Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept 2017 - May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Phosphoric acid, butyl ester (mono and di ester)
IUPAC Name:
Phosphoric acid, butyl ester (mono and di ester)
Test material form:
other: liquid
Details on test material:
no data

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding, RCC Laboratories India Private Limited, Genome Valley, Turkapally, Shameerpet (Mandal), Ranga Reddy District, Hyderabad - 500 078 India
- Age at study initiation: 12 -14 weeks
- Weight at study initiation: 242.1 - 282.6 g
- Housing: During acclimatization and randomization period all animals were housed in groups of two in polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with corn cob bedding. After randomization, males and females were housed individually. During the mating phase, animals were housed on one male: one female basis within each dose group.
- Diet (e.g. ad libitum): Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot Number: 2014C-040117MA) from ENVIGO was provided ad libitum.
- Water (e.g. ad libitum): Aquaguard filtered tap water was provided ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C to 22.3°C
- Humidity (%): 56 to 66%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 7. Nov. To: 19. Dec. 2017

Administration / exposure

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on preliminary solubility testing
- Concentration in vehicle: 5, 20 and 40 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography
Samples of the dose formulations from one set per dose group was taken immediately after preparation once before commencement of dosing (i.e. on day 5 of gestation and on day 19 of gestation (days were considered from first dosing of the group) for homogeneity and on day 11 of gestation for dose concentration (mean of homogeneity given as dose concentration). Analyses were performed.
The test item in corn oil was assayed on three occasions
01 Stability of test item at 0th, 2nd and 4th hour in corn oil matrix for the fortified concentrations of 5 mg/mL and 40 mg/mL.
02 Initial homogeneity test on Top, mid and bottom layers of corn oil fortified with of test item Viz., 5 mg/mL (Low dose), 20 mg/mL (Intermediate dose), and 40 mg/mL (High dose)
03 Dose concentration of test item in corn oil fortified with 5 mg/mL (Low dose), 20 mg/mL (Intermediate dose), and 40 mg/mL (High dose).
04 Final homogeneity test on Top, mid and bottom layers of corn oil fortified with of test item Viz., 5 mg/mL (Low dose), 20 mg/mL (Intermediate dose), and 40 mg/mL (High dose). Calibration solution, CS5 was used as bracketing standard.
Details on mating procedure:
Animals were paired on a one male: one female basis for a maximum period of fourteen days. In case pairing is unsuccessful, re-mating of female with proven male was considered. Each female was examined for vaginal smear or the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear and vaginal plug was taken as positive evidence of mating (Day 0 of gestation).
Duration of treatment / exposure:
day 5 of gestation and continued until day 19 of gestation
Frequency of treatment:
daily
Duration of test:
mating period + gestation period until gestation day 20
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
based on results of preliminary performed studies

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
twice daily for mortality
once daily for clinical signs

BODY WEIGHT: Yes
acclimatisation period: weekly
premating period: on first day of mating and weekly thereafter
gestation: on gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
On gestation day 3, 5, 8, 11, 14, 17 and 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
All female animals were weighed
- Organs examined:
The uteri of females were examined for the presence and number of implantation sites and the number of corpora lutea in the ovaries were determined
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
Gravid uteri including the cervix were weighed

- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes
The foetuses were removed, identified, weighed, sexed and evaluated for external malformation/variation. Each fetus was subject to external examination, which included all visible structures, surfaces and orifices (including the oral cavity)

- Soft tissue examinations: Yes
One-half of the fetuses (alternating foetuses within the litter) independent of sex were processed for visceral (soft tissue) alterations. All the foetuses selected for visceral examination were observed by Staple’s dissection technique for visceral malformations/alterations and discarded. The organs and structures of the head, neck, thorax and abdomen were observed. Organs and structures of live foetuses like skin, palate, nasal cavity, brain, eyeballs, olfactory bulb, lateral ventricle, internal ear, spinal cord, oesophagus, trachea, thoracic cavity, diaphragm, thyroid gland, thymus, lung, heart (blood vessels, atrium, ventricles, etc), liver, spleen, pancreas, stomach, intestinal tract, kidney, adrenal, ureters, urinary bladder and reproductive organs were observed under stereomicroscope for any malformations/variations.

- Skeletal examinations: Yes
One-half of the fetuses (alternating foetuses within the litter) independent of sex were processed for skeletal alterations. All the foetuses selected for skeletal examination were eviscerated, processed, stained with Alizarin red S (single staining) and examined for skeletal malformations/alterations by using stereomicroscope. All the bone structures of the head, spine, rib cage, pelvis and limbs were observed.

