" in the 1500 mg/kg bw group. There was no effect on any of there developmental parameters at 150 or 380 (500) mg/kg  bw.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 500 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Results summary:

DEA was administered to pregnant CD rats from gestation day 6 through day 15 at concentration of 0, 150, 500 and 1500 mg/kg bw.

At 500 and 1500 mg/kg bw moderate and severe skin irritation was caused, respectively. Maternal body weight gain was decreased in the

1500 mg/kg bw. Absolute and relative kidney weights were increased at 500 and 1500 mg/kg bw. Hematological effects including anemia,

abnormal red cell morphology (poikilocytosis, anisocytosis, polychromasia), and decreased platelet count were observed in all treatment

groups. The 1500 mg/kg bw group also had increased lymphocytes and total leukocytes.

In the fetuses, there were no effects of treatment on body weight or on incidence of external, visceral, or skeletal malformations/abnormalities.

Increased incidences of six skeletal variations involving the axial skeleton and distal appendages were observed in litters from the 1500 mg/kg bw

group. The skeletal variations included poor ossification in the parietal bones, cervical centrum #5, and thoracic centrum #10, lack of ossification

in all proximal hindlimb phalanges and some forelimb metacarpals, and callused ribs.

Consequently, the LOAEL for maternal toxicity was 150 mg/kg bw, while the NOAEL for prenatal developmental toxicity was adjusted to 380 mg/kg

bw due to dosing discrepancy at the 500 mg/kg bw group. The NOAEL for teratogenicity was >1500 mg/kg bw. Thus, signs of prenatal

developmental toxicity did only occur at clearly maternal toxic dose levels.

Detailed results:

Maternal, litter, fetal data - prenatal developmental toxicity (dermal) study in CD rats

Dose (mg/kg bw)

0

150

380 (500)

1500

n (females)

25

25

25

25

pregnant

23

24

25

22

mortality

0

0

0

0

body weight gain GD 6-15 (g)

49

51

50

45

corrected body weight (g)

307.6

308.3

307.5

293.8

skin irritation

0

0

1

10

blood parameters

RBC

6.22

6.01        

5.95        

5.69**

HGB

11.7        

11.1        

11.0*        

10.3**

HCT

34.2        

32.4*        

32.2*        

30.1**

MCV

55.1        

54.0*        

54.2*        

53.1**

MCH

18.7        

18.3*        

18.4        

18.0**

kidney weight absolute (g)

1.96        

2.01        

2.13**        

2.24**

% kidney weight (g)    

0.64        

0.65        

0.69**        

0.77**

gravid uterus (g)

116

119

114

118

corpora lutea  (mean)    

18.8        

17.1        

17.5        

17.4

implantation  (mean) 

16.2        

16.3        

15.6        

16.1

% resorption        

5.4

5.4        

4.6        

4.2

live fetuses  (mean)                

15.3        

15.4        

14.8        

15.4

fetal weight (g)

5. 4

5.5        

5.5        

5.4

Total external malformations (fetus/litter)        

0/0        

0/0        

0/0        

0/0        

Total external variations (fetus/litter)        

50/21        

57/22        

61/22        

47/19

Total soft tissue malformations (fetus/litter)      

5/4        

3/3        

9/7        

10/8

Total soft tissue variations (fetus/litter)

95/22

97/24        

110/25        

114/20

Total skeletal malformations (fetus/litter)

1/1

0/0        

0/0        

0/0        

Total skeletal variations (fetus/litter)

171/23

179/24      

180/25

163/22

Incidences for key skeletal variations expressed as No. of fetus (litter)

Cervical centrum (No.5) poorly ossified:

25(13)      

35(16)

26(14)

38(19)*

Thoracic centrum (No.10) poorly ossified:

1(1)

1(1)

6(5)

7(7)*

Parietal, reduced ossification:

7(3)

5(4)

10(6)

44(13)**

all proximal phalanges (hindlimb) poorly ossified:

71(17)

84(19)

104(23)

105(22)*

some metacarpals (forelimb) poorly ossified:

0(0)        

0(0)        

2(2)

4(4)

BWG = body weight gain, * p0.05 **p0.01

Remark:

Rats in various dose groups did not receive the total volume of DEA during dosing on GD 12-15. There was a possible 10-24% deficit in the expected doses delivered with the greatest difference seen in the 500 mg/kg bw dose group on GD 13. Assuming the maximum deficit in dose delivery (24%), the 500 mg/kg bw dose group still received an intermediate exposure to DEA about 380 mg/kg bw (relative to the 150 and 1500 mg/kg/day group) during this period; therefore, dose-response relationships were maintained.


Applicant's summary and conclusion