Phosphoric acid, iron(2+) lithium salt (1:1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2014 - 15 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: solid
- Appearance: homogeneous bright grey-brown powder
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: ca. 10 - 12 weeks
- Weight at study initiation: 148.4 – 191.4 g
- Housing: rats were housed individually in Polycarbonate cages type III (floor area about 800 cm²) furnished with dust-free wooden bedding. For enrichment, wooden gnawing blocks were offered
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An amount of test material was weighed, topped up with 1% carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogeniser. The aqueous test material preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept in a refrigerator.

VEHICLE (1% carboxymethylcellulose suspension in drinking water)
- Concentration of test material in vehicle: 0, 0.30, 1.00 and 3.00 g/100 mL
- Administered volume: 10 mL/kg bw (the calculation of the administration volume was based on the most recent individual body weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF THE TEST MATERIAL PREPARATIONS
Analytical verifications of the stability of the test material in drinking water containing 1% carboxymethylcellulose over a period of 8 days in a refrigerator had been verified prior to the start of the study (project No.: 14L00039; see PART III, Supplement).
Samples of the test material preparations were sent to the analytical laboratory once during the study period (at the beginning of administration) for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations (30 and 300 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

ANALYTICAL METHOD
> ICP-OES (after digestion with mineral acids)
- Apparatus: Inductively coupled plasma atomic emission spectrometer
- Sample preparation: Each sample was evaporated to dryness. After cooling down to room temperature the residue was moistened with 3 mL conc. sulphuric acid, covered with a watchglass and heated slowly on a heating plate. After the test material was cracked completely at a temperature of ca. 320 °C, the residue was cooled down and 5 mL conc. nitric acid was added in portions. The solution was heated slowly again and oxidised. After cooling down 5 mL of mixed acid was added in portions. TThe solution was repeatedly heated and oxidised until all organic components were destroyed. After oxidation the mineral acids were evaporated and the residue was taken up with 5 mL of concentrated hydrochloric acid anjd filled up to a volume of 50 mL with deionised water.
- Test parameters:
Wavelengths: Fe (238.204 nm), Li (670.780 nm), P (213.618 nm)
Calibration: external

Details on mating procedure:

Time-mated animals were paired by the breeder; the day of evidence of mating (detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

14 days (from implantation to one day prior to the expected day of parturition (GD 6 to GD 19)

Frequency of treatment:

Daily

Duration of test:

Surviving dams were sacrificed on GD 20.

No. of animals per sex per dose:

25 dams per dose

Control animals:

yes

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included checks for: signs of morbidity, pertinent behavioural changes and signs of overt toxicity. A check for mortality was performed twice a day on working days or once a day on Saturdays, Sundays or on public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.
The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Time schedule: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On GD 20, blood samples were obtained in a randomised order from all females by retrobulbar venous puncture
- How many animals: all dams
- Parameters checked included: leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On GD 20, blood samples were obtained in a randomised order from all females by retrobulbar venous puncture
- How many animals: all dams
- Parameters checked included: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), inorganic phosphate (INP), clacium (Ca), urea, creatine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomised order.
- Examination: After the dams had been sacrificed, they were necropsied and assessed for gross pathology

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (and distribution)
- Number of early resorptions: Yes (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
- Number of late resorptions: Yes (embryonic or fetal tissue in addition to placental tissue visible)
- Live foetuses: Yes
- Dead foetuses: Yes (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

FOETAL EXAMINATIONS
At necropsy each foetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the foetuses and the condition of placentas, umbilical cords, foetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the foetuses were sacrificed by a subcutaneous injection of pentobarbital. Approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live foetuses, proportions of pre-implantation loss, proportions of post-implantation loss, proportions of resorptions, proportion of live foetuses in each litter, litter mean foetal body weight, and litter mean placental weight were all analysed using the DUNNETT-test (two-sided) for the hypothesis of equal means.

Female mortality, females pregnant at terminal sacrifice, and number of litters with foetal findings were analysed using FISHER'S EXACT test (one-sided) for the
hypothesis of equal proportions.

Proportions of foetuses with malformations and variations and/or unclassified observations in each litter were analysed using the WILCOXON- test (one-sided) for the hypothesis of equal medians.

