Safety evaluation of calcium L-methylfolate (2024)

PMCID: PMC6816227

K.E. Niederberger,^a,⁎ I. Dahms,^b T.H. Broschard,^c R. Boehni,^d and R. Moser^d

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2.4.1. Bacterial reverse mutation assay

The Ames test [[19], [20], [21], [22]]) was conducted using two independent plate incorporation experiments with Salmonella typhimurium strains TA98, TA100, TA102, TA1535, TA1537 and Escherichia coli WP2 uvrA (pkM101). Both experiments were performed with and without metabolic activation by liver S9-mix prepared in-house from Aroclor 1254-pretreated male Wistar rats (S9-mix). L-5-MTHF-Ca was tested at concentrations of 5.00, 15.8, 50.0, 158, 1580 and 5000 μg/plate in experiment 1, and 50.0, 158, 158, 1580 and 5000 μg/plate in experiment 2. Double distilled water was the solvent (and negative control) and test concentrations were prepared by serial dilution. Positive controls in the absence of S9-mix were: daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide; and in the presence of S9-mix: 2-aminoanthracene and benzo[a]pyrene (BaP). All treatments were plated in triplicate, except that 6 plates were used for the negative control. Revertant colonies were scored using an electronic colony counter (Artek Systems Corp., Farmingdale, NY) or manually. Data were not analyzed statistically.

2.4.2. In vitro mammalian gene mutation test

Cell survival and mutation frequency of L5178Y TK^(+/−) mouse lymphoma cells exposed to L-5-MTHF-Ca were determined using a fluctuation protocol developed by Cole et al. (1983) [23]. Batches of L5178Y TK^(+/−) cells stored in liquid N₂ were purged of TK^(+/−) mutants and checked for spontaneous mutant frequency and absence of mycoplasma. For each experiment rapidly thawed cells were grown in RPMI 1640-medium with GlutaMAX 1 supplement (Gibco, Grand Island, NY) containing 10% heat-inactivated horse serum and 1% penicillin/streptomycin (designated RPMI-10). Subcultures were established when cells were growing well. S9-mix was prepared in house in male Wistar rats according to Ames et al. (1975) [22] except that Dulbecco’s phosphate buffered saline (PBS) containing 20 mM HEPES pH 7.4 was used instead of KCl solution.

To prepare cell cultures, at least 10⁷ L5178Y TK^(+/−) cells in RPMI 1640-medium with GlutaMAX 1 supplement with 5% heat-inactivated horse serum and 1% penicillin/streptomycin (designated RPMI-5) were exposed to 50–5000 μg/ml of L-5-MTHF-Ca while shaking at 37 °C. Exposure time was 3 h with S9-mix and 24 h without S9-mix in series 1. In series 2 and 3 in the absence of S9-mix exposure times were 3 and 24 h, respectively. Solvent and positive controls (4-nitroquinoline N-oxide or BaP in dimethyl sulfoxide (DMSO)) were included for each assay and each treatment was performed in duplicate, except positive controls were in single cultures only. After 3 or 24 h exposure, cells were centrifuged, washed, resuspended in 10 ml RPMI-10 and cell concentration adjusted to 2 × 10⁵/ml. Cultures were diluted to be plated for survival or transferred to flasks for growth through the expression period.

To test for survival an aliquot of each culture was diluted to 8 cells/ml with RPMI-20 (RPMI 1640-medium with GlutaMAX 1 supplemented with 20% heat-inactivated horse serum and 1% penicillin/streptomycin) and 0.2 ml/well was placed into microtiter plates and incubated until scorable (day 6–10). Wells with viable clones were stained with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenylbromide (MTT), identified by eye and counted.

