Slags, silicomanganese-manufg.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In accordance with the general rules for adaption set out in Annex XI the registrant justifies that further study of fertility (OECD 433, Extended One Generation Reproductive Toxicity Study) in vertebrates is not appropriate based on a weight of evidence approach, for the following reasons:

1.       The substance is of low toxicological activity and there is no systemic exposure. In a 90-day study in rats, plasma samples were analysed to quantify the degree of absorption of four major and representative elements present in the  Slags, silicomanganese-manufg.: Mn, Si, Al and Ba. Analysis was conducted using a validated ICP-MS method. Excellent linearity was demonstrated, with Limits of Quantitation (LoQs) at 0.0025 mg/L for Mn, 0.0125 mg/L far Ba, 0.025 mg/L for Al and 5 mg/L for Si. The highest dose administered in the 90-day study was 1000 mg/kg bw/day; blood samples were taken at 1, 4 and 24 hours after dosing during week 7 of the study. Analytical results for all blood samples were below the LoQ. The study provides a clear demonstration that there is no quantifiable systemic absorption of these marker elements even at the upper oral limit dose (1000 mg/kg bw/day). Mn and Al in these blood samples, at these LoQs, appeared comparable or lower than concentrations of these elements monitored in the municipal water supply of the test facility.

2.       The substance is UVCB of highly variable composition, being the raw material (ore) for an industrial extraction and refinement process; there is no requirement for quality control other than a high Mn content. Consequently, any given batch cannot be considered toxicologically representative of another. The consequent testing strategy must be either to test multiple batches, or to seek non-animal alternatives. The specified reproductive toxicity study (OECD 433, Extended One-Generation Reproductive Toxicity Study) uses a considerable number of animals, which would be multiplied if several “representative” batches required testing.  Commission Implementing Decision of 1.9.2017 requires the information requirement to be fulfilled by vertebrate testing only if no suitable alternative exists. A feasible non-animal alternative is to evaluate the potential for reproductive toxicity from the known components of the mixture; and information for all significant components of this slag are known to be submitted under REACH. The CLH Inventory has been searched for classification of the component metal oxides; none are proposed for classification for fertility (see Table 1). Further, none of the metal cations in isolation (Mn, Fe, Al, Mg, Ti) are classified for fertility. From the 90-day study, there is no demonstration that the components of the slag interact to affect absorption or bioavailability; hence no interference with the extrapolation is evident.

3.       A 90-day study of this substance Slags, silicomanganese-manufg. by the oral route in rats conducted to the upper limit dose (1000 mg/kg bw/day) demonstrated no sign of systemic toxicity, including to the reproductive tract of males and females.  The study was conducted to modern standards and to GLP, and included weights of testes, epididymides, ovaries and uterus; and histopathological examination of these tissues plus pituitary, prostate, seminal vesicles, and vagina.  There were some minor histological lesions in the wall of the stomach, attributed to site-of-contact effects of this granular slag applied by gavage.

4.       A modern, guideline and GLP-compliant study of developmental toxicity in the rat is available for a closely analogous material (Slags, ferromanganese-manufg., CAS No 69012-28-8 (EC No 273-728-1), registration number: 01-2119446651-40-0000).  The milled slag was administered in corn oil at doses up to 1000 mg/kg bw/day  during days 6-19 of pregnancy.  There were no signs of maternal toxicity, and no effect on pregnany or the offspring.  Fetal survival, growth and development were unaffected by treatment, even at the top limit dose.

5.       This Weight of Evidence clearly supports that an EOGRTS study of this systemically unavailable, and toxicologically inert slag is not an appropriate use of animals.  It can be safely predicted that no useful information will be obtained despite use of an excessive number of animals.

Existing classification of component chemicals

Substance       Classification (CL Inventory)

MnO2       Acute Tox 4 (oral and inhalation)

SiO2       Not classified

CaO       Eye damage 1, skin irritation 2, STOT-SE3

Al2O3       No harmonised classification – majority not classified

MgO       No harmonised classification – majority not classified

K2O       Eye Damage 1, Skin corrosion 1A

BaO       Not notified in isolation. Acute Tox 4 (BaO, SiO, MgO2)

S       Skin Irritation 2

FeO       No harmonised classification – majority not classified

TiO2       Not classified

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure

Justification for type of information:

In accordance with the general rules for adaption set out in Annex XI the registrant justifies that further study of fertility (OECD 433, Extended One Generation Reproductive Toxicity Study) in vertebrates is not appropriate based on a weight of evidence approach, for the following reasons:

1. The substance is of low toxicological activity and there is no systemic exposure. In a 90-day study in rats, plasma samples were analysed to quantify the degree of absorption of four major and representative elements present in the Slags, silicomanganese-manufg.: Mn, Si, Al and Ba. Analysis was conducted using a validated ICP-MS method. Excellent linearity was demonstrated, with Limits of Quantitation (LoQs) at 0.0025 mg/L for Mn, 0.0125 mg/L far Ba, 0.025 mg/L for Al and 5 mg/L for Si. The highest dose administered in the 90-day study was 1000 mg/kg bw/day; blood samples were taken at 1, 4 and 24 hours after dosing during week 7 of the study. Analytical results for all blood samples were below the LoQ. The study provides a clear demonstration that there is no quantifiable systemic absorption of these marker elements even at the upper oral limit dose (1000 mg/kg bw/day). Mn and Al in these blood samples, at these LoQs, appeared comparable or lower than concentrations of these elements monitored in the municipal water supply of the test facility.

2. The substance is UVCB of highly variable composition, being the raw material (ore) for an industrial extraction and refinement process; there is no requirement for quality control other than a high Mn content. Consequently, any given batch cannot be considered toxicologically representative of another. The consequent testing strategy must be either to test multiple batches, or to seek non-animal alternatives. The specified reproductive toxicity study (OECD 433, Extended One-Generation Reproductive Toxicity Study) uses a considerable number of animals, which would be multiplied if several “representative” batches required testing. Commission Implementing Decision of 1.9.2017 requires the information requirement to be fulfilled by vertebrate testing only if no suitable alternative exists. A feasible non-animal alternative is to evaluate the potential for reproductive toxicity from the known components of the mixture; and information for all significant components of this slag are known to be submitted under REACH. The CLH Inventory has been searched for classification of the component metal oxides; none are proposed for classification for fertility (see Table 1). Further, none of the metal cations in isolation (Mn, Fe, Al, Mg, Ti) are classified for fertility. From the 90-day study, there is no demonstration that the components of the slag interact to affect absorption or bioavailability; hence no interference with the extrapolation is evident.

