[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One of the 2 in vitro tests shows a positive results.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon and tryptophan operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1.2, 3.6, 12, 36, 120 µg/plate.
An additional concentration of 60 µg/plate was added in the second independent experiment for TA 1535 Salmonella typhimurium strain

Vehicle / solvent:

- solvent used: ethanol (60%)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

2-Nitrofluorene / sodium azide / 9-Aminoacridine / cis-Platinum (II) diammine dichloride / 2-Anthramine / Dimethyl-benzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar by direct incorporation or with pre-incubation for the second assay, if the first one is negative.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium):number of revertant colonies per plate

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 1-5 x 10 E 9

DETERMINATION OF CYTOTOXICITY
- Method: bacteriostatic activity - 3 assay

OTHER:
-Number of plates per assay: 3

Evaluation criteria:

R = (Number of revertant colonies wiith the test substance) / (Number of revertant colonies in the absence of the test substance)

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity: Bacteriostatic activity of 74-76% was observed with 120 µg/plate of test material. This bacteriostatic activity = the tolerated threshold of 75%.
No cytotoxic effect observed for the other doses.

COMPARISON WITH HISTORICAL CONTROL DATA:
Rate of spontaneous revertants and positive controls (with and without S9 mix) fall within the range of observed historical values at the facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For some strains with or without metabolic activation, a significative decrease of the number of revertants colonies is observed for the dose of 120 µg/plate.This finding is in accordance with the bacteriostatic activity observed for this concentration

STRAIN	DOSE/PLATE (µg)	R
		Assay 1: - S9 mix 10%	Assay 1: + S9 mix 10%	Assay 2: - S9 mix 10%	Assay 2: + S9 mix 10% and pre-incubation
TA 1535	120	0.20	0.18	0.35	0.93
	60	/	/	6.73	1.14
	36	3.53	0.93	4.73	0.93
	12	1.67	0.89	1.35	0.62
	3.6	0.93	1.36	0.77	0.62
	1.2	0.83	1.07	2.89	0.82
	Positive Control	55.03	13.31	62.09	4.89
TA 1537	120	0.35	0.31	0.45	0.79
	36	0.88	1.54	0.75	0.79
	12	1.53	1.12	1.45	0.93
	3.6	1.41	1.23	1.20	0.93
	1.2	1.18	1.19	0.15	0.90
	Positive Control	70.89	11.11	127.94	6.52
TA 98	120	0.22	0.27	0.36	1.36
	36	0.48	1.06	0.68	1.01
	12	0.89	1.00	0.96	1.12
	3.6	1.13	1.09	0.93	0.99
	1.2	1.15	1.15	1.00	0.94
	Positive Control	28.06	31.28	40.77	16.19
TA 100	120	0.24	0.39	0.33	0.54
	36	0.36	0.55	0.49	0.75
	12	0.52	0.95	0.70	0.92
	3.6	0.98	0.94	0.97	0.98
	1.2	1.05	1.01	0.90	0.96
	Positive Control	10.86	8.97	14.30	5.70
E.Coli	120	0.19	0.28	0.34	0.34
	36	0.43	1.14	0.54	1.26
	12	1.06	1.15	1.42	1.12
	3.6	0.86	1.12	1.11	1.19
	1.2	0.90	1.09	1.02	1.11
	Positive Control	6.87	5.44	13.44	4.66

R = (Number of revertant colonies in the presence of the test substance) / (Number of revertant colonies in the absence of the test substance)

Conclusions:

positive without metabolic activation in TA 1535 Salmonella typhimurium

At 36 µg/plate for TA 1535 Salmonella typhimurium strain a significant increase in revertant number is observed without metabolic activation. Results were confirmed in a second independent experiment in which a higher dose of 60 µg/plate also induced a significant increase in revertant number.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item (120, 36, 12, 3.6 and 1.2 µg/plate) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101). At 120 µg/plate for some strains a signiticant decrease in revertant number is observed with or without metabolic activation which is in accordance with the bacteriostatic activity observed for this concentration.

Executive summary:

The assessment of the potential mutagenic activity of the Substance was performed according to the Ames test (Salmonella His- and E.coli Trp- /microsome system) in compliance with the OECD Guideline 471 using the maximum tolerated concentration (standing below the threshold of 75%) recommended by OECD Guideline, i.e. 120 μg/plate for this toxicity assay. Four lower dilutions were chosen according to a geometrical (half-log) ratio were also tested.

