Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

EC Number:

241-922-5

EC Name:

Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Cas Number:

18015-76-4

Molecular formula:

C23H25N2 x C2HO4

IUPAC Name:

4-{[4-(dimethylamino)phenyl](phenyl)methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium hydrogen oxalate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In house bred animals
- Age at study initiation: 7 weeks for pre-study and the main studies
- Weight at study initiation:
Male – 186.24 to 225.53 g, Female – 186.04 to 220.75g (Pre study)
Male – 203.30 to 233.98 g, Female – 189.23 to 227.18g (Main Study)
Male – 181.23 to 187.51 g, Female – 181.09 to 186.97g (Follow up Main Study)

- Assigned to test groups randomly:
The animals were weighed and arranged in ascending order of their body weights. These stratified body weights were distributed to all the experimental groups using Microsoft Excel Spread sheet. In pre study, the body weight variations of animals selected for the experiment were -8.44 to +10.40 % and -6.70 to +8.31%. In main study, the body weight variations of animals selected for the experiment were -5.40 to +8.12 % and -8.73 to +8.90 % of the mean body weight in males and females, respectively. In follow up main study, the body weight variations of animals selected for the experiment were -1.76 to +2.00 % and -1.20 to +2.14 % of the mean body weight in males and females, respectively. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out the statistically significant differences between groups within each sex.

- Fasting period before study: none
- Housing: Maximum of three animals of same sex and group were housed together in a standard polypropylene cage (L 430 × B 285 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized corn cob was used as a bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) will be provided ad libitum to the animals throughout the experimental period. The contaminant analysis test reports of feed will be included in the study report.

- Water (e.g. ad libitum):Water will be provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit will be provided in plastic water bottles with stainless steel sipper tubes.

- Acclimation period: Healthy adult animals were acclimatized for five days to laboratory conditions and were observed for clinical signs daily. Veterinary examination of all the animals was performed on the day of receipt and on the day of randomization.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 22.9°C
- Humidity (%): relative humidity 48 to 67%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 28 July 2022 To: 14 November 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Solubility/miscibility test was performed using distilled water, 0.5% carboxymethyl cellulose and corn oil. Test item formed suspension in distilled water, uniform suspension in 0.5% carboxymethyl cellulose and suspension in corn oil. Corn oil was selected as vehicle and the same was used for the formulation preparation. The substance in fact can form the carbinol form in water that increases its concentration with increasing pH. Therefore corn oil was selected as the most appropriate vehicle.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Required quantity of test item was weighed as per the dose. The weighed test item was transferred to a clean mortar and ground using pestle by adding a small quantity of vehicle to get a uniform suspension. The content was transferred to a measuring cylinder. Again, a small quantity of vehicle was added to the mortar rinsed and transferred to the measuring cylinder. The rinsing procedure was repeated until the test item was transferred completely into the measuring cylinder. The final volume was made up with vehicle to get the desired concentration as per the dose requirement. Formulation of the test item was prepared freshly every day before dosing.

Doses selected for the dose range finding study: 0, 20, 60 and 180 mg/kg bw
Doses selected for the main study: 0, 20, 50 and 125 mg/kg bw
Doses selected for the main study follow up: 0, 10, 50 and 75 mg/kg bw

Concentration of test item in corn oil formulation: 0, 5, 15 and 45 mg/ml (for a dose volume of 4 ml/kg). For the stability and homogeneity and for method validation the concentrations at 0.5 mg/ml and 45 mg/ml were selected.

Stability of Dose Formulation
The stability of the test item in dose formulations was established under Bioneeds Study No.: BIO-ANM 1964.

Homogeneity and Dose Concentration Analysis
Homogeneity and Dose concentration analysis for concentration verification for study was done by Analytical Department of Bioneeds India Private Limited.
The analysis was done as per validated analytical methods detailed in the study plan of Study No.: BIO-ANM 1964. Sampling and analysis was performed for different dilutions/concentrations prepared for testing in study and the details was reported in the study report.
The samples prepared (top, mid and bottom layer) were transferred to Analytical Department of Bioneeds India Private Limited for dose concentration analysis. Only one set of aliquots of each concentration was analysed for concentration verification. The second aliquot was stored at suitable temperature and used for re-analysis (if required) otherwise was discarded after the confirmation of the results.

The validation of the analytical method was performed for the determination of content of Basic Green 4 Oxalate in Corn Oil using HPLC method. The validation was performed in terms of matrix effect, specificity, linearity, accuracy, precision, homogeneity and stability. The content of Basic Green 4 Oxalate was determined by using the validated method.
The method validation parameters evaluated for Basic Green 4 Oxalate meet the acceptance criteria. The results obtained are within the specified limits. See section 8 for further details. Thus, this method is suitable for the analysis of the Basic Green 4 Oxalate content in Corn Oil for dose formulation in the Genotoxicity studies

Duration of treatment / exposure:

2 days

Frequency of treatment:

once a day for two consecutive days for dose range finding study, main study and follow up study

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

No. of animals per sex per dose:

dose range finding: 3 animals/sex/dose
main study and follow up study: 5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

positive control: Ethyl methanesulfonate
Ethyl methanesulfonate will be dissolved in distilled water and will be administered at a dose of 250 mg/kg bw/day.