For the mutagenicity assay, cultures were maintained at cell densities below 1 × 10⁶ cells/ml for 2 or 3 days for expression of the TK⁻ mutation. Based on observed recovery and growth, cultures were selected to be plated for viability and mutagenicity. To test for viability, cell concentrations in the selected cultures were adjusted to 1 × 10⁴/ml and aliquots diluted to 8 cells/ml. Samples were dispensed on microtiter plates and incubated as described by Cole et al. (1983) [23] until scorable (d 7–10). Wells were stained with MTT and those containing viable clones were identified by eye and counted. Resistance to 5-trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 μg/ml) to cultures selected at the end of the expression period adjusted to cell densities of 1 × 10⁴/ml. TFT-treated cultures were dispensed into microtiter plates. Mutant selection started 2 d after start of the treatment in series 1 and 2 and 3 d after start in the series 3. Plates were incubated as described by Cole et al. (1983) [23] until scorable (d 10–14). For controls and positive test material effects mutation frequency was determined separately for small and large colonies in addition to the total mutation frequency. Data were not analyzed statistically.

2.4.3. In vivo mammalian erythrocyte micronucleus test

L-5-MTHF-Ca was given once by oral gavage to male rats at the limit dose of 2000 mg/kg body weight. This dose was selected based upon a preliminary range-finding experiment showing no toxic effects on three male or three female rats at this maximum dose. Negative control group rats received the solvent, i.e. an oral dose of 10 ml 0.25% aqueous Methocel K4M premium/kg bw. Positive control animals were treated with an oral dose of 16.5 mg cyclophosphamide/kg bw. Five animals per dose group were used. Rats were observed for clinical symptoms after treatment and weighed daily.

Bone marrow smears were prepared from one femur per animal according to a modified version of the method described by Schmid [24]. For the L-5-MTHF-Ca dose groups, preparation took place at 24 and 48 h after administration of the test material. For the positive and negative control groups, the preparation time was 24 h after treatment start. After euthanisation by CO₂, epiphyses were cut off the femur and bone marrow cells flushed out with foetal calf serum. The suspension was filtered according to Romagna (1988) and centrifuged for 5 min at 150 x g [25]. The sediment was resuspended in foetal calf serum and smears prepared. Slides were dried for 3 h then stained according to a modified Giemsa-staining method described by Gollapudi and Kamra (1979) using Giemsa's solution and Weise buffer solution and mounted in Entellan [26].

For microscopic investigation, one slide from each animal preparation was coded. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micronucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes 1000 erythrocytes was then calculated with the aid of the quotient.

2.4.4. Unscheduled DNA synthesis test

L-5-MTHF-Ca was tested for its ability to induce unscheduled DNA synthesis in hepatocytes of male Han Wistar (Crl:WI (Glx/BRL/Han) BR) rats using an in vivo/in vitro procedure [27]. L-5-MTHF-Ca was dissolved in the vehicle, 0.25% w/v aqueous Methocel K4M, and administered once via oral gavage at doses of 2000 or 800 mg/kg to groups of four rats. Dose volume was 10 ml/kg bw. Rats were sacrificed 12–14 h after dosing in experiment 1 and 2–4 h after dosing in experiment 2. Negative control groups for each exposure time received the vehicle at the same volume. Positive control groups were dosed (10 ml/kg bw) with of 10 mg/kg dimethylnitrosamine dissolved in purified water (2–4 h experiment) or 75 mg/kg 2-acetamidofluorene suspended in corn oil (12–14 h experiment).

Hepatocytes were prepared after perfusion of livers from three animals per dose group with buffer containing calcium and collagenase. Cultures were diluted to 1.5 × 10⁵ viable cells. Each well of a 6-well multiplate was filled with 3 ml of the hepatocyte suspension, and then plates were incubated at 37 ± 1 °C in 5% CO₂ in air (v/v) for at least 90 min.