 

Preparation of test material solution

The test material is diluted in ethanol 60%

 

Preliminary assay: Cytotoxicity

Five concentrations were studied. 0.1 mL of the bacterial suspension from a culture containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of the different concentrations of the test substance were successively added to 2 mL of overlay agar at 45°C, containing 10% (v/v) of a solution of L-Histidine-D-Biotine (2.5mM). After homogeneization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37°c, and the number of colonie counted.

A negative control containing the solvent alone was run in parallel.

 

Mutagenicity test

- Without metabolic activation:

Salmonella strains : for every strain, 0.1 mL of the bacterial suspension containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of every test substance concentration were successively added to 2 mL of overaly agar maintained surfusion in 45°C containing 10 % (v/v) of a L-Histidine-D-Biotin solution (0.5 mM).

 

E.Coli strain: in a test tube, 0.1 mL of the bacterial suspension containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of every test substance concentration were successively added to 2 mL of overaly agar maintained surfusion in 45°C containing 10 % (v/v) of Nutrient broth n°2 to which are added 5 µL of a L-Tryptophan solution at 2 mg/mL.

 

After homogeneization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37°c, and the number of colonie counted.

 

- With metabolic activation :

Two techniques have been used:

- direct plate incorporation: Same technique to that described above, except that immediately before pouring the mixture onto the plates, 0.5 mL of S9-mix metabolic activation system is quickly mixed,

 

- by pre-incubation: The test substance is preincubated with the test strain, and 0.5 mL of S9 -mix metabolic activation system at least for 20 min at 37°C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.

 

If the first assay gives a positive response, the incorporation plate method has been performed for the second S9 mix assay.

If the first assay gives a negative response, the pre-incubation method has been performed for the the second S9 mix assay.

Solvent controls, positive controls were performed like in the mutagenicity assay without and with metabolic activation.

 

Bacterioastatic activity

Bacterioastatic activity: Cytotoxic rate of 76% and 75% was observed with 120 µg/plate of test material. This rate = the threshold of 75%. No cytotoxic effect observed for the other doses.

For all strains without metabolic activation and for TA 100- E.coli with metabolic activation, a significative decrease of the number of revertants colonies is observed for the dose of 120 µg/plate.This finding is in accordance with the bacteriostatic activity observed for this concentration.

 

Mutagenicity assays

The mutagenic activity of the test item was assessed by means of the Ames’s test in the four Salmonella typhimurium strains

TA1535, TA1537, TA98, TA100 and in the E. Coli WP2 (uvr A-) (pKM 101) tested either in presence or in absence of metabolic activation, in two independent assays. A mutagenic activity was found without metabolic activation in the TA 1535 Salmonella typhimurium strains at, in both experiments. Values fall within the range of historical values observed at the facility.

 

 

Conclusion

At 36 µg/plate for TA 1535 Salmonella typhimurium strain a significant increase in revertant number is observed without metabolic activation. Results were confirmed in a second independent experiment in which a higher dose of 60 µg/plate also induced a significant increase in revertant number.

There is no evidence of any increase in the number of revertant colonies in the presence of the test item (120, 36, 12, 3.6 and 1.2 µg/plate) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101). At 120 µg/plate for some strains a significant decrease in revertant number is observed with or without metabolic activation which is in accordance with the bacteriostatic activity observed for this concentration.

 

In the assay conditions, the two concentrations of the test item 60 and 36 µg/plate induce reverse mutation on TA 1535 Salmonella typhimurium strain, without metabolic activation, according the OECD guidelines N°471 and to the method B13/B14 of the directive 2000/32/CE.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Because of the positivity of the Ames test, an in vivo test was been performed.

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-09-03 to 2015-10-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

no control of concentration in dosing formulations

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France, Saint-Germains-sur-l'Arbresle FRANCE
- Age at study initiation: 8 weeks old
- Weight at study initiation: between 185 g and 200 g
- Assigned to test groups randomly: yes, randpm-distribution
- Fasting period before study: A04C-10 from SAFE (batch 14294)
- Housing: bedding of dust-free, irradiated softwood pellets
- Diet (e.g. ad libitum): not fasted at the treatment time
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days for the gentoxicity study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3 °C
- Humidity (%): 55 +/- 15°C with minor exceptions
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: non toxic vehicle which allows an homogenous solution/suspension.
- Amount of vehicle (if gavage or dermal):
- Lot/batch no. (if required): MKBP7039V for toxicity assays and MKB6944V for main assay

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preliminary and confirmatory toxicity assays, suspensions at a maximum concentration of 200 mg/mL were prepared and administered to the animals at the dose volume of 10 mL/kg, giving a final dose of 2000 mg/kg. In the main genotoxicity assay, three suspensions of the test item at the concentrations of 200 - 100 and 50 mg/mL were prepared, giving final doses of 2000 - 1000 and 500 mg/kg, respectively when administered at 10 mL/kg.