Examinations

Tissues and cell types examined:

glandular stomach
duodenum
liver

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on dose range finding and first main study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were euthanized by cervical dislocation post 2 to 6 hours after the second treatment. Selected tissues (liver, glandular stomach and duodenum) were removed, dissected and a portion was collected for the comet assay. The tissues for the comet assay were placed into ice-cold mincing and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and were stored in ice-cold mincing buffer until processed.


-PREPARATION OF CELL NUCLEI OR SINGLE CELL
Single cell suspensions were prepared by chopping the tissue with scalpels in a small amount of ice-cold mincing solution. The scraped tissue solution was transferred into a homogenization tube and then gently homogenized using a homogenizer. Homogenate was filtered through a 70 micron pore nylon mesh and was then centrifuged at 800xg for 10 minutes. Single cells were suspended in DPBS. Cell suspensions were maintained at 2 to 8°C.

-PREPARATION OF SLIDES
Three slides were prepared for each tissue from each animal and labelled with the study number, animal no., tissue, slide number (e.g. 1/3 to 3/3) and sex of each animal using slide marker. To reduce the possibility of detachment of the agarose during the procedure, slides were pre coated with 100 µL of liquid agarose and the agarose was allowed to dry to a thin film. Approximately 75 µL of cell suspension with 75 µL of 1.0% low-melting agarose gel was mixed and rapidly pipette onto the surface of the pre coated slides and a coverslip was placed on it. Slides were placed on ice packs until the Agarose layer hardened (5 minutes). Slides were immersed in chilled lysing solution in the dark for overnight. After completion of lysing, the slides were rinsed in distilled water to remove residual detergent and salts prior to alkali unwinding step.


METHOD OF ANALYSIS:
The slides were placed onto a platform of submarine-type electrophoresis unit containing a chilled electrophoresis solution. Buffer reservoirs were filled with freshly made pH>13 Electrophoresis Buffer until the liquid level completely covers the slides.
Slides were allowed to sit in the alkaline buffer for 20 minutes to allow for unwinding of the DNA and the expression of alkali-labile damage. Power supply ~0.8 V/cm was turned on and current was adjusted to 300 milliamperes by raising or lowering the buffer level.
Electrophoresis solution was maintained a constant temperature usually between 2 to 10°C during electrophoresis under dimmed light. After completion of electrophoresis, power was turned off. Slides were removed from the buffer and place on a drain tray.
The slides were immersed in the neutralization buffer for 10 minutes. All slides were dehydrated by immersing the slides into absolute ethanol up to 10 minutes. Slides were stained with 80 μL 1X Ethidium. bromide for 5 minutes and then dipped in chilled distilled water to remove excess stain. Drained slides were kept in cold 100% methanol for 20 minutes for dehydration. Slides were air dried and then placed in an oven at 50±1°C for 30 minutes.

All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with Ethidium bromide, covered with a fresh coverslip and cells were scored under 400 X magnification.
At least 150 cells were analyzed per sample. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs were determined of at least 150 cells per sample.
After slide observation finished, slides were drained by keeping in cold 100% methanol for 20 minutes for dehydration. Slides were air dried and then placed in an oven at 50±1°C for 30 minutes

Evaluation criteria:

The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs were determined of at least 150 cells per sample.

Statistics:

Body weight were analyzed by SPSS at a 95% level of confidence (p<0.05) of significance. Inter group comparison of Body weight (day 1 and day 2) and percent tail DNA. The dose correlation was done using ‘t’ test. The statistical significances are designated by the superscripts as given below throughout the tables: *: Statistically significant (p<0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Rationale for exposure: the substance was tested for dose range finding and consequently tested. High toxicity was observed for both studies up to 120 mg/kg bw. Bioavailability of the substance at the selected organs is therefore demonstrated, due to the toxic effects recorded (stomach ballooning and mortality). The highest dose of the follow up main study was selected to be in the range 60 to 125 mg/kg bw, as being the maximum tolerated dose, i.e. 75 mg/kg bw, which is in very good agreement with the highest dose selected also for the in vivo studies reported in the pubblication "Genotoxicity of Malachite Green and Leucomalachite Green in female big blue B6C3F mice" (450 ppm in diet = 67.5 mg/kg for mouse).

