Cyanamide

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

endocrine system modulation

Remarks:

Hypothalamo-pituitary-adrenal axis activation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline available

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

neurotoxicity

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: 210-250 g
- Housing: individual cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): controlled
- Air changes (per hr): controlled
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intravenous

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single administration

Post exposure period:

4 h

Dose / conc.:

50 mg/kg bw/day

Remarks:

0.1 mL/ 100 g bw

Dose / conc.:

20 mg/kg bw/day

Remarks:

0.1 mL/ 100 g bw

Dose / conc.:

5 mg/kg bw/day

Remarks:

0.1 mL/ 100 g bw

Control animals:

yes

Details on study design:

Male rats were treated intravenously with 5, 20 or 50 mg/kg bw/day of cyanamide. Blood samples were taken before treatment and 30 min, 1, 2 h after treatment, as well as trunk blood samples taken after termination of the exposoure and sacrifice of the animals after 4 h. Brains and pituitaries were removed and collected. In situ hybridization histochemistry (ISHH) and a radioimmunoassay was performed (see below for further information). Statistical analysis was performed by one-way ANOVA followed by Fisher PLSD test. P<0.05 was considered significant.

Examinations:

SAMPLING
1. blood samples were taken imediately before i.v administration of cyanamide (t0), 30 min, 1 h and 2 h following administration
2. 4 h after administration, rats were sacrificed and brains and pituitaries were rapidly removed and trunk blood collected

Details on results:

The i.v. administration of saline (control) had no effect at any of the timepoints. Low, medium and high dose cyanamide-injected groups produced rapid increases in plasma corticosterone at 30 min (P<0.05, P<0.01 and P<0.01, respectively vs. control). In the low dose cyanamide group, plasma corticosterone concentrations returned to basal level after 120 min. A second dose dependent peak appeared at 4 h in both the medium and high dose groups (P< 0.05 and P<0.001, respectively vs. control). The area under the curve (AUC) of plasma corticosterone increased significantly in medium and high dose groups (P<0.05 and P<0.01). respectively vs. control), related to the dose of cyanamide. There are two peaks of corticosterone within 4 h after cyanamide administration. The first peak at 30 min is independent of the dose of cyanamide, while the latter at 240 min appears to be dose-dependent. The increase at 30 min is not due to a non-specific stress effect as plasma corticosterone in the saline injected control animals was not increased at any of the time-points studied. The plasma clearance of cyanamide differs dependent on the dose as the elimination half life is extended dose-dependently. The pharmacokinetics of cyanamide may offer an explanation for the latter peak of corticosterone release. Our present results indicate that cyanamide-induced changes in corticosterone levels can be seen for a considerable time after treatment dependent on the dose. The changes of CRF mRNA and AVP mRNA in the PVN, and POMC mRNA in the anterior pituitary, 4 h after cyanamide administration were analysed. We found a significant increase in the level of CRF mRNA in the medium and high dose groups (P<0.05 and P<0.01, respectively vs. control). In the low dose group, there was no significant difference compared to the control group.
The level of AVP mRNA in the PVN was significantly increased only in the high dose group (P<0.05). Similarly, only the high dose of cyanamide was able to significantly increase POMC mRNA, the ACTH precursor, in the anterior pituitary (P<0.01). Despite the activation of the axis, evidenced by the sustained release of corticosterone in response to the medium dose, this was without effect on POMC mRNA. This apparent lack of effect presumably reflects a balance between negative glucocorticoid feedback and releasing factor stimulation. The significant increase in CRF mRNA, but not AVP mRNA, noted with the medium dose of cyanamide suggests CRF mRNA may be more sensitive to the stimulatory effects of cyanamide than AVP mRNA.
The endpoint of HPA axis activation is an increase in corticosterone release from the adrenal gland.
The level of mRNA for releasing factors following acute stress is used as an index of hypothalamic activation instead of direct measurement of releasing factors in hypophysial portal blood. In this experiment, a typical stress response was observed. These results are the first to demonstrate that acute administration of cyanamide in freely moving rats results in a dose-dependent activation of the HPA axis.
The mechanism of HPA axis activation induced by cyanamide is not clear. One possible explanation may be due to the action of cyanamide on the central nervous system. Cyanamide has a simple structure (H2NCN: mol. wt.=42) and it has a property of rapid onset of inhibition of brain ALDH, and its inhibition depends on the dose. As described above, the physiological function of ALDH in brain may be related to maintaining optimum local aldehyde levels in the central nervous system. In the case of high dose cyanamide administration, it may cause a metabolic disturbance and may become a stressful stimulus to the central nervous system. To clarify the mechanism of cyanamide induced HPA axis activation additional studies will be required.
Cyanamide has not previously been reported to exert a direct effect on the HPA axis. The present results suggest that cyanamide is able to exert direct effects on central nervous system functioning. In conclusion, acute cyanamide administration resulted in a dose-dependent increase in plasma corticosterone concentrations and significant increases in not only CRF mRNA, but also AVP mRNA in the PVN and POMC mRNA in the anterior pituitary. These results suggest that cyanamide is able to activate the HPA axis at all levels of the axis. The mechanism underlying this activation is not clear and additional studies will be required to clarify the mechanism of cyanamide induced HPA axis activation.

Conclusions:

Under the reported conditions, cyanamide administration resulted in a dose-dependent increase in plasma corticosterone concentrations, significant increases in not only corticotrophin releasing factor (CRF) mRNA, but also arginine vasopressin (AVP) mRNA in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary. These results suggest that cyanamide is able to activate the HPA axis at all levels of the axis.

Executive summary:

For the investigation of the effects of cyanamide on hypothalamo-pituitary adrenal (HPA)-axis, male rats were treated intravenously with 5, 20 or 50 mg/kg bw/day of cyanamide. Blood samples were taken before treatment and 30 min, 1, 2 h after treatment, as well as trunk blood samples taken after termination of the exposoure and sacrifice of the animals after 4 h. Brains and pituitaries were removed and collected. In situ hybridization histochemistry (ISHH) and a radioimmunoassay was performed.

Cyanamide administration resulted in a dose-dependent increase in plasma corticosterone concentrations, significant increases in not only corticotrophin releasing factor (CRF) mRNA, but also arginine vasopressin (AVP) mRNA in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary. These results suggest that cyanamide is able to activate the HPA axis at all levels of the axis.

Reference 2

Endpoint:

hepatotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

See "details on study design"

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

other: hepatotoxicity

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Age at study initiation: 6 wks (Group III, IV, Control Taking Alcohol), 12 wks (Group Ia, Ib, IIa, IIb)
- Weight at study initiation: 196 - 260 g
- Fasting period before study: not specified
- Housing, diet and water: not specified
- Acclimation period: 7 to 15 days

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Cyanamide solution was prepared from Colme, which contains 6 g of cyanamide in 100 mL, diluted with saline, resulting in an end concentration of 16 mg of cyanamide per mL.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

15 wks

Frequency of treatment:

once daily

Dose / conc.:

8 mg/kg bw/day

Remarks:

before treatment: 6 wks 16% ethanol; after treatment 16% ethanol (group Ia); after treatment water (Group Ib)

Dose / conc.:

16 mg/kg bw/day

Remarks:

before treatment: 7 wks 16% ethanol; after treatment 16% ethanol (group IIa); after treatment water (Group IIb)

Dose / conc.:

8 mg/kg bw/day

Remarks:

+ water (Group III)

Dose / conc.:

16 mg/kg bw/day

Remarks:

+ water
(Group IV)

No. of animals per sex per dose:

Group Ia-IIb: 8 animals
Group III, I, control taking alcohol and matched control: 5 animals

Control animals:

yes, concurrent no treatment

Details on study design:

Fifty-five male Wistar rats were put into six groups, four experimental and two control groups. Animals of groups I , II , and control taking alcohol were accustomed to oral ethanol intake after an adaptative period of time ranging from 7 to 15 days. Ethanol was supplied in increasing concentrations (2, 4, 8, and 16 %, v/v). Cyanamide solution was prepared from Colme, which contains 6 g of cyanamide in 100 mL, diluted with saline, resulting in an end concentration of 16 mg of cyanamide per mL. It was administered daily intraperitoneally in doses of 8 and 16 mg/kg of body weight. Animals were examined daily and their body weights recorded.
Groups I and II were divided into two subgroups at the beginning of cyanamide treatment. Animals of subgroups la and IIa continued ethanol intake. In animals of subgroups lb and II b ethanol was withdrawn. Rats of groups III and IV never received ethanol.
Test and control animals were sacrificed from the 3rd to the 27th week of treatment.
Afte sacrificer, liver tissue was processed for light and electron microscopy. For light microscopy the material was fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, Masson's trichrome stain, periodic acid-Schiff, periodic acid-Schiff diastase, and Prussian blue.

Examinations:

1. Animal examinations for body weight and clinical symptoms
2. Liver tissue examinations via light and electron microscopy (stained with hematoxylin and eosin, Masson's trichrome stain, periodic acid-Schiff, periodic acid-Schiff diastase,
and Prussian blue)

Details on results:

MORTALITY
Three rats of the groups IIa , Ia , and IIb were found dead at the 3rd, 4th, and 22nd weeks of the experiment, respectively. No histologic study was performed on these rats.