Duration of treatment / exposure:

2 days

Frequency of treatment:

once per day

Post exposure period:

not applicable

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: methylmethanesulfonate
- Justification for choice of positive control(s): positive control substance listed within the OECD 489. this positive control targets the 3 tissues evaluated within this study (liver, stomach, testis).
- Route of administration: oral (gavage)
- Doses / concentrations: 100 mg/kg bw/day

Tissues and cell types examined:

Cells from liver, stomach and testis:
number of cells observed per animal: 150
number of cells observed per dose: 750.
Exminations performed on all aminals studied.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: One preliminary assay and one confirmatory assy were performed. The highest dose of 2000 mg/kg/day was the maximum tolerated dose, and in line with OECD 489 (2014).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
2 treatments at 24-hrs intervals.
Sampling 2 to 6 hr after the last administration

DETAILS OF SLIDE PREPARATION:
Befor use, a volume of 85 µL of 0.8% of Normal agarose (NA) was added on the microscope slide prelayered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardened. The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 μL/slide) kept at ca. 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Four slides per animal were prepared for the Comet assay.

METHOD OF ANALYSIS: Quantification of DNA damage

Evaluation criteria:

For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent negative control,
- This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.

When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage
in the tissues studied in this test system.


A test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.

The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskal-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control.

This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no marked clinical signs. Noticed that the feaces were dark blue. No mortality occured.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Preliminary study
- Dose range: 2000 mg/kg bw/day (x2) per os, on 2 animals.
- Clinical signs of toxicity in test animals: slight sniffing after 15 min of both treatment and dark blue faeces 24 hour after the first treatment.
No mortality

Confirmatory study:
- Dose range: 2000 mg/kg bw/day -x2) per os, on 5 animals.
- Clinical signs of toxicity in test animals: no marked clinical signs and dark blue faeces 6 and 24 hours after the first treatment.
No mortality

Results in Liver Cells
GROUP	TEST ITEM	DOSES in mg/kg/day (2x)	% of DNA in tail. Mean of medians per animal (/5 animals*)	NON PARAMETRIC statistical assessment		Hedgehogs
				p-Kruskall-Wallis	P Mann-Whitney	Relative ratio of hedgehogs	p
Negative control	Corn oil	0	0.36	N.S.	-	-	-
TREATED	Test item	2 000	0.27		N.S	1.15	N.S.
		1 000	1.69		N.S.	1.24	N.S.
		500	0.62		N.S.	1.35	N.S.
Positive control	Methylmethan Sulfonate	100	46.13		< 0.001	2.22	<0.05
Results in Stomach Cells
GROUP	TEST ITEM	DOSES in mg/kg/day (2x)	% of DNA in tail. Mean of medians per animal (/5 animals)	NON PARAMETRIC statistical assessment		Hedgehogs
				p-Kruskall-Wallis	P Mann-Whitney	Relative ratio of hedgehogs	p
Negative control	Corn oil	0	16.54	N.S.	-	-	-
TREATED	Test item	2 000	7.91		< 0.05	1.06	N.S.
		1 000	7.57		< 0.05	1.35	N.S.
		500	7.36		< 0.05	1.00	N.S.
Positive control	Methylmethan Sulfonate	100	70.45		< 0.01	0.66	< 0.05
Results in Testis Cells
GROUP	TEST ITEM	DOSES in mg/kg/day (2x)	% of DNA in tail. Mean of medians per animal (/5 animals)	NON PARAMETRIC statistical assessment		Hedgehogs
				p-Kruskall-Wallis	P Mann-Whitney	Relative ratio of hedgehogs	p
Negative control	Corn oil	0	2.58	N.S.	-	-	-
TREATED	Test item	2 000	1.05		< 0.05	0.94	N.S.
		1 000	1.03		< 0.05	0.96	N.S.
		500	1.27		N.S.	1.07	N.S.
Positive control	Methylmethan Sulfonate	100	38.26		< 0.001	0.59	<0.001

* for the high dose group in the liver analysis, only 4 animals were analysed

Conclusions:

The test item induced no statistically significant increases in DNA strand breaks whatever the doses in male rat isolated Liver. Stomach and Testis cells after oral administration. Therefore, the test item is considered having no genotoxic activity in these organs.