3. A 90-day study of this substance Slags, silicomanganese-manufg. by the oral route in rats conducted to the upper limit dose (1000 mg/kg bw/day) demonstrated no sign of systemic toxicity, including to the reproductive tract of males and females. The study was conducted to modern standards and to GLP, and included weights of testes, epididymides, ovaries and uterus; and histopathological examination of these tissues plus pituitary, prostate, seminal vesicles, and vagina. There were some minor histological lesions in the wall of the stomach, attributed to site-of-contact effects of this granular slag applied by gavage.

4. A modern, guideline and GLP-compliant study of developmental toxicity in the rat is available for a closely analogous material (Slags, ferromanganese-manufg., CAS No 69012-28-8 (EC No 273-728-1), registration number: 01-2119446651-40-0000). The milled slag was administered in corn oil at doses up to 1000 mg/kg bw/day during days 6-19 of pregnancy. There were no signs of maternal toxicity, and no effect on pregnany or the offspring. Fetal survival, growth and development were unaffected by treatment, even at the top limit dose.

5. This Weight of Evidence clearly supports that an EOGRTS study of this systemically unavailable, and toxicologically inert slag is not an appropriate use of animals. It can be safely predicted that no useful information will be obtained despite use of an excessive number of animals.

Existing classification of component chemicals

Substance Classification (CL Inventory)
MnO2 Acute Tox 4 (oral and inhalation)
SiO2 Not classified
CaO Eye damage 1, skin irritation 2, STOT-SE3
Al2O3 No harmonised classification – majority not classified
MgO No harmonised classification – majority not classified
K2O Eye Damage 1, Skin corrosion 1A
BaO Not notified in isolation. Acute Tox 4 (BaO, SiO, MgO2)
S Skin Irritation 2
FeO No harmonised classification – majority not classified
TiO2 Not classified

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Read-Across: Thacker (2016)

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

Read-Across: Stannard (2020)

Under the conditions of the study the No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was concluded to be 800 mg/kg/day. Due to the increased incidence of post implantation loss at 800 mg/kg/day, the dose level of 300 mg/kg/day was concluded to be the NOAEL for embryo-foetal survival. In view of the nature and incidence of major foetal abnormalities detected in all treated groups, which exceeded the concurrent control and historical control data ranges, a NOAEL for embryo-foetal development was not established.

It should be noted that the adverse effects reported in this study have only been observed in one species. There were confounding vehicle effects in this study diminishing its reliability to a certain extend and there are no maternal or developmental effects of prenatal exposure to the substance in any other species.  These factors are considered to lower the overall concern for human relevance.

Link to relevant study records

Reference

Reference 1

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

Justification for type of information:

This study is being used as read across to address the data requirement on the similar substance being registered. Please see attached RAAF document.

Reason / purpose for cross-reference:

other: read across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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

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

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.

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

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

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

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

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.

Key result

Abnormalities:

no effects observed

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.

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.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

 

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

There were clear vehicle tolerability issues at all dose levels, demonstrated by 5 premature deaths (23 %) in the vehicle (Corn oil; 2 mL/kg dose volume) control group due to gastro-intestinal disturbance, inappetence and embryo-foetal deaths, abortions and/or litter resorptions.

Reason / purpose for cross-reference:

other: Target

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau

Version / remarks:

November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Preliminary Phase: Approximately 19 to 23 weeks old. Main Phase: Approximately 27 - 31 weeks.
- Weight at study initiation: 2.59 to 4.62 kg.
- Housing: Suspended cages with shelves fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. The cages constituting each group were blocked by group and mounted in batteries.
Acclimation: One female per cage.
During mating: One stock male and one female per cage.
Gestation: One female per cage.
- Diet: Teklad 2930, pelleted diet. Restricted availability (initially 150 g/animal/day during acclimatisation up to six days prior to the onset of mating and 200 g/animal/day thereafter). The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed. If an individual showed a significant non-treatment related reduction in food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered and the consumption was recorded. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively. From 29 November 2016 all animals were provided with a portion of moistened diet and additional hay supplements on a daily basis for reasons of animal welfare.
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also used.
- Acclimation period: Preliminary Phase: Approximately three weeks. Main Phase: Approximately 11 weeks.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21 °C
- Humidity (%): 45 - 70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 14 hours light : 10 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Each week 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.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- The formulation was stored refrigerated (nominally 4 °C).

VEHICLE
- Concentration in vehicle: Formulated concentrations of 0, 50, 150 and 400 mg/mL.
- Amount of vehicle: 2mL/kg body weight. Calculated from the most recently recorded scheduled body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

FORMULATION ANALYSIS
- Stability: In view of the nature of the test material (by-product of the production of manganese alloys), assessment of stability within the vehicle was not possible/ relevant for this study.

- Homogeneity: Homogeneity was confirmed at concentrations of 10, 200, 350 and 500 mg/mL for 15 days when stored at refrigerated temperature (2 - 8 °C) and for one day when stored at ambient temperature (15 - 25 °C).

- Achieved Concentration: Samples of each formulation prepared for administration in the first and last week of treatment from each phase of the study were analysed for achieved concentration of the test material.

- Analytical Procedure: The analytical method involved adding acetone (8 mL) to the pre-weighed centrifuge tube of formulation and vortex mix to disperse the sample. The tubes are then placed in the centrifuge (10 minutes at 4 000 rpm). The solvent is removed by decantation (taking care not to disturb the pellet of test material at the bottom of tube). This step is repeated four times to ensure complete removal of the corn oil. The tubes are dried with the aid of nitrogen for 15 minutes (Dry Block, 30 °C). The tubes were allowed to reach room temperature and each tube was re-weighed (for information only). Tubes were then re-weighed after overnight drying and cooling, to obtain a final weight of the residue.

- Concentration of Dose Formulations: The formulations for the first week preliminary phase, last week preliminary phase, first week main phase and last week main phase were sampled. For Groups 2, 3 and 4, 6 × 1 mL (accurately weighed) were sampled from the middle of the formulation. For Group 1, 4 × 1 mL (accurately weighed) were sampled from the middle of the formulation. Three samples from Groups 2, 3 and 4 and two aliquots from Group 1 were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. Contingency analysis was performed during first week preliminary phase, first week main phase and last week main phase. Samples were disposed once confirmatory results were obtained.

- Results and Conclusions: The concentrations remained within the applied limits of ± 20 % from the nominal concentration, with the exception of Group 2 in the first week preliminary phase, and Groups 2 and 3 in the last week main phase. Contingency analysis of first week preliminary phase Group 2 confirmed the original result, during the contingency analysis one result was close to the nominal concentration of 50 mg/mL (49.3 mg/mL) however this was proven to be an outlier. This higher result was excluded as an outlier with 99 % confidence using the Dixon’s Q test. Contingency analysis of Group 2 and 3 in the last week main phase confirmed the original results therefore the mean of all six results is reported for both Groups 2 and 3.

