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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:
Circa 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
The effect of oral administrations of sodium dehydroacetate (DHA-NA) on pregnant mice and their fetuses
Author:
Shiobara S
Year:
1980
Bibliographic source:
Japanese Journal of Public Health Vol 27, No. 2

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Similar to Japanese MHW Teratogenicity Study
Deviations:
not specified
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate
EC Number:
224-580-1
EC Name:
Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate
Cas Number:
4418-26-2
Molecular formula:
C8H7O4.Na
IUPAC Name:
sodium 1-(6-methyl-2,4-dioxo-2H-pyran-3(4H)-ylidene)ethanolate
Specific details on test material used for the study:
Supplier: Taisho Technos Co. Ltd.

Test animals

Species:
mouse
Strain:
other: ICL-ICR Stated as JCL-ICR in publication mouse
Details on test animals or test system and environmental conditions:
The animals were reared in an animal laboratory under the following conditions: five to ten dams were housed in a single plastic cage and allowed free access to solid feed (CE-2, manufactured by CLEA Japan, Inc.) and drinking water, at a room temperature of 20 ± 1°C, and a humidity of 55 ± 5%. Animals were 8 weeks old and acclimatised for 1 to weeks before pairing.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Two females to one male, overnight mating confirmed by copulation plug.
Duration of treatment / exposure:
Day 6 to 15 of presumed gestation.
Frequency of treatment:
Daily during treatment period.
Duration of test:
From day 0 to day 17 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
The experimental animals used were 8-week-old, nulliparous male and female JCL-ICR mice . After 1 to 2 weeks of preliminary rearing, two female and one male mice were cohabitated one night for the purpose of mating. If a copulatory plug was confirmed on the following morning, this was designated as gestational day (GD) 0.

Examinations

Maternal examinations:
Body weight of the pregnant mice recorded every day for 17 days, from GD 0 until the end of the experiment in order to examine the effects on the dams, and the clinical course of pregnancy monitored daily.
Ovaries and uterine content:
Autopsies carried out on the pregnant mice in the control group and DHA-Na administration groups on GD 17. During the autopsies, blood tests for maternal hemoglobin (Hb) and hematocrit (Ht), performed macroscopic examination were undertaken and organ weight measurements for several internal organs, including the liver, kidneys and spleen. Histology specimens were prepared a histopathological examination undertaken. The same examinations were undertaken on non-pregnant mice in whom the presence of a copulatory plug was not observed after mating.
Fetal examinations:
Uterine weight measured, intrauterine fetuses assessed as were: the number of implantations sites, the number of live fetuses, body weight of the live fetuses, the number of dead fetuses (classified the dead fetuses into early and late deaths). Systematic macroscopic examination of the live fetuses from the cephalic to caudal regions, examination of congenital malformations, focusing on exencephaly, cleft palate, failed sternebral fusion, brachyury and knotty tails. Stained skeletal specimens were examined using the Dawson method and examined to determine the state of ossification of the skeletal system, as well as the presence of anomalies or malformations, using the calvarium, vertebrae, sternebrae, and ribs.
Statistics:
Yes, but methods not specified. Significance at the 5% level used for data.
Indices:
Not calculated but data clearly presented to illustrate the in utero findings.
Historical control data:
None specified.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day effects on maternal hemoglobin (Hb) and hematocrit (Ht), but no dose response relationship evident. See data table below.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
See data table below.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See data table below.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See data table below.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See data table below.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
See data table below.
At 200 mg/kg bw/day only. However this group had a significanlty higher number of live fetuses and implantations and therefore greater stress on the mother. The biological significance of the fetal deaths may therefore be questionable. See data table below.
Changes in pregnancy duration:
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): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): See data table below.
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
haematology
Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse maternal or ebmryo-fetal toxicity at 100 mg/kg bw/day.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day only. However this group had a significanlty higher number of fetuses which may have affected this parameter, the biological significance may therefore be questionable. No clear dose response relationship was evident. See data table below.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 200 mg/kg bw/day only. However this group had a significanlty higher number of fetuses which may have affected this parameter, the biological significance may therefore be questionable. No clear dose response relationship was evident. See data table below.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The number of live fetuses at 200 mg/kg bw/day was increased as was the number of dead fetuses. This apparent ambiguity may have been as a result of the greater number of implantations, thus the greater in utero burden to the mothers may have affected in utero development in this strain of mouse. See data table below.
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The number of fetuses with 14 ribs was higher in the treated groups, however this may be related to the number of dams with 14 ribs that were similarly higher in these groups compare to the control. Such skeletal finding may be regarded as a minor skeletal anomaly and thus a relationship to treament was cinsidered unlikely. See data table below. Compared to the control group, there was significantly delayed sternebral ossification observed in the DHA-Na administration groups. Based on the examination of metacarpal, metatarsal and coccygeal vertebral staining, overall, delayed ossification was observed in the DHA-Na 100 mg and 200 mg administration groups. Skeletal anomalies observed in the 50 mg and 100 mg groups included a number of cases with bifurcated and fused cervical vertebrae and ribs, although no significant difference was observed in comparison to the control group.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A single case with cleft palate in the 100 mg/kg bw/day administration group during the examination for congenital malformations, no anomalies were seen in the control group or any other administration groups. A relationship to treatment is considered unlikely. The authors state that cleft palate is a spontaneous malformation in this strain of mouse. See data table below.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Skeletal variants: number of metacarpals