Cells were radiolabelled and prepared for autoradiography as described by Kennelly et al., 1993 using Williams E medium-Incomplete (WE-I) containing 10 μCi/ml [³H] thymidine [27]. After 4 h incubation, cells were washed three times with WE-I containing 0.25 mM unlabelled thymidine and cultures were incubated overnight in the medium. Slides were coated in Ilford K2 liquid emulsion, gelled over ice for 10 min, incubated in a light-tight box at room temperature for 90 min., and then stored in the refrigerator for 14 d in sealed light-tight boxes with desiccant. Slides were developed in Kodak D19 developer and fixed using Ilford Hypam fixer. Cell nuclei and cytoplasm were stained with Meyers Haemalum/eosin Y and slides were dehydrated in ethanol, cleared in xylene and mounted with coverslips. Cells were scored. Nuclear and cytoplasmic grain counts were recorded and net grains/nucleus (NNG) calculated. Per animal 100 cells were analysed, using 2 or 3 slides in each case where possible.

2.5.1. Study design and test material administration

Test article in double distilled water was prepared daily at the testing facility and administered to animals via daily oral gavage (10 ml/kg bw) at concentrations of 0 (Control), 25 (low-dose), 100 (mid-dose) or 400 (high-dose) mg/kg bw/day. Dose levels were selected based upon publicly available information on toxicological studies performed with the racemate (D/L-5-MTHF-Ca) [28]. A 26-week study in rats showed deviations in body weight and clinico-chemical parameters in rats given daily oral doses of 120 and 360 mg/kg bw/d. Stability, hom*ogeneity and test article concentration in gavage formulations was verified by HPLC at RCC Ltd (Itingen, Switzerland) before treatment start and hom*ogeneity and concentration were analysed monthly. After 13 weeks (91 d for males, 92 d for females) ten animals per sex and group were sacrificed. Extra animals in the control and high-dose groups were retained for an additional 4-week treatment-free recovery period before being sacrificed.

2.5.2. Animal observations

Food consumption per cage and body weight (bw) per animal were measured weekly. Animals were observed twice daily for mortality and viability and once daily cageside for clinical signs. Detailed clinical observations in home cages, in a standard arena and in the hand were performed weekly. Forelimb and hind limb grip strength were measured in triplicate using a push-pull strain gauge (Mecmedin, AGF 25 N, Slinfold, UK) at weeks 13 and 17. Animals were placed with forepaws inside a triangular grasping ring and hind paws outside a triangular grasping ring. Using one hand the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Locomotor activity was measured quantitatively in randomised animals for 60 min in 15-min intervals during week 13 with an Activity Monitor 1052 System (Benwick Electronic Equipment Design Manufacture, Benwick, UK). Ophthalmoscopic examination of both eyes was performed on all animals prior to begin of the study and on control and high-dose animals at weeks 13 and 17. A mydriatic solution was applied followed by examination using a Miroflex 2 Ophthalmoscope (Heine, Herrsching, Germany).

2.5.3. Haematology and clinical biochemistry

Blood samples were drawn from all animals at 13 weeks (day 91/92) and from the recovery period animals after 17 weeks for haematology and plasma chemistry measurements. Samples were taken early in the working day from lightly anaesthetized rats after fasting for about 18 h. Samples were drawn from the retro-orbital plexus using a micro-haematocrit glass capillary tube. The anticoagulants EDTA-K2 (haematology), lithium heparin (methaemoglobin and clinical biochemistry samples), and sodium citrate (coagulation) were used during blood collection. Erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, reticulocyte count, reticulocyte fluorescence ratios, nucleated erythrocytes, Heinz bodies, methaemoglobin, total leukocyte counts, differential leukocyte counts, red blood cell morphology, thromboplastin time and activated partial thromboplastin time were measured. Clinical biochemistry measurements included glucose, urea, creatinine, uric acid, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl transferase, total protein and concentration of Ca, P, Na, K, Cl.

2.5.4. Urinalysis

Urine was collected into specimen vials using a metabolism cage during the 18 h fasting period prior to blood sampling after weeks 13 and 17. Analyses included evaluation of volume, specific gravity, osmolality, colour, appearance, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, sediment, red blood cells, and crystals.