MICROSCOPIC FINDINGS

From the histologic point of view, three types of lesions were found in hepatocytes.
1. Acute lesions: These lesions consisted of acidophilic bodies, hydropic degeneration, giant mitochondria, and occasionally steatosis. They were observed in rats receiving ethanol while undergoing cyanamide treatment (four animals of subgroup la and seven animals of subgroup IIa).
2. Homogeneous areas: The livers of the rats sacrificed from the 7th to 13th week of cyanamide treatment showed morphologic changes not present in the control groups. The hepatocytes displayed some clear and homogeneous areas in the cytoplasm, initially located near the space of Disse, but then enlarged and became confluent. These areas were distributed in the entire lobule. On electron microscopy these homogeneous areas showed a marked increase of glycogen disposed in granules, hyperplasia of smooth endoplasmic reticulum (SER), and a decrease in the number of other organelles. This lesion was found in all animals sacrificed from the 7th to the 13th week (belonging to groups la, lb , lb ; Figs. 1 to 6). In addition, in one rat of group lb the liver tissue also presented a few inclusion bodies following sacrifice in the 7th week.
3. Inclusion bodies: This lesion was observed from the 13th to 27th week in the cytoplasm of the rat liver cells. It consisted of round or kidney-shaped inclusion bodies that, gave the cytoplasm a ground-glass appearance. Initially, the inclusion body-bearing hepatocytes were located in the periportal area and coexisted with homogeneous areas in the rest of the hepatocytes, but in more affected animals, the lesion extended throughout the Lobule. The inclusion bodies were well demarcated from the rest of the cytoplasm, sometimes separated by a clear halo that was not seen in Epon-embedded material. The bodies were intensely positive with periodic acid-Schiff and silver methenamine stains. In Masson trichrome stain, the bodies were gray-blue, which contrasted with the reddish stain of the rest of the cytoplasm. In semithin sections of Epon-embedded material, small vacuoles and dense granules were observed in the inclusion bodies. On electron microscopy, the inclusion bodies were not membrane bound and contained principally glycogen disposed in /3-granules (Fig. 10). a-Granules were also seen around less developed inclusion bodies. In more developed inclusion bodies, the glycogen deposits inside the inclusions were denser, and secondary lysosomes containing lamellar structures appeared. The components of SER, distributed between the glycogen granules, gave a mesh-like appearance. The bodies also contained lipid vacuoles, with heavy deposits of the glycogen granules around them. Inclusion bodies were observed in all of the rats sacrificed between the 13th and 27th weeks, except in one animal of group lb that was sacrificed i n the 27th week and presented homogeneous areas.

Conclusions:

Under the reported experimental conditions, the results indicate that cyanamide triggers the formation of inclusion bodies in liver cells of rats after exposure to cyanamide and ethanol in combination, similiar to the hepatocyte inclusion bodies described in alcoholics treated with cyanamide.

Executive summary:

Hepatocyte inclusion bodies similar to those described in alcoholics treated with cyanamide were reproduced in the rat. For this cause male Wistar rats were put into six groups, four experimental and two control groups. Animals of groups I, II, and control taking alcohol were accustomed to oral ethanol intake, group I and II before the cyanamide treatment.

The characteristic inclusions were developed in animals receiving 8 or 16 mg/kg of body weight, whether they were prepared previously with ethanol intake or not. Inclusion bodies consist of round, well-demarcated cytoplasmic areas, which contain a large amount of glycogen disposed in Beta-granules, lipid droplets, and secondary lysosomes. They appeared at the 13th week of cyanamide treatment onward. Initially, hepatocytes bearing inclusion bodies are located predominantly at the periportal areas, but the lesion later progresses toward the center of the lobule. Prior to the inclusion body development, cyanamide induces another morphologic change in liver cells, consisting of cytoplasmic homogeneous areas, made up of glycogen disposed in agranules and smooth endoplasmic reticulum tubules. This change is described for the first time in relation to this drug.

Reference 3

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-12-09 to 2022-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
1. XTT Test
With the XTT test, cell proliferation as well as the viability of the cells after treatment with the test item are determined colorimetrically.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [(sodium-3’-(1-phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate] to form an orange water-soluble formazan dye by dehydrogenase activity in active mitochondria.

2. BCA Test
- Principle of test: The BCA test predicts cytotoxic or necrotic effects. Cytotoxic effects lead to a reduction of the proliferation rate of the cells. This results in a reduction of the protein content of the cell culture as compared to the control cultures and is detected colorimetrically after an incubation period via the BCA test.
The BCA reagents are comprised of water-soluble and stable BCA (bicinchoninic acid) and an alkaline Cu2+ solution. The amino acids cysteine, cystine, tryptophan and tyrosine, which are a constituent of every cell, bind to these reagents, i.e. these amino acids reduce Cu2+ to Cu+ which then binds to bicinchoninic acid to form a water-soluble violet dye. The intensity of the dye correlates with the cell number in the culture.


- Short description of test conditions:
10 µg/mL of Cycloheximide was used as positive control. The positive control was dissolved in DMSO at a concentration of 10 mg/mL.
DMSO at a final assay concentration of 0.1% was used as solvent control.
FRTL-5 cells were used in a single cell suspension at a density of 4.0 x 105 cells per mL. Sextuplicates were used as the number of replicates for test item concentrations and controls and the exposure period was 24 h.
The treated cells were either incubated with the XTT or BCA staining solution and then evaluated spectrophotometrically.


- Parameters analysed / observed:
-Absorbance (XTT: at 450 nm; BCA: 550 nm); % viability

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Endpoint addressed:

other: cytotoxicity

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

other: in medium

Vehicle:

DMSO

Details on exposure:

CELLS
The test was carried out with FRTL-5 cells (ECACC, cat.-no. 91030711, cells provided by CLS, art.no. 500407). Cells were cultured in 75 cm² culture flasks (Greiner) in Coon’s modified F12 medium, supplemented with 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL insulin, 10 nM hydrocortisone, 10 ng/mL Gly-His-Lys acetate, 1 mU/mL thyroid-stimulating hormone (TSH), and 5 µg/mL transferrin (= culture medium) at 37 °C and 5% CO2.

-Seeding:
Log phase FRTL-5 cultures were washed and trypsinized with Trypsin EDTA for approximately 3 minutes. The enzymatic reaction was stopped with cell culture medium and a single cell suspension was made at a density of 4.0 x 105 cells per mL. 50 µL of the cell suspension was seeded in all cavities of a 96 well plate (Greiner). The cell culture plate was incubated for 24 +/- 2 h at 37 +/- 1°C, 5.0% CO2 / 95% air to achieve approximately 60 - 70% confluence in each cavity. Subsequently, the cell culture plate was examined microscopically for consistent cell quantity.


DOSE GROUPS
1. Solvent control: 0.1% (v/v) DMSO in cell culture medium
2. Positive control: Cycloheximide at a final assay concentration of 10 µg/mL in 0.1% (v/v) DMSO in cell culture medium
3. Test material: 7 concentrations: 10000; 1000; 100; 10; 1; 0.1 and 0.01 µM (active ingredient)


EXPOSURE / TREATMENT
The cell culture medium of the previously seeded cells was removed and 200 µL of the dilutions of the test item or the controls, respectively, were added in sextuplicates (see Table 1). The cell culture plate was incubated for 24 +/- 2 h in humidified air (5.0% CO2 / 95% air) at 37 +/- 1°C.


XTT MEASUREMENT
1 – 2 h before the end of the incubation period, the cell cultures were examined microscopically for cell growth inhibition and cell lysis. After that, 50 µL of the XTT labelling mixture (Roche Diagnostics, Lot No. 45022000) were added to each well. This mixture consists of the XTT labelling reagent (5 mL) and of the electron coupling reagent (0.1 mL). The cells were incubated for further 1 – 2 h and the plate was subsequently transferred to a microplate reader equipped with a 450 nm filter to read the absorbance (reference wavelength 650 nm).

BCA MEASUREMEMT
At the end of the incubation period, the cell cultures will be examined microscopically for cell growth inhibition and cell lysis. After that, the cells were washed 2 times with cold PBS buffer. Thereafter, the microplate was incubated for at least 30 min at < -15° C. 200 µL of the BCA labelling mixture (BC ASSAY Protein Quantitation Kit; Interchim Uptima, Reagent A: Lot No. W03L150, Reagent B: Lot No. W04L138-02) was added to each well. This mixture consists of the BCA labelling reagent A (5 mL) and the BCA labelling reagent B (0.1 mL). The cells were incubated for further 0.5 – 2 h and the plate was subsequently transferred to a microplate reader equipped with a 550 nm filter to read the absorbance.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 h

Frequency of treatment:

once per replicate

Post exposure period:

XTT: 1 - 2 h
BCA: 0.5 - 2 h

Dose / conc.:

10 000 other: µM

Dose / conc.:

1 000 other: µM

Dose / conc.:

100 other: µM

Dose / conc.:

10 other: µM

Dose / conc.:

1 other: µM

Dose / conc.:

0.1 other: µM

Dose / conc.:

0.01 other: µM

No. of animals per sex per dose:

not applicable

Details on study design:

Refer to "Principles of method if other than guideline"

Examinations:

XTT EVALUATION
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of the orange formazan formed, as monitored by the absorbance.
The results for the dose-response cytotoxicity curves are presented as the arithmetic mean  standard deviation. The mean absorption (A490 nm) was determined for each set of wells. Mitochondrial dehydrogenase activity was calculated as follows:

% viability = 100 x ((A490 nm sample)/(A490 nm control))

A490 nm sample: Absorption value of treated cells (Test item or controls)
A490 nm control: Absorption value of the solvent control

BCA EVALUATION
The mean absorption (A550 nm) and standard deviation of the parallel cultures were calculated and used for assessing the percentage of growth inhibition (% G.I.) following the depicted formula:

% viability = 100 x ((A550 nm sample)/(A550 nm control))

A550 nm sample: Absorption value of treated cells (Test item or controls)
A550 nm control: Absorption value of the solvent control


ACCEPTANCE CRITERIA
The lower the dehydrogenase activity % value, the higher the cytotoxic potential of the test item is. Dehydrogenase activity or protein content of less than 70% compared to untreated control cultures (solvent control) is considered as a clear cytotoxic effect.