Dose Range Finding Study (Pre study)
-Clinical Signs of Toxicity and Mortality
No clinical signs or mortality were observed in animals treated with test item at 20 and 60 mg/kg and vehicle control in either sex.
At 180 mg/kg, on day 2 lethargy was observed in all animals. After dosing on day 2 lethargy was observed in all animals and nasal discharge was observed in one male and two females . Mortality was recorded for one female at 180 mg/kg.

-Body Weight
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

-Gross Pathology
No gross pathological findings were observed in any of the animals dosed at 20 and 60 mg/kg and vehicle control. Stomach ballooning was observed in all animals dosed at 180 mg/kg.


Main Study
Clinical Signs of Toxicity and Mortality
In main study, no clinical sign was observed in any of the animals dosed 20, 50 mg/kg and no mortality was observed in any of the animals in both sexes.
On day 2 lethargy and nasal discharge was observed in all animals dosed at 125 mg/kg. One female animal found dead and one female animal found in the moribund status; and were sacrificed humanely.

-Body Weight
There was no statistically significant change in mean body weight among the positive control dosed animals and Basic Green 4 oxalate dosed animals in comparison with vehicle dosed animals in both the sexes.

-Gross Pathology
No gross pathological findings were observed in any of the animals dosed at 20, 50 and 125 mg/kg, vehicle control and positive control.


Follow up Main Study
-Clinical Signs of Toxicity and Mortality
In main study, no clinical sign was observed in any of the animals dosed 10, 50 and 75 mg/kg and no mortality was observed in any of the animals in both sexes.

-Body Weight
There was no statistically significant change in mean body weight among the positive control dosed animals and Basic Green 4 oxalate dosed animals in comparison with vehicle dosed animals in both the sexes.

-Gross Pathology
No gross pathological findings were observed in any of the animals dosed at 10, 50 and 75 mg/kg, vehicle control and positive control.

DOSE CONCENTRATION ANALYSIS
In the comet assay, the percent recovery of Basic Green 4 oxalate at the tested concentrations on Day 1 (24/08/2022) ranged from 94.30 to 98.58 % and on Day 2 (25/08/2022) ranged from 91.10 to 99.12 % of the nominal concentrations. The results obtained indicate recovery was within the acceptance criteria of ±15% of the nominal concentrations .

Any other information on results incl. tables

Sex	Group & Dose (mg/kg)	Animal No.		Organ
			Liver	Glandular Stomach	Duodenum
Male	G1 & 0	Rh3765	2.085	1.837	2.047
		Rh3766	3.520	5.594	4.254
		Rh3767	4.740	5.464	4.052
		Rh3768	3.807	4.945	4.467
		Rh3769	4.461	5.43	5.233
		Mean	3.72	4.65	4.01
		±SD	1.04	1.59	1.19
	G2 & 10	Rh3770	2.527	3.033	1.971
		Rh3771	3.208	6.819	4.000
		Rh3772	4.774	4.113	4.174
		Rh3773	4.283	5.175	4.412
		Rh3774	4.477	5.94	5.344
		Mean	3.85	5.02	3.98
		±SD	0.95	1.49	1.24
	G3 &50	Rh3775	3.164	2.875	2.232
		Rh3776	3.262	6.577	3.934
		Rh3777	4.820	4.029	4.43
		Rh3778	4.283	4.965	4.135
		Rh3779	4.514	6.129	5.327
		Mean	4.01	4.92	4.01
		±SD	0.75	1.52	1.13
	G4 & 75	Rh3780	3.176	3.084	2.243
		Rh3781	3.312	6.861	3.876
		Rh3782	4.935	4.126	4.354
		Rh3783	4.382	5.275	4.518
		Rh3784	4.517	5.948	5.591
		Mean	4.06	5.06	4.12
		±SD	0.78	1.49	1.22
	G5 & 250  (EMS)	Rh3785	5.837	8.199	7.064
		Rh3786	5.336	8.281	6.334
		Rh3787	5.391	7.619	6.388
		Rh3788	5.567	8.216	6.488
		Rh3789	5.193	8.222	6.536
		Mean	5.46*	8.11*	6.56*
		±SD	0.25	0.27	0.29

 

