Reaction mass of Octyl-3-[3-tert-butyl-4-hydroxy-5-(5-chloro-2H-benzotriazole-2-yl)phenyl]propionate and 2-Ethylhexyl-3-[3-tert-butyl-4-hydroxy-5-(5-chloro-2H-benzotriazole-2-yl)phenyl]propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Eversorb 109 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate in the absence and presence of S9 metabolic activation (OECD TG471).

 

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 24, 2017 to July 11, 2017

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 activation

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: Acridine mutagen ICR 191, 2-Aminofluorene, 2-Aminoanthracene

Evaluation criteria:

Tester Strain Background Revertants
Without S9 With S9
TA98 5-60 10-60
TA100 40-240 40-240
TA102 200-550 200-600
TA1535 2-45 2-45
TA1537 2-25 2-35

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Genotype Confirmation Test of Salmonella typhimurium Tester Strains

Genotype character	Phenotypic observation	Tester Strains
		TA98	TA100	TA102	TA1535	TA1537
Histidine requirement	growing on biotin plate	－	－	－	－	－
	growing on histidine/biotin plate	+	+	+	+	+
rfa mutation	inhibition zone of crystal violet	+	+	+	+	+
ΔuvrB mutation	growing on non UV-irradiated plate	+	+	+	+	+
	growing on UV-irradiated plate	－	－	+	－	－
R-factor	ampicillin resistance	+	+	+	－	－
Genotype confirmed		Passed	Passed	Passed	Passed	Passed

+: the presence

－: the absence

 

Table 2. Mutagenicity Test of Eversorb 109 in Salmonella typhimurium Strains without S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537
Replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	26	15	26	76	81	66	301	334	208	13	23	21	9	17	6
	M^f	22 ± 6			74 ± 8			281 ± 65			19 ± 5			11 ± 6
50	I^e	24	30	23	98	76	63	271	294	299	9	24	15	13	15	8
	M^f	26 ± 4			79 ± 18			288 ± 15			16 ± 8			12 ± 4
150	I^e	26	28	18	61	57	62	330	317	297	10	14	22	16	13	12
	M^f	24 ± 5			60 ± 3			315 ± 17			15 ± 6			14 ± 2
500	I^e	32	26	41	75	66	54	332	313	273	12	13	16	10	13	17
	M^f	33 ± 8			65 ± 11			306 ± 30			14 ± 2			13 ± 4
1500	I^e	14	25	14	76	63	64	318	276	297	15	15	12	14	14	12
	M^f	18 ± 6			68 ± 7			297 ± 21			14 ± 2			13 ± 1
5000	I^e	18	27	25	62	69	66	238	276	282	15	11	25	12	20	17
	M^f	23 ± 5			66 ± 4			265 ± 24			17 ± 7			16 ± 4
Positive control^b	I^e	257	233	250	650	474	471	1318	1198	1416	420	434	423	174	162	133
	M^f	243^c± 18			532^c± 102			1311^c± 109			426^d± 7			156^d± 21

a: Negative control was DMSO.

b: Positive controls: 1 μg/plate 2-nitrofluorene for TA98

0.5 μg/plate sodium azide for TA100

0.1 μg/plate mitomycin C for TA102

0.1 μg/plate sodium azide for TA1535

0.5 μg/plate acridine mutagen ICR 191 for TA1537

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate.

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated.

 

Table 3. Mutagenicity Test of Eversorb 109 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537
Replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	36	34	49	67	62	67	328	386	378	8	6	14	4	10	15
	M^f	40 ± 8			65 ± 3			364 ± 31			9 ± 4			10 ± 6
50	I^e	31	39	23	67	69	66	444	419	353	14	10	9	15	22	17
	M^f	31 ± 8			67 ± 2			405 ± 47			11 ± 3			18 ± 4
150	I^e	41	23	35	57	70	69	378	261	375	11	14	13	17	17	10
	M^f	33 ± 9			65 ± 7			338 ± 67			13 ± 2			15 ± 4
500	I^e	44	36	34	94	75	77	255	319	259	9	11	14	15	9	16
	M^f	38 ± 5			82 ± 10			278 ± 36			11 ± 3			13 ± 4
1500	I^e	35	47	48	77	72	73	325	318	382	13	9	10	10	10	14
	M^f	43 ± 7			74 ± 3			342 ± 35			11 ± 2			11 ± 2
5000	I^e	42	48	33	80	74	63	311	333	302	7	11	17	9	10	15
	M^f	41 ± 8			72 ± 9			315 ± 16			12 ± 5			11 ± 3
Positive control^b	I^e	160	145	137	487	458	482	1672	1606	1346	130	120	112	165	188	199
	M^f	147^c± 12			476^c± 16			1541^c± 172			121^d± 9			184^d± 17

a: Negative control was DMSO.

b: Positive controls: 0.5 μg/plate 2-aminofluorene for TA98

4 μg/plate 2-aminofluorene for TA100

4 μg/plate 2-aminoanthracene for TA102

1 μg/plate 2-aminoanthracene for TA1535

2 μg/plate 2-aminoanthracene for TA1537

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate.

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated.

Conclusions:

According to OECD 471 test method, Eversorb 109 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316023-GT which is based on the SOP for the OECD 471 (CTPS-TE00201) and OECD 471 (OECD, 1997). The results of this OECD 471 test for Eversorb 109 show that test validity criteria was met.

Based on the preliminary assay results, 5000 μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of Eversorb 109 at 50, 150, 500, 1500 and 5000 μg/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. No cytotoxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that Eversorb 109 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/plate either in the absence or in the presence of metabolite activation. Based on the data obtained from this study, it was concluded that under the test condition, Eversorb 109 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate in the absence and presence of S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Based on the preliminary assay results, 5000 μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of Eversorb 109 at 50, 150, 500, 1500 and 5000 μg/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. No cytotoxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that Eversorb 109 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/plate either in the absence or in the presence of metabolite activation. Based on the data obtained from this study, it was concluded that under the test condition, Eversorb 109 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate in the absence and presence of S9 metabolic activation.