Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to OECD 471 test method, Eversorb EP4 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

According to OECD 473 test method, Eversorb EP4 was negative effect under the condition of in vitro mammalian chromosome aberration test.

According to OECD 490 test method, Eversorb EP4 is not mutagenic in the mouse lymphoma L5178Y test system under the experimentalc ondition of In Vitro Mammalian Cell Gene Mutation Tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 08, 2016 to December 28, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Characteristics of five Salmonella typhimurium strains

Test strain	Histidine requirement	uvrB mutation	rfa mutation	Ampicillin resistance
TA98	+	+	+	+
TA100	+	+	+	+
TA102	+	ϴ	+	+
TA1535	+	+	+	ϴ
TA1537	+	+	+	ϴ

+ means had the characteristic; ϴ means did not have the characteristic

Table 2. Toxicity of the test article of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)
With S9 Mix	Negative control group	236
	Positive control group^a	1532
	Test group
	5	264
	2.5	327
	1.25	283
	0.625	369
	0.3125	326
Without S9 Mix	Negative control group	182
	Positive control group	725
	Test group
	5	151
	2.5	185
	1.25	150
	0.625	154
	0.3125	164

^a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

 

Table 3. Reverse mutation test of Salmonella typhimurium TA98

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	37	36	58	44 ± 12
	Positive control group^b	376	405	355	379 ± 25*
	Test group
	5	98	117	85	100 ± 16*
	2.5	71	87	81	80 ± 8
	1.25	59	42	37	46 ± 12
	0.625	44	44	72	53 ± 16
	0.3125	24	40	48	37 ± 12
Without S9 Mix	Negative control group	33	41	36	37 ± 4
	Positive control group	381	345	341	356 ± 22*
	Test group
	5	37	39	39	38 ± 1
	2.5	37	53	44	45 ± 8
	1.25	28	27	41	32 ± 8
	0.625	41	40	49	43 ± 5
	0.3125	30	48	36	38 ± 9

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.             

^b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).

Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 4. Reverse mutation test of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	216	221	243	277 ± 14
	Positive control group^b	1386	1535	1485	1469 ± 76*
	Test group
	5	232	322	363	306 ± 67
	2.5	249	253	249	250 ± 2
	1.25	214	198	269	227 ± 37
	0.625	240	271	259	257 ± 16
	0.3125	282	269	253	268 ± 15
Without S9 Mix	Negative control group	215	190	175	193 ± 20
	Positive control group	638	629	677	648 ± 26*
	Test group
	5	132	152	152	145 ± 12
	2.5	153	192	194	180 ± 23
	1.25	158	186	178	174 ± 14
	0.625	153	144	154	150 ± 6
	0.3125	166	250	248	221 ± 48

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 5. Reverse mutation test of Salmonella typhimurium TA102

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	482	416	464	454 ± 34
	Positive control group^b	945	968	984	966 ± 20*
	Test group
	5	437	446	472	452 ± 18
	2.5	470	426	453	450 ± 22
	1.25	512	489	478	493 ± 17
	0.625	498	473	515	495 ± 21
	0.3125	451	517	521	496 ± 39
Without S9 Mix	Negative control group	407	416	382	402 ± 18
	Positive control group	838	861	802	834 ± 30*
	Test group
	5	347	334	388	356 ± 28
	2.5	381	430	420	410 ± 26
	1.25	344	353	376	358 ± 17
	0.625	393	378	346	372 ± 24
	0.3125	443	391	371	402 ± 37

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).

Without S9 Mix: Mitomycin C (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 6. Reverse mutation test of Salmonella typhimurium TA1535

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	23	33	24	27 ± 6
	Positive control group^b	363	348	325	345 ± 19*
	Test group
	5	32	25	45	34 ± 10
	2.5	15	23	20	19 ± 4
	1.25	12	18	16	15 ± 3
	0.625	20	17	15	17 ± 3
	0.3125	14	17	24	18 ± 5
Without S9 Mix	Negative control group	30	26	24	27 ± 3
	Positive control group	224	230	154	236 ± 16*
	Test group
	5	19	26	21	22 ± 4
	2.5	24	22	18	21 ± 3
	1.25	17	17	26	20 ± 5
	0.625	18	18	16	17 ± 1
	0.3125	17	28	35	27 ± 9