For blood parameters with bidirectional changes non-parametric one-way analysis was performed using the KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
For blood parameters with unidirectional changes a pairwise comparison of each dose group was performed with the control group using the WILCOXON-
test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians

In all cases * for p ≤ 0.05; ** for p ≤ 0.01

Indices:

Conception rate (%) = (number of pregnant females / number of fertilised animals) x 100

Pre-implantation loss (%) = [(number of corpora lutea – number of implantations) / number of corpora lutea] x 100

Post-implantation loss (%) [(number of implantations – number of live foetuses) / number of implantations] x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
> IN LIFE EXAMINATIONS
MORTALITY
There were no test material-related or spontaneous mortalities in any females of the test groups (0, 30, 100 or 300 mg/kg bw/day).

CLINICAL FINDINGS
No clinical signs or changes of general behaviour, which may be attributed to the test material, were detected in any female at dose levels of 30, 100 or 300 mg/kg bw/day during the entire study period.

BODY WEIGHTS
The mean body weight and the mean body weight gain of the test material-treated dams in test groups 1-3 (30, 100 or 300 mg/kg bw/day) were generally comparable to the controls throughout the entire study period.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of the test groups 1-3 (30, 100 or 300 mg/kg bw/day) was not influenced by treatment with the test material. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

FOOD CONSUMPTION
The mean food consumption of the dams in all test groups (30, 100 and 300 mg/kg bw/day) was generally comparable to the concurrent control throughout the study period. If calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the treated dams consumed a comparable amount of food as the controls.

HAEMATOLOGY
At gestation day 20 total white blood cell (WBC) and absolute neutrophil counts were increased in dams of test groups 2 and 3 (100 and 300 mg/kg bw/day). In dams of test group 3 (300 mg/kg bw/day) relative neutrophil counts were increased and relative lymphocyte counts were decreased in addition.
Total white blood cell (WBC) counts were already higher in dams of test group 1 (30 mg/kg bw/day), but the mean was within the historical control range (WBC 4.04-5.84 Giga/L). Relative reticulocyte counts were higher in dams of test groups 1, 2 and 3 (30, 100 and 300 mg/kg bw/day), but the values were within the historical control range (relative reticulocyte counts 1.2-3.1%). Therefore, these alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
At gestation day 20 aspartate aminotransferase (AST) activities were decreased and urea and total bilirubin levels were increased in dams of test group 3 (300 mg/kg bw/day). Total bilirubin values were already higher in dams of test group 2 (100 mg/kg bw/day). However, all mentioned values were within historical control ranges (AST 0.87-1.54 μkat/L; urea 4.52-7.14 mmol/L; total bilirubin 1.00-1.65 μmol/L). Therefore, these changes were regarded as incidental and not treatment-related.

> TERMINAL EXAMINATIONS
UTERUS WEIGHT
The mean gravid uterus weights of the animals of test group 1-3 (30, 100 and 300 mg/kg bw/day) were not influenced by the test material. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

NECROPSY FINDINGS
No test material-related or spontaneous findings occurred at necropsy in any dam.

REPRODUCTION DATA
The conception rate reached 96% in the mid-dose group (100 mg/kg bw/day) and 100% in the control, low- and high-dose groups (0, 30 and 300 mg/kg bw/day). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
There were no test material-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 30, 100 and 300 mg/kg bw/day) with regard to the conception rate, the mean number of corpora lutea and implantation sites or the values calculated for the pre- and the post-implantation losses, the number of resorptions and viable foetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain
and age.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical biochemistry

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other:

Remarks:

NOAEL was highest dose tested

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
> EXAMINATION OF THE FOETUSES
SEX DISTRIBUTION
The sex distribution of the foetuses in test groups 1-3 (30, 100 and 300 mg/kg bw/day) was comparable to the control foetuses.

WEIGHT OF THE PLACENTAE
The mean placental weights of test material-treated groups 1-3 (30, 100 and 300 mg/kg bw/day) were comparable to the corresponding control group.

WEIGHT OF THE FOETUSES
The mean foetal weights were similar in all test groups (0, 30, 100 and 300 mg/kg bw/day) and did not show any biologically relevant differences.

> EXTERNAL EXAMINATION OF THE FOETUSES
FOETAL EXTERNAL MALFORMATIONS
One foetus of test group 2 (100 mg/kg bw/day) showed two external malformations and had associated skeletal findings (Table 2). The incidences of these malformations were neither significantly different from control nor dose-dependent and, therefore, not considered biologically relevant (Table 3).

FOETAL EXTERNAL VARIATIONS
No external variations were recorded.

FOETAL EXTERNAL UNCLASSIFIED OBSERVATIONS
No unclassified external observations were recorded.

> SOFT TISSUE EXAMINATION OF THE FOETUSES
FOETAL SOFT TISSUE MALFORMATIONS
No soft tissue malformations were recorded.

