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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:
Initiated 11 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau
Version / remarks:
24 November 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Alternative name: Ferromanganese slag milled
- Appearance: Grey/green solid
- Storage conditions: At ambient temperature
- Expiry date: As the test Item is a UVCB an expiry date has not been applied

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD)
- Age at study initiation: 71 to 78 days old
- Weight at study initiation: 232 to 299 g
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. During acclimation up to four animals were housed per cage; during pairing one (stock) male and one female were housed per cage. During gestation each female was housed alone. An aspen soft white untreated wood block was provided to each cage throughout the study (except during pairing) and replaced when necessary. A plastic shelter was provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
- Diet (e.g. ad libitum): Non-restricted certified pelleted diet
- Water (e.g. ad libitum): Potable water from the public supply was provided as libitum via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20 - 24 °C. There were no deviations.
- Humidity (%): Monitored and maintained within the range of 40 - 70 % (relative). There were no deviations.
- Air changes (per hr): Not specified; filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting; 12 hours of light:12 hours of darkness.

IN-LIFE DATES
- From: 13 July 2016
- To: 08 to 11 August 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was weighed, transferred to a mortar and ground to a fine powder using a pestle. Small amounts of the pre-weighed vehicle were added and mixed to form a smooth paste. The suspension was poured into a measuring cylinder which had been wetted with the vehicle and the mortar was rinsed thoroughly with the vehicle and the rinsed residue was added to the measuring cylinder. The required volume was achieved by addition of the remaining vehicle and the suspension was transferred to a beaker for mixing. The final suspension was transferred to the issue container using a syringe. The formulations were prepared in ascending group order. The formulation was prepared weekly and refrigerated (nominally +4 °C).

VEHICLE
- Concentration in vehicle: 0, 20, 66 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was confirmed at concentrations of 10 and 200 mg/kg/day for 15 days when stored at refrigerated temperature and 1 day when stored at ambient temperature.
Specimen formulations at 350 and 500 mg/mL were prepared. Only those formulations which could be drawn up a rabbit oral gavage dosing syringe were analysed to assess the homogeneity of the test material in the liquid matrix.
Samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test material.

FORMULATION ANALYSIS
- Preparation of Test Samples
A representative sample (1 mL, accurately weighed) of test formulation was mixed by vortex with acetone (8 mL) to disperse the sample. The sample was centrifuged (4000 rpm, 10 minutes) to separate the test material sediment from the corn oil/acetone supernatant. The supernatant was decanted off and the process of centrifuging and decanting (using gentle mixing rather than vortexing, with the exception of the final wash where vortex mixing was again used) was repeated a further three times with acetone (8 mL). The samples were dried by nitrogen blow-down in a dry block (30 °C) for 15 minutes and weighed for information only. The samples were re-weighed to determine the mass of the sample after leaving overnight at ambient temperature.

- Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (corn oil) with known amounts of test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

- Calculations
The following equations were used to calculate the test material concentration:
Analysed concentration, mg/mL = [(W2 - W1)/ W3] x D
Procedural recovery analysed concentration, mg/mL = (W2 - W1) / V

Procedural recovery values were determined using the following equation:
Procedural recovery (%) = (Analysed concentration, mg/mL/ Fortified concentration, mg/mL) / 100

Where
W1 = Original mass of centrifuge tube (mg)
W2 = Final mass of centrifuge tube and solid residue (mg)
W3 = Weight of sample taken (g)
V = Nominal volume of recovery sample (1.0 mL)
D = Density of sample (g/mL)

- Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters: The specificity of the analysis (test material analysed weight should be <0.01g) and the method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 10, 200, 350 and 500 mg/mL during the method validation.

- Homogeneity in Corn Oil Formulations
The homogeneity of the test material in corn oil formulations was assessed at nominal concentrations of 10, 200, 350 and 500 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided (4 × 100 mL) into 4 amber glass screw top bottles and submitted for analysis.

- Ambient Temperature Storage (+15 to +25 °C)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. At 350 and 500 mg/mL formulations were also hand mixed using a spatula. After stirring for 20 minutes (representing 0 hours), 1 hour and 2 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 1 day storage the contents were remixed and sampled as detailed above.

- Refrigerated Storage (+2 to +8 °C)
The remaining bottles were refrigerated on receipt and on Days 1, 8 and 15; the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion, hand mixed using a spatula (350 and 500 mg/mL only) followed by magnetic stirring for 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.
- Concentration of Dose Formulations
For the first and last occasions of treatment, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed for the control group and 6 × 1 mL, accurately weighed for all treated groups) and submitted for analysis. Triplicate samples (duplicate samples for control groups) were analysed and the remaining samples were retained for contingency.
For the first occasion contingency samples were analysed for Groups 2 and 3. Samples were disposed of once satisfactory results were achieved.