2.5.5. Histopathology

After 13 or 17 weeks of treatment, animals were euthanised by exsanguination following anaesthetisation via intraperitoneal injection of sodium pentobarbitone. Animals were weighed and necropsied. Weights were recorded for brain, heart, liver, thyroids and parathyroids, thymus, kidneys, adrenals, uterus, spleen, testes, epididymides and ovaries. Relative organ to terminal body weight ratios and organ to brain ratios were calculated. All macroscopic abnormalities were recorded. Samples of the weighed organs listed above and the following organs and tissues were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (except where indicated): aorta, bone (sternum, femur including joint), bone marrow (femur), cecum, colon, duodenum, esophagus, exorbital lacrimal glands, eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), ileum, with Peyer's patches, jejunum with Peyer's patches, larynx, lacrimal gland exorbital, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), mammary gland area, nasal cavity (turbinates), pancreas, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), stomach, tongue, trachea, urinary bladder (infused with formalin at necropsy), vagin* and any gross lesions. Organ and tissue samples were processed, embedded, cut to ca. 2–4 μm thickness and stained with haematoxylin and eosin. Histological examinations were performed on preserved organs and tissues from all control and high dose animals, and all gross lesions noted in any of the test groups at the time of terminal sacrifice.

2.6.1. Study design and test material administration

The test article in 0.25% aqueous hydroxypropyl methylcellulose was prepared daily at the testing facility under subdued lighting and kept in brown glass bottles at room temperature and stirred continuously during administration. Formulation concentrations were verified once during the first week of treatment at the Institute of Toxicology, Merck KGaA using a validated spectrophotometric method. Test article was administered to animals via daily oral gavage (10 ml/kg bw) on 15 consecutive days from gestation day (GD) 5 to 19 at concentrations of 0 (Control), 100 (low-dose), 300 (mid-dose) or 1000 (high-dose) mg/kg bw.

2.6.2. Animal observations and examinations

Behaviour and appearance were checked twice daily. Body weight of female rats was measured on GD 0, and then daily from GD 5 to 20. Food consumption was measured on GD 5, 10, 15, 20. Water consumption was measured on GD 3, 6, 9, 12, 15, 18, 20.

On GD 20 all females were anaesthetised by inhalation, abdominal cavities were opened and ovaries and uteri removed. Gravid uteri were weighed. Bodies without uteri and foetuses were weighed and recorded as terminal body weights. The number of corpora lutea in both ovaries was determined and the number of live and dead foetuses and resorptions were determined. Resorptions were recorded as: complete (occurred shortly after implantation and detected after staining of uteri with ammonium sulphide), early (diameter ≤0.5 cm, no macroscopic differentiation between foetal and placental residues possible) or late (diameter usually >0.5 cm, clear macroscopic differentiation between foetal and placental residues possible). The total number of implantations was determined by ammonium sulphide staining. Females without implantation sites were recorded as being non-pregnant.

Foetuses were examined macroscopically for abnormal deviations and weighed. Sex was determined based on anogenital distance and inspection of gonads. In each treatment group all foetuses were numbered and those with uneven numbers of each litter were scheduled for transverse section. Foetuses were fixed in formalin and dissected transversely (method of Wilson, 1965) and inspected for organ malformations (modified Barrow and Taylor, 1969) [29,30]. Even numbered foetuses were eviscerated and examined for organ malformations. Eviscerated foetuses were fixed in alcohol and treated according to the double straining method of Whitaker and Dix (1979), i.e. the soft parts were cleared with KOH solution, skeletons were stained with Alizarin Red S, and the cartilage with Alcian Blue. Foetuses were then examined for skeletal abnormalities and malformations [31].

2.7.1. In vivo mammalian erythrocyte micronucleus test

Statistical analyses were performed on the number of micronuclei-containing polychromatic erythrocytes per animal. Each treatment group was compared to the negative control. For comparisons, the exact Mann-Whitney-test (SPSS-System (Version 9.0) under Windows NT) was used against one-sided alternatives.

