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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:
6 May 1986 to 8 January 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was not performed according to OECD guideline and it is not reported if it was performed according to GLP.

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:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethylene glycol diethyl ether
- Physical state:clear colourless liquid
- Analytical purity:>99% by GC
- Impurities: 0.57% monoethyl ether and 0.33% unknown impurity by GC.
- Lot/batch No.:Manufacturer's Lot No. 2110KL; 3309KL (purified at Research Triangle Institute. NIH Contract No. NOlES-45061. RTI Project 31IU-3050).
- Stability under test conditions: Bulk chemical stability was 2 weeks when stored at temperature below 60°C and protected from light. Aqueous solutions (10 mg/ml) indicated no significant loss after 3 weeks of storage in sealed glass bottles.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: reputable breeder
- Age at study initiation: six month old
- Weight at study initiation: approximately 3.6 kg (2860 g to 4710g)
- Fasting period before study:
- Housing: housed singly in stainless steel cages with· mesh flooring.
- Diet: Purina Certified Rabbit Chow@ ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.7-23.9°C
- Humidity (%): 3 - 78%
- Air changes (per hr): 12 to 14 times per hour
- Photoperiod (hrs dark / hrs light): 12hrs/12hrs

IN-LIFE DATES: From 6 May 1986 To 8 January 1987

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DOSE PREPARATION
- Rate of preparation of diet (frequency): every 14 days
- Storage temperature :room temperature

VEHICLE
- Concentration in vehicle (mg/mL): dose level (mg/kg)/dose volume (3 mL/kg)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A 5 ml aliquot of each formulation of test substance and vehicle was analyzed to verify the concentration of the test compound prior to dosing on gestational day 6. Additional samples of each formulation were analyzed after use at the end of the dosing period to confirm stability
Details on mating procedure:
- Impregnation procedure: artificial insemination (induced ovulation by injection of chorionic gonadotropin prior insemination)
Duration of treatment / exposure:
Gestational day 6 through day 19
Frequency of treatment:
Once daily
Duration of test:
31 days, from day 0 of pregnancy to sacrifice on gestational day 30
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 400 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
36, 36, 36 and 41 females were used in the 0, 50, 200 and 400 mg/kg bw/day dose groups, respectively
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a preliminary developmental toxicity study in which significant maternal mortality and decreased embryo/fetal viability occurred at 500 and 600 mg DEGDEE/kg bw/day.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during treatment (gestational days 6-19) at the time of dosing

BODY WEIGHT: Yes
- Time schedule for examinations: gestational days 0, 6-19 (before dosing) and 30 (following sacrifice).
At sacrifice, the liver and gravid uterus were also weighed.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #30
- Organs examined: Uterus and foetus

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 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:
Prior to General Linear Models (GLM) analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homogeneity of variance was performed on all data analysed by ANOVA. Appropriate GLM procedures were used to determine the significance of the dose-response relationship (Test for Linear Trend), and to determine whether significant dose effects, replicate effects or dose X replicate interactions had occurred for selected measures (ANOVA). When a significant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test and Williams' Test were used to compare DGDEE-exposed versus vehicle control groups for that measure. A one-tailed Dunnett's test was employed for all pairwise comparisons except that a two-tailed test was utilized for maternal and foetal body weight parameters. In addition, the data for any measure which exhibited a significant (p<0.05) dose X replicate interaction in a two-way (dose X replicate) ANOVA is presented as mean ± S.E.M. for each cell in the ANOVA design, and dose effects within each replicate were further evaluated using a one-way ANOVA, Test for Linear Trend. Dunnett's Test and Williams' Test. Non-significant (i.e., p>0.05) replicate effects or dose X replicate interactions on selected measures warranted pooling data across replicates for nonparametric analysis. Nominal scale measures were analysed by a Chi-Square Test for Independence for differences among treatment groups and by a Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, then a one-tailed Fisher Exact Probability Test was used for pairwise comparisons between each DGDEE-treated group and control.
Indices:
Embryotoxicity indices:

Corporea lutea per doe
Implantation sites per litter
% preimplantation loss
Resorptions per litter (%)
No. litters with resorptions (%)
Dead foetuses per litter (%)
No.litters with dead foetuses (%)
Non live implants per litter (%)
Adversely affected implants per litter (%)
No. litters with adversely affected implants (%)
No. live litters with live foetuses
Live foetuses per litter
Male foetuses per litter (%)
Average foetal body weight (g) per litter

