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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:
2016-09-02 to 2017-02-21
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted by the Council on 22nd January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: ESD0021175
- Expiration date of the lot/batch: 2017-11-27
- Apperance: colourless waxy solid
- Storage condition of test material: Refrigerator (2 to 8 °C)

- Purity: 35% (w/v) solution in water
- Date of arrival: 14 September 1989
- Storage conditions: room temperature

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: ESD0021175
- Expiration date of the lot/batch: 2017-11-27
- Apperance: colourless waxy solid
- Storage condition of test material: Refrigerator (2 to 8 °C)

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Distilled water
Details on exposure:
The dose formulations were prepared shortly before each dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dose formulations from one set per dose group will be taken immediately after preparation once before commencement of dosing i.e. on day 5 of gestation and on day 19 of gestation (i.e. day 1 and day 15 of treatment) or homogeneity (mean of homogeneity will be given as dose concentration). On day 11 of gestation (e.e. day 7 of treatment), samples of all dose formulations will be analysed for dose concentrations by analysing triplicate samples.
Analysis was performed according to the validated analytical method provided by the sponsor.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one female
- Length of cohabitation: maximum period of fourteen days
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: Each female examined for presence of vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
Duration of treatment / exposure:
day 5 of Gestation to day 19 of Gestation
Frequency of treatment:
daily
Duration of test:
Animals were sacrificed on day 20 of gestation by cesariean section.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
females: 24 / group
males: 50 males in total were used for mating
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results from a dose range finding study in Wistar rats with the test substance (RCC study number 6265), conducted at RCC Laboratories India Private Limited, the 4 doses were selected.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Once on day of receipt and twice daily thereafter. Once on day of necropsy
-
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Acclimatization period once daily. Twice daily on initial 3 days after treatment; once daily thereafter

BODY WEIGHT: Yes
Time schedule for examinations:
- Acclimatization Period: Once weekly
- Premating: On first day of pairing and weekly thereafter (if any)
- Gestation: On gestation day 0, 3, 5, 8, 11, 14, 17, 19 and 20 and at death, if any