2.7.2. Subchronic oral toxicity study

Statistical analyses were performed by RCC on body and organ weights, all ratios, and clinical chemistry parameters. Means and standard deviations were calculated for quantitative data, with male and female rats evaluated separately. Treatment groups were compared to control groups using a one-way analysis of variance (ANOVA). The Dunnett-test based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. The Steel-test was applied when the data could not be assumed to follow a normal distribution. Locomotor activity and grip strength data were analyzed using a Student’s T-Test. Fisher’s exact-test was applied to opthalmoscopy data and macroscopic findings.

2.7.3. Developmental study

To compare parameters to the control the two sided Dunnett test was used for body weight, body weight gain, terminal body weight, foetus weight, and food and water consumption. The Fisher-Pitman Permutation test with adjustment according to Bonferroni-Holm under consideration of k≥2 treatment groups was used to statistically analyse corpora lutea implantations, live foetuses, percent life foetuses, percent resorptions, dead and malformed foetuses, and percent post implantation loss [32].

3.1.1. Bacterial reverse mutation assay

L-5-MTHF-Ca did not induce a clear or dose-dependent increase in the number of revertant colonies in any of the tester strains with- or without S9-metabolic activation in either of the two Ames test experiments (data not shown). To reach the highest concentrations, i.e. 500–5000 μg/plate, the material was plated as a suspension. In both experimental series there were no signs of cytotoxicity of L-5-MTHF-Ca to the bacteria (i.e. no reduction in number of spontaneous revertants and no clearing of the background lawn was observed). Negative control mutant frequencies were within acceptable ranges designated according to Levin et al. (1982) and Kier et al. (1986), and the historical controls of the laboratory (TA 98: 15–60; TA 100: 75–200; TA 102: 200–450; TA 1535: 3–37; TA 1537: 4–31; WP2: 50–200) (slight deviations in only one test series were accepted when all other parameters of that series were in the regular range) [33,34]. Positive control compounds induced a clear increase in the number of revertants over the negative control, thereby showing the expected reversion properties of all strains and good metabolic activation of the S9-mix. Results obtained for the negative and positive controls confirmed the validity of the assay. L-5-MTHF-Ca did not cause an increase in mean number of revertant colonies in any of the tester strains with or without metabolic activation and there were no dose-related increases in mutations with- or without S9-metabolic activation. Therefore, L-5-MTHF-Ca was considered to be non-mutagenic under the experimental conditions.

3.1.2. In vitro mammalian gene mutation test

L-5-MTHF-Ca was found to be non-mutagenic in mouse lymphoma cells at concentrations of 50.0, 158, 500, 1580, 2810 and 5000 μg L-5-MTHF-Ca/ml. The maximum concentration was selected after a preliminary range-finding assay showed reduced cell relative survival at the highest concentration tested without a relevant change in pH and osmolarity. Mean mutant frequencies of negative controls met validity requirements of less than 2-fold the historical mean values (130 × 10⁻⁶ without S9, 146 × 10⁻⁶ with S9) in series 1 and series 2 (Table 1), but not in series 3 (2.2-times the historical mean). The positive control in series 1 without S9 and series 2, met the validity requirements of a mutation frequency at least 2-fold above the highest value of the historical negative controls (241 × 10⁻⁶) and at least a 4-fold increase in mutation frequency of the actual negative control. The positive control in series 1 with S9 gave a mutation frequency 2-fold above the highest value of the historical negative controls but only 2.5-fold higher than the actual negative control, thereby not meeting validity requirements. Validity requirements for the assay were only met by series 1 without S9 and series 2 experiments. Colony characterisation by colony size of the negative and positive controls and the highest concentrations of L-5-MTHF-Ca confirm that both the large and small colony mutants were adequately detected.