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 was summarized in tabular form. Statistical analysis was performed using Statplus program. All the data was checked for Normality with Shapiro-Wilk W test and for Homogeneity with Bartlett Chi-Square test. Each group of animals was subjected to Analysis of Variance (ANOVA) among the groups, and Bonferroni Test for unequal replications. For discontinuous data, nonparametric test (Mann-Whitney U-Test) was used. Values are given as mean ± standard deviation (SD).
Indices:
Pre–implantation loss (%)
(Number of Corpora Lutea - number of implantation sites)/ Number of Corpora Lutea x100
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 x100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference in the body weight were observed in any of the low, intermediate and high dose group animals when compared with control group, whereas the body weight gain (%) was significantly decreased in the intermediate and high dose group when compared to control group on day 14 and 17 of gestation. These changes could not be attributed to toxicity due to test item exposure since, there were no change in absolute body weight of dam even though increase in body weight gain in percentage on day 20.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Weight of cervix and gravid uterus did not show significant difference between the control and treated groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
please refer to description of findings

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
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
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
no changes reported

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse changes up to highest dose tested observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant difference observed in litter weight in all treatment groups as compared to control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External examination of foetuses revealed small cranium head in one foetus of control group. Small size and pale body was observed in one pub of group 1 (dam no. 27) and in two pubs each in group 2 (dam no. 34 and 36), group 3 (dam no. 55 and 62) and group 4 (dam no. 95 and 96). Additionally, in one pub of dam no. 96 (group 4) head abnormalities (absence of eye bulge, Naris atresia Nose, absence of tongue, absence of mandible, absence of oral opening and smaller upper jaw in mouth) were observed. The findings of small size and pale bodies are not dose related and in line with historical control data. Likewise, the head abnormalities in one single pub out of a total of 208 pubs are within historical control ranges and hence considered a spontaneous finding without toxicological relevance.
No significant difference was observed in external findings in all treatment groups when compared with control group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal examination of foetuses revealed non ossified sternal center twenty seven in control, twelve in low and intermediate, twenty six in high dose group. While non ossified Xyphoid six in control, five in low and nine in high dose group were observed.
Type and distribution of variations noted during skeletal examination at the dose levels of 50, 200 and 400 mg/kg body weight did not indicate any test item-related effects.
Visceral malformations:
no effects observed
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
no changes reported

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed up to highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

MORTALITY AND CLINICAL SIGNS

 

Group

1

2

3

4

Dose (mg/kg bw)

0

50

200

400

Mortality

0/24

0/24

0/24

0/24

Clinical signs

0/24

0/24

0/24

0/24

Expressed as number of animals affected/Total number of animals

  SUMMARY OF BODY WEIGHT

BODY WEIGHTS (G) – SUMMARY - FEMALE

Group

Body weights (in gram)

Gestation Day

 

0

3

5

8

11

14

17

20

Group 1

Mean

248.0

253.0

257.7

266.4

276.8

287.7

299.3

319.2

SD

8.9

9.8

10.6

11.4

11.5

11.7

13.0

17.2

Min

232.8

237.4

242.1

251.4

258.6

265.1

269.1

272.6

Max

266.1

270.4

276.4

286.5

299.4

312.2

328.9

354.5

N

24

24

24

24

24

24

24

24

Group 2

Mean

250.8

255.6

260.7

268.5

277.7

287.0

298.9

319.7

SD

7.8

7.9

8.4

8.9

11.1

12.4

14.0

18.7

Min

236.4

240.1

246.3

254.8

263.7

268.4

271.6

275.8

Max

268.4

274.2

282.6

290.5

302.3

315.6

325.9

348.6

N

24

24

24

24

24

24

24

24

Group 3

Mean

250.4

255.5

260.2

267.0

275.8

285.3

296.9

318.5

SD

8.9

9.0

9.4

10.4

12.2

12.1

12.8

16.4

Min

239.2

243.1

246.4

251.8

259.5

270.0

273.9

276.5

Max

268.5

272.1

275.8

284.9

297.3

308.9

322.8

345.2

N

24

24

24

24

24

24

24

24

Group 4

Mean

250.5

255.4

260.7

267.6

275.5

283.9

294.5

315.1

SD

7.6

7.8

8.3

9.5

10.0

10.4

11.5

17.4

Min

236.8

242.1

248.8

256.5

263.8

268.9

272.3

275.8

Max

265.9

271.2

278.9

290.6

298.4

306.5

315.3

338.2

N

24

24

24

24

24

24

24

24

BODY WEIGHT GAIN (%) – SUMMARY

FEMALE

Group

Body weight Gain %

Gestation Day

 