The coefficient of variation was within the acceptance criteria ≤ 10 % for all samples confirming the accuracy of analysis, with the exception of Group 3 in the last week main phase. Original analysis of the first week main phase had a coefficient of variation greater than 10 %, re-analysis confirmed that the low result of 37.1 mg/mL was an outlier. This low result was excluded as an outlier with 95 % confidence using the Dixon’s Q test. Procedural recovery values were within the acceptable limits confirming the continued accuracy of the analytical procedure.

Details on mating procedure:

MATING
- Impregnation procedure: Natural mating observed. A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
- M/F ratio per cage: 1:1
- After mating: Each female was injected intravenously with 25 i.u. luteinizing hormone. Where possible only females mating at least twice were allocated to the study.
- Day 0 of gestation: On the day of mating.
- Verification of same strain and source of both sexes: Males were stock New Zealand White bucks. Allocation was according to group and cage position in the sequence of mating. 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 treated group and to prevent more than one sibling female in each group, where possible.

Duration of treatment / exposure:

The test material was administered to females orally (by gavage) from day 6 to day 28 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

29 days.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Preliminary Phase and Main Phase.

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Preliminary Phase and Main Phase.

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

Preliminary Phase and Main Phase.

No. of animals per sex per dose:

Preliminary phase: 6 females per dose
Main phase: 16 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As no previous studies had been conducted in the rabbit on the registered substance or any other inorganic Mn-based substance, doses of 0, 100, 300 and 800 mg/kg/day were selected for the preliminary phase of the study following consultation with the sponsor, and based on results from a main rat embryo-foetal study in which treatment with the test material at doses up to 1 000 mg/kg/day was well tolerated by pregnant females and did not affect embryo-foetal survival, growth or external foetal morphology. However, since the rabbit gut is more sensitive than the rat gut, the highest dose was reduced by 20 %. Since these dose levels were tolerated in the preliminary phase of the study in pregnant rabbits, the same dose levels were employed in the main phase.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: A viability check was performed near the start and end of each working day. Where necessary, animals were prematurely killed for reasons of animal welfare or if they exhibited pregnancy loss. A complete necropsy was performed in all cases.
- Cage side observations checked: Mortality.

DETAILED CLINICAL 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. 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. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.
- Signs associated with dosing: Detailed observations were recorded daily at the following times in relation to dose administration: One to two hours after completion of dosing and as late as possible in the working day.
- Clinical signs: A detailed physical examination was performed on each animal on days 0, 6, 12, 18, 24 and 29 after mating to monitor general health. Clinical observations were presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes.
- Time schedule for examinations: The weight of each adult was recorded weekly during acclimatisation, on the day of mating (day 0) and on days 3 and 6 - 29 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from day 1 after mating.

WATER CONSUMPTION AND COMPOUND INTAKE: No.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Animals surviving until the end of the scheduled study period were killed on day 29 after mating. Females that exhibited pregnancy loss were killed on the day of detection. The sequence to allow satisfactory inter-group comparison.
- Method of kill for all adult animals: Intravenous injection of sodium pentobarbitone.
- 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:
The following were recorded for all animals (including those prematurely sacrificed, where possible) for each ovary/uterine horn:
- Gravid uterus weight: Yes, including cervix and ovaries.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live foetuses: Yes
- Apparently non pregnant animals and for apparently empty uterine horns: The absence or number of uterine implantation sites was confirmed.
- Females exhibiting pregnancy loss: Expelled uterine contents were identified and examined, as appropriate.

Fetal examinations:

REPRODUCTIVE ASSESSMENT
- Method of kill for foetuses: Subcutaneous injection of sodium pentobarbitone.
- External examinations: Yes. Examination of all viable foetuses and placentae. Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded.
- Fixation: One half of eviscerated foetuses were decapitated; heads were initially stored in Bouin’s fluid. The remaining eviscerated foetuses and torsos were fixed in industrial methylated spirit. Industrial methylated spirit-fixed foetuses and torsos were processed and stained with Alizarin Red.
- Soft tissue examinations: Yes. All foetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each foetus was also recorded.
- Skeletal examinations: Yes. Alizarin Red stained foetuses and torsos were assessed for skeletal development and abnormalities.
- Head examinations: Yes. Bouin’s fixed foetal heads were subject to free-hand serial sectioning.

DATA EVALUATION
Findings observed were classified, according to severity and incidence, as:
- Major abnormalities: Normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. partially open eyelids, absent kidney/ureter.
- Minor abnormalities: Minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
- Variants: Alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs and thoracolumbar vertebra, incomplete ossification of 5th and 6th sternebrae.

Indices:

REPRODUCTIVE ASSESSMENT
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.

All group values and SD (as appropriate) were calculated from the individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test material-related clinical signs observed at routine physical examination comprised changes in faecal output and thin build; these signs were considered secondary to the reductions in food consumption and were not attributed to the test material. The incidence of general coat staining and/or hair loss showed no relationship to treatment and these signs are commonly observed in rabbits at this laboratory.
- Control female number 14: Ante mortem clinical signs of reduced faecal output.
- Control female number 12: Ante mortem clinical signs of reduced faecal and urine output, low hay intake and thin build.
- Control female number 3: No ante mortem clinical signs.
- Control number 7: Clinical signs of underactive behaviour, low hay and water intake, thin build, hunched posture and reduced faecal and urine output apparent on days 20 and 21 of gestation.
- Control number 19: Ante mortem clinical signs of low hay intake, thin build, reduced faecal and urine output and small faecal pellets.
- Female number 35 (100 mg/kg/day): Clinical signs of low hay intake, reduced faecal output and small faecal pellets.
- Female number 85 (800 mg/kg/day): Clinical signs of reduced faecal and urine output.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were seven premature deaths during the course of the study, none of which were attributable to the test material:
- Control female number 14 was found dead on day 26 of gestation.
- Control female number 12 was killed for welfare reasons on day 21 of gestation due to a decline in general clinical condition.
- Control female number 3 aborted its litter on day 21 of gestation.
- Control number 7 was killed on day 21 of gestation after red staining was found in the cage under tray.
- Control number 19 aborted its litter on day 27 of gestation after red staining, three placentae and four foetuses were found in the cage under tray.
- Female number 35 receiving 100 mg/kg/day was killed on day 24 of gestation after red staining and foetal material was found in the cage under tray.
- Female number 85 receiving 800 mg/kg/day was killed on day 20 of gestation after red staining was found in the cage under tray.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean body weight loss was recorded for all groups of females, including controls, following the start of dosing on day 6 of gestation. Body weight performance during the dosing period was variable however no effect of treatment with the test material was inferred.
There was no effect of treatment on gravid uterine weights or net mean body weight losses after the weight of the uterus was excluded.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related effect on food consumption at any dose level investigated.
A marked reduction in food consumption was observed following the start of dosing on day 6 of gestation in all groups, including controls; this effect was partly attributable to the calorific content of the corn oil vehicle that all animals were receiving and was considered not to represent an effect of the test material.