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
other: skeletal variants
Description (incidence and severity):
see tabulated data

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

Effect of DHA-Na on pregnant mice

Dose (mg/kg)

 

0

50

100

200

Mated

 

25

25

25

25

Pregnant (%)a

 

20 (80.0)

18(72.0)

21(84.0)

20(80.0)

Premature delivery

 

0

0

0

0

Maternal death

 

0

0

0

0

Total litter loss

 

0

0

0

0

Dams with alive fetuses (%)b

20(100.0)

18(100.0)

21(100.0)

20(100.0)

Numerals in parentheses indicate 

a: percentages to the number of mated dams.

b: percentages to the number of pregnant dams

Effect of DHA-Na on pregnant mice (continued)

 

Dose (mg/kg)

0

50

100

200

Body weight (g)

(m) (σ)

 

 

 

at 0 day

25.2 ± 0.8

24.9 ± 1.0

25.0 ± 1,2

25.3 ± 1.2

at 17 day

41.5 ± 4. 5

41.0 ± 5.7

42.6 ± 4.0

40. 5 ± 4. 2

B.W. gain rate (%)

165.0 ± 18.24

164.9 ± 20.87

170.4 ± 14. 85

159.8 ± 14.46

Uterus (g)

11.00 ± 4.18

12.19 ± 4.76

12.39 ± 3. 36

11.06 ± 3.43

Organ weight (g)

 

 

 

 

Liver

1.88 ± 0.21(4.5)

1.88 ± 0.32 (4. 6)

1.90 ± 0.17 (4.5)

1.95 ± 0.20 (4.8)

Kidneys

0.33 ± 0.03 (0.8)

0.32 ± 0.03 (0.8)

0.32 ± 0.03 (0.8)

0.33 ± 0.03 (0.8)

Spleen

0.10 ± 0.03 (0.2)

0.11 ± 0.03 (0.3)

0.13 ± 0.04 (0.3)

0.12 ± 0.03 (0.3)

Hb (g/d/)

13.5 ± 0.81

13.7 ± 0.94

13.5 ± 0.64

13.6 ± 0.59

Ht (%)

38.5 ± 3.3

41.0 ± 2.4*

37.8 ± 3.1

39.1 ± 2.4

 

(m); mean           (σ); S.D.

* Significant at 5% level in comparison with the control group
Numerals in parentheses indicate relative weight (%)

Tab. 4 Effect of DHA-Na administered to the dams on embryonic development in mice

Dose (mg/kg)

0

50

100

200

Dams

20

18

21

20

No. of implantations

165

174

191

195

 

(m) (σ)

 

 

 

 

8.25 ± 3.45

9.67 ± 4.03

9.10 ± 2.57

9. 75+3.06

Alive fetuses

 

 

 

 

No. of fetuses

161 (97.6)

166 (95.4)

187 (97.9)

178 (91.3)**

Litter size

8.05 ± 3.32

9.22 ± 3.84

8.90 ± 2.64

8.90 ± 3.06

Body weight (g)

0.93 ± 0.12

0.86 ± 0.11**

0.92 ± 0.11

0.76 ± 0.11**

Dead fetuses

 

 

 

 

No. of fetuses

4 (2.4)

8 (4.6)

4 (2.1)

17 (8.9)**

Early

3 (1.8)

6 (3.4)

2 (1.0)

7 (3.6)

Late

1 (0.6)

2 (1.1)

2 (1.0)

10 (5.1)

(m); mean (σ); S.D.

* Significant at 5% level in comparison with the control group
Numerals in parentheses indicate relative weight(%)

Effect of DHA-Na administered to the dams on anomalies in fetal mice

Dose (mg/kg)

0

50

100

200

Dams

20

18

21

20

No. of fetuses examined

161

166

187

178

No. of fetuses with malformation

 

 

 

 

Cleft palate

0

0

1 (0.5)

0

Exencephaly

0

0

0

0

Numerals in parentheses indicate percentage to the number of total fetuses examined

Tab. 6 Effect of DHA-Na administered to the dams on skeletal variations in fetal mice

Dose (mg/kg)

0

50

100

200

Dams

20

18

21

20

No. of fetuses examined

161

166

187

178

No. of fetuses with variations

 

 

 

 

Cervical vertebrae

0

1

2

0

Thoracic vertebrae

0

0

0

0

Rib

1

2

2

0

Lumbar vertebrae

0

0

0

0

Sacral vertebrae

0

0

0

0

Extremities

0

0

0

0

Total

1

3

4

0

No. of fetuses with cervical ribs

2

0

0

0

No. of fetuses with 14 ribs

44 (27.3)