Positive control:

Cycloheximide (10 µg/mL)

Details on results:

1. XTT Measurement - test item
6 replicates per concentration of Cyanamide were incubated in parallel. Prior to staining with the XTT-labelling reagent, the cells were observed microscopically on growth inhibition and cell lysis. At a concentration of 1000 µM and above, cytotoxic effects were visible.
The calculated mean OD measured after XTT staining, standard deviation, Coefficient of Variation (CV) and calculated viability are shown in Table 1 (see "Any other information on results incl. tables").

1. XTT Measurement - positive control
In the XTT staining, cytotoxic were detected at concentrations of 1000 µM and above. The positive control showed a clear cytotoxic effect, verifying the responsiveness of the cells (see Table 2, "Any other information on results incl. tables").

2. BCA Measurement - test item
6 replicates per concentration of Cyanamide were incubated in parallel. Prior to freezing cells for the BCA staining, the cells were observed microscopically on growth inhibition and cell lysis. At a concentration of 10000 µM, cytotoxic effects were visible.
The calculated mean OD measured after XTT staining, standard deviation, Coefficient of Variation (CV) and calculated viability are shown in Table 3 ("Any other information on results incl. tables")

3. BCA Measurement - positive control
The positive control showed a clear cytotoxic effect, verifying the responsiveness of the cells (see Table 4, "Any other information on results incl. tables" ).

Table 1: Results of the cytotoxicity test for the test item (XTT)

Concentration [µM]	Mean OD	SD	CV	Viability [%]
10000	0.0948	0.0049	5.2	34
1000	0.1941	0.0102	5.3	70
100	0.2864	0.0520	18.2	103
10	0.2995	0.0700	23.4	108
1	0.3296	0.0488	14.8	119
0.1	0.3581	0.0419	11.7	129
0.01	0.3444	0.0238	6.9	124

 

Table 2: Results of the cytotoxicity test for the positive control (XTT)

	Mean	SD	CV	Viability [%]
Cycloheximide (10 µg/mL)	0.115	0.004	3.49	42

 

 

Table 3: Results of the cytotoxicity test for the test item (BCA)

Concentration [µM]	Mean OD	SD	CV	Viability [%]
10000	0.3986	0.0808	20.3	48
1000	0.8857	0.0575	6.5	107
100	1.0149	0.0358	3.5	123
10	0.9412	0.0474	5.0	114
1	0.9302	0.0460	4.9	112
0.1	0.9367	0.0527	5.6	113
0.01	0.9028	0.0485	5.4	109

 

Table 4: Results of the cytotoxicity test for the positive control (BCA)

	Mean	SD	CV	Viability [%]
Cycloheximide (10 µg/mL)	0.299	0.033	11.16	36

 

 

Conclusions:

In conclusion it can be stated, that the test item shows cytotoxic concentrations in FRTL-5 cells at a concentration of 1000 µM and above, as verified via XTT and BCA staining.

Therefore, 100 µM will be chosen as maximum starting concentration for subsequent assays employing FRTL-5 cells.

Executive summary:

In the present study, FRTL-5 cells were assessed on their sensitivity towards rising concentrations of the test item. The vitality of the cells or potential cytotoxic effects of the chemicals were registered via the mitochondrial dehydrogenase activity of the cell culture via the XTT assay and via the reaction of the cellular proteins with copper ions and BCA.

Hereby, the test item was mixed with cell culture medium at rising concentrations and FRTL-5 cells were incubated for 24 ± 2h with this mixture. Prior to staining with XTT for mitochondrial dehydrogenase analysis, the cells were inspected microscopically for cytotoxic effects. The decrease in the activity of mitochondrial dehydrogenases of the individual cultures was then analysed as a measure for cytotoxicity and compared to those of the respective controls.

 

Cytotoxic effects were detected microscopically at a concentration of 1000 µM and above.

The XTT staining showed reduced viability at a concentration of 1000 µM and above.

The BCA assay showed reduced protein content at the highest tested concentration of 10000 µM.

In conclusion it can be stated, that the test item shows cytotoxic concentrations in FRTL-5 cells at a concentration of 1000 µM and above, as verified via XTT and BCA staining.

Therefore, 100 µM will be chosen as maximum starting concentration for subsequent assays employing FRTL-5 cells.

Reference 4

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-03-30 to 2022-04-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
The activity of thyroid peroxidase (TPO) is considered one of the key events during thyroid hormone homeostasis and signalling. The TPO assay uses the human thyroid follicular cell line Nthy-ori 3-1, which is capable of expressing high amounts of the enzyme. Mixing lysates of these cells with Luminol, a specific substrate for peroxidases, and hydrogen peroxide, results in the production of luminescence which can be quantified via integrative luminescence measurement. The activity of TPO is directly proportional to total luminescence. Chemicals, which interfere with TPO activity, can therefore directly be identified when co-incubated with the lysate and substrate.

- Short description of test conditions:
Lysate with a protein concentration of 0.2 mg/mL was mixed with six concentrations of the test item (50% aqueous cyanamide solution) in a (concentration range 0.001 to 1000 µM) and incubated for 30 min. Subsequently, luminol was added to the mixture and the luminescent reaction was initiated by the addition of hydrogen peroxide immediately prior to measurement. The relative activity to SC was evaluated in two independent runs.

- Parameters analysed / observed:
Chemical interference, relative TPO activity to solvent control for the test item, negative control and positive control

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Endpoint addressed:

other: endocrine disruption

Species:

human

Strain:

not specified

Sex:

not specified

Route of administration:

other: in medium

Vehicle:

DMSO

Details on exposure:

CELLS
The test will be carried out with the thyroid follicular cell line Nthy-ori 3-1, obtained from the European Collection of Authenticated Cell Cultures (ECACC 90011609). These cells are characterized as mycoplasma-free and will be cultured in 75 cm² culture flasks (Greiner) at 37 +/- 1°C and 5.0% CO2. The used media will be RPMI 1640 + 2mM Glutamine + 10% Foetal Bovine Serum (FBS). Upon reaching 75 - 90% confluence, cells will be subcultured at 0.4 x 105 - 0.8 x 105 cells/mL. To maintain the integrity of the response, the cells should be grown for more than one passage from the frozen stock in the conditioned media.

DOSE GROUPS
Blank: solvent for controls without luminol added prior to measurement
Solvent control (SC): solvent for test item and PC, respectively (at least n=3 per plate)
Reference chemical 1: 6 concentrations of MMI (n=3)
Reference chemical 2: 6 concentrations of AMI (n=3)
Test item: 6 concentrations of the test item (n=3)

EXPERIMENTAL PROCEDURE
Based on the solubility observed in the preliminary test, the highest acceptable test item concentration was chosen and serially diluted in a 1:10 ratio in the respective solvent.
For preparing the cell lysate used in the assay, cells were cultured as described above. Upon reaching 75 – 90% confluence, cells were harvested by scraping them off the flask bottom, resuspended in PBS, collected by centrifugation and lysed in 0,1% Na-Deoxycholate (Alfa Aesar, Lot Q18F040) in PBS. The obtained cell lysate was cleared from debris by centrifugation and either used directly in the assay or stored at -80°C until use.
On each day of use in the TPO assay the protein concentration of the cell lysate preparation was determined (BCA Protein Assay Kit, Uptima, Lot No. A: W09L198, W03L150; B: V06L07-02).
For the TPO assay, Potassium phosphate-buffer (pH 7.4) was added to the cell lysate to obtain a lysate preparation with a protein concentration of 0.1-0.2 mg/mL. 693 µL of this solution were mixed with 7 µL of the stock solutions of the test item, the reference chemicals or solvent control. 200 µL per well of this mixture were transferred to a microtiter plate and incubated at 37°C for 30 min with gentle shaking.

Following the incubation period, 20 µL 400 µM Luminol working solution was added (35.6 µM final concentration). Immediately prior to the measurement of each well, 5 µL 80 mM H2O2 were added to initiate the reaction (1.8 mM final concentration). The luminescence was integrated over a period of 10 seconds at a luminescence plate reader (Tecan infinite 200 Pro, Tecan).

CHEMICAL INTERFERENCE TEST
To exclude the possibility of a false negative result, an interference test of the test item with the used chemicals, in particular the hydrogen peroxide and luminol, was performed. 693 µL Glycine-NaOH buffer (pH 9.0) were mixed with 7 µL of the stock solutions of the test item, the reference chemicals or solvent control. 200 µL per well of this mixture were transferred to a microtiter plate (according to the scheme shown below) and incubated at 37°C for 30 min with gentle shaking.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

30 min

Frequency of treatment:

once per replicate

Dose / conc.:

0.001 other: µM

Dose / conc.:

0.01 other: µM

Dose / conc.:

0.1 other: µM

Dose / conc.:

1 other: µM

Dose / conc.:

10 other: µM

Dose / conc.:

100 other: µM

Dose / conc.:

1 000 other: µM

No. of animals per sex per dose:

not applicable

Control animals:

other: not applicable

Details on study design:

refer to "Principles of method if other than guideline"

Examinations:

EVALUATION
The relative TPO activity to solvent control was obtained by subtracting the mean luminescence value of the Blank from the luminescence value of each well to normalize the data and, afterwards, dividing the normalized value of each well in the plate by the mean value of the normalized solvent control (solvent control = 100%). The mean values and standard deviations of the relative transcriptional activity for each concentration of the test chemical were calculated.