0

3

5

8

11

14

20

Group 1

Mean

0.0

2.0

3.9

7.4

11.6

16.0

28.7

SD

0.0

0.7

0.9

1.3

1.8

2.3

5.8

N

24

24

24

24

24

24

24

Group 2

Mean

0.0

1.9

4.0

7.1

10.7

14.4

27.5

SD

0.0

0.5

1.0

1.6

2.6

3.1

6.3

N

24

24

24

24

24

24

24

Group 3

Mean

0.0

2.1

3.9

6.7

10.2*

13.9*

27.2

SD

0.0

0.4

0.7

1.2

2.2

2.6

5.4

N

24

24

24

24

24

24

24

Group 4

Mean

0.0

2.0

4.1

6.8

10.0*

13.4*

15.8

SD

0.0

0.4

0.8

1.3

1.7

2.4

6.8

N

24

24

24

24

24

24

24

* Significant at p ≤ 0.05 level with g

PREGNANCY STATUS

GROUP

G1

G2

G3

G4

DOSE (mg/kg bw)

0

50

200

400

FEMALE ANIMALS MATED

24

24

24

24

PREGNANT FEMALE ANIMALS

21

21

22

20

MACROSCOPIC FINDINGS

GROUP

G1

G2

G3

G4

DOSE (mg/kg bw)

0

50

200

400

ANIMALS EXAMINED

24

24

24

24

ANIMALS WITHOUT ABNORMALITY

24

24

24

24

ANIMALS AFFECTED

0

0

0

0

SUMMARY OFGRAVID UTERINE WEIGHT

Group

G1

G2

G3

G4

Dose (mg/kg bw)

0

50

200

400

No. of Dams

21

21

22

20

Mean

54.99

53.18

56.63

52.38

SD

19.64

19.13

20.27

14.55

  SUMMARY OF MATERNAL DATA

Group

No. of CL

Live Implants

Dead Implants

Early

Resorption[w1] 

Late Resorption

Combined Implants

Pre Implantation Loss (%)

Post Implantation Loss (%)

G1

Total

275

212

1

12

4

229

364

209

Mean

13.10

10.10

0.05

0.57

0.19

10.90

17.34

9.94

SD

2.32

3.58

0.22

0.81

0.87

3.39

22.73

14.86

G2

Total

276

214

0

4

1

219

429

147

Mean

13.14

10.19

0.00

0.19

0.05

10.43

20.43

7.02

SD

2.46

3.82

0.00

0.40

0.22

3.63

24.97

21.94

G3

Total

322

235

1

12

1

249

511

156

Mean

14.00

10.22

0.04

0.52

0.04

10.83

22.21

7.11

SD

2.04

4.40

0.21

0.95

0.21

4.42

30.31

16.69

G4

Total

278

208

0

9

1

218

403

70

Mean

13.90

10.40

0.00

0.45

0.05

10.90

20.16

3.50

SD

2.17

3.03

0.00

1.15

0.22

3.43

24.21

7.77

 

 SUMMARY OF LITTER DATA

Group

 

Mean Foetuses Weight

(g)

Mean Total Litter Weight

(g)

Total Number Foetuses

Male Foetuses (%)

Female Foetuses (%)

Control

Mean

3.59

36.49

 

212

 

51.7

 

48.3

SD

0.77

16.48

N

21

21

Low Dose

Mean

3.48

36.72

 

214

 

43.9

 

56.1

SD

0.58

11.22

N

20

20

Medium Dose

Mean

3.50

36.09

 

235

 

50.6

 

49.4

SD

0.77

14.63

N

22

22

High Dose

Mean

3.37

33.75

 

208

 

55.8

 

44.2

SD

0.84

10.11

N

20

20

Expressed values are statistically not significant at p ≤ 0.05. All values within historical control range.