RESULTS
- Method Validation
The analytical procedure was successfully validated for the test material in corn oil with respect to the specificity of analysis, method accuracy and precision.
The specificity of the assay was demonstrated by the absence of test material in the control sample (<0.01g).
Method accuracy and precision were confirmed; a mean procedural recovery value of 107.6 % (CV = 2.69 %, n = 5) was obtained for 10 mg/mL, 100.4 % (CV = 0.05 %, n = 5) was obtained for 200 mg/mL, 100.3 % (CV = 0.13 %, n = 5) was obtained for 350 mg/mL and 100.5 % (CV = 0.30 %, n = 5) was obtained for 500 mg/mL.

- Homogeneity of Dose Formulations
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 10 % of the initial time zero value. This is with the exception of the Day 8 value at 350 mg/mL where the mean analysed concentration was -11.0 % of the initial time zero value. No analytical reason could be found for this but the result was considered acceptable as the Day 15 value and the Day 8 value at 500 mg/mL were within limits.
The coefficient of variation was less than 10 %. This is with the exception of the 2 hour result at 350 mg/mL where the coefficient of variation was 10.71 %. The 2 hour ambient stir was repeated on Day 8 and this was found to be within acceptable limits.
Recovery results during the trial remained within ±7.5 % of the mean recovery found during validation showing the continued accuracy of the method. This is with the exception of 1 recovery at 10 mg/mL on Days 8 and 15. As the remaining three recoveries performed on these occasions were within acceptable limits the results were considered valid.

- Concentration of Dose Formulations
The mean concentrations were within applied limits of ±20 %, confirming the accuracy of formulation. The precision from mean for Group 2 for the first preparation was outside of the 10 % limit. The contingency samples were analysed and the mean %CV of all 6 results were reported. The overall CV was 11.06 %. The contingency samples for the first preparation of Group 3 were analysed in error. The mean and %CV of all 6 results were reported.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of analysis, method accuracy and precision.
The homogeneity was confirmed for the test material in corn oil formulations at nominal concentrations of 10, 200, 350 and 500 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days.
The mean concentrations in test formulations analysed for the study were within ±10 % of nominal concentrations, confirming accurate formulation.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. Only females showing at least two copulation plugs were allocated.
- Day 0 of gestation: When positive evidence of mating was detected.
Duration of treatment / exposure:
Females: Day 6 to 19 after mating
Frequency of treatment:
Once daily
Duration of test:
Animals were sacrificed on Day 20 after mating
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Formulated concentration: 20 mg/mL
Dose / conc.:
330 mg/kg bw/day (actual dose received)
Remarks:
Formulated concentration: 66 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Formulated concentration: 200 mg/mL
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected in conjunction with the Sponsor. Dose levels were selected based on the effects of a preliminary embryo-foetal study at the testing laboratory which investigated dose levels of 100, 500 or 1000 mg/kg/day. In that study there were no unscheduled deaths, no signs associated with dosing, no treatment related clinical signs and no findings detected at necropsy of the dams or external examination of the foetuses. Overall body weight gain Days 6 - 20 of gestation and food consumption for the days 9 - 19 of gestation were low/slightly low for females receiving the test material at all dose levels. Females receiving 1000 mg/kg/day had low mean adjusted body weight gain when compared to Controls. The differences from Control were minor and considered not to be dose limiting at the degree observed.
There was considered to be no dose limiting effect of the test material on reproductive parameters of implantations, resorptions (early or late), live young, sex ratio or placental, litter and foetal weights.
As a result the limit dose of 1000 mg/kg/day was considered appropriate for use on a preliminary study with low and intermediate dose levels of 100 or 330 mg/kg/day utilising a common ratio of 3 to investigate the dose response of any potential toxicity observed.
- Rationale for animal assignment: Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
Detailed observations were recorded daily during the treatment period one to two hours after completion of dosing and as late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6 - 20 after mating.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0 - 2, 3 - 5, 6 - 9, 10 - 13, 14 - 17 and 18 - 19 after mating inclusive.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals were killed on Day 20 after mating by carbon dioxide asphyxiation
- Organs examined: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries)
- Number of corpora lutea: Yes (for each ovary/uterine horn)
- Number of implantations: Yes (for each ovary/uterine horn)
- Number of early resorptions: Yes (for each ovary/uterine horn)
- Number of late resorptions: Yes (for each ovary/uterine horn)
- Number of foetuses (live and dead): Yes (for each ovary/uterine horn)
- Other: For any apparently non pregnant animals, the number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, 1964).
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No