FOOD CONSUMPTION GESTATION: Yes
- on Gestation day 3, 5, 8, 11, 14, 17 and 19

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
All animals including those found dead or sacrificed in moribund condition were subject of complete gross necropsy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead during study)
- 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
- Soft tissue examinations: Yes: half per littera
- Skeletal examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the body weight, body weight change, feed consumption, reproduction and external, visceral and skeletal alterations/variations.
•Data were summarized in tabular form. Statistical analysis were performed using statplus program. Values were given as mean ± standard deviation (SD).
•All the data were checked for Normality with Shapiro-Wilk W test.
•All the data were checked for Homogeneity with Bartlett Chi-Square test.
•Data were subjected to perform Analysis of Variance (ANOVA) for continuous data.
•Discontinuous data were subjected to nonparametric test (Mann-Whitney U-Test).
Indices:
The following formulae were used for calculating the indices:
Pre–implantation loss (%)
(Number of Corpora Lutea - number of implantation sites) / Number of Corpora Lutea x 100
Post–implantation loss (%)
(Number of implantation sites - Total number of live foetuses) / Number of implantation sites x 100
Sex Ratio (% males)
Number of male foetuses (Day 0) / Total number of foetuses (Day 0) x 100
Variation Incidence (%)
Number of foetuses with variation/ Total Number of foetuses examined x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs observed in all females up to 80 mg/kg bw/d. Prior to death of all group 120 mg/kg bw/d animals, these animals showed diarrhoea, dullness and piloerection. Two animals additionally showed epistaxis.
Mortality:
mortality observed, treatment-related
Description (incidence):
No Mortalities observed up to the dose of 80 mg/kg bw/d. All females of 120 mg/kg bw dose group died beginning on treament day 4 until day 12 of treatment. Prior to death, all animals showed diarrhoea, dullness and piloerection. Two animals additionally showed epistaxis.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference was observed in the body weight and body weight gain (%) of pregnant females in low, intermediate and medium high dose group animals as compared with control group animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant difference was observed in the feed consumption of pregnant females in low, intermediate and medium high dose group animals when compared with control group animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopy did not reveal any abnormalities during last week in all animals of groups 1 and 4. In the last week of treatment, all the animals in group 5 were found dead. Hence, ophthalmoscopy was performed for group 4 animals.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters such as Erythrocyte count (RBC), Hemoglobin (Hb), Hematocrit (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Total leucocyte count (WBC), Differential leukocyte count (DC), Prothrombin time (PT) and Activated Partial Thromboplastin time (APTT) did not show any toxicologically relevant findings in the treated groups when compared with control group.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters like Glucose (GLU), Urea, Creatinine (CREA), Triglycerides (TRIGL), Alkaline phosphatase (ALP), Bilirubin (BIL), Sodium (Na), Potassium (K), Chloride (Cl), Total protein (TPO), Albumin (ALB), Globulin (GLB) and Albumin and Globulin ratio (A/G) did not show any toxicologically relevant findings in treatment groups when compared with control group. Where as in cholesterol, there was significant decrease in medium high as compared with intermediate dose. In aspartate aminotransferase, there was significant increase in medium high as compared with intermediate dose and significant decrease in intermediate dose as compared with low dose. In alanine aminotransferase, there was significant increase in low, intermediate and medium high dose as compared with control and significant increase in medium high as compared with low and intermediate dose.
The significant changes observed in the clinical pathology parameters were marginal and could not be attributed to the test item administration as these values were within clinical and biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related macroscopic findings were observed at necropsy on day 20 of gestation in any of the animals up to 80 mg/kg bw/d. All Females of 120 mg/kg bw dose group died beginning on treatment day 4 until day 12 of treatment. Findings at necropsy of 120 mg/kg bw/d dose group females: Thymus - small in size, slightly; Adrenals – enlarged, slightly; Spleen – enlarged, slightly; Stomach – distended, moderately to markedly; Fore stomach raised whitish foci, moderately to markedly; Ulceration moderately to markedly; Mucous membrane peeled off in some females; Glandular stomach – reddened, slightly to moderately.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions occurred during the treatment period.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups. No litter loss occurred
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
With the exception of one dead fetus out of 170 fetuses at the 80 mg/kg bw/d dose group, no dead fetuses were observed in any of the dose groups (0, 20, 40, 80 mg/kg bw/d).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Due to the study design of an OECD 414 study, pregnancy is terminated by caesarian section at day 20 of gestation. No changes were observed at any dose group.
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): Due to the study design of an OECD 414 study, pregnancy is terminated by caesarian section at day 20 of gestation.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All female animals of control, low, intermediate, medium high and high dose groups showed positive evidence of mating. At necropsy, three females each in control and 40 mg/kg bw/d group, four females in 20 mg/kg bw/d dose group and two females in the 80 mg/kg bw/d dose group were found non pregnant. In high dose group (i.e. 120 mg/kg bw/d) three animals were found non pregnant during necropsy.
Other effects:
no effects observed
Details on maternal toxic effects:
All Females of 120 mg/kg bw dose group died beginning on treatment day 4 until day 12 of treatment. Findings at necropsy of 120 mg/kg bw/d dose group females: Thymus - small in size, slightly; Adrenals – enlarged, slightly; Spleen – enlarged, slightly; Stomach – distended, moderately to markedly; Fore stomach raised whitish foci, moderately to markedly; Ulceration moderately to markedly; Mucous membrane peeled off in some females; Glandular stomach – reddened, slightly to moderately. At necropsy no abnormality was detected in all females of dose groups 0, 20, 40, 80 mg/kg bw/d.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
Remarks on result:
other:
Remarks:
At high dose of 120 mg/kg bw/d all dams died

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
other: none

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
At time of ceasarian section and thus time point of weighing all foetuses, no significant difference was observed in foetal weight between control and treated groups. Foetal body wheight change is not an aim of the study design.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At time of ceasarian section which is part of the OECD 414 study design and thus time point of weighing all foetuses, no significant difference was observed in foetal weight between control and treated groups. Foetal body wheight change is not an aim of the study design.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No reduction of live foetuses was observed throughout the dose groups 0, 20, 40 and 80 mg/kg bw/d. The high dose group (i.e. 120 mg/kg bw/d) could not be evaluated as all dams of this dosing group died until treatment day 12.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was calculated as percent (%) male and no significant difference was observed in sex ratio in treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No changes in litter size and litter weights could be determined in all treated groups compared to control group.
Changes in postnatal survival:
not specified
Description (incidence and severity):
Postnatal survival is not an aim of the study design of an OECD 414 study.
External malformations:
no effects observed
Description (incidence and severity):
There were no test item related variations/malformations observed in foetuses subjected for external and visceral (soft tissue) examination.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of foetuses showing skeletal variation in treatment groups were comparable with control group. There were no test item related skeletal variations/malformations in any of the treated group. Most of the observations recorded were minor abnormalities and considered as routine variables.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test item related variations/malformations observed in foetuses subjected for external and visceral (soft tissue) examination.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
<= 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the medium high dose of 80 mg/kg bw/d.
Remarks on result:
other:
Remarks:
At high dose of 120 mg/kg bw/d all dams died