- Log IC50 induction considerations
For positive chemicals, the concentrations that induce the IC50 were provided.
The ICx value was calculated by interpolating between 2 points on the X-Y coordinate, one immediately above and one immediately below an ICx value. Where the data points lying immediately above and below the ICx values have the coordinates (c,d) and (a,b) respectively, the ICx values may be calculated using the following equation:

log⁡[ICx]=a-[(b-(100-x))/(b-d)]*(a-c)


ACCEPTANCE CRITERIA
The results will be based on two (or three) independent runs. If two runs give comparable and therefore reproducible results, it is not necessary to conduct a third run. To be acceptable, the results should:
(1) Meet the performance standard requirements:
- The results of 3 reference chemicals should be within the acceptable range

Criteria of the reference chemicals for the assay:
Name Judgement Test range [µM]
Methimazole (MMI) Positive: IC50 should be calculated 0.01 - 1000
Amiodarone (AMI) Negative: IC50 should not be calculate 0.01 - 1000

(2) Be reproducible.


INTERPRETATION
Positive results will be characterised by both the magnitude of the effect and the concentration at which the effect occurs. Expressing results as a concentration at which a 50% (log IC50) reduction is reached accomplishes this goal. However, a test chemical is determined to be positive if the log IC50 could be calculated in at least two of two or two of three runs, whereas a test chemical is considered as negative if the log IC50 could not be calculated in two of two or two of three runs.

- Positive and negative decision criteria in TPO inhibition assay
Positive If the IC50 is calculated in at least two of two or two of three runs.
Negative If the IC50 fails to calculate in two of two or two of three runs.

Positive control:

Methimazole (MMI)

Details on results:

1. Chemical interference test
The test item and the controls were incubated with Glycine-NaOH-Buffer (pH9.0) and luminescence was measured and relative luminescence intensity to solvent control was calculated. The mean luminescence activities were calculated and are summarized in Table 1 (see "Any other information on results incl. tables"). The measured luminescence intensities in the interference test were subtracted from the measured intensities during the two experiments. The relative TPO activities shown are therefore based on the interference corrected data.

2. Test item
The relative TPO activity to solvent control for the test item was calculated. The mean values obtained during the two independent runs are shown in Table 2 (see "Any other information on results incl. tables"). For the test item no IC50 values could be determined since the dose response curve did not cross 50% activity.

3. Positive control: Methimazole
The relative TPO activity to solvent control for the test item was calculated. The mean values were obtained during the two independent runs and are shown in Table 3 (see "Any other information on results incl. tables").

4. Negative control: Amiodarone
The relative TPO activity to solvent control for the test item was calculated. The mean values obtained during the two independent runs are summarized in Table 4 (see "Any other information on results incl. tables").

5. Acceptance criteria
The TPO assay is functioning correctly if the following criteria are met:
(1) The results of the reference chemicals should be within the acceptable range. During both runs the criterion was fulfilled. For results, refer to Table 5 (see "Any other information on results incl. tables").
(2) The results should be reproducible.
In both experiments, the controls, as well as the test item showed comparable results. Therefore, the results are considered reproducible.

Table 1:  Summary of measured Luminescence during the interference test

Log Concentration [M]	Measured Luminescence (RLU) for
	Methimazole		Amiodarone		Cyanamid L500P
	Mean	SD	Mean	SD	Mean	SD
-3	-252.67	3.00	2103.00	147.60	1491.67	43.84
-4	247.00	21.50	5292.00	171.16	1519.67	202.16
-5	915.33	12.53	1630.00	28.75	1553.67	113.71
-6	1236.00	61.58	1566.33	60.23	1604.67	120.14
-7	1224.33	119.31	1547.33	38.51	1534.67	64.70
-8	1419.67	146.77	1581.00	33.01	1535.67	25.03
-9	-252.67	3.00	2103.00	147.60	1491.67	43.84
Solvent control	1702.44	177.37

 

Table 2:  Summary of relative TPO activity for the test item

Log Concentration [M]	Relative TPO activity to solvent control [%] for
	Run 1		Run 2
	Mean	SD	Mean	SD
-3	78.2	3.3	67.3	8.8
-4	79.0	0.9	60.4	14.4
-5	76.6	7.0	69.3	2.6
-6	79.5	10.8	77.4	7.3
-7	92.1	5.4	82.6	1.3
-8	87.5	1.8	84.0	6.4
-9	78.2	3.3	67.3	8.8
Log IC50 [M]:	Could not be calculated		Could not be calculated

 

Table 3:   Summary of relative TPO activity for the positive control

Log Concentration [M]	Relative TPO activity to solvent control [%] for
	Run 1		Run 2
	Mean	SD	Mean	SD
-3	0.9	0.0	0.6	0.4
-4	4.4	0.5	5.0	3.1
-5	26.5	2.4	25.8	5.9
-6	67.2	5.0	66.2	0.9
-7	84.9	3.1	79.8	6.7
-8	87.1	9.7	95.5	5.3
Log IC50 [M]:	-5.58		-5.60
IC50 [M]:	2.68 x 10^-6		2.51 x 10^-6

 

Table 4:  Summary of relative TPO activity for the negative control

Log Concentration [M]	Relative TPO activity to solvent control [%] for
	Run 1		Run 2
	Mean	SD	Mean	SD
-3	72.1	4.5	62.7	3.9
-4	62.0	5.3	69.1	7.3
-5	84.6	3.2	66.4	7.3
-6	84.5	3.3	80.1	1.6
-7	81.5	4.7	78.0	10.1
-8	99.7	4.3	88.8	0.4
Log IC50 [M]:	Could not be calculated		Could not be calculated

 

Table 5: Reliability check: reference chemicals

Name	Log IC50 [M] in
	Run 1	Run 2
Methimazole	-5.50	-5.57
Amiodarone	Could not be calculated	Could not be calculated

 

Table 6: Historical data

Chemical	Literature			Eurofins (2019-2022)
	Mean	Lower limit	upper limit	Mean	SD	Min	Max	n
Methimazole	-5.40	-5.80	-5.00	-5.57	0.58	-6.55	-4.88	13
Amiodarone	-	-	-	-	-	-	-	13

 

 

Conclusions:

In this study under the given conditions cyanamide was considered to not inhibit TPO activity, since no IC50 could be calculated.

Executive summary:

In this study the ability of cyanamide (test item Cyanamid L500 P, a 50% aqueous cyanamide solution) to inhibit TPO activity was investigated using the lysate of the human thyroid follicular cell line Nthy-ori 3-1. Lysate with a protein concentration of 0.2 mg/mL was mixed with seven concentrations of the test item (Cyanamid L500 P) in a concentration range of 0.001 to 1000 µM and incubated for 30 min. Subsequently, Luminol was added to the mixture and the luminescent reaction was initiated by the addition of hydrogen peroxide immediately prior to measurement. The relative activity to SC was evaluated in two independent runs. To assess the possibility of false negative data, an interference test was conducted, where the same concentrations of the test item were mixed with Glycine-NaOH buffer instead of cell lysate. In both runs no IC₅₀-value could be determined. Moreover, the interference test showed, the test item did not induce a luminescence signal on its own, or in conjunction with Luminol and H₂O₂. Therefore, the measured signal was only TPO-dependent luminescence, and the test item was determined to not inhibit TPO.

The controls confirmed the validity of the test. In this study under the given conditions the active ingredient cyanamide was considered to not inhibit TPO activity, since no IC₅₀ could be calculated

Reference 5

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-03-30 to 2022-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
In the present study the ability of cyanamide, the active ingredient of Cyanamid L500 P to inhibit the activity of the Sodium-Iodide-Symporter (NIS) was investigated. The activity of the Sodium-Iodide-Symporter (NIS), a membrane protein responsible to import iodide into thyroid cells for the production of thyroid hormones is considered one of the key events during thyroid hormone homeostasis and signalling. The NIS assay uses the rat thyroid cell line FRTL-5, which expresses high amounts of the symporter. Treating the cells with a Sodium-Iodide solution results in uptake of iodide into the cells. The amount of iodide can be quantified using the “Sandell-Kolthoff reaction”, an iodide-catalysed reduction of yellow Ce4+-Ions to colourless Ce3+-Ions. The activity of NIS is directly correlated with the amount of iodide within the cells. Chemicals, which interfere with NIS activity, can therefore directly be identified when co-incubated with the cells prior to treatment with an iodide-solution.

- Short description of test conditions:
As maximum concentration of 1000 µM of the active ingredient cyanamide was found to be both soluble and compatible with the survival of the FRTL-5 cell line. In total, 6 concentrations, ranging from 1000 µM to 0.01 µM were tested.
As positive control NaClO4 and as negative control NaAc was used.

- Parameters analysed / observed:
The amount of Iodide, taken up by the cells was determined. Iodide catalyses the reduction of the coloured Ce4+ to colourless Ce3+, the loss of absorbance in solution is therefore directly correlated to NIS activity.

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Endpoint addressed:

other: endocrine desruption

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

other: in medium

Vehicle:

other: HBSS

Details on exposure:

CELLS
The test was carried out with the rat thyroid cell line FRTL-5, obtained from CLS Cell Lines Service GmbH (Art.-No. 500407, Lot 500407-919). These cells were characterized as mycoplasma-free and were cultured in 75 cm2 culture flasks (Greiner) at 37 1°C and 5.0% CO2. The used media was Coon’s modified F12 medium (PAN Biotech, Lot 7151121) supplemented with 5% heat-inactivated FBS (Bio&Sell, Lot BS.301657.5), 2 mM L-glutamine (Gibco, Lot 2329655), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco, Lot 149237), 10 µg/ml insulin (Sigma, Lot SLCK4397), 10 nM hydrocortisone (Sigma, Lot SLBV4565), 10 ng/ml Gly-His-Lys acetate (Sigma, Lot SLBW3410), 1 mU/ml thyroid-stimulating hormone (TSH, Sigma, Lot MKCJ9988), and 5 µg/ml transferrin (Sigma, BCCB0367).
Upon reaching 75 - 90% confluence, cells were subcultured at 0.4 x 105 - 0.8 x 105 cells/mL. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock in the conditioned media.