- Examination of all viable foetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter. Examined externally with abnormalities recorded. The sex of each foetus was recorded.
- Examination of nominally 50 % of foetuses in each litter: Sexed internally and eviscerated.
- Fixation: Foetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining foetuses were fixed whole in Bouin’s fluid.
- Processing: Bouin’s fixed foetuses were subject to free-hand serial sectioning. IMS fixed foetuses were processed and stained with Alizarin Red.
- Foetal Pathology Examination: for the Bouin’s fixed foetuses, serial sections were examined for visceral abnormalities. The Alizarin Red stained foetuses were assessed for skeletal development and abnormalities.
Statistics:
The following data types were analysed at each time point separately:
- Body weight, using absolute weights and gains over appropriate study periods
- Gravid uterine weight and adjusted body weight
- Food consumption, over appropriate study periods
- Litter size and survival indices
- Foetal, placental and litter weight

The following comparisons were performed:
Group 1 vs Groups 2, 3 and 4

Significant differences between the groups compared were expressed at the 5 % (p<0.05) or 1 % (p<0.01) level.
Indices:
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilisation of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea - Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = [(Number of implantations - Number of live foetuses) / Number of implantations] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs considered to be related to treatment with the test material at 100, 330 or 1000 mg/kg/day.
There were no signs associated with administration. Noisy breathing/rales was observed in four females receiving the low dose on a single occasion, in the absence of similar findings at the intermediate or high dose this is considered to have arisen by chance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Overall body weight gain during Days 6 - 20 of gestation of females receiving the test material at all dose levels was lower than Control (maximum effect 91 % of control), however there was no dose relationship apparent. Differences in body weight performance were apparent from before the start of dosing and the difference was considered not to be adverse at the degree observed and may not be attributable to the test material.
There was no clear effect on gravid uterine weights. When gravid uterine weight was taken into account, low adjusted body weight (maximum effect 95 % of Control) and adjusted body weight gain (maximum effect 76 % of control) remained attaining statistical significance at the 330 or 1000 mg/kg/day, indicating the weight differences are due to maternal differences rather than that of the pregnancy.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Following the start of treatment, during gestation days 6 - 9, food consumption of females at all dose levels was lower than Control (maximum effect 91 % of control), however a dose response was not apparent. However prior to the start of dosing, food intake in all groups of females subsequently treated with the test material was consistently marginally lower than in Controls, so these findings are considered to be a continuation of that rather than an effect of treatment.
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):
There was no clear effect on gravid uterine weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal any gross abnormalities for maternal females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
There were considered to be no effects on numbers of implantations, or pre- or post-implantation losses.

Occasional parameters did attain statistical significance, including high post implantation loss in females receiving 330 mg/kg/day and post implantation loss at 100 mg/kg/day but these were not observed at the high dose level of 1000 mg/kg/day and were considered not to be adverse at the degree observed.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
There were considered to be no effects on numbers of resorptions (early or late).

Occasional parameters did attain statistical significance, including high early and total resorptions in females receiving 330 mg/kg/day, but this was not observed at the high dose level of 1000 mg/kg/day and was considered not to be adverse at the degree observed.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were considered to be no effects on numbers of live young
Changes in pregnancy duration:
not examined
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
Description (incidence and severity):
All females receiving the test material at 100, 330 or 1000 mg/kg/day from gestation Day 6 were found to be pregnant at necropsy on Day 20 of gestation. One control female (no. 4) was non pregnant.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on placental weights in animals receiving the test material at doses up to 1000 mg/kg/day.
High placental weights (up to 111 % of control) were recorded attaining statistical significance at the 330 and 1000 mg/kg/day dose levels, 5/20 were above the concurrent control range in each group. The historical group mean range for placental weights is 0.51 - 0.58 g, which is only marginally lower than the value attained at the high dose level.
These differences are therefore considered not to be adverse at the degree observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: The test material was well tolerated.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on lfoetal weights in animals receiving the test material at doses up to 1000 mg/kg/day.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on numbers of live young at doses up to 1000 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were considered to be no effects on sex ratio at doses up to 1000 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on litter weights in animals receiving the test material at doses up to 1000 mg/kg/day.
External malformations:
no effects observed
Description (incidence and severity):
There were no major abnormalities which were attributed to the maternal administration of the test material.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no skeletal abnormalities or changes in the incidence of skeletal variants which were attributed to the maternal administration of the test material.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no minor visceral abnormalities which were attributed to the maternal administration of the test material.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no adverse effect on embryo-foetal survival, development or growth

