Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to OECD guideline OECD 414.

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
please refer to chapter 13: category justification document

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Category justification attached to chapter 13

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
Category justification attached to chapter 13
REPORTING FORMAT FOR THE ANALOGUE APPROACH
please refer to chapter 13: read across justification document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Read-across justification attached to chapter 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read-across justification attached to chapter 13

3. ANALOGUE APPROACH JUSTIFICATION
Read-across justification attached to chapter 13

4. DATA MATRIX
Read-across justification attached to chapter 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']manganate(2-)

EC Number:

239-407-5

EC Name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']manganate(2-)

Cas Number:

15375-84-5

Molecular formula:

C10H12MnN2O8.2Na

IUPAC Name:

disodium [2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetato(4-)]manganate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: EDTA-MnNa2
Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Appearance: Off-white powder
Batch: CFC 9380A-retest
Purity: 91.0%
Test substance storage: At room temperature
Stable under storage conditions until: 30 October 2017 (expiry date)
Purity/composition correction factor: Yes, correction factor according to purity/active ingredient
Test substance handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Disodium; 2-[2-(bis(carboxylatomethyl)amino)ethyl(carboxylate-methyl)¬amino]acetate; manganese(+2) cation
CAS Number: 15375-84-5
Molecular formula: C10H12MnN2O8.2Na
Molecular weight: 389.1
pH: 6.6 at concentration of 1%
Solubility in water: 412 g/L
Stability in vehicle: stable

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females were used at initiation of the study.
Stock male NZW rabbits were used for mating with the females. These males were adult and proven fertile. After mating these males were placed back in their stock and might be used for future studies.

Source: Charles River, Chatillon sur Chalaronne, France.
Number of animals: F0-generation: 110 females, F1-generation: 974 fetuses.
Age at delivery: Females were approximately 16 weeks.
Acclimatization: At least 5 days prior to mating.
Randomization: The animals were allocated to the groups by computer-generated random algorithm according to body weight, with all animals within ± 20% of the mean. Upon observation of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Identification: By tattoo in the ear.
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C (range of actual daily mean: 18.4 – 19.8°C), a relative humidity of 40 to 70% (range of actual daily mean: 54 – 78%), and at least 10 air changes/hour.
Accommodation: Females were individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
Diet: During acclimatization until Day 5 post-coitum, free access to standard pelleted diet for rabbits, which had a zinc content of circa 73 ppm (K-H from SSNIFF® Spezialdiäten GmbH, Soest, Germany). From Day 6 post-coitum onwards, free access to standard pelleted diet for Groups 1-4, or standard pelleted diet supplemented with zinc carbonate (approximately 200 ppm zinc, i.e. approximately 400 ppm zinc carbonate) for Group 5. Data on the actual concentration of zinc (determined by analysis) in both the standard diet and the diet supplemented with zinc carbonate were provided by SSNIFF® Spezialdiäten GmbH, Soest, Germany.
Water: Free access to tap-water.
In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were provided during the study period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method: Oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (10 November 2015), according to a validated method (Project 509386).Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 (lowest concentration) and Group 4 (highest concentration) were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Details on mating procedure:

One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

From Days 6 to 28 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

From Days 6 to 28 post-coitum, inclusive.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 30 and 100 mg/kg bw (with and without extra zinc)
Basis:
actual ingested

No. of animals per sex per dose:

22
5 groups of 22 = 110 in total

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were based on the results of a tolerability study in non-pregnant rabbits (total number of 6 animals at levels of 300-750 mg/kg bw) and a dose range finding study in pregnant rabbits (6 animals/group at levels of 0, 80, 250 and 750 mg/kg bw)

Examinations

Maternal examinations:

Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Body weights: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.
Food consumption: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.
Water consumption: Subjective appraisal was maintained during the study.