DOSE GROUPS
1. Blank: Water or respective matrix (n=8 per plate)
2. Solvent control (SC1/SC2): solvent for test item and PC, respectively (n=3 per plate)
3. Positive control (PC): 6 concentrations of sodium perchlorate (n=3)
4. Negative control (NC): 6 concentrations of sodium acetate (n=3)
5. Test item (TI): 6 concentrations of the test item (n=3)
6. NaI-Standard: 8 concentrations (S0-S7) (n=2)


PREPARATION OF STOCK SOLUTIONS FOR SANDELL-KOLTHOFF-REACTION

1. Ammoniumcer(IV)-sulfate solution (4x)
1.253 g of Ce(NH4)4(SO4)4 x 2 H2O (CAS-No.: 10378-47-9; Sigma, Lot MKCJ4871) was dissolved in 20 mL ultrapure water (Sigma, Lot RNBK1826). Slowly, 5mL concentrated Sulfuric acid (H2SO4, Sigma, Lot SZBE1120V) was added, during this procedure, the solution was cooled on ice. Afterwards, the solution was brought to 50 mL with ultrapure water. The concentration of the solution was 42 mM of Ammoniumcer(IV)-sulfate. The solution is stable for 6 months when kept at 4°C in the dark. Prior to use, the solution was diluted fourfold. This solution was labelled reagent A.

2. Sodium arsenite solution (4x)
0.475 g As2O3 (CAS-No.: 7784-46-5; Sigma, Lot BCCB5045) and 2.4 g NaCl (CAS-No.: 7647-14-5; VWR, 21E054106) was dissolved in 5 mL 2 M NaOH (Alfa Aesar, Lot P19G502). The solution was brought to 50 mL with ultrapure water. Insoluble residues were removed via centrifugation. The concentration of this solution was 96 mM of sodium arsenite. The solution is stable for 6 months when kept at 4°C in the dark. Prior to use, the solution was diluted fourfold. This solution was labelled reagent B.

3. NaI-Standard solutions
29.98 mg NaI (CAS-No.: 7681-82-5; Alfa Aesar, Lot no. M22F016) was dissolved in 100 mL ultrapure water in a volumetric flask. The resulting solution had a concentration of 2 mM NaI. The solution was diluted 1000 times to prepare a 2 µM NaI-stock solution.


EXPERIMENTAL PROCEDURE
For performance of the assay, 200 µL of a FRTL-5 cell-solution (125.000 cells/mL) were seeded in a clear 96-well microtiter plate (flat bottom, column 4-12). After culturing the cells for three days, the assay was performed.
Based on the solubility observed in the preliminary test, the highest acceptable test item concentration was chosen and serially diluted in a 1:10 ratio in the respective solvent.
On the day of the assay the cells were be approximately 80% confluent in the 96-well microtiter plate. The culture medium was removed via aspiration and the cells were washed with uptake buffer (HBSS + 10 mM HEPES). After washing, 80 µL of uptake buffer were dispensed atop the cells. 10 µL of controls and test chemicals were added to the cells. Immediately afterwards, 10 µL of a 100 µM NaI-solution were added.

Following an incubation period of 60 min at 20°C, the assay plate was washed with cold (4°C) uptake buffer, and residual supernatant was immediately discarded by inverting the assay plate on absorbent paper.
For the blank, 200 µL of water were added to the first column of the assay plate. Sodium iodide standards (S1–S7) and water (S0) were distributed in duplicate (100 µL each) in the second and third column of the assay plate. Water (100 µL) was added to columns 3–12.
100 µL of reagent A were added to all wells except the blank wells. 100 µL of reagent B were added to all wells. Subsequently, the plate was covered and incubated for 30 min in the dark.
The bottom of the plate was wiped to remove any liquid and the plate was read in a plate reader at a wavelength of 420 nm.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

60 min

Frequency of treatment:

once per replicate

Post exposure period:

30 min

Dose / conc.:

1 000 other: µM

Dose / conc.:

100 other: µM

Dose / conc.:

10 other: µM

Dose / conc.:

1 other: µM

Dose / conc.:

0.1 other: µM

Dose / conc.:

0.01 other: µM

No. of animals per sex per dose:

not applicable

Control animals:

no

Details on study design:

refer to "Principles of method if other than guideline"

Examinations:

EVALUATION
To obtain relative NIS activity to solvent control the mean absorbance value of the blank was subtracted from each well absorbance value to normalize the data. Afterwards, the data was logarithmized (log A) and, for the standard values, the log A was plotted against the concentration, using a semi-log graph. Using linear regression, a standard curve was fitted and the iodide-concentration of the test item was calculated via the regression. The absolute concentrations of iodide were calculated. For the determination of NIS activity, the concentration of iodide that was measured in the solvent control was set to 100% and the concentrations that were measured at the respective of controls and test item were set into relation to the solvent control. The relative amount of iodide was considered to be equivalent to the relative NIS activity.

- Log IC50 induction considerations
For positive chemicals, the concentrations that induce the IC50 was provided. To this end, the relative NIS activity was plotted against the respective logarithmic concentration.
The IC50 value was calculated by interpolating between 2 points on the X-Y coordinate, one immediately above and one immediately below the IC50 value. Where the data points lying immediately above and below the IC50 values have the coordinates (c,d) and (a,b) respectively, the IC50 values may be calculated using the following equation:

log⁡[IC50]=a-[(b-(100-50))/(b-d)]*(a-c)


ACCEPTANCE CRITERIA
The results were based on two (or three) independent runs. If two runs gave comparable and therefore reproducible results, it was not necessary to conduct a third run.
To be acceptable, the results should:
(1) Meet the performance standard requirements:
- The regression coefficient (r²) should be above 0.98
- The absorbance of S0 should be ≥ 0.800
- The results of positive and negative control should be correctly classified according to the following table

Name Judgment Test range [µM]
NaClO4 Positive: IC50 should be calculated 0.001 - 100
NaAc Negative: IC50 should not be calculated 0.01 - 1000

(2) Be reproducible.


INTERPRETATION
Positive results were characterised by both the magnitude of the effect and the concentration at which the effect occurs. Expressing results as a concentration at which a 50% (log IC50) reduction is reached accomplished this goal. However, a test chemical was determined to be positive if the log IC50 could be calculated in at least two of two or two of three runs, whereas a test chemical was considered as negative if the log IC50 could not be calculated in two of two or two of three runs.

Positive and negative decision criteria in NIS assay:
Positive: If the IC50 is calculated in at least two of two or two of three runs.
Negative: If the IC50 fails to calculate in two of two or two of three runs.

Positive control:

Sodium perchlorate

Details on results:

SOLUBILITY TEST
The test item was found to be soluble in HBSS at a highest concentration of 100 mM. In conjunction with the results of a previously performed Cytotoxicity study with the test item and FRTL-5 cells, the highest final concentration tested in the main experiment was 1000 µM. A correction factor of 1.97 was applied to correct for the content of the active ingredient.

MAIN NIS TEST
The relative NIS activity to solvent control for the test item was calculated. The mean values obtained during two independent runs are shown in Table 1 (refer to Any other information on results incl. tables).
Since the inhibition of NIS activity is less than 50%, no IC50 values can be determined for the test item. The test item will therefore be judged negative with regard to inhibition of NIS activity.

The assay is functioning correctly if the following criteria are met:
- The regression coefficient (r²) should be above 0.98
- The absorbance of S0 should be ≥ 0.800
- The results of positive and negative control should be correctly classified.

The regression coefficient (r²) was calculated to be 0.93 for run 1 and 0.96 for run 2. Therefore, this validity criterion was not met during both runs. However, the linearity was given and the calculated results fit well within the historical data. Therefore, the deviation was accepted and the runs regarded valid.
The mean absorbance of S0 was 1.042 in run 1 and 1.010 in run 2. Therefore, this validity criterion was fulfilled during both runs.
The summary of calculated IC50 for the controls is shown in Table 2 (refer to Any other information on results incl. tables).
Both controls were correctly classified in both runs. Therefore, this validity criterion was fulfilled during both runs.

Table 1:   Summary of relative NIS activity for the test item

Log Concentration [M]	Relative NIS activity to solvent control [%] for
	Run 1		Run 2
	mean	SD	mean	SD
-3	129.6	14.4	77.7	3.8
-5	114.8	18.8	93.5	3.2
-6	153.7	8.5	96.7	16.6
-7	118.4	18.8	92.2	6.9
-8	102.7	11.7	110.9	8.9
-9	99.5	5.4	92.9	17.7
log IC50 [M]:	Could not be calculated		Could not be calculated

 

Table 2:   Summary of calculated IC₅₀ for the controls

Chemical	Calculated IC50 for
	Run 1	Run 2
NaClO4	0.14 µM	0.15 µM
NaAc	-	-

- = not applicable

 

Table 3: Historical Data (since 2019)

Chemical	IC50 [µM] (acc. to Waltz et al)	Eurofins Data mean IC50 [µM]	n
NaClO4	0.10 ± 0.027	0.093	10
NaCOOCH3	-	-	10

 

Conclusions:

In this study under the given conditions the active ingredient cyanamide was classified as negative in the NIS assay, since an IC50 value could not be calculated in both runs.