In the main phase of the study (day 19 - 23 after mating) all females were given moistened diet for welfare reasons due to several females in all groups showing low consumption of the dry pelleted diet. For the majority of animals in all groups the provision of moistened diet resulted in an improvement in food consumption.

- Control female number 14: Before death this animal had shown negligible food intake since the start of treatment, with no clear improvement following the provision of moistened diet from day 21 of gestation, an associated progressive weight loss of 0.73 kg during days 6 - 25 of gestation.
- Control female number 12: Before termination this animal had shown negligible food intake since the start of treatment, an associated progressive weight loss of 0.57 kg during days 6 - 21 of gestation.
- Control female number 3: Before termination this animal had shown weight loss of 0.32 kg during days 17 - 21 of gestation and low food intake since day 18 of gestation.
- Control number 7: Before termination this animal had shown negligible food intake and progressive weight loss of 0.57 kg since the start of treatment. On day 20 of gestation, due to its clinical condition this female was not dosed, and was given biolapis on the hay and extra vegetables in an attempt to improve food intake.
- Control number 19: Before termination this animal had shown negligible food intake since the start of treatment, with no clear improvement following the provision of moistened diet from day 20 of gestation, an associated progressive weight loss of 0.88 kg during days 6 - 27 of gestation.
- Female number 35 (100 mg/kg/day): Before termination this animal had shown negligible food intake from day 6 until day 22 of gestation and progressive weight loss of 0.69 kg since the start of treatment.
- Female number 85 (800 mg/kg/day): Before termination this animal had shown negligible food intake and progressive weight loss of 0.44 kg since the start 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):

effects observed, treatment-related

Description (incidence and severity):

Signs in relation to dose administration were limited to one control female (number 14) and one female receiving 800 mg/kg/day (number 69). The high dose group female became stressed after dosing on day 7 after mating, with signs of underactive behaviour, increased body temperature and fast respiration observed. The Control female showed similar signs of underactive and unresponsive behaviour on day 25 after mating.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic abnormalities detected in the parent females that were considered to be related to treatment with the test material.
- Control female number 14: Macroscopic abnormalities comprised a reddened caecum with dehydrated contents and a distended gallbladder.
- Control female number 12: Macroscopic abnormalities comprised pale liver, fluid caecal contents, dehydrated faecal pellets in the colon and a large amount of fur in the stomach.
- Control female number 3: No macroscopic abnormalities were detected.
- Control number 7: Macroscopic abnormalities comprised pale kidneys and enlarged and pale liver.
- Control number 19: Macroscopic abnormalities comprised pale contents in the jejunum and caecum.
- Female number 35 (100 mg/kg/day): Macroscopic examination revealed that abortion all seven implantations had occurred, the uterus was found to be thickened with a dark internal wall, and the colon contained dehydrated faecal pellets.
- Female number 85 (800 mg/kg/day): Macroscopic abnormalities comprised poorly defined dark areas on the lungs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Control female number 3: Aborted its litter on day 21 of gestation. Placental material was found in the cage on the morning of Day 21 of gestation and at necropsy only a single empty implantation site was apparent.
Control female number 19: Aborted its litter on day 27 of gestation.
Female number 35 (100 mg/kg/day): On day 24 of gestation foetal material was found in the cage under tray. Since an abortion occurred in the Control group, this single incidence of abortion in the 100 mg/kg/day group was considered unrelated to treatment with the test material.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of maternal treatment with the test material on the mean numbers of implantations or pre-implantation loss.

On both the preliminary and main phases of the study there was a clear increase in the mean percentage of post implantation loss (mainly due to an increase in early resorptions) at 800 mg/kg/day when compared with controls; this consistency/reproducibility across the two independent and temporally separated phases of the study gives cause for concern and a relationship between the higher level of post implantation loss and treatment with the test material cannot be completely discounted. Analysis of the distribution of litters with 0, 1, 2, 3, 4 or ≥ 5 resorptions demonstrates far fewer litters with 0 or only 1 resorption in the 800 mg/kg/day group compared with the other three study groups.

At 300 mg/kg/day, the overall mean percentage of post implantation loss was similar to controls.

At 100 mg/kg/day, the mean percentage of post implantation loss on the preliminary phase of the study was low but was much higher on the main phase resulting in the overall mean level of post implantation being higher than in controls. In the absence of an increase in post implantation loss at 300 mg/kg/day, in the absence of consistency/reproducibility across the two phases of the study, and since the number of litters with no resorptions was similar to controls, no effect of treatment was considered to be inferred.

Control female 3 had only a single empty implantation site apparent at necropsy.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Control female number 14: Uterine examination revealed total litter resorption (one early resorption and 7 late resorptions).
Control number 7: Examination of the uterus revealed that total litter resorption of 8 implantations had occurred.
Female number 85 (800 mg/kg/day): Macroscopic examination revealed that total litter resorption of the seven implantations had occurred. As a total litter resorption occurred in the control group, this single incidence of total litter resorption in the 800 mg/kg/day group was considered unrelated to treatment with the test material.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Control female number 12: Uterine examination revealed five live foetuses and 2 early resorptions.
Control number 19: The uterus contained two early resorptions and four empty implantation sites.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

In addition to premature deaths, at scheduled termination on day 29 after mating, two control females (numbers 15 and 21), two low dose group females (numbers 29 and 36), one intermediate dose group female (number 52) and three high dose females (numbers 69, 73 and 80) were found not to be pregnant. In addition, female No. 83 in the 800 mg/kg/day had a total litter resorption which was considered unrelated to treatment since a total litter loss occurred in the control group. Therefore, a total of 15, 19, 21 and 17 litters were available for assessment at 0, 100, 300 and 800 mg/kg/day, respectively.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Increased incidence of post-implantation loss at 800 mg/kg bw/day.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean foetal weights were considered unaffected by maternal treatment with the test material at all dose levels investigated.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment with the test material on the sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter weights were considered unaffected by maternal treatment with the test material at all dose levels investigated.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of major abnormalities in all treated groups compared with concurrent control. Although the total number of foetuses with major abnormalities in the 100 mg/kg/day group (seven foetuses in four litters) was essentially similar to control (six foetuses in four litters), the nature of the major abnormalities detected in the 100 mg/kg/day group was distinctly different to the control group and the historical control data range (HCD) but was similar to those detected in the 300 or 800 mg/kg/day groups.

The major abnormalities that were observed were diverse in nature, however several litters (Group 2: 2 animals; Group 3: 1 animal; Group 4: 3 animals) had one or more foetuses affected with abnormalities, including anasarca and open eyelids, and the majority of these abnormalities had not previously been recorded in the HCD population. As a consequence, the major abnormalities detected in the treated groups were considered to be attributable to the test material.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Several litters had one or more foetuses affected with major abnormalities including bent long bones.