In addition, blood and urine samples were collected from the first 10 animals/group surviving to planned necropsy. In case sampling of urine was unsuccessful, the next animal in that group was selected for urine sampling until 10 acceptable samples were collected. The animals were not fasted before blood sampling.
On the day of planned necropsy, prior to necropsy, blood samples (1.0 mL) were drawn from the ear artery using vacutainers (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with Li-heparin for determination of clinical biochemistry parameters.
Urine (with a maximum of 10 mL) was collected in a plastic 12-ml tube from the bladder during scheduled necropsy.

All animals surviving to the end of the observation period and all animals showing premature delivery were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol®20%; AST Farma B.V., Oudewater, The Netherlands) and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
Terminal body weight and kidney weight were recorded from all surviving animals.
All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). In addition, the kidneys, ureters, urinary bladder and urethra of all animals were collected and fixed in 10% buffered formalin.

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity using a small flexible plastic or metal feeding tube. For late resorptions a gross external examination was performed.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Ref.1). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (Ref. 2). The sex of all fetuses was determined by internal examination.
 
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique (Ref. 3). After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Ref. 4). Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. As a possible treatment related effect was suspected in the high dose group, skeletal examination was extended to all fetuses from Groups 2, 3 and 5.

All specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 5) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (Ref. 6 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 7) was applied to frequency data.
- The Mann Whitney test (Ref. 8) was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 9) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Ref. 10) was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) x 100 / (number of corpora lutea)

Post-implantation loss (%) = (number of implantation sites - number of live fetuses) x 100 / (number of implantation sites)

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter) x 100 / (number of viable fetuses/litter)

Historical control data:

Included in the report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No mortality occurred in this study.
Two females delivered early on Day 29 post-coitum (one 10 mg/kg treated female and one 30 mg/kg treated female; nos. 34 and 50). By mistake, one female (no. 106) from Group 5 (100 mg/kg with zinc) was terminated on Day 25 post-coitum instead of Day 29 post-coitum.
There was a high incidence and persistence of gray discoloration of the urine in all treated groups, compared to the control group. Gray discoloration was noted in 15 females of the 10 mg/kg group and in all animals of the 30 mg/kg group and 100 mg/kg groups, both with and without extra zinc. In addition, diarrhea was observed in 9 females in the 10 mg/kg group, 16 females in the 30 mg/kg group, and 20 and 21 females in the 100 mg/kg groups without extra zinc and with extra zinc, respectively. Furthermore, faeces containing mucus was noted in 1, 2, 7 and 5 females in the 10 mg/kg, 30 mg/kg, 100 mg/kg without extra zinc and 100 mg/kg with extra zinc groups, respectively. Reduced faeces production was noted in a dose related manner at 30 mg/kg and 100 mg/kg (both with and without extra zinc).
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. These signs did not show any apparent dose-related trend and/or were also observed during the pre-treatment period. At the incidence observed, these signs were considered to be of no toxicological relevance.
Females in the 100 mg/kg groups (both with and without extra zinc) showed on average a body weight stasis on Day 9 post-coitum. This recovered during the remainder of treatment, however for Group 4 (100 mg/kg without extra zinc) this did not recover to control values. Mean body weight and body weight gain of females at 10 and 30 mg/kg remained in the same range as in the control group. Corrected body weight gain was unaffected by treatment up to 100 mg/kg (both with and without extra zinc).
Mean food consumption (before and after correction for body weight) was significantly reduced between Days 9-13 post-coitum at 30 mg/kg and between Days 6-16 post-coitum at 100 mg/kg (both with and without extra zinc). This was not statistically significant on Days 13-16 post-coitum at 100 mg/kg with zinc.
At 10 mg/kg, mean food consumption before and after correction for body weight remained in the same range as controls.
In general, relatively large variations in food consumption were observed among individual animals of all groups (including controls). This is more often seen for rabbits of this age and strain which are housed and treated under the conditions in this study.
The mean levels of creatine kinase were increased in all treated groups (measured in 10 animals per group). This increase was not statistically significant compared to the controls as a high variation within the groups was observed. At 10 mg/kg, this increase was due to two females with a value of 1135 and 3827 U/L. At 30 mg/kg, this was due to five females with values between 1227 and 3498 U/L. At 100 mg/kg without extra zinc, this was due to five females with values between 1204 and 19,240 U/L. And at 100 mg/kg with extra zinc, this was due to five females with values between 1269 and 25,280 U/L. In addition, individual females of the 30 mg/kg and 100 mg/kg groups (both with and without extra zinc) showed increased LDH concentrations. The mean levels of the remaining clinical biochemistry parameters were unaffected by treatment up to 100 mg/kg (with and without extra zinc).
In all treated groups, the colour of the urine was affected (i.e. brown discolouration for most animals; ocher or beige for some animals). The mean levels of the remaining urinalysis parameters were unaffected by treatment up to 100 mg/kg. Any statistically significant changes were not considered treatment related as no dose response was noted or the change was due to relatively high control values.
In the treated groups, there were 7, 14, 14 and 13 females with discoloured contents in the urinary bladder for Groups 2, 3, 4 and 5, respectively. For most of these animals this content was gray, for one animal it was black and for another animal it was black-brown. The incidence of the remaining necropsy findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show any apparent dose related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
The mean weight of the kidneys was unaffected by treatment up to 100 mg/kg (with and without extra zinc).
There were no effects on the number of pregnant females, number of corpora lutea, implantation sites, or pre- or post-implantation loss noted following treatment up to 100 mg/kg. Two treated females (nos. 34 and 50) at 10 and 30 mg/kg had an early delivery on Day 29 post-coitum). Female no. 34 delivered 7 viable fetuses and 3 dead fetuses and female no. 50 delivered 10 viable fetuses and 1 dead fetus showing cannibalism. There were 1, 2, 1, 1 and 1 non-pregnant females in the control, 10, 30, 100 mg/kg without extra zinc and 100 mg/kg with extra zinc groups, respectively. All remaining females were pregnant and had litters with viable fetuses.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on litter size in any group. Mean litter sizes were 9.7, 9.8, 9.5, 8.8 and 9.1 fetuses/litter for the control, 10, 30, 100 mg/kg without extra zinc and 100 mg/kg with extra zinc groups, respectively.
There were no treatment related effects on the sex ratio between control and treated animals. Mean sex ratios (males:females) were 45:55, 55:45, 48:52, 50:50 and 44:56 for the control, 10, 30, 100 mg/kg without extra zinc and 100 mg/kg with extra zinc groups, respectively.