Sex	Group & Dose (mg/kg)	Animal No.		Organ
			Liver	Glandular Stomach	Duodenum
Female	G1 & 0	Rh3790	3.111	4.016	2.635
		Rh3791	4.602	6.016	3.768
		Rh3792	4.421	4.325	5.511
		Rh3793	4.428	5.008	4.936
		Rh3794	4.063	4.921	5.372
		Mean	4.13	4.86	4.44
		±SD	0.60	0.77	1.22
	G2 & 10	Rh3795	3.144	2.881	3.007
		Rh3796	3.701	7.964	5.322
		Rh3797	5.046	4.216	5.766
		Rh3798	4.647	3.774	3.382
		Rh3799	4.608	7.707	5.035
		Mean	4.23	5.31	4.50
		±SD	0.78	2.36	1.23
	G3 & 50	Rh3800	2.752	3.960	3.256
		Rh3801	3.884	7.791	3.417
		Rh3802	5.102	4.331	5.808
		Rh3803	5.023	5.523	5.138
		Rh3804	4.620	7.596	5.483
		Mean	4.28	5.84	4.62
		±SD	0.98	1.79	1.20
	G4 & 75	Rh3805	2.926	3.861	3.175
		Rh3806	3.931	7.070	3.764
		Rh3807	5.154	4.468	5.954
		Rh3808	4.709	6.986	5.242
		Rh3809	4.633	7.476	5.623
		Mean	4.27	5.97	4.75
		±SD	0.87	1.67	1.22
	G5 & 250  (EMS)	Rh3810	6.075	8.929	6.550
		Rh3811	6.117	8.220	8.328
		Rh3812	6.826	8.613	6.129
		Rh3813	6.172	9.968	10.487
		Rh3814	6.139	10.434	6.309
		Mean	6.27*	9.23*	7.56*
		±SD	0.32	0.93	1.86

Applicant's summary and conclusion

Conclusions:

The substance was tested for in vivo gene mutation following OECD489. Under the experimental conditions the substance did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 75 mg/kg.

Executive summary:

The test item was evaluated in the Comet Assay in Wistar Rats as per OECD Guideline No. 489, “In Vivo Mammalian Alkaline Comet Assay”, adopted on 29 July 2016.

This study was conducted to determine if the test item caused an increase in DNA damage in cells from specific organs. The COMET assay detects single and double stranded breaks when DNA is analyzed under alkaline conditions (>pH 13). These strand breaks, when they occur in vivo, may be repaired, resulting in no persistent effect, may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. The pre study consisted of four groups, vehicle control, 20, 60 and 180 mg/kg; the test item was administered at a dose volume of 4 mL/kg in corn oil. In the pre study, each group of rat consisted of 3 males and 3 females. Clinical signs like lethargy, nasal discharge was observed at 180 mg/kg, mortality was observed in the animals dosed at 180 mg/kg, gross pathological findings like stomach ballooning  was observed at 180 mg/kg. For animals dosed at 20 and 60 mg/kg found normal, no body weight variations and no gross pathological findings were observed in any of the animals.

The main study consisted of 5 groups of rats and each group consisted of 5 males and 5 females. G1 group was administered with vehicle, G2 group was administered with 20 mg/kg, G3 group was administered with 50 mg/kg and G4 group was administered with 125 mg/kg of test item and G5 group was administered with positive control Ethyl methane sulfonate [EMS] at 250 mg/kg for two consecutive days by the oral route using a gavage cannula. Clinical signs like lethargy and nasal discharge at 125 mg/kg was observed for all animals; one female animal was found dead and one female animal was in the moribund status and sacrificed humanely; hence follow up main study was conducted with the dose levels of 10, 50 and 75 mg/kg.

The follow up main study consisted of 5 groups of rats and each group consisted of 5 males and 5 females. G1 group was administered with vehicle, G2 group was administered with 10 mg/kg, G3 group was  administered with 50 mg/kg, G4 group was administered with 75 mg/kg of test item and G5 group animals was administered withpositive control Ethyl methane sulfonate [EMS] at 250 mg/kg for two consecutive days by the oral route using a gavage cannula. Approximately 2 to 6 hours after the last dosing, all rats were euthanized by cervical dislocation and the designated organs (liver, glandular stomach and duodenum) were collected. Tissues were processed, single cells were isolated and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with Ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode.

The results for the assessment of the test item, to cause DNA strand breaks are provided for the doses of 10 [G2], 50 [G3] and 75 [G4] mg/kg, respectively, in male and female Wistar rats. The average % tailing for DNA from male liver cells was 3.72, 3.85, 4.01 and 4.06 and % tailing for DNA from female liver cells was 4.13, 4.23, 4.28 and 4.27. In cells from the glandular stomach, the observed average % tailing for DNA was 4.65, 5.02, 4.92 and 5.06 for males and for 4.86, 5.31, 5.84 and 5.97 females. The average % tailing of DNA observed in cells from the duodenum was 4.01, 3.98, 4.01 and 4.12 for males, and 4.44, 4.50, 4.62 and 4.75 for females. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any organ for any of the Basic Green 4 oxalate  groups when compared to the vehicle control group.

The positive control [G5], Ethyl methane sulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the COMET assay for this test of Basic green 4 oxalate were fully adequate.

Dose concentration analyses conducted for dose verification were found to be within the acceptable range of ± 15% of the nominal concentration. These data support the conclusion that the nominal doses of the test item accurately represent the conditions for this COMET assay.

The data obtained under the conditions employed during this experiment support the conclusion that the test item did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 75 mg/kg.