In addition to the major abnormalities, at 800 mg/kg/day there was an increased incidence of unossified areas within the cranial bones and mandibles, thoracic vertebral defects, various sternebral/rib and costal cartilage abnormalities, delayed/incomplete ossification of fontanelles, cranial centres, sternebrae, vertebrae, pelvic bones, epiphyses and digit bones with the incidences exceeding the HCD range. At 300 mg/kg/day there was an increased incidence of unossified areas within the cranial bones and mandibles, delayed/incomplete ossification of fontanelles, cranial centres, sternebrae, vertebrae (litter 54), epiphyses, digit bones, pectoral and pelvic girdle and long bones (litter 54) which exceeded the HCD range.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Several litters had one or more foetuses affected with major abnormalities including various heart and blood vessel abnormalities.

Other effects:

no effects observed

Description (incidence and severity):

Mean placental weights were considered unaffected by maternal treatment with the test material at all dose levels investigated.

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

Major foetal abnormalities detected in all treated groups exceeding concurrent and historical control data ranges.

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: vertebra

skeletal: hindlimb

visceral/soft tissue: cardiovascular

other: anasarca

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

800 other: mg/kg/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Foetal Examinations: Major Abnormality Findings - Group Incidences

Group	1	2	3	4
Compound	Control	Test material	Test material	Test material
Dose (mg/kg/day)	0	100	300	800

 

		Foetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		112	99	157	105		15	19	21	17
Total Number Affected		6	7	13	19		4	4	6	6
Head
Skeletal	Cleft premaxillae	0	0	0	1		0	0	0	1
	Multiple folded retina	0	0	1	0		0	0	1	0
	Small misshapen lens	1	0	0	0		1	0	0	0
	Protrusion cerebral hemisphere into oral cavity	0	0	0	1		0	0	0	1
	Fused major upper incisors	1	0	0	0		1	0	0	0
External	Acephaly	0	0	1	0		0	0	1	0
	Open eye lid(s)	0	2	4	0		0	2	1	0
	Partially open eye lid(s)	0	0	1	0		0	0	1	0
	Punctate opening of eye lid(s)	0	1	1	0		0	1	1	0
	Cleft upper lip	0	0	0	1		0	0	0	1
	Cleft palate	0	1	1	1		0	1	1	1
	Single medial lower incisor	0	0	0	1		0	0	0	1
Cervical/Thoracic
Skeletal	Thoracic kyphosis	0	0	1	0		0	0	1	0
	Cervical/thoracic scoliosis	1	1	0	1		1	1	0	1
	Multiple cervical vertebral abnormalities	1	0	0	0		1	0	0	0
	Multiple cervicothoracic vertebral/rib abnormalities	0	0	0	2		0	0	0	2
	Distorted rib cage	0	2	7	0		0	2	1	0
	Partially split sternum	1	0	0	0		1	0	0	0
Visceral	Dilated ascending aorta/aortic arch	0	2	2	1		0	2	2	1
	Narrow pulmonary trunk	0	1	1	0		0	1	1	0
	Dilated pulmonary trunk	0	2	1	1		0	2	1	1
	Persistent truncus arteriosus	0	0	1	0		0	0	1	0
	Muscular ventricular septal defect	0	0	1	0		0	0	1	0
	Misshapen heart	0	0	1	0		0	0	1	0
	Large ventricle/thickened wall	0	0	1	0		0	0	1	0
	Thickened pericardium/fluid filled	0	1	0	0		0	1	0	0
Lumbar (and abdominal)/Sacral/Caudal
Skeletal	Sacral kyphosis	0	0	1	0		0	0	1	0
	Thoracolumbar scoliosis	0	0	0	1		0	0	0	1
	Lumbar/sacral/caudal spina bifida/occulta	0	1	1	0		0	1	1	0
	Misshapen/disorganised caudal vertebrae	2	0	0	0		1	0	0	0
	Partially fused caudal vertebrae	1	0	0	0		1	0	0	0
	Brachyury	1	0	0	1		1	0	0	1
External	Thoracogastroschisis	0	0	1	0		0	0	1	0
	Gastroschisis	1	0	0	0		1	0	0	0
	Omphalocele	0	1	1	2		0	1	1	1
	Anasarca	0	1	7	0		0	1	1	0
Visceral	Absent kidney(s)	1	0	1	6		1	0	1	3
	Absent ureter(s)	1	0	0	6		1	0	0	3
	Absent gonad(s)	0	0	0	2		0	0	0	1
Appendicular
Skeletal	Bent scapula	1	0	1	2		1	0	1	1
	Misshapen scapula	0	0	1	0		0	0	1	0
	Bent clavicle(s)	0	1	5	0		0	1	1	0
	Bent long bones	0	4	7	9		0	2	1	3
	Absent fibula	0	0	0	1		0	0	0	1
	Absent pubis	0	0	0	1		0	0	0	1
	Misshapen/wide ischium	0	0	0	1		0	0	0	1
	Ectrodactyly forepaw(s)	0	0	1	0		0	0	1	0
	Brachydactyly forepaw(s)	0	0	1	0		0	0	1	0
	Ectrodactyly hindpaw(s)	0	0	1	0		0	0	1	0
	Brachydactyly hindpaw(s)	0	0	1	0		0	0	1	0
External	Hyperflexion forelimb(s)	1	0	0	0		1	0	0	0
	Malrotated hindlimb(s)	0	0	2	1		0	0	2	1

Note: Individual foetuses/litters may occur in more than one category.  

 