Teratogenicity indices:

No. foetuses with gross/skeletal/visceral malformations
Gross/skeletal/visceral malformations per litter
No. litters with gross/skeletal malformations
No. foetuses with variations
No. litters with variations
Foetuses malformed per litter (%)
No. foetuses malformed (%)
No. litters with malformations (%)
Males malformed per litter (%)
Females malformed per litter (%)
Historical control data:
None reported

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
DEGDEE treatment did not adversely influence maternal viability with the exception that one of the 27 confirmed pregnant does (3.7%) in the 400 mg/kg group died after dosing on gd 15.Necropsy performed on the same day of death revealed hemorrhagic lining of the trachea and slightly hemorrhagic lungs which was judged a consequence of DEGDEE exposure.The pregnancy incidence was similar across doses and ranged from 85.7% to 88.6%.
The greatest occurrence of toxic manifestations was observed in the high dose group e.g., ataxia, coma, dyspnea and postdosing vocalization predominated at 400 mg DEGDEE/kg/day.
Maternal body weight was similar among dose groups on gd 0 as well as throughout the treatment and post-treatment periods. In addition, corrected weight gain and gravid uterine and liver weights did not differ among dose groups. Maternal weight gain measured during gestation varied significantly among dose groups but no statistical differences were detected beween the control and treatment values. Exposure to 400 mg DEGDEE/kg significantly reduced weight gain measured during the treatment period.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The number of implantation sites per litter did not vary with treatment. Embryo/foetal viability was not affected by the test substance as indicated by dose independent incidences of resorptions and foetal deaths. In addition. the number of live foetuses per litter was not affected by treatment. Foetal body weights were slightly (but not significantly) decreased in the high dose group. When foetal bodyweights were analysed by sex, however, foetal female body weight exhibited a
significant decreasing trend which was entirely related to the statistically nonsignificant weight reduction in the 400 mg/kg/day group.
Examination of the gd 30 foetuses indicated that embryo/foetal development was not altered by exposure. External, visceral and skeletal evaluations identified structural anomalies whose expressions were unrelated to treatment.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Maternal toxicity

 

Diethylene glycol diethyl ether (mg/kg/day)

0

50

200

400

Maternal toxicity

Total does treated

36

36

36

41

No removed

1a

2b

1c

9d

No dead

0

0

0

1e

Non pregnant at sacrifice

5

4

4

4

No pregnant at sacrifice (%)

30 (85.7)

30(88.2)

31(88.6)

27(87.1)

Maternal weight gain (gestation) (g)f

175.33± 39.71

278.67 ± 32.70

261.61 ± 35.70

121.85 ± 53.71

Maternal weight gain (treatment) (g)f

-10.33 ± 41.34§§

32.33 ± 24.77

11.61 ± 36.73

-138.50 ± 51.19*

Clinical signs

Alopecia and Abrasion on Neck

1(gd6) 1(gd7)

 

 

 

Alopecia on face

1(gd15) 1(gd16) 1(gd17) 1(gd18)

 

 

 

Alopecia under chin

1(gd12)

 

 

 

Alopecia on forepaws

 

1(gd30)

1(gd 17)

 

Exophthalmos

1(gd6)

 

 

 

Cloudy eye

1(gd6) 1(gd17)

 

 

 

Weight lossj

1(gd7)(170) 1(gd9)(240) 2(gd11)(180 to 330)

1(gd17)(150)

1(gd18)(170)

1(gd19)(310)

1(gd8)(180)

1(gd9)(210)

1(gd11)(160)

3(gd12)(150 to 230)

1(gd17)(160)

1(gd7) (280)

1(gd10)(170)

1(gd12)(180)

1(gd14)(150)

1(gd17) (190)

1(gd18)(170)

1(gd19)(170)

1(gd11)(160) 1(gd14)(250)

1(gd16) (150)

1(gd18) (220)

Vocalisation postdosing

 

1(gd17)

2(gd12) 2(gd13) 1(gd14) 1(gd15) 1(gd18) 1(gd19)

1(gd8) 3(gd11) 2(gd12) 2(gd13) 2(gd15) 1(gd17) 1(gd19)

Ataxia

 