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Group Mean Body Weight Values During Gestation (g)

Day

 

Dose Group (mg/kg)

0

100

330

1000

0

Mean

SD

N

259

10

19

260

14.2

20

259

11.9

20

254

12.2

20

3

Mean

SD

N

278

12.9

19

275

15.2

20

274

11.6

20

272

14.9

20

6

Mean

SD

N

295

11.4

19

292

18.9

20

290

13.5

20

285

16.1

20

7

Mean

SD

N

299

13.1

19

293

16.6

20

291

12.9

20

287*

16.2

20

8

Mean

SD

N

304

13.1

19

296

18.3

20

295

14.2

20

292*

16.7

20

9

Mean

SD

N

309

12.5

19

302

18.5

20

300

14.1

20

296*

16.9

20

10

Mean

SD

N

314

13.1

19

306

19.2

20

304

15.7

20

301*

16.6

20

11

Mean

SD

N

323

13.5

19

315

18.9

20

312*

15.0

20

309**

17.1

20

12

Mean

SD

N

329

14.7

19

319

18.4

20

318

14.5

20

314**

18.7

20

13

Mean

SD

N

331

14.2

19

323

20.9

20

322

15.6

20

320

19.1

20

14

Mean

SD

N

338

13.4

19

329

22.7

20

329

16.8

20

325*

19.3

20

15

Mean

SD

N

349

16.4

19

338

21.9

20

338

16.4

20

336*

19.3

20

16

Mean

SD

N

360

15.8

19

348

24.0

20

349

17.9

20

346*

20.9

20

17

Mean

SD

N

375

16.5

19

362

25.0

20

363

17.7

20

359*

22.0

20

18

Mean

SD

N

391

18.0

19

378

25.9

20

377

20.2

20

376*

22.1

20

19

Mean

SD

N

407

19.2

19

390*

26.6

20

391*

21.4

20

392*

26.6

20

20

Mean

SD

N

423

19.9

19

407

29.0

20

407

22.4

20

407

27.3

20

Change 0- 6

Mean

SD

N

37

7.1

19

32

6.9

20

31

7.3

20

31

8.0

20

Change 6 - 20

Mean

SD

N

128

12.3

19

116*

15.5

20

117*

14.6

20

122*

14.5

20

*p<0.05

**p<0.01

 

Table 2: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) on Day 20 of gestation

Parameter

 

Dose Group (mg/kg)

0

100

330

1000

Body weight on Day 6

Mean

SD

N

295

11.4

19

292

18.9

20

290

13.5

20

285

16.1

20

Terminal body weight on Day 20

Mean

SD

N

425

20.2

19

409*

29.7

20

408*

23.1

20

407*

27.0

20

Body weight change Days 6 - 20

Mean

SD

N

130

12.7

19

117*

16.8

20

118*

15.4

20

122*

14.3

20

Gravid uterine weight

Mean

SD

N

96

9.7

19

89

14.1

20

93

10.9

20

95

11.8

20

Adjusted body weight Day 20

Mean

SD

N

329

14.4

19

320

23.0

20

315*

16.2

20

312**

18.3

20

Adjusted body weight change days 6 - 20

Mean

SD

N

33

8.6

19

28

9.6

20

25*

10.7

20

27*

6.0

20

*p<0.05

**p<0.01

 

Table 3: Placental weight - group mean values (g) on Day 20 of gestation

Parameter

 

Dose Group (mg/kg)

0

100

330

1000

Placental weight

Mean

SD

N

0.53

0.044

19

0.55

0.060

20

0.57*

0.055

20

0.59**

0.065

20

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the maternal and foetal NOAEL were both considered to be 1000 mg/kg/day.
Executive summary:

An assessment of the influence of the test material on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy in the rat was carried out in accordance with the standardised guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700 and JMAFF 12 Nohsan No. 8147 under GLP conditions.

Three groups of 20 females received the test material at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and foetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All foetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

There were no unscheduled deaths, no signs associated with dosing, no adverse treatment related clinical signs, effects on body weight or food consumption and no findings detected at necropsy of the dams.

All animals receiving the test material were pregnant. There was no effect on embryo-foetal survival growth or development. High placental weights (up to 111 % of control) were recorded attaining statistical significance at the 330 and 1000 mg/kg/day dose levels, but the differences were considered not to be adverse at the degree observed.

Under the conditions of this study, the test material was well tolerated and the maternal NOAEL was considered to be 1000 mg/kg/day. There was no adverse effect on embryo-foetal survival, development or growth, therefore the foetal NOAEL was considered to be 1000 mg/kg/day.