Foetal Examinations: Minor Skeletal Abnormality Finding – Group Incidences

		Foetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		112	99	157	105		15	19	21	17
Number Intact		51	47	73	49		15	19	21	17
Minor Skeletal Abnormalities
Cranial	Unossified area(s)	1	2	11	18		1	2	3	8
	Sutural bone(s)	0	0	0	1		0	0	0	1
	Fissure(s)	0	0	1	1		0	0	1	1
	Additional suture(s)	2	1	0	1		1	1	0	1
	Fissured interparietal	1	0	1	2		1	0	1	2
	Small interparietal	0	0	2	2		0	0	1	1
	Bent cornu(a) of hyoid	2	2	1	2		1	2	1	2
	Misshapen hyoid	0	0	0	1		0	0	0	1
	Unossified area(s) mandible(s)	1	2	7	11		1	2	3	5
Vertebral element abnormalities	Small/misshapen1st cervical vertebral arch(es)	0	1	0	3		0	1	0	3
	Cervical	3	0	0	3		1	0	0	2
	Thoracic	1	1	0	5		1	1	0	3
	Lumbar	0	0	0	1		0	0	0	1
	Sacral/caudal	1	2	1	0		1	1	1	0
	Scoliosis, minimal	1	0	0	0		1	0	0	0
Ribs	Short	0	0	0	1		0	0	0	1
	Branched	1	1	0	1		1	1	0	1
	Fused/partially fused	1	1	0	3		1	1	0	3
	Additional	0	0	0	1		0	0	0	1
	Misshapen	1	0	0	0		1	0	0	0
	Malpositioned	0	0	0	2		0	0	0	2
	Thickened	0	0	0	1		0	0	0	1
	Distally thickened	0	0	0	3		0	0	0	2
	Interrupted ossification 13th	0	1	1	0		0	1	1	0
	Interrupted ossification	0	0	0	1		0	0	0	1
	Absent articulating surface	0	1	0	1		0	1	0	1
	Misaligned articulating surface	0	1	0	0		0	1	0	0
Sternebrae	Partially fused	5	5	1	5		4	4	1	4
	Hemisternebra	1	1	0	0		1	1	0	0
	Bipartite ossified	2	1	0	4		1	1	0	4
	Misaligned ossification sites	3	0	0	1		3	0	0	1
	Misaligned hemicentres	1	2	0	4		1	2	0	4
	Isolated ossification site	1	0	0	1		1	0	0	1
	Misshapen	1	1	0	0		1	1	0	0
Costal Cartilage	Partially fused	1	0	1	10		1	0	1	8
	Misaligned	2	2	0	5		1	2	0	5
	Branched	0	1	0	0		0	1	0	0
	Additional	0	1	0	2		0	1	0	2
	7th not connected to sternum	3	6	4	3		3	3	4	3
Appendicular	Long acromion/metacromion process	0	0	0	1		0	0	0	1
	Misshapen clavicle(s)	0	0	1	0		0	0	1	0
	Flexure forepaw(s)	1	0	0	0		1	0	0	0
	Additional unossified phalanx forepaw	1	0	0	0		1	0	0	0
Tail	Kinked	0	0	1	0		0	0	1	0
	Short	1	0	0	0		1	0	0	0
Total affected by one or more of the above		20	19	20	40		10	9	10	14
Rib and vertebral configuration
Cervical rib	Short supernumerary	0	0	5	2		0	0	4	2
1st rib	Short	0	0	2	0		0	0	2	0
Number of 13th ribs	Short supernumerary	16	27	29	22		8	14	13	12
	Full supernumerary	75	55	72	70		14	18	20	17
	Total	82	73	95	81		14	19	21	17
Thoracolumbar vertebrae	18	0	0	0	1		0	0	0	1
	20	35	17	26	30		11	10	10	13
Pelvic girdle	Unilateral caudal shift	5	6	7	3		3	5	6	3
Delayed/incomplete ossification/unossified
Cranial	Large anterior fontanelle	3	3	7	8		1	3	4	6
	Large posterior fontanelle	3	3	5	8		1	3	4	5
	Large lacrimal fossa	0	2	6	3		0	2	3	3
	Large nasofrontal suture	0	2	3	1		0	2	2	1
	Large frontal suture	0	0	3	0		0	0	1	0
	Large frontal parietal suture	0	1	2	5		0	1	1	2
	Large parietal suture	0	0	2	0		0	0	1	0
	Cranial centres	1	3	6	7		1	3	3	4
Sternebrae	5th	11	9	19	11		6	5	10	7
	Other	22	17	41	36		9	6	13	13
	Total	31	24	57	42		11	8	17	13
Vertebrae	Cervical (includes odontoid process)	6	3	9	18		4	2	8	12
	Thoracic	1	1	7	3		1	1	2	3
	Lumbar	0	0	8	1		0	0	2	1
	Sacral/caudal	0	1	7	2		0	1	2	2
Ribs	1st rib(s)	1	0	0	0		1	0	0	0
	Ribs (any)	0	1	7	2		0	1	1	1
Appendicular	Epiphyses	12	9	27	23		5	4	9	9
	Talus	0	1	1	2		0	1	1	2
	Metacarpals/phalanges	14	13	32	37		7	5	11	11
	Metatarsals/ phalanges	3	5	13	15		3	4	4	6
	Pelvic girdle	1	1	8	2		1	1	3	2
	Long bones	0	0	7	2		0	0	1	1
	Scapula(e)	0	2	7	2		0	1	1	1
Increased ossification
Cranial	Partially fused jugal to maxilla	3	0	1	1		3	0	1	1

Note: Individual foetuses/litters may occur in more than one category.

Foetal Examinations: Minor Visceral Abnormality and Necropsy Findings – Group Incidences

		Foetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		112	99	157	105		15	19	21	17
Number of Heads Examined at Detailed Visceral Examination		61	52	84	56		15	18	21	17
Head abnormalities (fixed visceral)
Eyes	Folded retina	1	0	0	0		1	0	0	0
Brain	Subdural haemorrhage	2	0	1	0		1	0	1	0
	Dilated interventricular foramen	1	0	0	0		1	0	0	0
	Dilated lateral ventricle	1	0	1	0		1	0	1	0
	Misshapen pituitary	0	0	0	1		0	0	0	1
Total affected by one or more of the above		3	0	2	1		2	0	2	1
Necropsy observations (fresh visceral)
Eye(s)	Small	0	1	0	1		0	1	0	1
Lungs	Absent accessory lobe	0	0	0	2		0	0	0	2
	Small	0	2	4	0		0	1	1	0
	Dark	3	0	0	0		1	0	0	0
	Large	0	1	0	0		0	1	0	0
	Pale	0	1	1	0		0	1	1	0
Liver	Pale	0	0	1	0		0	0	1	0
	Swollen	0	0	2	0		0	0	2	0
Gall bladder	Bilobed	0	0	1	2		0	0	1	2
Stomach	Clear fluid filled cyst attached to	0	2	6	0		0	1	1	0
Total affected by one or more of the above		3	3	9	5		1	2	3	3
Necropsy observations (external)
Limb(s)	Flexure forepaw(s)	2	3	6	3		2	2	2	2
	Protrusion between Hindlimb/groin	0	0	0	1		0	0	0	1
Tail	Hooked	2	0	0	0		2	0	0	0
Skin	Subcutaneous oedema	0	1	0	0		0	1	0	0
Total affected by one or more of the above		4	3	6	4		4	2	2	3

Note: Individual foetuses/litters may occur in more than one category.