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Tab. 1            Mortality and Clinical Signs Females - Summary

 

Group

G1

G2

G3

G4

G5

Dose (mg/kg bw)

0

20

40

80

120

Mortality

0/24

0/24

0/24

0/24

24/24

Clinical signs

0/24

0/24

0/24

0/24

Dullness

-

(24/24)

Diarrhoea

-

(24/24)

Piloerection

-

(24/24)

Epistaxis

-

(02/24)

 

 

Tab. 2            Pregnancy Status - Summary

 

Group

G1

G2

G3

G4

G5*

Dose (mg/kg bw)

0

20

40

80

120

Females mated

24

24

24

24

24

Pregnant females

21

20

21

22

21

* All the animals were found dead between day 8 and 16 of gestation period (i.e. day 4 and day 12 of treatment)

 

 

Tab. 3            Body weight (G) dams - Summary

Group

Body weights (G)

Gestation Day

 

0

3

5

8

11

14

17

20

Group 1

Mean

253.6

261.8

269.0

276.5

288.0

303.2

319.1

329.4

SD

7.44

8.67

10.44

11.42

13.34

15.01

14.82

14.04

N

21

21

21

21

21

21

21

21

Group 2

Mean

255.8

261.7

267.7

274.7

283.8

297.0

313.5

327.1

SD

9.27

9.25

9.80

11.75

13.47

13.35

14.11

15.30

N

20

20

20

20

20

20

20

20

Group 3

Mean

256.8

262.6

268.5

275.1

286.2

302.3

322.6

338.4

SD

8.25

6.97

6.68

7.87

7.35

10.08

11.49

15.02

N

21

21

21

21

21

21

21

21

Group 4

Mean

265.0

262.5

269.7

277.3

287.8

301.0

319.1

333.4

SD

8.14

7.91

9.44

11.30

12.41

13.04

14.59

16.26

N

22

22

22

22

22

22

22

22

 

 

Tab. 4            Body weight Gain (%) dams - Summary

Group

Body weights Gain (%)

Gestation Day

-

0

3

5

8

11

14

17

20

Group 1

Mean

0.0

3.2

6.1

9.0

13.6

19.6

25.9

29.9

SD

0.00

1.91

3.38

4.31

4.78

5.44

5.52

5.38

N

21

21

21

21

21

21

21

21

Group 2

Mean

0.00

2.3

4.7

7.5

11.0

16.2

22.7

28.0

SD

0.00

1.47

2.66

3.90

4.52

4.56

5.93

7.10

N

20

20

20

20

20

20

20

20

Group 3

Mean

0.0

2.3

4.6

7.2

11.5

17.8

25.7

31.9

SD

0.00

1.30

2.54

3.90

3.92

5.21

6.42

7.73

N

21

21

21

21

21

21

21

21

Group 4

Mean

0.0

2.6

5.4

8.4

12.5

17.6

24.8

30.4

SD

0.00

1.77

3.18

4.51

5.07

5.51

7.15

8.75

N

22

22

22

22

22

22

22

22

 

 

Caesarean Data

Tab. 5            Maternal Data - Summary

 

Parameter

Group

G1

G2

G3

G4

Dose (mg/kg bw)

0

20

40

80

No. of Dams

21

20

21

22

Gravid Uterine Weight (G)

Mean

41.5978

38.2861

50.8140

41.9012

SD

19.0702

20.3954

15.4402

20.3170

Total Foetuses

Number

163

136

183

170

Mean

7.76

6.80

8.71

7.73

SD

3.83

4.09

3.15

4.08

No. of CL

Mean

12.48

11.60

12.14

11.05

SD

3.37

2.64

2.52

4.71

No. of Implantation

Mean

8.48

7.90

9.48

8.82

SD

3.92

4.36

3.03

4.26

Early Resorption

Mean

0.67

0.90

0.76

1.09

SD

0.86

1.12

1.22

1.31

Late Resorption

Mean

0.05

0.20

0.00

0.00

SD

0.22

0.89

0.00

0.00

Pre implantation loss

Mean

4.0

3.7

2.7

2.2

SD

3.44

3.34

3.26

2.20

%

30.85

34.23

20.75

18.38

Post implantation loss

Mean

0.7

1.1

0.8

1.1

SD

0.90

1.29

1.22

1.39

%

9.07

14.19

8.75

14.75

Total Number of resorption/s

Number

15

22

16

24

 