Executive summary:

In this study the ability of cyanamide (test item Cyanamid L500 P, a 50% aqueous cyanamide solution) to inhibit the activity of the Sodium/Iodide symporter (NIS) was investigated using the rat thyroid cell line FRTL-5. Employing the Sandell-Kolthoff-reaction, the amount of Iodide, taken up by the cells was determined. Iodide catalyses the reduction of the coloured Ce⁴⁺ to colourless Ce³⁺, the loss of absorbance in solution is therefore directly correlated to NIS activity. As maximum concentration of 1000 µM of the active ingredient cyanamide was found to be both soluble and compatible with the survival of the FRTL-5 cell line. In total, 6 concentrations, ranging from 1000 µM to 0.01 µM were tested. For the test item no IC₅₀ values could be calculated, since the inhibitory effect of the test item was less than 50% in the tested concentration range. The controls showed the validity of the assay. NaClO₄ was correctly classified as Inhibitor of the NIS, while Sodium-Acetate did not cause inhibition of NIS activity. In this study under the given conditions the active ingredient cyanamide was classified as negative in the NIS assay, since an IC₅₀ value could not be calculated in both runs.

Reference 6

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

other: endocrine disruption

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

not specified

Details on exposure:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

not applicable

Frequency of treatment:

once

Post exposure period:

30 min

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

not specified

Control animals:

yes, concurrent no treatment

Details on study design:

One set of animals was given 125-Iod 15 min before measurement of protein-bound iodine. The thyroids of a second set were assayed for TPO-catalyzed guaiacol oxidation.

Examinations:

Presence of active TPO and incorporaion of 125-Iod was measured in thyroids:
Protein-bound iodine is expressed as the percentage of incorporation of 125-Iod- into trichloracetic acid-insoluble thyroid homogenate proteins in 15 min. For guaiacol assays, the 105,000 x g microsomal pellets were homogenized in 0.5 mL 50 mM K-phosphate buffer, pH 7.4, and assayed for TPO activity at two levels of enzyme to establish linearity with respect to TPO.

Details on results:

The investigation of the inhibitory effect of cyanamide on TPO activity showed that both guaiacol and iodide peroxidation as well as iodination were inhibited by NH2CN. Fifty percent inhibition occurred at assay concentrations between 1-4 µM, depending on the assay. The preincubation experiments yielded results which were identical to those obtained when cyanamide was added directly in the assay systems. This indicated that the extent of inhibition by cyanamide was governed by its final concentration in the assay systems. To distinguish reversible from irreversible inhibition, preincubation of TPO with NH2CN, followed by gel separation of enzyme from NH2CN, was carried out. There was 20% recovery from the G-25 column of TPO preincubated with H2O2 and cyanamide compared an average (IdU and GU) recovery of 50% for TPO with Cyanamide in the absence of H2O2 and with the recoveries of 52% and 58% for the control preincubations. Inactivating concentrations of cyanamide were the same magnitude as inactivating concentrations of thiourea in the presence of iodide and H2O2.

Experiment	Initial activity	Initial activity	Activity recovered from G-25	Activity recovered from G-25	Recovery from G-25 (% of initial activity)	Recovery from G-25 (% of initial activity)
	GU^a	IdU^b	GU^a	IdU^b	GU^a	IdU^b
1. TPO	13.9	0.58	6.8	0.32	49	55
2. TPO + H2O2	11.0	0.53	7.0	0.28	63	52
3. TPO + NH2CN	12.0	0.49	5.5	0.26	46	53
4. TPO + NH2CN + H2O2	13.9	0.58	3.07	0.12	22	20

 

^aPeroxidation: guaiacol units (micromoles of guaiacol oxidized per min).

^bIodination: iodination units (micromoles of monoiodo Glu-Tyr-Glu formed per min).

Conclusions:

Under the reported experimental conditions of time and dosage of cyanamide, thyroidal protein iodination was 97-99% inhibited, while the peroxidase was inhibited by 25-35% only as measured by guaiacol oxidation.

Reference 7

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
Thyroperoxidase (TPO) activity in Beagle dog and Han Wistar rat thyroid microsomes was determined by the method of Chang HC and Doerge DR (2000), as described by Paul KB et al. (2014). In brief, this method exploits the ability of TPO to catalyse the oxidation of guaiacol to a coloured product. The rate of production of this coloured product can be determined spectrophotometrically (OD450), and TPO activity is then expressed in units of AOD450 /min/mg protein.

GLP compliance:

no

Type of method:

in vitro

Remarks:

/ex vivo

Endpoint addressed:

other: endocrine disruption

Species:

other: dog and rat

Strain:

other: rat: Han Wistar; dog: Beagle

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
1. Han Wistar rats (n=100 individuals, Envigo, UK);
2. Beagle dogs (n=5 individuals, BioIVT, UK)

Vehicle:

DMSO

Details on exposure:

Frozen thyroids were harvested from non-juvenile, male Han Wistar rats and male Beagle dogs.

PRELIMINARY STUDIES
Concentration ranges for cyanamide and the positive control, 6-Propyl-2-thiouracil (PTU) were determined through preliminary range-finding experiments.

TEST AND CONTROL ITEMS
-DMSO (vehicle control)
-Cyanamide (test item)
-6-Propyl-2-thiouracil ((PTU), positive control)

PREPARATION OF THYROID MICROSOMES
Individual thyroids were weighed and a 10% (w/v) homogenate prepared in ice-cold SET buffer containing 500 U/mL catalase. The homogenate was processed to a microsomal fraction by differential centrifugation, according to Laboratory Method Sheet (LMS) Cent001. The microsomal fractions were resuspended in SET buffer lacking catalase, pooled, aliquoted, frozen, and stored at approximately -70 °C until required for protein content determination (Lowry) and TPO analysis.

THYROID MICROSOMAL DETERMINATION
The protein content in each pooled microsomal preparation was determined using a modification of the method of Lowry et al. (Lowry et al., 1951) with bovine serum albumin standards.

TESTING PROCEDURE
Microsomes were pre-incubated with 35 mM guaiacol in assay buffer (100 mM sodium/potassium phosphate buffer, pH 7.4) at 37 °C in a 96-well plate. Reactions were initiated by addition of 300 µM H2O2 . The total assay volume was 200 µL.
To measure inhibitory potential, reference and test items were added to the assay system at a range of at least 9 concentrations along with a control incubation where DMSO only was added. Six technical replicate incubations were run per concentration tested.

DATA MANIPULATION AND EVALUATION
A linear model was fitted to the TPO activity versus microsomal protein data set. Linear regression was performed using GraphPad Prism (Version 7.02, GraphPad Software Inc, San Diego, California, USA). TPO activity (expressed as a percentage of control activity) was determined as a function of test substance or positive control substance concentration, and IC50 parameters were estimated by fitting a four-parameter logistic model to the resulting data set. Model fitting was performed using GraphPad Prism (Version 7.02, GraphPad Software Inc, San Diego, California, USA).

Duration of treatment / exposure:

120 seconds

Frequency of treatment:

once

Dose / conc.:

0 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

0.01 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

0.1 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

1 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

2.5 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

5 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

10 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

25 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

50 other: µM

Remarks:

dog and rat microsomes

Dose / conc.:

100 other: µM

Remarks:

dog and rat microsomes

Examinations:

TPO activities at 37 °C were determined by monitoring the rate of production of the coloured guaiacol oxidation product (OD450) over time. Activities were expressed as AOD450 /min/mg protein. IC50 values for cyanamide and PTU were calculated by fitting a four-parameter logistic model to the plot of TPO activity versus inhibitor concentration.

Positive control:

6-propyl-2-thiouracil (PTU) at following concentrations:
0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50 and 100 µM (rat);
0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, 100 and 200 µM (dog)

Details on results:

PROTEIN DEPENDENCY
1. Han Wistar Rat: TPO activity was linear with regard to protein concentration, up to 60 µg protein per reaction. A final protein content of 40 µg per reaction was used in subsequent TPO assays, as this amount allowed a constant rate of guaiacol oxidation for up to 60 seconds.
2. Beagle Dog :TPO activity was linear with regard to protein concentration, up to 100 µg protein per reaction. A final protein content of 75 µg per reaction was used in subsequent TPO assays, as this amount allowed a constant rate of guaiacol oxidation for up to 60 seconds.

POSITIVE CONTROL ITEM
1. Han Wistar Rat: The positive control inhibitor, 6-propyl-2-thiouracil (PTU), was included in the TPO activity assay at the following final concentrations: 0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50 and 100 µM. These concentrations were chosen for the preliminary dose range finding according to the referenced IC50 value (Paul et al., 2013) and were deemed appropriate for reporting. PTU was a potent inhibitor of TPO activity, exhibiting an IC50 value of 6 µM, 95% CI: 4 to 11 µM. This estimate is similar to a previously published estimate of 1.3 µM, 95% CI: 0.5 to 3.2 µM (Paul et al., 2013).
2. Beagle Dog: The positive control inhibitor, 6-propyl-2-thiouracil (PTU), was included in the TPO activity assay at the following final concentrations: 0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, 100 and 200 µM. These concentrations were chosen for the preliminary dose range finding and were deemed appropriate for reporting. PTU was a potent inhibitor of TPO activity, exhibiting an IC50 value of 31 µM, 95% CI: 25 to 41 µM.

-TEST ITEM
1. Han Wistar Rat: Cyanamide was included in the TPO activity assay at final concentrations of: 0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, and 100 µM. These concentrations were chosen for the preliminary dose range finding and were deemed appropriate for reporting. Using this range of concentrations, the IC5 0 value of cyanamide for rat TPO was calculated to be 7 µM (95% CI: 5.0 to 10 µM)
2. Beagle Dog: Cyanamide was included in the TPO activity assay at final concentrations of: 0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, and 100 µM. These concentrations were chosen for the preliminary dose range finding and were deemed appropriate for reporting. Using this range of concentrations, the IC50 value of cyanamide for dog TPO was calculated to be 6 µM (95% CI: 5 to 7 µM).