FOETAL SOFT TISSUE VARIATIONS
Two soft tissue variations were detected in all test groups including the control (0, 30, 100 or 300 mg/kg bw/day), i.e. dilated renal pelvis and dilated ureter. The incidences of these variations were neither significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at higher incidences.

FOETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS
No unclassified soft tissue observations were detected.

> SKELETAL EXAMINATION OF THE FOETUSES
FOETAL SKELETAL MALFORMATIONS
Skeletal malformations were detected in test groups 1-3 (30, 100 or 300 mg/kg bw/day), as shown in Table 5. One foetus had associated external findings. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data. None of these individual findings is considered to be related to treatment.

FOETAL SKELETAL VARIATIONS
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of foetal skeletons and appeared without a relation to dosing (Table 7). The overall incidences of skeletal variations were comparable to the historical control data. For a better overview, all skeletal variations with statistically significant differences between control and the treated groups were compiled in the table below (Table 8). All incidences were expressed on a foetus per litter basis and any statistically significant differences were shown.
The mid-dose finding ‘supernumerary thoracic vertebra’ and the low-dose finding ‘supernumerary rib (14th) (cartilage present)’ were not considered to be treatment-related, because there is no dose-response. Also, as can be seen from Table 6, the increased incidences of ‘incomplete ossification of skull; unchanged cartilage’ and ‘wavy rib’ were well inside the historical control range. Thus, despite of an apparent dose response, these findings were not considered as adverse events.

FOETAL SKELETAL UNCLASSIFIED CARTILAGE OBSERVATIONS
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (Table 9). The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing. The incidence of notched cartilage between basisphenoid and basioccipital was significantly increased in the low-dose group (30 mg/kg bw/day). Since there is no dose-response relationship and other test material-treated groups were unaffected, an association with the treatment as well as a toxicological relevance were not assumed.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

prenatal development

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no developmental effects observed

Remarks:

NOAEL was highest tested dose

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analyses

The concentration of the test material in the samples were calculated by means of its iron, lithium and phosphorus contents.

For the samples 3 - 9 the recoveries found are in good compliance with the expected concentrations (91 - 98 %). The test material was found to be homogeneously distributed in the vehicle (related to the samples 3 - 5 and 7 - 9).

Table 1: Analytical Results

Sample no.	Values found (g/100 g)	Value of test material found (g/100 mL)	Expected concs. of test material (g/100 mL)	Recovery found/expected values (%)
1 (test material)	Fe: 35.0 LI: 4.4 P: 19.7	-	-	-
2 (vehicle)	Fe: < 0.001 Li: < 0.001 P: < 0.001	-	-	-
3	Fe: 0.099 Li: 0.012 P: 0.054	Fe: 0.283 Li: 0.273 P: 0.274	0.3	Fe: 94 Li: 91 P: 91
4	Fe: 0.100 Li: 0.012 P: 0.055	Fe: 0.286 Li: 0.273 P: 0.279	0.3	Fe: 95 Li: 91 P: 93
5	Fe: 0.098 Li: 0.012 P: 0.054	Fe: 0.280 Li: 0.273 P: 0.274	0.3	Fe: 93 Li: 91 P: 91
6	Fe: 0.330 Li: 0.13 P: 0.180	Fe: 0.943 LI: 0.932 P: 0.914	1.0	Fe: 94 Li: 93 P: 91
7	Fe: 1.00 Li: 0.13 P: 0.57	Fe: 2.857 Li: 2.955 P: 2.893	3.0	Fe: 95 Li: 98 P: 96
8	Fe: 1.00 Li: 0.13 P: 0.58	Fe: 2.857 Li: 2.955 P: 2.944	3.0	Fe: 95 Li: 98 P: 98
9	Fe: 1.00 Li: 0.13 P: 0.57	Fe: 2.857 Li: 2.955 P: 2.893	3.0	Fe: 95 Li: 98 P: 96

 

Table 2: Individual Foetal External Malformations

Test group	Dam no. - Foetus no., Sex	Finding
0 (0 mg/kg bw/day)	none
1 (30 mg/kg bw/day)	none
2 (100 mg/kg bw/day)	61-03 F^†	mandibular micrognathia, aglossia
3 (300 mg/kg bw/day)	none

  ^† foetus with additional skeletal malformation

Table 3: Total External Malformations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Litter Foetuses	N N	25 258	25 258	24 251	25 261
Foetal incidence	N (%)	0.0	0.0	1 (0.4)	0.0
Litter incidence	N (%)	0.0	0.0	1 (4.2)	0.0
Affected foetuses/litter	Mean (%)	0.0	0.0	0.5	0.0