Formulation Analysis

Analysed Concentrations for the Test Material in Corn Oil

Occasion	Group	Nominal Concentration (mg/mL)	Analysed Concentration (mg/mL)							A	B	Procedural Recovery (%)
			Analysis
			1	2	3	4	5	6	Mean
First Week Preliminary Phase	1	0	ND	ND						-	-
	2	50	34.9	33.5	35.5	35.7	35.2	49.3**	35.0	- 30.0	2.44	100.3
	3	150	148	145	147	-	-	-	147	- 2.0	0.93	100.1
	4	400	391	384	392	-	-	-	389	- 2.8	1.14	100.1
Last Week Preliminary Phase	1	0	ND	ND	-	-	-	-	-	-	-	-
	2	50	47.3	48.1	54.7	-	-	-	50.0	+ 0.1	8.16	101.1
	3	150	144	153	152	-	-	-	150	+ 0.2	3.43	100.3
	4	400	399	415	392	-	-	-	402	0.5	2.93	100.1
First Week Main Phase	1	0	ND	ND	-	-	-	-	-	-	-	-
	2	50	37.1*	44.0	46.8	47.7	47.8	46.5	46.1	- 7.8	2.99	99.8, 101.8
	3	150	139	144	141	-	-	-	141	- 6.0	1.50	100.1, 101.5
	4	400	381	391	396	-	-	-	390	- 2.6	1.96	100.1, 100.9
Last Week Main Phase	1	0	ND	ND	-	-	-	-	-	-	-
	2	50	30.5	32.4	35.7	35.3	36.3	36.0	34.4	- 31.2	6.86	100.7, 101.1, 98.2, 98.9
	3	150	94.6	90.1	86.3	88.5	98.6	114	95.3	- 36.5	10.48	100.3, 100.2, 98.6, 99.9
	4	400	354	353	361	-	-	-	356	- 11.0	1.21	100.0, 101.1

A: Relative mean error, representing the deviation from nominal (%).

B: Coefficient of variation (%).

ND: Not detected.

*: Excluded as an outlier with 95 % confidence using the Dixon’s Q test.

**: Excluded as an outlier with 99 % confidence using the Dixon’s Q test.

Mean analysed concentrations and coefficients of variation were calculated using unrounded figures.

Conclusions:

Under the conditions of the study the No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was concluded to be 800 mg/kg/day. Due to the increased incidence of post implantation loss at 800 mg/kg/day, the dose level of 300 mg/kg/day was concluded to be the NOAEL for embryo-foetal survival. In view of the nature and incidence of major foetal abnormalities detected in all treated groups, which exceeded the concurrent control and historical control data ranges, a NOAEL for embryo-foetal development was not established.

It should be noted that the adverse effects reported in this study have only been observed in one species. There were confounding vehicle effects in this study diminishing its reliability to a certain extend and there are no maternal or developmental effects of prenatal exposure to the substance in any other species. These factors are considered to lower the overall concern for human relevance.

Executive summary:

The developmental toxicity of the test material was assessed according to the EU, OECD 414 and the EPA (OPPTS 870.3700) Guidelines and the Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000 and in compliance with GLP.

The purpose of this study was to assess the influence of the test material, a by-product of the production of manganese alloys, on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy in the New Zealand White rabbit.

The study was conducted in two phases (preliminary phase and main phase). In total, three groups of 22 females received the test material at doses of 100, 300 or 800 mg/kg/day and at a dose volume of 2 ml/kg by oral gavage administration, from day 6 to 28 after mating inclusive. A similarly constituted control group received the vehicle, corn oil, at the same volume dose and for the same duration as treated groups. Animals were killed on day 29 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 29 after mating and the gravid uterus weight was recorded. All foetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

There were seven premature deaths during the course of the study (five control females, one female receiving 100 mg/kg/day and one female receiving 800 mg/kg/day), none of which were attributable to the test material. Treatment at dose levels up to and including 800 mg/kg/day was generally well tolerated, with no test material-related signs observed in relation to dose administration or changes in clinical condition; the incidences of reductions in faecal output and thin build were secondary to the reductions in food consumption observed in all groups, including controls, and were not attributed to the test material administration. Maternal body weight performance, gravid uterine weights and net body weight losses and food consumption were unaffected by the test material administration at all dose levels investigated and there were no test material-related macroscopic abnormalities detected at scheduled termination.

There was no effect of maternal treatment with the test material on the mean numbers of implantations, pre-implantation loss or sex ratio at any dose level investigated. At 800 mg/kg/day, a consistent and reproducible increase in the percentage of post-implantation loss was evident in both phases of the study, with fewer litters with 0 or 1 resorption present compared to the other three study groups; a relationship with the test material could not be discounted. Post implantation loss was considered unaffected by maternal treatment at 100 or 300 mg/kg/day, and mean placental, litter and foetal weights were essentially similar to controls in all treated groups.

There was an increased incidence of major abnormalities in all treated groups compared with concurrent control. Although the total number of foetuses with major abnormalities in the 100 mg/kg/day group (seven foetuses in four litters) was essentially similar to control (six foetuses in four litters), the nature of the major abnormalities detected in the 100 mg/kg/day group was distinctly different to the control group and the historical control data range (HCD) but was similar to those detected in the 300 or 800 mg/kg/day groups. The major abnormalities that were observed were diverse in nature, however several litters (Group 2, two animals; Group 3, one animal; Group 4, three animals) had one or more foetuses affected with abnormalities including anasarca, open eyelids, various heart and blood vessel abnormalities and bent long bones, and the majority of these abnormalities had not previously been recorded in the HCD population. As a consequence, the major abnormalities detected in the treated groups were considered to be attributable to the test material.

In addition to the major abnormalities, at 800 mg/kg/day there was an increased incidence of unossified areas within the cranial bones and mandibles, thoracic vertebral defects, various sternebral/rib and costal cartilage abnormalities, delayed/incomplete ossification of fontanelles, cranial centres, sternebrae, vertebrae, pelvic bones, epiphyses and digit bones with the incidences exceeding the HCD range. At 300 mg/kg/day there was an increased incidence of unossified areas within the cranial bones and mandibles, delayed/incomplete ossification of fontanelles, cranial centres, sternebrae, vertebrae (litter 54), epiphyses, digit bones, pectoral and pelvic girdle and long bones (litter 54) which exceeded the HCD range.

Under the conditions of the study the No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was concluded to be 800 mg/kg/day. Due to the increased incidence of post implantation loss at 800 mg/kg/day, the dose level of 300 mg/kg/day was concluded to be the NOAEL for embryo-foetal survival. In view of the nature and incidence of major foetal abnormalities detected in all treated groups, which exceeded the concurrent control and historical control data ranges, a NOAEL for embryo-foetal development was not established. The aetiology of these foetal malformations remains unclear and will be further investigated outside of the scope of this study. Based on these, a  Category 2 (H361) for developmental toxicity has been assigned as these adverse effects were only observed in one species with confounding vehicle effects and no maternal or developmental effects of prenatal exposure to the registered substance in the other species.  This is considered to lower the concern for human relevance.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Increased incidence of post-implantation loss at 800 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

Major foetal abnormalities detected in all treated groups exceeding concurrent and historical control data ranges.