 

Table 1. Inhibition of TPO Activity by PTU and Cyanamide in Male Han Wistar Rat and Male Beagle Dog Thyroid Microsomes

Species	Substance	IC50 Value (µM)	95 % Confidence Interval (µM)
Rat	PTU	5.682	3.916 - 10.8
	Cyanamide	6.923	4.971 - 10.42
Dog	PTU	30.77	25 - 40.71
	Cyanamide	6.241	5.326 - 7.362

 

Conclusions:

Cyanamide is a potent inhibitor of canine and rat TPO according to the described in vitro test system and their underlying experimental conditions.

Executive summary:

The objective of this study was to assess a putative potential for the test item cyanamide to inhibit thyroperoxidase (TPO) activity in two separate test systems:
1. pooled thyroid microsomes prepared from male Han Wistar rat
2. pooled thyroid microsomes prepared from male Beagle dog.

The guaiacol assay of TPO activity was used for this study. The known TPO inhibitor 6-propyl-2-thiouracil (PTU) was used as positive control (reference) substance. The potency of the test/Reference items as inhibitors of TPO was quantified in terms of half maximal inhibitory concentrations (IC50) values.
The positive control substance PTU caused the expected inhibition of TPO in both test systems, thus confirming the sensitivity of the test systems. Against rat TPO, its estimated IC50 was 6 µM (95% confidence interval (CI): 4 - 11 µM). Against dog TPO, its estimated IC50 was 31 µM (95% CI: 25 - 41 µM).
Against rat TPO, the test item cyanamide displayed an estimated IC50 value of 7 µM (95% CI: 5 to 10 µM). Against dog TPO, cyanamide displayed an estimated IC50 6 µM (95% confidence interval (CI): 5 to 7 µM).
Based on the results obtained under the conditions of this study it is concluded that cyanamide potently inhibits both dog and rat TPO in vitro.

Reference 8

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
Thyroperoxidase (TPO) activity in Human thyroid microsomes was determined by the method of Chang HC and Doerge DR (2000), as described by Paul KB et al. (2014). In brief, this method exploits the ability of TPO to catalyse the oxidation of guaiacol to a coloured product. The rate of production of this coloured product can be determined spectrophotometrically (OD450), and TPO activity is then expressed in units of AOD450 /min/mg protein.

GLP compliance:

no

Type of method:

in vitro

Remarks:

/ex vivo

Endpoint addressed:

other: endocrine disruption

Species:

other: human

Sex:

male

Details on test animals or test system and environmental conditions:

Frozen thyroids harvested from non-juvenile, male humans (n=3 individuals, Capital Biosciences Inc., USA)

Details on exposure:

PRELIMINARY STUDIES
Concentration ranges for cyanamide and the positive control, 6-Propyl-2-thiouracil (PTU) were determined through preliminary range-finding experiments.

PREPARATION OF THYROID MICROSOMES
Individual thyroids were weighed and a 10% (w/v) homogenate prepared in ice-cold SET buffer containing 500 U/mL catalase. The homogenate was processed to a microsomal fraction by differential centrifugation. The microsomal fractions were resuspended in SET buffer lacking catalase, pooled, aliquoted, frozen, and stored at approximately -70 °C until required for protein content determination (Lowry) and TPO analysis.

TEST AND CONTROL ITEMS:
-DMSO (vehicle control)
-Cyanamide (test item)
-6-Propyl-2-thiouracil ((PTU), positive control)

THYROID MICROSOMAL PROTEIN DETERMINATION:
The protein content in the pooled microsomal preparation was determined using a modification of the method of Lowry et al. (Lowry et al., 1951) with bovine serum albumin standards.

INHIBITION OF TPO-CATALYSED GUAIACOL OXIDATION:
TPO-catalysed guaiacol oxidation, in the presence or absence of inhibitors, was determined by the method of Chang HC and Doerge DR (2000), as described by Paul KB et al. (2014).
1. A preliminary protein-dependency experiment was carried out to determine the optimal concentration of microsomes for use in the subsequent TPO inhibition assays with cyanamide or PTU.
2. Inhibition assay:
a. Human thyroid microsomes were pre-incubated with multiple concentrations of test or reference items at 37 °C in 100 mM Na/K phosphate buffer, pH 7.4, containing 35 mM guaiacol.
b. The enzyme reaction was initiated by addition of 300 µM H2O2.
c. TPO activities at 37 °C were determined by monitoring the rate of production of the coloured guaiacol oxidation product (OD450) over time.
d. Activities were expressed as AOD450 /min/mg protein.
e. IC50 values for cyanamide and PTU were calculated by fitting a four-parameter logistic model to the plot of TPO activity versus inhibitor concentration.

DATA MANIPULATION AND EVALUATION
A linear model was fitted to the TPO activity versus microsomal protein data set. Linear regression was performed using GraphPad Prism (Version 7.02, GraphPad Software Inc, San Diego, California, USA). TPO activity (expressed as a percentage of control activity) was determined as a function of test substance or positive control substance concentration, and IC50 parameters were estimated by fitting a four-parameter logistic model to the resulting data set. Model fitting was performed using GraphPad Prism (Version 7.02, GraphPad Software Inc, San Diego, California, USA).

Duration of treatment / exposure:

60 seconds

Dose / conc.:

0 other: µM

Dose / conc.:

0.01 other: µM

Dose / conc.:

0.1 other: µM

Dose / conc.:

1 other: µM

Dose / conc.:

2.5 other: µM

Dose / conc.:

5 other: µM

Dose / conc.:

10 other: µM

Dose / conc.:

25 other: µM

Dose / conc.:

50 other: µM

Dose / conc.:

100 other: µM

Dose / conc.:

200 other: µM

Examinations:

TPO activity was determined by means of the ability of TPO to catalyse the oxidation of guaiacol to a coloured product. The rate of production of this coloured product can be determined spectrophotometrically (OD450), and TPO activity is then expressed in units of AOD450 /min/mg protein.

Positive control:

6-Propyl-2-thiouracil (PTU) at following concentrations:
0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, 100 and 200 µM

Details on results:

Protein dependency:
TPO activity was linear with regard to protein concentration, up to 150 µg protein per reaction. A final protein content of 50 µg per reaction was used in subsequent TPO assays, as this amount allowed a constant rate of guaiacol oxidation for up to approximately 60 seconds.

Positive Control Item:
The positive control inhibitor, 6-propyl-2-thiouracil (PTU), was included in the TPO activity assay at the following final concentrations: 0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, 100 and 200 µM. These concentrations were chosen for the preliminary dose range finding and were deemed appropriate for reporting. PTU was a potent inhibitor of TPO activity exhibiting an IC50 value of 12 µM, 95% CI: 11 to 14 µM.

Test Item:
Cyanamide was included in the TPO activity assay at final concentrations of: 0, 0.01, 0.1, 1, 2.5, 5, 10, 25, 50, and 100 µM. These concentrations were chosen for the preliminary dose range finding and were deemed appropriate for reporting. Using this range of concentrations, the IC50 value of cyanamide for human TPO was calculated to be 2.2 µM (95% CI: 2.0 to 2.4 µM).

Tab. 1: Inhibition of TPO Activity by PTU and Cyanamide in Male Human
Thyroid Microsomes

Species	Substance	IC50 Value (µM)	95% Confidence Interval (µM)
Human	PTU	12.36	11.22-13.61
	Cyanamide	2.177	1.994-2.364

Conclusions:

In this test system, cyanamide is a potent inhibitor of human TPO activity.

Executive summary:

The objective of this study was to assess a putative potential for the test item cyanamide to inhibit thyroperoxidase (TPO) activity in the following test system:

 

1. pooled thyroid microsomes prepared from non-juvenile male human

 

The guaiacol assay of TPO activity was used for this study. The known TPO inhibitor 6-propyl-2-thiouracil (PTU) was used as positive control (reference) substance. The potency of the test/reference items as inhibitors of TPO was quantified in terms of half maximal inhibitory concentrations (IC50) values. The positive control substance PTU caused the expected inhibition of TPO in both test systems, thus confirming the sensitivity of the test systems. Against human TPO, its estimated IC50 was 12 µM (95% confidence interval (CI): 11 - 14 µM). Against human TPO, the test item cyanamide displayed an estimated IC50 value of 2.2 µM (95% CI: 2.0 to 2.4 µM). Based on the results obtained under the conditions of this study it is concluded that cyanamide inhibits human TPO in vitro.

Description of key information

Based on the currently available information including positive as well as negative evidence, it can be concluded, that given the complexity of the ED topic, a scientifically reliable decision as to the fulfilment of the ED criteria cannot be taken for cyanamide for humans and mammals at this stage. The newly performed in vitro TPO assays raise uncertainties regarding an endocrine mechanism based on TPO-inhibition. Further data should be generated to fill identified data gaps in order to come to a scientifically sound conclusion applying a WoE approach.

Additional information

Experimental study results of the investigations on the Thyroperoxidase (TPO), Sodium-Iodide-Symporter (NIS), liver cells and the Hypothalamo-Pituitary-Adrenal Axis are presented in the following.

 

The potential of cyanamide to inhibit the Thyroperoxidase (TPO) activity was assessed by means of the Guiacol Assay of TPO using canine, rodent and human thyroid microsomes as well as by means of the Luminol Assay of TPO using the human thyroid follicularcell line Nthy-ori 3-1. Four studies are reported for this cause.