 

Pup Data

Tab 6             Litter data – Summary

 

Parameter

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Dams

24

24

24

24

No. of Litters

21

20

21

22

Total No. of Foetuses

163

136

183

170

Mean Litter Size

7.76

6.80

8.71

7.73

No. of live foetuses

Number

163

136

183

169

Mean

7.76

6.80

8.71

7.68

SD

3.83

4.09

3.15

4.09

(%)

100

100

100

99.4

No. of dead foetuses

Number

0

0

0

1

Mean

0.00

0.00

0.00

0.05

SD

0.00

0.00

0.00

0.21

No. of live male foetuses

Number

70

72

80

84

Mean

3.33

3.60

3.81

3.82

SD

1.85

2.48

1.91

2.56

No. of live female foetuses

Number

93

64

103

85

Mean

4.43

3.20

4.90

3.86

SD

2.60

1.91

2.26

2.53

 

 

Tab.7             Group Mean Foetal weight

 

Group

Foetal weight /

Litter (G)

Foetal weight (G)

Male

Female

Group 1

Mean

3.5834

3.6996

3.4960

SD

0.6132

0.6325

0.5866

N

163

70

93

Group 2

Mean

3.6514

3.7097

3.5859

SD

0.5052

0.5193

0.4845

N

136

72

64

Group 3

Mean

3.8994

4.0176

3.8076

SD

0.6315

0.5102

0.7004

N

183

80

103

Group 4

Mean

3.7112

3.7236

3.6990

SD

0.5006

0.4952

0.5085

N

170

84

86

 

 

Tab. 8            Group Mean Sex Ratio

 

Group

Sex ratio (%)

Male

Sex ratio (%)

Female

Group 1

Mean

41.75

58.25

SD

15.73

15.73

N

163

Group 2

Mean

47.02

52.98

SD

25.65

25.65

N

136

Group 3

Mean

41.50

58.50

SD

19.44

19.44

N

183

Group 4

Mean

44.38

51.31

SD

23.66

24.21

N

170

 

 

Effects on Embryos/Foetuses

Tab. 9            External findings - Summary

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Litter Examined

21

20

21

22

No. of Foetuses Examined

163

136

183

170

Variation Incidence – Number (%)

No. of Foetus with Variations

Total Variations

1(0.61)

1(0.74)

2(1.10)

1(0.59)

Body Small in size

1(0.61)

1(0.74)

1(0.55)

1(0.59)

Body Pale

0(0.00)

0(0.00)

1(0.55)

0(0.00)

 

 

Tab. 10           Visceral Findings - Summary

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Litter Examined

21

20

21

22

No. of Foetuses Examined

76

63

87

81

Variation Incidence – Number (%)

No. of Foetus with Variations

Total Variations

7(9.21)

1(1.59)

0(0.00)

2(2.47)

Ureters: Convoluted

4(5.26)

0(0.00)

0(0.00)

2(2.47)

Spleen: Pale

2(2.63)

0(0.00)

0(0.00)

0(0.00)

Spleen: Small in size

1(1.32)

1(1.59)

0(0.00)

0(0.00)

 

 

Tab. 11           Skeletal Findings - Summary

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Litter Examined

21

20

21

22

No. of Foetuses Examined

87

73

96

89

Variation Incidence – Number (%)

Nasal

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Premaxilla

Not Ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Mandible

Short

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Exoccipital

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Interparietal

Incompletely ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Supraoccipital

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Hyoid

Incompletely ossified

1(1.16)

0(0.00)

1(1.04)

0(0.00)

Vertebrae - Thoracic

Dumbbell shaped

7(8.14)

13(17.81)

14(14.58)

14(15.73)

Bipartite

2(2.33)

0(0.00)

2(2.08)

5(5.62)

Cervical

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Caudal

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Lumbar

Dumbbell shaped

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Hemivertebra

0(0.00)

1(1.37)

0(0.00)

0(0.00)

Misaligned

0(0.00)

1(1.37)

0(0.00)

0(0.00)

Sacral

Dumbbell shaped

0(0.00)

1(1.37)

0(0.00)

0(0.00)

Sternal Centers

Not ossified

9(10.47)

2(2.74)

2(2.08)

2(2.25)

Manubrium

Not ossified

0(0.00)

0(0.00)

1(1.04)

0(0.00)

Xyphiod

Not ossified

6(6.98)

2(2.74)