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: vertebra

skeletal: hindlimb

visceral/soft tissue: cardiovascular

other: anasarca

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

800 other: mg/kg/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Reference 4

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted on a read across substance.

Justification for type of information:

Read across to similar substance. Please see attached RAAF document.

Reason / purpose for cross-reference:

read-across source

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.

Key result

Abnormalities:

no effects observed

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the maternal and foetal NOAEL were both considered to be 1000 mg/kg/day for the read across material.

Executive summary:

An assessment of the influence of the read across 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Two studies were conducted in accordance with standardised guidelines under GLP conditions. The quality of the database is therefore considered to be high. The studies were awarded a reliability score of 2 on the basis of read across.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Read-Across: Thacker (2016)

An assessment of the influence of the 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.

Read-Across: Stannard (2020)

The developmental toxicity of the test material was assessed according to the EU, OECD 414 and the EPA (OPPTS 870.3700) Guidelines and the Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The purpose of this study was to assess the influence of the test material, a by-product of the production of manganese alloys, on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy in the New Zealand White rabbit.

The study was conducted in two phases (preliminary phase and main phase). In total, three groups of 22 females received the test material at doses of 100, 300 or 800 mg/kg/day and at a dose volume of 2 ml/kg by oral gavage administration, from day 6 to 28 after mating inclusive. A similarly constituted control group received the vehicle, corn oil, at the same volume dose and for the same duration as treated groups. Animals were killed on day 29 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 29 after mating and the gravid uterus weight was recorded. All foetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

There were seven premature deaths during the course of the study (five control females, one female receiving 100 mg/kg/day and one female receiving 800 mg/kg/day), none of which were attributable to the test material.Treatment at dose levels up to and including 800 mg/kg/day was generally well tolerated, with no test material-related signs observed in relation to dose administration or changes in clinical condition; the incidences of reductions in faecal output and thin build were secondary to the reductions in food consumption observed in all groups, including controls, and were not attributed to the test material administration. Maternal body weight performance, gravid uterine weights and net body weight losses and food consumption were unaffected by the test material administration at all dose levels investigated and there were no test material-related macroscopic abnormalities detected at scheduled termination.

There was no effect of maternal treatment with the test material on the mean numbers of implantations, pre-implantation loss or sex ratio at any dose level investigated.At 800 mg/kg/day, a consistent and reproducible increase in the percentage of post-implantation loss was evident in both phases of the study, with fewer litters with 0 or 1 resorption present compared to the other three study groups; a relationship with the test material could not be discounted. Post implantation loss was considered unaffected by maternal treatment at 100 or 300 mg/kg/day, and mean placental, litter and foetal weights were essentially similar to controls in all treated groups.

There was an increased incidence of major abnormalities in all treated groups compared with concurrent control. Although the total number of foetuses with major abnormalities in the 100 mg/kg/day group (seven foetuses in four litters) was essentially similar to control (six foetuses in four litters), the nature of the major abnormalities detected in the 100 mg/kg/day group was distinctly different to the control group and the historical control data range (HCD) but was similar to those detected in the 300 or 800 mg/kg/day groups.The major abnormalities that were observed were diverse in nature, however several litters (Group 2, two animals; Group 3, one animal; Group 4, three animals) had one or more foetuses affected with abnormalities including anasarca, open eyelids, various heart and blood vessel abnormalities and bent long bones, and the majority of these abnormalities had not previously been recorded in the HCD population. As a consequence, the major abnormalities detected in the treated groups were considered to be attributable to the test material.

In addition to the major abnormalities, at 800 mg/kg/day there was an increased incidence of unossified areas within the cranial bones and mandibles, thoracic vertebral defects, various sternebral/rib and costal cartilage abnormalities, delayed/incomplete ossification of fontanelles, cranial centres, sternebrae, vertebrae, pelvic bones, epiphyses and digit bones with the incidences exceeding the HCD range. At 300 mg/kg/day there was an increased incidence of unossified areas within the cranial bones and mandibles, delayed/incomplete ossification of fontanelles, cranial centres, sternebrae, vertebrae (litter 54), epiphyses, digit bones, pectoral and pelvic girdle and long bones (litter 54) which exceeded the HCD range.

Under the conditions of the study the No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was concluded to be 800 mg/kg/day. Due to the increased incidence of post implantation loss at 800 mg/kg/day, the dose level of 300 mg/kg/day was concluded to be the NOAEL for embryo-foetal survival. In view of the nature and incidence of major foetal abnormalities detected in all treated groups, which exceeded the concurrent control and historical control data ranges, a NOAEL for embryo-foetal development was not established. The aetiology of these foetal malformations remains unclear and will be further investigated outside of the scope of this study.

Category 2 (H361) for developmental toxicity has been assigned as these adverse effects were only observed in one species with confounding vehicle effects and no maternal or developmental effects of prenatal exposure to the registered substance in the other species.  This is considered to lower the concern for human relevance.

Mode of Action Analysis / Human Relevance Framework

A guideline and GLP compliant prenatal development toxicity study on the read-acros substance identified no effects of the test material on the pregnant rat or on survival or development of foetuses after dosing up to the limit dose of 1000 mg/kg/day.

Contrary to the rat prenatal development data, in the rabbit prenatal development study, adverse foetal effects (increased overall incidence of major visceral abnormalities) were observed at all dose levels (100, 300 or 800 mg/kg/day), with minor skeletal ossification delays also apparent at 300 or 800 mg/kg/day. In view of the nature and incidence of major foetal abnormalities detected in all treated groups, a NOAEL for embryo-foetal development was not established. However, it should be noted that the morphological changes in foetuses were disparate and without a clear dose-response when the individual incidences of anomalies were considered on a litter or foetal basis. With regard to maternal toxicity, effects were largely comparable to controls at all dose levels, however, clear vehicle tolerability issues were apparent at all dose levels, demonstrated by 5 premature deaths (23 %) in the vehicle (corn oil; 2 mL/kg dose volume) control group due to gastro-intestinal disturbance, inappetence and embryo-foetal deaths, abortions and/or litter resorptions. Though maternal clinical signs, body weight gains and food intake were comparable to controls in all groups, poor diet consumption reflecting GI disturbance in all groups, including controls, reduces the reliability of these data. Whilst post-implantation loss was marginally increased in doe’s given 800 mg/kg/day, the higher incidence of deaths in the controls which also had marked embryo-foetal deaths has biased these data as the premature decedent data are excluded from means. 

Cassification as Category 2 (H361) for developmental toxicity effects is adequate, to reflect uncertainty in the reliability of the rabbit prenatal evidence, with adverse effects only observed in one species with confounding vehicle effects and no maternal or developmental effects of prenatal exposure to the test material in the other species. This is considered to lower the concern for human relevance.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification as Category 2 (H361) for developmental toxicity effects.