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (0.4μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 7. Reverse mutation test of Salmonella typhimurium TA1537

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	26	12	13	17 ± 8
	Positive control group^b	486	438	422	449 ± 33*
	Test group
	5	17	25	18	20 ± 4
	2.5	22	14	25	20 ± 6
	1.25	13	15	12	13 ± 2
	0.625	18	14	12	15 ± 3
	0.3125	11	12	13	12 ± 1
Without S9 Mix	Negative control group	17	15	19	17 ± 2
	Positive control group	1013	1036	874	974 ± 88*
	Test group
	5	6	8	6	7 ± 1
	2.5	5	11	7	8 ± 3
	1.25	8	2	6	5 ± 3
	0.625	2	5	7	5 ± 3
	0.3125	4	9	15	9 ± 6

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: 9-Aminoacridine (50.0μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Conclusions:

According to OECD 471 test method, Eversorb EP4 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100161001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997). The results of this OECD 471 test for Eversorb EP4 show that test validity criteria was met.

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of Eversorb EP4 at 0.3125, 0.625, 1.25, 2.5 and 5mg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. At the highest dose with S9 Mix in tester strains of Salmonella typhimurium TA98 of test groups, the results showed that the number of revertants increase twice more than negative controls group. Based on the data obtained from this study, it was concluded that under the test condition, Eversorb EP4 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 11, 2016 to March 1, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague-Dawley rats

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Karyology Analysis of Chinese Hamster Ovary Cells

No. of chromosome	<18	18	19	20	21	22	>22
No. of cells	0	0	14	31	5	0	0

Table 2. Concurrent Cytotoxicity Analysis of Eversorb EP4 in Chinese Hamster Ovary Cells

Concentration (μg/mL)	Cell Number (× 10^5cells)	ICC^a N-N0	RICC^b (%)	Cytotoxicity^c (%)
Before Treatment
Untreated (-S9)	26.6
Untreated (+S9)	23.0
After Treatment
Scheme I (-S9, 3h)
Negative Control	58.4	31.8	100.0	0.0
7.5	59.8	33.2	104.4	0.0
21.44	56.6	30.0	94.3	5.7
61.25	50.2	23.6	74.2	25.8
175	37.0	10.4	32.7	67.3
500	30.4	3.8	11.9	88.1
Positive Control^d	48.0	21.4	67.3	32.7
Scheme II (+S9, 3h)
Negative Control	156.2	133.2	100.0	0.0
7.5	153.6	130.6	98.0	2.0
21.44	162.6	139.6	104.8	0.0
61.25	136.0	113.0	84.8	15.2
175	21.0	-2.0	0.0	100.0
500	35.8	12.8	9.6	90.4
Positive Control^e	31.2	8.2	6.2	93.8
Scheme III (-S9, 20h)
Negative Control	59.2	32.6	100.0	0.0
7.5	56.8	30.2	92.6	7.4
21.44	53.2	26.6	81.6	18.4
61.25	43.0	16.4	50.3	49.7
175	32.4	5.8	17.8	82.2
500	27.4	0.8	2.5	97.5
Positive Control^f	49.0	22.4	68.7	31.3

a ICC: increased in cell counts = Cell No._{After treatment}(N) - Cell No._{Before treatment}(N₀)

b RICC: relative increase in cell counts; RICC = (ICC_treatment/ICC_control) × 100

c Cytotoxicity (%) = 100 – RICC

d Positive control was 0.33mg/mL mitomycin C (MMC)

e Positive control was 11.2mg/mL cyclophosphamide (CPP)

f Positive control was 0.2mg/mL mitomycin C (MMC)

 

Table 3. Summary of Chromosome Aberrations in Chinese Hamster Ovary Cells for Eversorb EP4