 

 

1(gd13)

Comatose

 

 

1(gd16)

2(gd10) 2(gd12) 1(gd13) 1(gd14) 2(gd15)

Convulsions

 

 

 

1(gd12)

Dyspnea

 

 

 

1(gd12) 1(gd19)

Epistaxis

1(gd16)

 

1(gd12)

2(gd13)

Congested

 

 

 

1(gd13)

Blood from mouth post dosing

1(gd14)

 

 

 

Diarrhoea

 

 

 

1(gd14) 1(gd15) 1(gd16)

Holding Head Up Postdosing

 

1(gd16)

 

1(gd14)

Lacrimation

 

1(gd14) 1(gd17) 1(gd18) 1(gd19)

 

1(gd14) 1(gd15)

Dead

 

 

 

1(gd15)

Necropsy (gd30)

 

 

 

 

Cyst on left fallopian tube

 

1

 

 

Cyst on right fallopian tube

 

1

 

 

Gallbladder twice normal size

 

 

 

1

Hemorrhagic lining of uterus

 

1

 

 

Pale liver

1

 

 

 

White mass in wall of uterus

 

1

 

 

aDoe was removed because she delivered early.

bOne doe delivered early and one doe aborted.

cDoe delivered early.

dTwodoes delivered early. One doe was removed because her oesophagus was punctured during treatment, resulting in her death. Four does showed evidence of having aspirated the dosing compound at necropsy. Two does were removed because their palates were punctured at treatment, resulting in their death.

eDeath of confirmed pregnant doe was compound related.

fIncludes all does pregnant at sacrifice; mean± S.E.M.

gDOSExDAY interaction was significant(p≤0.0001);See above for results of two-way ANOVA.

hweight gain during gestation minus gravid uterine weight.

IOne doe's liver weight was not recorded.

§§ p<0.01; Test for Linear Trend.

*P<0.05 (William’s test)

jClinical weight loss of equal to or greater than 150 g in 24 hours; range of weight loss (g) in parentheses.


 

Table 2: Embryo toxicity

 

Diethylene glycol diethyl ether (mg/kg/day)

0

50

200

400

Embryo toxicity

All littersa

30

30

31

27

Corporea lutea per doeb

9.47 ± 0.36

9.77 ± 0.39

9.50 ± 0.43

10.12 ± 0.47

Implantation sites per litterb

6.90 ± 0.66

7.00 ± 0.54

7.23 ± 0.48

7.89 ± 0.55

% preimplantation lossb, d

28.12 ± 5.74

29.35 ± 4.81

23.74 ± 4.20

23.10 ± 4.47

Resorptions per litterb(%)

0.93 ± 0.30

(16.27 ± 5.53)

0.43 ± 0.21

(8.63 ± 4.62)

0.19 ± 0.09

(2.59 ± 1.16)

1.00 ± 0.46

(13.88 ± 6.09)

No. litters with resorptions (%)

12f(40.0)

7g(23.3)

5 (16.1)

8h(29.6)

Dead foetuses per litterb(%)

0.07 ± 0.05

(0.70 ± 0.49)

0.00 ± 0.00

(0.00 ± 0.00)

0.03 ± 0.03

(0.32 ± 0.32)

0.00 ± 0.00

(0.00 ± 0.00)

No.litters with dead foetuses (%)

2

(6.7)

0

(0.0)

1

(3.2)

0

(0.0)

Non live implants per litterb,f(%)

1.00 ± 0.30

(16.97 ± 5.53)

0.43 ± 0.21

(8.63 ± 4.62)

0.23 ± 0.09

(2.92 ± 1.18)

1.00 ± 0.46

(13.88 ± 6.09)

No. litters with non live implantsf(%)

13

(43.3)

7

(23.3)

6

(19.4)

8

(29.6)

Adversely affected implants per litterb,g(%)

2.03 ± 0.42

(32.70 ± 6.32)

1.33 ± 0.32

(19.92 ± 5.24)

1.23 ± 0.26

(19.35 ± 4.23)

2.07 ± 0.47

(27.84 ± 6.04)

No. litters with adversely affected implantsg(%)

21

(70.0)

17

(56.7)

19

(61.3)

18

(66.7)