In the different experimental series precipitation of the test material occurred at concentrations ≥158 or ≥500 μg/ml and relative total growth values were very low for concentrations of L-5-MTHF-Ca above 1580 μg/ml. In series 1 without S9, mean mutation frequencies at 2810 and 5000 μg/ml L-5-MTHF-Ca were between 2- and 4-fold the mean actual negative control, thereby showing a weak effect on mutation frequency in this experiment. Mutation frequency in series 2 was less than 2-fold the actual negative control and L-5-MTHF-Ca was determined to have no effect on the mutation frequency. According to the evaluation criteria, test materials showing a weak effect in one series and no effect in another series are assessed as negative or non-mutagenic and it can be concluded that L-5-MTHF-Ca did not induce mutagenic effects at the TK locus in mouse lymphoma cells.

3.1.3. In vivo mammalian erythrocyte micronucleus test

L-5-MTHF-Ca did not induce micronucleus formation in the immature erythrocytes of rats (data not shown). No clinical symptoms were observed in any of the animals receiving 2000 mg/kg bw L-5-MTHF-Ca and there was no treatment-related decrease in body weight. Mean number of micronuclei-containing cells per 1000 polychromatic erythrocytes for negative controls (0.50–2.00) were within the range of historical controls of the laboratory for male rats (0.5–2.2). The positive control resulted in an increase in polychromatic erythrocytes with micronuclei (p < 0.01) and values (10.0–20.5) were within the range of historical controls of the laboratory (8.0–22.0). L-5-MTHF-Ca did not induce a statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei compared to the negative control and the number of normochromatic cells with micronuclei, calculated per 1000 normochromatic erythrocytes, was not increased. There was a significant (p < 0.05) increase in the quotient of normochromatic to polychromatic erythrocytes in the L-5-MTHF-Ca 48 h exposure time group, 2.50 compared to 1.32 of the negative control group, a possible result of depressed erythroblast proliferation as a result of cytotoxicity, and a valuable indication of bone-marrow exposure by the test agent. L-5-MTHF-Ca did not induce micronucleus formation in the immature erythrocytes of rats in the study, therefore, L-5-MTHF-Ca was considered to be non-genotoxic under the conditions of the study.

3.1.4. Unscheduled DNA synthesis test

L-5-MTHF-Ca did not induce DNA damage that could be repaired by unscheduled DNA synthesis in liver cells of rats given oral gavage doses of 800 and 2000 mg/kg bw, neither at 2–4 h, nor at 12–14 h sampling time (Table 2). No clinical signs of toxicity or weight loss were observed in any main study animal. Negative (vehicle) controls gave a group mean NNG value below zero with only 0.3-0.7% of cells in repair; both parameters were within the range of historical controls. Positive controls induced increases in NNG to more than 17 and more than 50% of cells were in repair. The assay was therefore accepted as valid. Treatment with 800 or 2000 mg/kg L-5-MTHF-Ca did not produce a group mean NNG value greater than -1.9 nor were more than 2.7% cells found in repair at either dose. These data confirm that L-5-MTHF-Ca does not have genotoxic effects on rat livers.

3.1.5. Subchronic oral toxicity study

There were no mortalities related to the administration of L-5-MTHF-Ca. A single female in the high-dose treatment group died following a gavage error which was confirmed at necropsy. All other animals survived until scheduled necropsy.

Analysis of L-5-MTHF-Ca formulations in distilled water confirmed the stability and hom*ogeneity of the gavage test solutions at room temperature over a period of 2 h.

Clinical observations were limited to bright faeces observed in the mid- and high-dose L-5-MTHF-Ca groups. These were attributed to the beige-yellow colour of the test article and considered toxicologically irrelevant. Daily and weekly clinical and functional observations revealed isolated incidences reported equally in control and test item-treated animals and not related to L-5-MTHF-Ca administration. Locomotor activity in 15-min time intervals was different for some of the treatment groups compared to the control, but differences did not occur in both sexes, did not show a clear dose relationship and total activity over the 60-min interval was not different between experimental groups, therefore, differences in locomotor activity were considered incidental. Lower mean hind limb grip strength in the mid-dose (p < 0.01) and high-dose (p < 0.05) L-5-MTHF-Ca females at Week 13 (587g and 622g, respectively verses 700g in the control) was considered incidental as it was not accompanied by differences in forelimb strength, a clear dose relationship was lacking, and no other signs of muscular weakness or motor neurotoxicity in either sex were observed.