Treatment	Concentration (μg/mL)	Treating Hour	S9 (-/+)	Aberrant Cells (%)
Scheme I (-S9, 3h)
Negative Control^a	0	3	-	0.00
Test Article	7.5	3	-	0.00
	21.44	3	-	0.00
	61.25	3	-	0.00
	175	3	-	0.67
	500	3	-	-*
Positive Control^b	0.33	3	-	21.67**
Scheme II (+S9, 3h)
Negative Control^c	0	3	+	0.00
Test Article	30	3	+	0.33
	85.75	3	+	1.00
	245	3	+	0.33
	700	3	+	3.00
	2000	3	+	0.00
Positive Control^d	11.2	3	+	30.67**
Scheme III (-S9, 20h)
Negative Control^a	0	20	-	0.00
Test Article	7.5	20	-	0.33
	21.44	20	-	0.00
	61.25	20	-	1.00
	175	20	-	-*
	500	20	-	-*
Positive Control^b	0.2	20	-	36.33**

All data were scored from 300 metaphase cells of each treatment (duplicate cultures).

a Negative control was 1% DMSO in McCoy’s 5A medium.

b Positive control was mitomycin C (MMC).

c Negative control was 1% DMSO in S-9 mixture medium.

d Positive control was cyclophosphamide (CPP).

*: Too few metaphases

**: The frequency of aberrant cells was significantly higher than that of the negative control (One-tailed binomial test, α = 0.01).

Conclusions:

According to OECD 473 test method, Eversorb EP4 was negative effect under the condition of in vitro mammalian chromosome aberration test.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316025-GT and OECD 473 (OECD, 2016). The results of this OECD 473 test for Eversorb EP4 show that test validity criteria was met.

Based on the results of a preliminary concentration-range finding cytotoxicity test, 500 and 2000 μg/mL as the highest dosage were used to perform the test respectively in 3-hour or 20-hour treatment without S9 metabolic activation (Schemes I and III) and 3-hour treatment with S9 metabolic activation (Scheme II). In the chromosome aberration test, five doses of Eversorb EP4, negative and positive controls were tested in duplicate with or without S9 Mix. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition, Eversorb EP4 was negative effect in mammalian chromosome aberration test (in vitro).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 09, 2018 to Sep. 11, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

L5178Y/TK+/--3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

L5178Y/TK+/--3.7.2C

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1

Dose-range Finding Test: Cytotoxicity of Eversorb EP4 (3 Hour Treatment)

Dose     (μg/ml)	cell count after 24 hours of subculture (cells/ml x10^5)	cell count after 48 hours of subculture (cells/ml x10^5)	SG^(1)     (x10^5cells/ml)	RSG^(2)     (%)
without metabolic activation
SC	5.2	7.4	19	100
16	4.8	8.4	20	105
31	2.2	8.8	10	50
63	1.6	8.0	6	33
125	0.8^(4)	4.8	2	10
250^(3)	0.5^(4)	1.6	0	2
with metabolic activation
SC	2.9	5.4	8	100
16	3.8	5.0	10	121
31	3.1	5.3	8	105
63	2.9	6.6	10	122
125	3.0	6.6	10	126
250^(3)	3.3	6.3	10	133

Note: all calculations were made without rounding off

SC = solvent control = dimethyl sulfoxide

(1)= suspension growth

(2)= relative suspension growth

(3)= the test item precipitated in the exposure medium

(4)= since less than 1.25 x 105c/ml were present, no subculture was performed

SG = Suspension growth = [Cell count after 24 hour of subculture (Day 1) /1.6 x 105] x [Cell count after 48hours of subculture (Day 2)/1.25 x 105]*

* Or appropriate cell concentration

RSG = [SG(test)/SG(control)] x 100

Table 2

Dose-range Finding Test: Cytotoxicity of Eversorb EP4 (24 Hour Treatment)

Dose     (μg/ml)	cell count after 24 hours of subculture (cells/ml x10^5)	cell count after 24 hours of subculture (cells/ml x10^5)	SG^(1)     (x10^5cells/ml)	RSG^(2)     (%)
without metabolic activation
SC	9.7	6.3	39	100
16	9.8	6.7	42	107
31	8.8	6.9	39	99
63	5.2	5.9	20	50
125	^1.9	2.5	3	8
250^(3)	0.2^(4)	0.2	0	0