No. live litters with live foetusesh

27

28

31

24

Live foetuses per litterb, h

6.56 ± 0.57

7.04 ± 0.49

7.00 ± 0.46

7.75 ± 0.54

Male foetuses per litterb(%)

2.96 ± 0.41

(41.57 ± 4.60)

3.43 ± 0.39 (49.07 ± 6.32)

3.35 ± 0.32

(51.17±6.32)m

3.46 ± 0.34

(47.39 ± 4.38)

Average foetal body weight (g) per litterb, h

45.22 ± 1.60

46.36 ± 1.40

46.12 ± 1.52

42.26 ± 1.86

aIncludes all does pregnant at sacrifice; litter size = no. implantation sites per doe.

bReported as mean ± S.E.M.

c anddThe corpora lutea for one doe were not recorded.

e% preimplantation loss = [corpora lutes per doe (CL)-implantationsites per litter (IMP)]/CL(100): a 0% implantation loss value was assigned to does if CL < IMP for statistical analysis.

fThree confirmed-pregnant females had 100% resorptions.

gTwo confirmed-pregnant females had 100% resorptions.

hThree confirmed-pregnant females had 100% resorptions

iZero variance in one or more group – test not conducted

jnon live implants = dead plus resorbed.

kAdversely affect = non live implants plus malformed

lIncludes only does with live foetuses: litter size = no. live fetuses per doe.

mone foetus in one litter was not sexed.

§p<0.01; Test for Linear Trend.

 

 

 

Table 3: Teratogenicity

 

 

Diethylene glycol diethyl ether (mg/kg/day)

0

50

200

400

Teratogenicity

Total foetuses examineda

177

197

217

186

Total litters examinedb

27

28

31

24

No. foetuses with gross malformationsd

0

3

4

2

No. litters with gross malformationse

0

3

3

1

Gross malformations per litterc

0.00 ± 0.00

0.11 ± 0.06

0.13 ± 0.08

0.08 ± 0.08

No. foetuses with skeletal malformationsd

19

17

15

10

No. litters with skeletal malformationse

11

9

8

4

Skeletal malformations per litterc

0.70 ± 0.27

0.61 ± 0.23

0.048 ± 0.18

0.42 ± 0.22

No. foetuses with visceral malformationsd

16

9

15

17

No. litters with visceral malformationse

8

4

10

8

Visceral malformations per litterc

0.59 ± 0.20

0.32 ± 0.18

0.48 ± 0.18

0.71 ± 0.24

No. foetuses with variationsf

108

118

157

152

No. litters with variationsg

8

4

10

8

Foetuses malformed per litter (%)c, d

1.15 ± 0.31

(19.29 ± 5.00)

0.96 ± 0.27

(12.42 ± 3.56)

1.00 ± 0.23

(16.89 ± 4.20)

1.21 ± 0.28

(16.08 ± 3.80)

No. foetuses malformed (%)d

31

(17.51)

27

(13.71)

31

(14.29)

29

(15.59)

No. litters with malformations (%)e

15

(55.56)

13

(46.43)

17

(54.84)

13

(54.17)

Males malformed per litter (%)c

0.46 ± 0.16

(17.54 ± 6.65)

0.38 ± 0.15

(10.35 ± 4.62)

0.26 ± 0.09

(12.90 ± 4.98)

0.58 ± 0.17

(21.53 ± 6.89)

Females malformed per litter (%)c

0.74 ± 0.22

(19.27 ± 5.27)

0.63 ± 0.23

(12.44 ± 4.25)

0.75 ± 0.21

(16.85 ± 4.52)

0.68 ± 0.18

(14.59 ± 4.16)

aOnly live foetuses were examinedfor malformations.

bIncluded only litters with live foetuses.

cReported as mean ± S.E.M. for all live foetuses

dFoetuses with one or more malformations.

eLitter with one or more malformed foetuses.

fFoetuses with one or more variations.

gLitters with one or more foetuses with variations

Applicant's summary and conclusion

Conclusions:
Development of the New Zealand White rabbit is not sensitive to Diethylene glycol diethyl ether (DEGDEE) administered by gavage at maternally nontoxic doses. A NOAEL for DEGDEE-induced developmental toxicity was 400 mg/kg/day, a dose which produced maternal CNS depression and lethality (3.7%). The lowest dose given, 200 mg/kg/day, represented a NOAEL for DEGDEE-induced maternal toxicity.