Executive summary:

The genotoxic potential of the test item was investigated in the in vivo comet assay performed under alkaline conditions,i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in the male rat, in isolated Liver, Stomach and Testis cells, after two treatments at 24-hour interval by oral route (gavage) at 3 dose levels (2000, 1000 and 500 mg/kg), followed by one expression time of 2 to 6 hours after the last treatment.

A preliminary assay was performed on 2 male rats at the highest dose of 2000 mg/ kg/ day (x2) per os. It induced slight sniffing 15 minutes after both treatments.

It was noticed that, 24 hours after the first treatment, the feces were dark blue. No mortality was observed. These clinical signs were considered as acceptable and the dose of 2000 mg/ kg/ day (x2) per os was thus assessed for the induction of clinical signs during the confirmatory toxicity assay.

The confirmatory toxicity assay was performed on 5 male rats at the highest dose of 2000 mg/ kg/ day (x2) per os. It induced no marked climical signs. It was noticed that, 6 and 24 hrs after the first treatment, the feces were dark blue. No mortality was observed.

These clinical signs were considered as acceptable and the dose of 2000 mg/ kg/ day (x2) per os was thus retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2) per os were also tested.

The comet assay was performed on 5 males/group (4 analysed for the high dose group for liver), at 0 mg/kg b.w./day (vehicle alone - negative control), 500 mg/kg b.w./day, 1000 mg/kg b.w./day and 2000 mg/kg b.w./day.

5 additional aminals were treated orally twice with a positive control, the Methylmethane sulfonate, at a dose of 100 mg/kg

The rats were treated 2 times at 24 hours-intervals with the test item at the different doses. Two to six hours after the second treatment, the rats are euthanized and cells from the selected target organs (i.e. liver, stomach and testis) are isolated.

After isolation, single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unvinding and electrophoresis in alkaline conditions and under dimmedlight to prevent any additional DNa dmage. After neutralization, slides are dried and could therefore be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows indeed the calculation of metrics for DNA migration.

Results in liver

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 or 500 mg/kg/day (x2).

At the intermediary dose of 1000 mg/kg/day (x2), the mean of medians of percentage of DNA in tail was over the upper bound of historical data,i.e.1.69 vs.1.01. Nevertheless, this effect was due to heterogeneity between the 5 animals of this group. Indeed, one of the animals presented a median of percentage of DNA in tail higher than the other,i.e. 5.23vs. 0.19-1.58 while it was noticed that its liver presented an irregular appearance.

The test item was thus considered as not genotoxic toward the liver.

Results in stomach

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 or 500 mg/kg/day (x2).

Statistically significant decreases in the percentage of DNA in tail were noted at all the tested doses without any dose-effect relationship. However, it has no meaning in terms of genotoxic hazard.

 

The test item was thus considered asnot genotoxic toward the stomach.

Results in testis

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 or 500 mg/kg/day (x2).

Statistically significant decreases in the percentage of DNA in tail were noted at the two highest tested doses of 2000 and 1000 mg/kg/day (x2). However, it has no meaning in terms of genotoxic hazard.

 

The test item was thus considered as not genotoxic toward the testis.

CONCLUSION

The test item was investigated by the means of the in vivo comet assay under alkaline conditions (SCGE) in the Liver, Stomach and Testis of male rat treated orally twice with 2000, 1000 and 500 mg/kg/day, with one sampling time 2 to 6 hours after the last treatment according to OECD guideline (OECD 489, 2014).

 

The validity criteria for the study were met. The current study is thus considered as valid.

Under these experimental conditions, the test item induced no statistically significant increases in DNA strand breaks whatever the doses in male rat isolated Liver, Stomach and Testis cells after oral administration. Therefore, the test item is considered having no genotoxic activity in these organs.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The Ames test performed on the Substance shows a positive result without metabolic activation on one strain (TA 1535) whereas the in vitro micronucleus test gives negative results.

 

Therefore, an in vivo test, a comet assay, has been performed in the liver, stomach and testis of male rats treated orally. Under the experimental conditions, the Substance did not induced statistically significant increases in DNA strand breaks whatever the doses in male rats isolated liver, stomach and testis cells.

 

So, the Substance is considered having no genotoxic activity in these organs.