Table 4: Total Soft Tissue Variations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/day)	Test group 3 (300 mg/kg bw/day)
Litter Foetuses	N N	25 123	25 125	24 120	25 124
Foetal incidence	N (%)	5 (4.1)	5 (4.0)	8 (6.7)	5 (4.0)
Litter incidence	N (%)	4 (17)	3 (12)	5 (21)	4 (16)
Affected foetuses/litter	Mean (%)	4.5	4.7	6.7	4.2

Table 5: Individual Foetal Skeletal Malformations

Test group	Dam no. - Foetus no., Sex	Finding
0 (0 mg/kg bw/day)	none
1 (30 mg/kg bw/day)	44-06 M	cleft sternum
2 (100 mg/kg bw/day)	61-03 F^†	severely malformed skull bones
3 (300 mg/kg bw/day)	81-05 M	exoccipital fused with 1st cervical arch, misshapen basisphenoid, branched rib

^† foetus with additional external malformation

Table 6: Total Skeletal Malformations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Litter Foetuses	N N	25 135	25 133	24 131	25 137
Foetal incidence	N (%)	0.0	1 (0.8)	1 (0.8)	1 (0.7)
Litter incidence	N (%)	0.0	1 (4.0)	1 (4.2)	1 (4.0)
Affected foetuses/litter	Mean (%)	0.0	0.7	0.7	0.6

Table 7: Total Foetal Skeletal Variations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Litter Foetuses	N N	25 135	25 133	24 131	25 137
Foetal incidence	N (%)	134 (99)	130 (98)	129 (98)	136 (99)
Litter incidence	N (%)	25 (100)	25 (100)	24 (100)	25 (100)
Affected foetuses/litter	Mean (%)	99.0	98.0	98.3	99.3

Table 8: Occurrence of Statistically Significantly Increased Skeletal Variations (expressed as mean percentage of affected foetuses/litter)

Finding	Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/day)	Test group 3 (300 mg/kg bw/day)	HCD Mean % (range)
Incomplete ossification of skull; unchanged cartilage	3.1	9.0	12.7**	23.9**	7.6 (0.0 - 25.9)
Supernumerary thoracic vertebra	2.4	3.8	6.3*	6.3	4.1 (0.0 - 19.1)
Supernumerary rib (14th); cartilage present	5.8	13.6*	9.3	5.6	5.7 (0.0 - 27.3)
Wavy rib	3.1	11.5*	12.6**	16.3**	4.9 (0.0 - 17.0)

 HCD = Historical control data

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 9: Total Unclassified Cartilage Observations

		Test group 0 (0 mg/kg bw/day)	Test group 1 (30 mg/kg bw/day)	Test group 2 (100 mg/kg bw/day)	Test group 3 (300 mg/kg bw/day)
Litter Foetuses	N N	25 135	25 133	24 131	25 137
Foetal incidence	N (%)	101 (75)	101 (76)	108 (82)	106 (77)
Litter incidence	N (%)	24 (96)	24 (96)	24 (100)	25 (100)
Affected foetuses/litter	Mean (%)	72.7	76.1	82.5	77.6

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test material to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused signs of maternal toxicity at dose levels of 100 and 300 mg/kg bw/day taking clinical pathology parameters into account. However, there were no toxicologically relevant adverse foetal findings evident.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day.
The NOAEL for prenatal developmental toxicity was 300 mg/kg bw/day, the highest dose tested.

Executive summary:

The prenatal developmental toxicity of the test material was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414 and EPA OPPTS 870.3700.

During the study the test material was administered as an aqueous suspension to groups of 25 time-mated female Wistar rats by gavage at dose levels of 0, 30, 100 and 300 mg/kg bw/day on gestation days (GD) 6 through 19. The control group was dosed with the vehicle in parallel (1% carboxymethylcellulose suspension in drinking water). A standard dose volume of 10 mL/kg bw was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead foetuses) were determined. The foetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the foetuses of each litter were examined for soft tissue findings and the remaining foetuses for skeletal (inclusive cartilage) findings.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test material to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused signs of maternal toxicity at dose levels of 100 and 300 mg/kg bw/day taking clinical pathology parameters into account. However, there were no toxicologically relevant adverse foetal findings evident.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day.

The NOAEL for prenatal developmental toxicity was 300 mg/kg bw/day, the highest dose tested.