84 (50.6)**

140 (74.9)**

141 (79.2)**

No. of dams of fetuses with 14 ribs

12 (60.0)

17 (94.4)*

21 (100.0)*

20 (100.0)*

in parentheses indicate percentages to the number of total fetuses examined Significant at 5% and 1% level in comparison with the control group

Effect of DHA-Na administered to the dams on skeletal development in fetal mice

Dose (mg/kg)

 

0

50

100

300

Dams

20

18

21

20

No. of fetuses examined

 

161

166

187

178

No. of fetuses with deformed sternebrae

10 (6.1)

57 (34.3)**

34 (18.1)**

127 (71.3)**

No. of metacarpals

 

 

 

 

 

Left

0

1 (0.6)

2 (1.2)

2 (1.1)

5(2.8)

 

1

0

0

0

3 (1.7)

 

2

1 (0.6)

2 (1.2)

3 (1.6)

59 (33.1)**

 

3

56 (34.8)

120 (72.3)**

138 (73.8)**

108 (60.7)**

 

4

103 (64.0)

42 (25.3)**

42 (22.5)**

3 (1.7)**

 

5

0

0

0

0

Right

0

1 (0.6)

2 (1.2)

2 (1.1)

5 (2.8)

 

I

0

0

0

3 (1.7)

 

2

1 (0.6)

2 (1.2)

3 (1.6)

59 (33.1)**

 

3

58 (34.8)

120 (72.3)**

138 (73.8)**

108 (60.7)**

 

4

103 (64.0)

42 (25.3)**

42 (22.5)**

3 (1.7)**

 

5

0

0

2 (1.1)

0

No. of metatarsals

 

 

 

 

 

Left

0

1 (0.6)

2 (1.2)

4 (2.1)

6 (3.4)

 

1

0

1(0.6)

0

3 (1.7)

 

2

1 (0.6)

0

0

7 (3.9).

 

3

1 (0.6)

31 (18.7)**

7 (3.7)*

88 (49.4)**

 

4

144 (89.4)

130 (78.3)**

172 (92.0)

74 (41.6)**

 

5

14 (8.7)

2 (1:2)**

4 (2.1)**

0**

Right

0

1 (0.6)

2 (1.2)

4 (2.1)

6 (3.4)

 

1

0

1 (0.6)

0

3 (1.7)

 

2

1 (0.6)

0

0

7 (3.9)*

 

3

1 (0.6)

31 (18.7)**

7 (3.7)*

88 (49.4)**

 

4

144 (89.4)

130 (78.3)**

172 (92.0)

74 (41.6)**

 

5

14 (8.7)

2 (1.2)**

4 (2.1)**

0**

No. of coccygeal

 

 

 

 

 

 

0

4 (2.5)

9 (5.4)

6 (3.2)

78 (43.8)**

 

1

5 (3.1)

26 (15.7)**

11 (5.9)

32 (18.0)**

 

2

51 (31.7)

43 (25.9)

46 (24.6)

58 (32.6)

 

3

65 (40.4)

69 (41.6)

102 (54.5)**

10 (5.6)**

 

4

25 (15.5)

16 (9.6)

17 (9.1)

0**

 

5

9 (5.6)

3 (1.8)

5 (2.7)

0**

 

6

1 (0. 6)

0

0

0

 

7

1 (0.6)

0

0

0

*** Significant at 5% and 1% level in comparison with the control group Numerals in parentheses indicate percentages to the number of examined

Applicant's summary and conclusion

Conclusions:
Maternal toxicity was evident at 200 mg/kg bw/day as changes in haematological parameters with increases in haemoglobin and haematocrit. Some fetal effects were noted as an increase in fetal mortality and body weight at 200 mg/kg bw/day. Increased incidences of fetal 14th (a common anomaly in this mouse strain) were closely associated with the same rib count in the dams. Some delayed sternebral ossification was observed in all the DHA-Na administration groups. Skeletal an
omalies observed in the 50 or 100 mg/kg bw/day but there was no significant difference observed in comparison to the control group.
Executive summary:

In this developmental toxicity study DHA-Na was administered daily by oral gavage to pregnant JCLICR mice from GD 6 to GD 15 at concentrations of 0, 50, 100 or 200 mg/kg bw/day. Maternal toxicity was evident at 200 mg/kg bw/day as changes in haematological parameters with increases in haemoglobin and haematocrit. Some fetal effects were noted as an increase in fetal mortality and body weight at 200 mg/kg bw/day. This may have reflected the increased number of implantations and live foetuses in this group. Increased incidences of fetal 14thwere closely associated with the same rib count in the dams. Some delayed sternebral ossification was observed in all the DHA-Na administration groups. Skeletal anomalies observed in the 50 or 100 mg/kg bw/day groups included a few cases of bifurcated and fused cervical vertebrae and ribs, however there was no significant difference observed in comparison to the control group.