Slightly lower mean male and female fetal body weights were noted at 100 mg/kg. This was more severe for Group 4 (without extra zinc) than Group 5 (with extra zinc). These changes were not statistically significant. Mean combined (male and female) fetal body weights were 38.1, 38.6, 37.4, 34.6 and 36.1 grams for the control, 10, 30, 100 mg/kg without extra zinc and 100 mg/kg with extra zinc groups, respectively.
The numbers of fetuses (litters) available for complete fetal morphological examination were 204(21), 196(20), 200(21), 184(21) and 189(21) in Groups 1, 2, 3, 4 and 5, respectively. External, visceral and skeletal examinations were done for all fetuses and soft tissue cephalic examination was done for approximately half of the fetuses for all groups. Two dams (34 in Group 2 and 50 in Group 3) delivered on Day 29 and dam 106 in Group 5 was accidentally euthanized on Day 25.
There were no treatment related effects on fetal external morphology following treatment up to 100 mg/kg (with and without extra zinc). Five different malformations were observed in this study. These occurred singly in 1, 2, 1 and 1 fetuses of Groups 1, 2, 3 and 4 respectively. None were noted in Group 5. Due to this group distribution of external malformations, all were considered to be chance findings. There were no external variations seen for any fetus in any group.
There were no treatment related effects on visceral morphology following treatment up to 100 mg/kg (with and without extra zinc). Visceral malformations occurred in 4(3), 6(6), 4(4), 2(2) and 3(3) fetuses (litters) of Groups 1, 2, 3, 4 and 5, respectively. This group distribution of total malformations and neither the distribution of each of the 13 individual malformations indicate a possible treatment relationship. Visceral variations were noted for 6.6%, 9.4%, 8.3%, 14.3 and 9.5% of the fetuses per litter in Groups 1, 2, 3, 4 and 5, respectively. Of all variations observed, only the finding of left carotid originating from the brachiocephalic trunk had a somewhat notable group distribution: 1.9%, 1.9%, 1.9%, 5.5% and 1.7% of fetuses per litter in groups 1, 2, 3, 4 and 5 respectively. The litter incidence in Group 4 was slightly increased, but since it was far below the historical control maximum value (29.5% for malpositioned left carotid), this increase was not considered treatment related. Remaining visceral variations noted were not considered to be treatment related as they occurred across all groups, at a very low incidence, in the absence of a dose-dependent distribution and/or were within the range of available historical control data.
An increased incidence was observed in Groups 4 and 5 for caudal shift of pelvic girdle. The mean litter incidences of this skeletal variation were 16.7%, 19.6%, 15.6%, 30.7% and 26.2% per litter in Groups 1, 2, 3, 4 and 5, respectively. There was no dose-response relationship and the incidences in Groups 4 and 5 were not statistically significantly increased, but were approximately 1.6-1.8 times the incidence of the control value. These values were above the historical control 95% confidence interval (20.8% per litter) but still below the maximum historical control value of 38.5%. In addition, ‘caudal shift’ is often seen together with an increase in the number of ‘13th full ribs’ which was not the case in this study. Group 4 fetuses (100 mg/kg without extra zinc) showed signs of retarded skeletal ossification compared to the other groups. This was evidenced by higher (but not statistically significant) incidences for unossified metacarpals and/or metatarsals and reduced ossification of the skull. Mean litter incidences of these variations were for unossified metacarpals (in all cases the metatarsals were ossified) 4.2%, 6.0%, 8.2%, 13.1%, 6.5% and for reduced ossification of the skull 0.8%, 1.8%, 0.0%, 2.4% and 1.6% per litter in Groups 1, 2, 3, 4 and 5, respectively. Compared to historical control data, the Group 4 incidence for unossified metacarpals was above the maximum value (13.1% versus 7.7% per litter). This also applied to the Group 3 incidence (8.2% per litter), but this incidence was only slightly above the maximum and it was more in line with the Group 2 and 5 incidences for unossified metacarpals (6.0% and 6.5% per litter, respectively). Therefore, only the increased incidence of unossified metacarpals in Group 4 was considered to be toxicologically relevant. For the finding of reduced ossification of the skull it is noteworthy that the concurrent control incidence was above the historical maximum value (0.8% versus 0.6% per litter). Therefore, and taken into account the absence of a dose-related response, only reduced ossification of the skull in Group 4 was considered to be meaningful. The two signs of retarded skeletal ossification in Group 4 were considered to be treatment related and can be attributed to the lower fetal body weights (mean of 34.6 grams versus control value 38.1 grams).
Skeletal malformations occurred in 3(2), 3(3) 4(4), 3(2) and 2(2) fetuses (litters) in Groups 1, 2, 3, 4 and 5, respectively. The ones that were observed in the treated groups occurred singly in one or a few fetuses and therefore were not considered to be treatment related.
The remaining skeletal variations noted were not considered treatment related as they occurred in the absence of a dose-related incidence trend and/or occurred infrequently.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for EDTA-MnNa2 could not be established due to the occurrence of discoloured urine and increased creatine kinase levels for animals treated at 10 mg/kg and higher. At 30 mg/kg and higher reduced food intake was seen; at 100 mg/kg (with and without extra zinc) reduced body weight gain was seen which did not fully recover in animals of Group 4 (100 mg/kg without extra zinc). The developmental NOAEL was established as being 30 mg/kg; the developmental findings at 100 mg/kg were observed in the presence of maternal toxicity.