Note: all calculations were made without rounding off

SC = solvent control = dimethyl sulfoxide

(1)= suspension growth

(2)= relative suspension growth

(3)= the test item precipitated in the exposure medium

(4)= since less than 1.25 x 105c/ml were present, no subculture was performed

SG = Suspension growth = [Day 0 cell count/1.25 x 10⁵]* x [Day 1 cell count/1.25 x 10⁵]*

* Or appropriate cell concentration

RSG = [SG(test)/SG(control)] x 100

Table 3

Experiment 1: Cytotoxic and Mutagenic Response of Eversorb EP4 in the Mouse Lymphoma L5178Y Test System

dose   (μg/ml)	RSG   (%)	CE day2   (%)	RCE   (%)	RTG   (%)	mutation frequency per 10^6survivors total    ( small   large )
without metabolic activation 3 hour treatment
SC1	100	97	100	100	83   ( 27   53 )
SC2		90			70   ( 25   43 )
3.1	89	98	105	93	99   ( 42   53 )
6.3	98	102	110	107	75   ( 37   35 )
12.5	114	85	91	104	69   ( 46   21 )
25	94	99	107	100	79   ( 36   40 )
50	101	113	121	122	68   ( 26   39 )
100	70	88	94	66	68   ( 32   34 )
125	46	94	101	46	60   ( 23   36 )
175^(1)	30	70	75	23	95   ( 35   57 )
MMS	95	41	44	42	505   ( 194  286 )
with metabolic activation 3 hour treatment
SC1	100	91	100	100	76   ( 16   58 )
SC2		97			67   ( 21   44 )
3.1	98	85	90	89	81   ( 19   61 )
6.3	93	91	97	90	93   ( 24   65 )
12.5	100	111	119	119	89   ( 35   49 )
25	105	102	109	114	81   ( 40   37 )
50	100	58	61	61	100   ( 26   72 )
100	91	93	99	90	80   ( 24   53 )
125	103	69	74	76	91   ( 12   78 )
175^(1)	98	93	99	97	68   ( 11   56 )
CP	68	64	68	46	602   ( 134  424 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;

SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1) = the test item precipitated in the exposure medium

Table 4

Experiment 2: Cytotoxic and Mutagenic Response of Eversorb EP4 in the Mouse Lymphoma L5178Y Test System

dose   (μg/ml)	RSG   (%)	CE day2   (%)	RCE   (%)	RTG   (%)	mutation frequency per 10^6survivors total    ( small   large )
without metabolic activation 24 hour treatment
SC1	100	93	100	100	132   ( 61   63 )
SC2		76			161   ( 73   79 )
3.1	101	97	115	116	128   ( 50   70 )
6.3	89	88	104	92	129   ( 50   72 )
12.5	93	94	112	104	140   ( 60   71 )
25	88	95	113	100	104   ( 33   66 )
50	70	76	90	63	199   ( 140   47 )
100	38	83	98	37	214   ( 116   79 )
125	19	72	86	17	176   ( 92   73 )
175^(1)	9	86	102	9	180   ( 98   68 )
MMS	88	60	72	63	938   ( 321  491 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;

SC = Solvent control = DMSO; MMS = Methylmethanesulfonate

Conclusions:

According to OECD 490 test method, Eversorb EP4 is not mutagenic in the TK mutation test system under the experimental condition of In Vitro Mammalian Cell Gene Mutation Tests.

Executive summary:

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

The study procedures described in this report were based on the most recent OECD guideline.

In the first experiment, Eversorb EP4 was tested up to concentrations of 175 μg/mL in the absence and presence S9-mix. The incubation time was 3 hours. The Relative total growth (RTG) was reduced to 23% in the absence of S9-mix and no toxicity was observed at this

dose level in the presence of S9-mix. Eversorb EP4 precipitated in the culture medium at this dose level.

In the second experiment, Eversorb EP4 was tested up to concentrations of 115 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 9%.

Eversorb EP4 did not precipitate in the culture medium at this dose level.

In the absence of S9-mix, Eversorb EP4 did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, Eversorb EP4 did not induce a biologically relevant increase in the mutation frequency.

In conclusion, Eversorb EP4 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental condition of In Vitro Mammalian Cell Gene Mutation Tests.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available