 SUMMARY OF EXTERNAL FINDINGS

Group

G1

G2

G3

G4

Dose (mg/kg bw)

0

50

200

400

No. of Dam Examined

24 (21)

24 (21)

24 (22)

24 (20)

No. of Foetuses Examined

210

213

235

208

Variation Incidence – Number (%)

 

No. of Foetus with Variations

Small in size

0(0.00)

3(1.41)

1(0.43)

2(0.96)

Head small cranium

1(0.48)

0(0.00)

0(0.0)

0(0.0)

Absent eye bulge

0(0.0)

1(0.53)

0(0.0)

1(0.48)

Nose: Naris atresia

0(0.0)

0(0.0)

0(0.00)

1(0.48)

Body pale

0(0.0)

3(1.41)

1(0.43)

1(0.48)

Mouth: Absence of tongue

0(0.0)

0(0.0)

0(0.00)

1(0.48)

Mouth: Absence of oral opening

0(0.0)

0(0.0)

0(0.00)

1(0.48)

Mouth: Smaller upper jaw

0(0.0)

0(0.0)

0(0.00)

1(0.48)

All observed head abnormalities are related to one single pub (foetus no. 2 of dam no. 96) of group 4.

  SUMMARY OF VISCERAL FINDINGS

Group

G1

G2

G3

G4

Dose (mg/kg bw)

0

50

200

400

No. of Litter Examined

21

21

22

20

No. of Foetuses Examined

100

103

112

101

Variation Incidence – Number (%)

No. of Foetus with Variations

Ureters: Convoluted

1(1.0)

1(0.97)

0(0.0)

0(0.0)

SUMMARY OF SKELETAL FINDINGS

Group

G1

G2

G3

G4

Dose (mg/kg bw)

0

50

200

400

No. of Dam Examined

21

21

22

20

No. of Foetuses Examined

110

110

123

107

Variation Incidence – Number (%)

Sternal Centers (5)

Not Ossified

27(24.55)

12(10.91)

12(9.76)

26(24.3)

Xyphiod

Not Ossified

6(5.45)

5(4.55)

0(0.00)

9(8.41)

HISTORICAL CONTROL DATA

Type of study – Prenatal and Developmental Toxicity Studies
Species – Wistar Rat

No of studies – 7
Total no of pups - 1231

Serial No.

Mean Foetus Weight (g)

No. of Litters (Dam)

Number of Foetus

1

37.1831

±

9.735

10

99

2

32.6552

±

13.545

20

193

3

25.9775

±

10.873

18

142

4

27.6178

±

14.556

21

163

5

36.7318

±

16.766

21

212

6

40.0196

±

16.206

22

244

7

28.0719

±

10.401

20

178


Type of study – Prenatal and Developmental Toxicity Studies
Species – Wistar Rat

No of studies – 7                    
Total no of pups - 1171

Foetal abnormalities

Incidence

Small in size

18

Domed head

1

Head – small cranium

2

Spleen – small in size

8

Pale spleen

5

Uterus - convoluted

6

Pale body

3

Nose - misshapen

1

Absence of tongue and mandible

1

Applicant's summary and conclusion

Conclusions:
Based on the findings from this developmental toxicity study, it is considered that the test item is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 400 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal and foetal toxicity was considered as 400 mg/kg bw/day.
Executive summary:

The test item, Hordaphos MDB dissolved in refined groundnut oil (Arachis oil) was administered once daily by oral gavage to three treatment groups of twenty four pregnant female Wistar rats each from day 5 to day 19 of gestation at dose levels of 50, 200 and 400 mg/kg body weight/day. A control group of twenty four females was administered with vehicle (refined groundnut oil) alone.

The treatment with test item resulted in no mortalities. All the dams survived till the scheduled sacrifice.

No test item related clinical signs were observed in any of the pregnant female animals of low, intermediate and high dose groups as well as in control group. The body weight and feed consumption during gestation period in all treatment groups were comparable with thre control group. The mean body weight gain (%) was slightly reduced in the high dose animals on gestation days 14 and 17.

No treatment-related effects were observed in reproduction parameters such as litter weight, corpora lutea count, early and late resorption and number of live and dead foetuses, pre-implantation loss, post-implantation loss as well as sex ratio at any dose level in the female animals treated with 50, 200 and 400 mg/kg body weight/day. The mean fetal weight of high dose pups was slightly decreased. The external and visceral variations observed in foetuses were randomly distributed across the groups. Therefore, these findings were considered as incidental findings and not test item-related.

Type and distribution of variations noted during skeletal examination at the dose levels of 50, 200 and 400 mg/kg body weight/day did not indicate any test item-related effects.

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