 

Davidson, 1979

In this study, the TPO inhibition potential of cyanamide in rat thyroids was investigated by means of the Guiacol Assay. One set of animals was given 125-Iod 15 min before measurement of protein-bound iodine. The thyroids of a second set were assayed for TPO-catalyzed guaiacol oxidation. Triiodide formation was assayed by a modification of a previously described method. The 1.0-mL assay system contained 1 mM KI, 0.088 mM H2O2, 50 mM sodium citrate buffer (pH 5.6), and 6.67 µg TPO. Initial rates were measured at 353 nm (0-0.1 absorbance = full scale). Guaiacol oxidation was assayed using 33 mM guaiacol and a 3.0-mL system. When indicated, 3.3 mM guaiacol were used. Iodination of Glu-Tyr-Glu. Iodination of Glu-Tyr-Glu was measured spectrophotometrically at 290 nm, as an initial rate of formation of monoiodo Glu-Tyr-Glu. It was shown that the TPO retained from 65-75% of its activity as measured by guaiacol oxidation.

 

Haines, 2018a

In this study the putative potential of the test item cyanamide to inhibit TPO activity in two separate test systems was assessed: 1. pooled thyroid microsomes prepared from male Han Wistar rat 2. pooled thyroid microsomes prepared from male Beagle dog. The guaiacol assay of TPO activity was used for this study. The known TPO inhibitor 6-propyl-2-thiouracil (PTU) was used as positive control (reference) substance. The potency of the test/Reference items as inhibitors of TPO was quantified in terms of half maximal inhibitory concentrations (IC50) values. The positive control substance PTU caused the expected inhibition of TPO in both test systems, thus confirming the sensitivity of the test systems. Against rat TPO, its estimated IC50 was 6 µM (95% confidence interval (CI): 4 - 11 µM). Against dog TPO, its estimated IC50 was 31 µM (95% CI: 25 - 41 µM). Against rat TPO, the test item cyanamide displayed an estimated IC50 value of 7 µM (95% CI: 5 to 10 µM). Against dog TPO, cyanamide displayed an estimated IC50 6 µM (95% confidence interval (CI): 5 to 7 µM). Based on the results obtained under the conditions of this study it is concluded that cyanamide potently inhibits both dog and rat TPO in vitro.

 

Haines, 2018b

In this study the putative potential of the test item cyanamide to inhibit TPO activity in the following test system was assessed: pooled thyroid microsomes prepared from non-juvenile male human. The guaiacol assay of TPO activity was used for this study. The known TPO inhibitor 6-propyl-2-thiouracil (PTU) was used as positive control (reference) substance. The potency of the test/reference items as inhibitors of TPO was quantified in terms of half maximal inhibitory concentrations (IC50) values. The positive control substance PTU caused the expected inhibition of TPO in both test systems, thus confirming the sensitivity of the test systems. Against human TPO, its estimated IC50 was 12 µM (95% confidence interval (CI): 11 - 14 µM). Against human TPO, the test item cyanamide displayed an estimated IC50 value of 2.2 µM (95% CI: 2.0 to 2.4 µM). Based on the results obtained under the conditions of this study it is concluded that cyanamide inhibits human TPO in vitro.

 

Brinkmann, 2022a

In this study the ability of cyanamide (test item Cyanamid L500 P, a 50% aqueous cyanamide solution) to inhibit TPO activity was investigated using the lysate of the human thyroid follicular cell line Nthy-ori 3-1. Lysate with a protein concentration of 0.2 mg/mL was mixed with seven concentrations of the test item (Cyanamid L500 P) in a concentration range of 0.001 to 1000 µM and incubated for 30 min. Subsequently, Luminol was added to the mixture and the luminescent reaction was initiated by the addition of hydrogen peroxide immediately prior to measurement. The relative activity to SC was evaluated in two independent runs. To assess the possibility of false negative data, an interference test was conducted, where the same concentrations of the test item were mixed with Glycine-NaOH buffer instead of cell lysate. In both runs no IC50-value could be determined. Moreover, the interference test showed, the test item did not induce a luminescence signal on its own, or in conjunction with Luminol and H2O2. Therefore, the measured signal was only TPO-dependent luminescence, and the test item was determined to not inhibit TPO. The controls confirmed the validity of the test. In this study under the given conditions the active ingredient cyanamide was considered to not inhibit TPO activity, since no IC50 could be calculated.

 

 

Potential inhibitory effects on the NIS were investigated in FRTL-5 rat cells. A preliminary study was performed to assess cytotoxicity as the basis for the maximum concentration in the main test.

 

Brinkmann, 2022b

In this study, FRTL-5 cells were assessed on their sensitivity towards rising concentrations of the test item. The vitality of the cells or potential cytotoxic effects of the chemicals were registered via the mitochondrial dehydrogenase activity of the cell culture via the XTT assay and via the reaction of the cellular proteins with copper ions and BCA. Hereby, the test item was mixed with cell culture medium at rising concentrations and FRTL-5 cells were incubated for 24 ± 2h with this mixture. Prior to staining with XTT for mitochondrial dehydrogenase analysis, the cells were inspected microscopically for cytotoxic effects. The decrease in the activity of mitochondrial dehydrogenases of the individual cultures was then analysed as a measure for cytotoxicity and compared to those of the respective controls. Cytotoxic effects were detected microscopically at a concentration of 1000 µM and above. The XTT staining showed reduced viability at a concentration of 1000 µM and above. The BCA assay showed reduced protein content at the highest tested concentration of 10000 µM. In conclusion it can be stated, that the test item shows cytotoxic concentrations in FRTL-5 cells at a concentration of 1000 µM and above, as verified via XTT and BCA staining. Therefore, 100 µM will be chosen as maximum starting concentration for subsequent assays employing FRTL-5 cells.

 

Brinkmann, 2022c

In this study the ability of cyanamide (test item Cyanamid L500 P, a 50% aqueous cyanamide solution) to inhibit the activity of the Sodium/Iodide symporter (NIS) was investigated using the rat thyroid cell line FRTL-5. Employing the Sandell-Kolthoff-reaction, the amount of Iodide, taken up by the cells was determined. Iodide catalyses the reduction of the coloured Ce⁴⁺ to colourless Ce³⁺, the loss of absorbance in solution is therefore directly correlated to NIS activity. As maximum concentration of 1000 µM of the active ingredient cyanamide was found to be both soluble and compatible with the survival of the FRTL-5 cell line. In total, 6 concentrations, ranging from 1000 µM to 0.01 µM were tested. For the test item no IC₅₀ values could be calculated, since the inhibitory effect of the test item was less than 50% in the tested concentration range. The controls showed the validity of the assay. NaClO₄ was correctly classified as Inhibitor of the NIS, while Sodium-Acetate did not cause inhibition of NIS activity. In this study under the given conditions the active ingredient cyanamide was classified as negative in the NIS assay, since an IC₅₀ value could not be calculated in both runs.

 

Investigations on liver cells and Hypothalamo-Pituitary-Adrenal Axis are reported below.

Guillen, 1984

For the investigation of hepatocyte inclusion bodies in the rat similar to those described in alcoholics treated with cyanamide. For this cause male Wistar rats were put into six groups, four experimental and two control groups. Animals of groups I, II, and control taking alcohol were accustomed to oral ethanol intake, group I and II before the cyanamide treatment. The characteristic inclusions were developed in animals receiving 8 or 16 mg/kg of body weight, whether they were prepared previously with ethanol intake or not. Inclusion bodies consist of round, well-demarcated cytoplasmic areas, which contain a large amount of glycogen disposed in Beta-granules, lipid droplets, and secondary lysosomes. They appeared at the 13th week of cyanamide treatment onward. Initially, hepatocytes bearing inclusion bodies are located predominantly at the periportal areas, but the lesion later progresses toward the center of the lobule. Prior to the inclusion body development, cyanamide induces another morphologic change in liver cells, consisting of cytoplasmic homogeneous areas, made up of glycogen disposed in agranules and smooth endoplasmic reticulum tubules. This change is described for the first time in relation to this drug. Under the reported experimental conditions, the results indicate that cyanamide triggers the formation of inclusion bodies in liver cells of rats after exposure to cyanamide and ethanol in combination, similiar to the hepatocyte inclusion bodies described in alcoholics treated with cyanamide.

 

Kinoshita, 2000

For the investigation of the effects of cyanamide on hypothalamo-pituitary adrenal (HPA)-axis, male rats were treated intravenously with 5, 20 or 50 mg/kg bw/day of cyanamide. Blood samples were taken before treatment and 30 min, 1, 2 h after treatment, as well as trunk blood samples taken after termination of the exposoure and sacrifice of the animals after 4 h. Brains and pituitaries were removed and collected. In situ hybridization histochemistry (ISHH) and a radioimmunoassay was performed. Cyanamide administration resulted in a dose-dependent increase in plasma corticosterone concentrations, significant increases in not only corticotrophin releasing factor (CRF) mRNA, but also arginine vasopressin (AVP) mRNA in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary. Under the reported conditions, these results suggest that cyanamide is able to activate the HPA axis at all levels of the axis.

 

 

Conclusion

Based on the currently available information including positive as well as negative evidence, it was considered that in vitro assays not to be the first choice to further assess potential ED properties of cyanamide, due to major differences between in vitro and in vivo metabolism. Nevertheless, they are useful to further investigate the MoA. The new ED studies demonstrate no inhibitory potential of cyanamide regarding NIS and TPO. The available evidence is insufficient to propose a plausible link between EATS‑mediated adversity and endocrine activity and thus, no conclusion can be drawn if cyanamide meets the ED criteria.

Consequently, to avoid misidentification of cyanamide as an EDC for human health, additional studies are necessary to clearly identify primary endocrine effects and to distinguish them from those that are consequent to generalized stress responses or indirect toxicities.

Taken together, it can be concluded, that given the complexity of the ED topic, a scientifically reliable decision as to the fulfilment of the ED criteria cannot be taken for cyanamide for humans and mammals at this stage. The newly performed in vitro TPO assay raise uncertainties regarding an endocrine mechanism based on TPO-inhibition. Further data should be generated to fill identified data gaps in order to come to a scientifically sound conclusion applying a WoE approach.