4(4.17)

1(1.12)

Ribs (Left)

Waviness

0(0.00)

0(0.00)

2(2.08)

3(3.37)

Supernumerary

12(13.95)

11(15.07)

14(14.58)

11(12.36)

Ribs (Right)

Waviness

0(0.00)

1(1.37)

2(2.08)

5(5.62)

Supernumerary

11(12.79)

11(15.07)

11(11.46)

12(13.48)

Ischium (Left)

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Ischium (Right)

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Pubis (Left)

Not ossified

2(2.33)

0(0.00)

0(0.00)

0(0.00)

Pubis (Right)

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

 


 

 

Individual Macroscopic Findings Females

 

Group / Dose

Dam Number

Necropsy Findings

1/

0 mg/kg

 

01

No Abnormality Detected

02

No Abnormality Detected

03

No Abnormality Detected

04

No Abnormality Detected

05

No Abnormality Detected

06

No Abnormality Detected

07

No Abnormality Detected

08

No Abnormality Detected

09

No Abnormality Detected

10

No Abnormality Detected

11

No Abnormality Detected

12

No Abnormality Detected

13

No Abnormality Detected

14

No Abnormality Detected

15

No Abnormality Detected

16

No Abnormality Detected

17

No Abnormality Detected

18

No Abnormality Detected

19

No Abnormality Detected

20

No Abnormality Detected

21

No Abnormality Detected

22

No Abnormality Detected

23

No Abnormality Detected

24

No Abnormality Detected

Group / Dose

Dam Number

Necropsy Findings

2/

20 mg/kg

 

25

No Abnormality Detected

26

No Abnormality Detected

27

No Abnormality Detected

28

No Abnormality Detected

29

No Abnormality Detected

30

No Abnormality Detected

31

No Abnormality Detected

32

No Abnormality Detected

33

No Abnormality Detected

34

No Abnormality Detected

35

No Abnormality Detected

36

No Abnormality Detected

37

No Abnormality Detected

38

No Abnormality Detected

39

No Abnormality Detected

40

No Abnormality Detected

41

No Abnormality Detected

42

No Abnormality Detected

43

No Abnormality Detected

44

No Abnormality Detected

45

No Abnormality Detected

46

No Abnormality Detected

47

No Abnormality Detected

48

No Abnormality Detected

Group / Dose

Dam Number

Necropsy Findings

2/

20 mg/kg

 

25

No Abnormality Detected

26

No Abnormality Detected

27

No Abnormality Detected

28

No Abnormality Detected

29

No Abnormality Detected

30

No Abnormality Detected

31

No Abnormality Detected

32

No Abnormality Detected

33

No Abnormality Detected

34

No Abnormality Detected

35

No Abnormality Detected

36

No Abnormality Detected

37

No Abnormality Detected

38

No Abnormality Detected

39

No Abnormality Detected

40

No Abnormality Detected

41

No Abnormality Detected

42

No Abnormality Detected

43

No Abnormality Detected

44

No Abnormality Detected

45

No Abnormality Detected

46

No Abnormality Detected

47

No Abnormality Detected

48

No Abnormality Detected

 

Group / Dose

Dam Number

Necropsy Findings

 

5/

120 mg/kg

 

97

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Urinary bladder - distended, slightly;

Adrenals - enlarged, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci;

                 Ulcers, slightly;

Glandular stomach – reddened, slightly

 

98

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Urinary bladder - distended, slightly;

Adrenals - enlarged, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci  

                 moderately; Ulcers slightly;

Glandular stomach – reddened, slightly

 

99

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                 markedly; reddish fluid content present; Ulceration,

                 moderately;

Glandular stomach – reddened, slightly

 

100

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach - distended slightly; Fore stomach raised whitish foci

                 moderately; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

101

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Lungs – reddened, slightly;

Spleen - enlarged in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                 moderately; Ulceration, moderately;

Glandular stomach – reddened, moderately

 

5/

120 mg/kg

 

102

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size. slightly;

Stomach - distended slightly; Fore stomach raised whitish foci

                 markedly; reddish fluid content, present; Ulceration,

                 moderately;

Glandular stomach – reddened, slightly

 

103

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately; Mucous membrane 

                  peeled off;

Glandular stomach – reddened, moderately

 

104

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals - enlarged slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

105

External:

Perineum soiled

 

Internal:

 

Lungs reddened all lobes slightly; Autolytic changes were set in

 

106

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach – reddened, moderately

 

5/

120 mg/kg

 