In females administered L-5-MTHF-Ca, mean weekly body weights, body weight gains and feed consumption were comparable to those in the control during both the main treatment and the recovery periods. In males, mean body weight of the mid-dose group was numerically lower compared to the other groups at the study start and significantly lower (p < 0.05) than the control on days 22, 29, 36 and 43. At all other time points during the main study and recovery period, the mean body weight of mid-dose males was not significantly different from the control. Furthermore mean body weight was not reduced in the high-dose group and there were no differences in body weight gain or feed consumption in the main or recovery periods between any treatment groups. Therefore, the transient reduction in the mean body weight of the mid-dose was considered incidental.

Ophthalmological examination (Week 13) identified two incidences of corneal opacity in the control (one per sex) and three in high-dose males, in addition four incidences of persistent pupillary membrane in the control (one male, three females) and the high-dose treatment group (three males, one female) were observed. Both optical abnormalities have been reported to occur spontaneously and in high frequencies in Wistar and Han rats [35,36]. Ocular examination results were not statistically significant for either sex during the pre-test and at the end of the treatment period; thus the isolated incidences were not attributed to L-5-MTHF-Ca administration.

There were no statistically significant changes in haematology or coagulation parameters in males at the end of the treatment (Table 3). In high-dose females at Week 13 the relative segmented neutrophil count increased and the relative lymphocyte count decreased (p < 0.05; data not shown). The differences in the relative counts of the two haematological parameters stems from the numerical but not significant increase in absolute neutrophil counts in high-dose females (Table 3). The parameters were not significantly different after the recovery period (not shown). As total blood cell counts were unchanged, there were no accompanying histopathological effects or a dose-response effect, all haematology and coagulation means were within the laboratory’s historical control range observed in the age and strain of rat used, and there was no evidence of infection or inflammation, the differences were considered to be not toxicologically relevant.

Statistically significant changes in clinical chemistry results from Week 13 included lower uric acid and triglycerides (p < 0.05) in mid-dose males and lower aspartate aminotransferase (AST) (p < 0.01) and alanine aminotransferase (ALT) (p < 0.05) in mid-dose females compared to controls (Table 4). These differences were not dose-related and were within the laboratory’s historical range for the sex, species and age of rat used and were, therefore, judged not to be toxicologically relevant. Cholesterol and phospholipid levels were higher in high-dose females compared to the controls (p < 0.05), whereas levels in males were numerically higher but did not reach statistical significance. Values were well within the laboratory’s historical range, and findings were not accompanied by any histopathological changes or other indicators of hepatocellular or cholestatic liver injury and effects were reversible, i.e. differences did not persist through the recovery period (Table 5). Consequently, differences were considered to be not toxicologically relevant. Aspartate aminotransferase levels were lower in high-dose males (p < 0.05) but were within the historical range, were not accompanied by any other indications of toxicological relevance and were note different after the recovery period, therefore were considered not toxicologically relevant. Mean lactate dehydrogenase (LDH) levels in all male L-5-MTHF-Ca-treatment groups were numerically lower than the control group, reaching statistical significance in the high-dose group. The measured mean LDH level for the control group was outside the laboratory’s historical range (1.09 to 5.72 μkat/l) and the value for the mid-dose group was slightly outside the historical range, whereas mean values for low- and high-dose groups were within the historical range. After the recovery phase LDH levels of the control and high-dose group were not significantly different and were both within the historical range (Table 5). Similarly, creatine kinase levels were numerically lower than those in the control group in all L-5-MTHF-Ca-treated males, reaching significance in the low- and high dose groups. Again, the control group was outside the laboratory’s historical range (1.31 to 5.36 μkat/l) while mean values in the test article-treated groups were within the historical range, and after the recovery phase creatine kinase levels of the control and high-dose group (2.66 and 2.12, respectively) were not significantly different and were both within the historical range. Female rats in the high dose group had significantly higher creatine kinase levels after the recovery period although treatment groups weren’t significantly different following the main study. As all measured parameters indicated healthy liver, kidneys and heart, it was concluded that the LDH and creatine kinase measurements in the control group were spuriously high, and differences were not toxicologically relevant in relation to the test article. In both sexes calcium levels increased with L-5-MTHF-Ca concentration reaching significance in the high-dose group and sodium levels were significantly higher in mid- and high-dose animals. The differences did not persist during the recovery period and all values were within the historical range of the laboratory, therefore, the increase was seen as reversible and not adverse.