Executive summary:

Dose levels were based on results of the dose range finding study and in consultation with the Sponsor.

One hundred ten mated female New Zealand White rabbits were assigned to five groups of 22 animals each. The test item, EDTA-MnNa2, was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 10, 30, 100 and 100 mg/kg bw/day (Groups 2, 3, 4 and 5, respectively). Rabbits of the control group received the vehicle, water, alone. Groups 1 till 4 received the standard pelleted diet, whereas females of Group 5 received standard diet supplemented with zinc carbonate. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. On Day 29 post-coitum, blood and urine samples were collected from 10 pregnant animals/group for determination of clinical biochemistry and urine parameters. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S for skeletal examinations. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

In all treated groups, there was a high incidence and persistence of gray discoloration of the urine. In addition, diarrhea and faeces containing mucus were noted in a dose related manner at all dose levels. The discolouration of urine from 10 mg/kg onwards was confirmed at urinalysis and macroscopic examination. Moreover, mean serum levels of creatine kinase were increased in all treated groups. This increase was not statistically significant compared to the controls as a high variation within the groups was observed. In addition, individual females at 30 mg/kg and 100 mg/kg (both with and without extra zinc) showed increased serum LDH concentrations. Food consumption (before and after correction for body weight) was reduced between Days 9-13 postcoitum at 30 mg/kg and between Days 6-16 post-coitum at 100 mg/kg (both with and without extra zinc). In line with this, a reduced faeces production was noted in a dose related manner at 30 mg/kg and 100 mg/kg (both with and without extra zinc). At 100 mg/kg (both with and without extra zinc), females showed on average a body weight stasis on Day 9 post-coitum. This recovered during the remainder of treatment, however for Group 4 (100 mg/kg without extra zinc) this did not recover to control values.

At 100 mg/kg (with and without extra zinc), there was a non-statistically significantly increase in the incidence of caudal shift of pelvic girdle. The mean litter incidences of this variation were 16.7%, 19.6%, 15.6%, 30.7% and 26.2% per litter in Groups 1, 2, 3, 4 and 5, respectively. However, the number of litters in which this finding was seen, was not different, viz. 15, 15, 11, 16 and 15 litters, respectively. These values were above the historical control 95% confidence interval (20.8% per litter) but still below the maximum historical control value of 38.5%. In addition, ‘caudal shift’ is often seen together with an increase in the number of ‘13th full ribs’ which was not the case in this study. In addition, at 100 mg/kg without extra zinc (Group 4), the fetuses showed signs of retarded skeletal ossification. This was evidenced by higher (but not statistically significant) incidences of unossified metacarpals and reduced ossification of the skull. Mean litter incidences of these variations were for unossified metacarpals 4.2%, 6.0%, 8.2%, 13.1%, 6.5% and for reduced ossification of the skull 0.8%, 1.8%, 0.0%, 2.4% and 1.6% per litter in Groups 1, 2, 3, 4 and 5, respectively. The two signs of retarded skeletal ossification in Group 4 were attributed to the lower fetal body weights (means of 34.6 grams versus control value 38.1 grams) and are in line with the effect on maternal body weights. At 100 mg/kg with extra zinc (Group 5), no delay in skeletal ossification was observed despite a slightly lower fetal body weight (mean of 36.1 grams versus control value 38.1 grams) and a slightly lower maternal body weight gain. These results showed that zinc supplementation in Group 5 likely amended the slight developmental delay noted in Group 4. Litter size, sex ratio, fetal malformations, external variations and visceral variations were unaffected up to 100 mg/kg (with and without extra zinc). No developmental toxicity was observed at 10 and 30 mg/kg; the developmental effects observed at 100 mg/kg were seen in the presence of maternal toxicity.

CONCLUSION

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for EDTA-MnNa2 is close to 10 mg/kg bw/d due to the occurrence of discoloured urine and increased creatine kinase levels for animals treated at 10 mg/kg and higher. At 30 mg/kg and higher reduced food intake was seen; at 100 mg/kg (with and without extra zinc) reduced body weight gain was seen which did not fully recover in animals of Group 4 (100 mg/kg without extra zinc). The developmental NOAEL was established as being 30 mg/kg; the developmental findings at 100 mg/kg were observed in the presence of maternal toxicity.