107

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration markedly; Mucous membrane peeled off;

Glandular stomach – reddened, moderately

 

108

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach – reddened, moderately

 

109

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach - reddened moderately;

Autolytic changes were set in

 

110

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus – small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

5/

120 mg/kg

 

111

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach –reddened, moderately;

Autolytic changes were set in

 

112

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; reddish fluid content present; Ulceration

                  moderately;

Glandular stomach – reddened, slightly

 

113

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

114

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals - enlarged in size, slightly;

Stomach - non glandular mucosa thickened slightly; raised whitish foci moderately; Ulceration slightly; Glandular stomach - reddened slightly

 

115

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; Ulceration moderately;

Glandular stomach - reddened slightly

 

5/

120 mg/kg

 

116

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen - enlarged in size, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci

                 markedly; Ulceration, markedly;

Glandular stomach - reddened moderately

 

117

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals - enlarged in size, slightly;

Stomach - non glandular mucosa thickened slightly; raised whitish foci moderately; Ulceration, moderately; Glandular stomach - reddened slightly

 

118

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach - reddened moderately;

Autolytic changes were set in

 

119

External:

Perineum soiled; Rigor mortis present; Nostrils reddish discharge slightly

 

Internal:

 

Thymus - small in size, moderately;

Adrenals – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular & non glandular – reddened, moderately

 

120

External:

Perineum soiled; Rigor mortis present; Nostrils reddish discharge slightly

 

Internal:

 

Thymus - small in size, moderately;

Adrenals – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular & non glandular – reddened, moderately

 

Applicant's summary and conclusion

Conclusions:
Pregnant females (24/dose group) were dosed by oral gavage at 0, 20, 40, 80 and 120 mg/kg bw /d solved in water as vehicle beginning at day 6 of pregnancy up to day 19 of pregnancy. On day 20 all females were sacrificed by caesarian section. Fetuses were counted, weighted, sex determined and divided in two groups by randomization. One group of fetuses was selected for visceral examination the second group was prepared for skeletal examination.

Dullness, diarrhoea, piloerection and epistaxis (two females) were observed in 24 out of 24 female animals treated at 120 mg/kg body weight (high dose). All animals of the high dose group died during day 8 to day 16 of gestation (i.e. between day 4 and day 12 of treatment). Gross pathological local findings of the decedent dams at stomach, fore-stomach and glandular stomach are considered a direct consequence of the chemical property of the test substance, namely being a cationic surfactant. These findings are:
- Stomach – distended, moderately to markedly;
- Fore stomach raised whitish foci, moderately to markedly;
- Ulceration moderately to markedly;
- Mucous membrane peeled off in some females;
- Glandular stomach – reddened, slightly to moderately.
No mortality or clinical signs of toxicity or macroscopic changes were observed in all animals dosed up to 80 mg/kg bw/d.

The evaluation of the reproductive organs of all dams dosed up to 80 mg/kg bw/d, pre- and post-implantation loss, gravid uterine weight, litter size, viability of the fetuses, foetus weight per litter and foetus weight, sex ratio of the fetuses and the external, visceral and skeletal examination of the fetuses revealed that HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the dose of 80 mg/kg body weight. Hence, under the present experimental conditions, HYEQS is not a teratogen.
Executive summary:

Pregnant females (24/dose group) were dosed by oral gavage at 0, 20, 40, 80 and 120 mg/kg bw /d solved in water as vehicle beginning at day 6 of pregnancy up to day 19 of pregnancy. On day 20 all females were sacrificed by caesarian section. Fetuses were counted, weighted, sex determined and divided in two groups by randomization. One group of fetuses was selected for visceral examination the second group was prepared for skeletal examination.

Dullness, diarrhoea, piloerection and epistaxis (two females) were observed in 24 out of 24 female animals treated at 120 mg/kg body weight (high dose). All animals of the high dose group died during day 8 to day 16 of gestation (i.e. between day 4 and day 12 of treatment). Gross pathological local findings at stomach, forestomach and glandular stomach are considered a direct consequence of the chemical property of the test substance. No mortality or clinical signs of toxicity were observed in all animals dosed up to 80 mg/kg bw/d.

The evaluation of the reproductive organs of the dams, pre- and post-implantation loss, gravid uterine weight, litter size, viability of the foetuses, foetus weight per litter and foetus weight, sex ratio of the foetuses and the external, visceral and skeletal examination of the foetuses revealed that HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the dose of 80 mg/kg bw/d. Hence, under the present experimental conditions HYEQS is not a teratogen.