Urinalysis parameters of L-5-MTHF-Ca-treated female rats were not different from the control group. In males, specific gravity and osmolality were significantly reduced in some test article-treated groups accompanied by significantly higher pH in high-dose males (Table 6). The significant differences did not persist through the recovery period (not shown). All values remained within the 95% tolerance limits of the historical control data, were transient, and no morphological changes were observed, therefore, these findings were considered to be non-adverse. There were no differences between treatment groups for scores of protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, sediment, red blood cells, and crystals in either sex (not shown).

Absolute organ weights and organ-to-body/brain weight ratios did not support any toxicologically relevant effects related to the test article in male (Table 7) or female (Table 8) rats. At the end of the treatment period the only difference in organ weights in male rats was observed in the mid-dose group which had significantly (p < 0.05) lower mean absolute epididymes weight than the control group. Significant differences were not observed when calculated relative to body or brain weigh and no dose-related effect was seen. After the recovery period, the epididymus in high-dose males was numerically heavier than the control, reaching significance when relative weight compared to the brain weight was calculated (data not shown). In females none of the treatment group absolute organ weights were different from the control group. Calculated relative to body weight, the ovaries were significantly smaller in low-dose females and kidneys were larger in mid-dose females. When calculated relative to brain weight, treatment high-dose females had larger liver and kidneys compared to the control. The differences in liver and kidney relative weight did not persist throughout the recovery period. Taken together, there were no differences in organ or relative organ weights attributed to treatment with the test article.

Histological sections of the tissues identified in the methods section for histopathology were examined by light microscopy and findings were graded in severity using a five point system of minimal, slight, moderate, marked or severe. Occasional findings were recorded as present or as unilateral or bilateral. Findings in tissues are reported in Table 9. Tissues identified for histopathology and not included in the table did not have findings. Two high-dose females had moderate or slight centriacinar (periportal) intrahepatic brown pigmentation and increased Kupffer cell pigment. This finding was occasionally observed in control rats at the laboratory and therefore its presence in these animals was considered to be fortuitous rather that a result of treatment with the test article. There were no treatment-related macroscopic or microscopic findings related to exposure to the test substance.

3.1.6. Prenatal developmental toxicity study

There were no maternal deaths during the study. Of the 25 rats per group, 24 were pregnant in the control group, 23 in the low-dose group, 22 in the mid-dose group and 22 in the high-dose group on GD 20. There were two reported incidences of hair loss in the high-dose group and one in each of the other groups. One incidence of skin lesion was observed in the mid-dose group and one incidence of discharge from the nose in the high-dose group. Observed incidences were considered not treatment related.

None of the treatment groups differed from the control in respect to body weight, body weight gain or food consumption. Water consumption in the low- and mid-dose groups was comparable to the control. In the high-dose group water consumption increased slightly but was statistically significant between GD 15 and GD 20. This was considered to be non-adverse.

There were no abnormal findings in dams sacrificed on GD 20. Gravid uterus weight, number of corpora lutea and number of implantations in the treated groups were not significantly different from the control group. There were no treatment related effects on resorptions, number of foetuses, mean foetus weight or sex distribution. No differences in type and frequency of skeletal variations, soft tissue malformations or ossification status were observed in the treated groups compared to the control (Table 10). In addition, all evaluated parameters were in the normal range of the historical control.

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