[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Controls

Untreated negative controls:

yes

Remarks:

Dimethyl sulphoxide

Positive controls:

yes

Remarks:

TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli

Details on test system and experimental conditions:

TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli

Rationale for test conditions:

The results of the study from both the initial and confirmatory mutation assay showed that, Licocare RBW 300 FL TP did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

Evaluation criteria:

Tester strain integrity
All Salmonella typhimurium tester strains and the Escherichia coli tester strain used in this assay must demonstrate their growth potential in the presence of histidine and tryptophan, respectively.

All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation, respectively.

Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of uvrA mutation.

Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.

• The spontaneous reversion rates in the vehicle control must be in the range of in-house historical data.
• There must be at least three non-toxic dose levels.
• The top dose selected should demonstrate toxicity. In case of non-toxic test items, the top dose tested should be 5000 µg/plate.
• All tester strain culture titers must be in the range of 1-2 x 109 cells/mL to ensure that appropriate numbers of bacteria are used for plating.
• The positive control substances should produce a more than 3-fold increase in mutant colony frequencies when compared to the respective vehicle control plates.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Results of Preliminary Toxicity Test

Treatment (mg/plate)

TA 100 revertant colonies/plate^*

Presence of Metabolic Activation

Absence of Metabolic Activation

Mean

Background lawn Intensity**

Precipitation**

Mean

Background lawn Intensity**

Precipitation**

DMSO

112

4+

NPO

113

4+

NPO

50

111

4+

NPO

107

4+

NPO

100

112

4+

NPO

107

4+

NPO

200

109

4+

NPO

108

4+

NPO

400

106

4+

NPO

107

4+

NPO

800

107

4+

NPO

105

4+

NPO

1600

108

4+

NPO

107

4+

NPO

3200

108

4+

NPO

107

4+

NPO

5000

106

4+

NPO

107

4+

NPO

*: Mean of two replicates                           **: Refer Appendix 7

               

Table 2. Viable Counts of the Overnight Culture of the Tester Strains

Tester Strains		Viable Counts (x 10^9CFU/mL*)
		Initial Mutation Assay		Confirmatory Mutation Assay
TA98		1.54		1.56
TA100		1.58		1.57
TA1535		1.55		1.56
TA1537		1.56		1.58
WP2uvrA (pKM101)		1.63		1.62

 

* Required Cell count: 1-2x10⁹Colony Forming Units (CFU)/mL

 

 

 

 

 

 

TALBE 3. Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	2	NA	108	3	NA	16	3	NA	13	3	NA	148	3	NA
50	25	2	0.96	108	2	1.00	16	2	1.02	13	2	1.00	146	3	0.99
158	26	2	1.00	105	2	0.97	15	2	0.94	13	2	0.95	147	4	0.99
500	26	2	1.01	105	3	0.98	14	2	0.88	12	1	0.90	146	4	0.99
1581	25	2	0.95	105	3	0.97	13	3	0.83	12	2	0.88	147	4	0.99
5000	25	3	0.95	106	3	0.98	13	2	0.83	11	1	0.83	146	2	0.99
Positive controls	364^c	8^c	14.01^c	765^c	15^c	7.08^c	163^c	6^c	10.21^c	164^c	9^c	12.33^c	576^d	8^d	3.89^d
^aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                                               ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                                         SD: Standard deviation

 

 

 

 

 

 

Table 4. Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	2	NA	107	2	NA	14	2	NA	14	2	NA	146	3	NA
50	24	3	0.95	107	3	1.00	15	2	1.07	12	3	0.90	148	2	1.01
158	25	2	0.97	107	5	1.00	15	2	1.02	12	2	0.90	148	4	1.02
500	25	2	0.97	104	3	0.98	14	3	0.98	13	2	0.93	145	3	0.99
1581	25	3	0.97	106	4	0.99	14	3	0.95	13	3	0.93	146	3	1.00
5000	24	2	0.93	105	4	0.98	14	2	1.00	13	2	0.93	144	3	0.99
Positive controls	273^c	9^c	10.65^c	562^d	7^d	5.26^d	152^d	10^d	10.63^d	163^e	12^e	11.92^e	566^f	14^f	3.89^f
^aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number  ^bRatio of treated/vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98: 2-Nitrofluorene (2 µg/plate),                     ^dTA100, TA1535: Sodium azide (1 µg/plate),       ^eTA1537: 9-Aminoacridine (50 µg/plate),             ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                       NA: Not applicable                                                         SD: Standard deviation

 

 

 

 

 

 

Table 5. Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	3	NA	112	3	NA	17	2	NA	15	3	NA	147	5	NA
50	26	2	0.99	107	4	0.96	16	3	0.98	13	2	0.86	148	4	1.00
158	25	2	0.95	109	3	0.97	16	3	0.94	14	2	0.95	148	3	1.01
500	25	3	0.95	104	4	0.93	16	2	0.94	13	2	0.89	146	3	1.00
1581	26	2	1.00	107	3	0.96	15	3	0.88	13	2	0.86	146	4	1.00
5000	25	3	0.94	108	3	0.96	14	3	0.82	13	3	0.89	144	4	0.98
Positive controls	372^c	15^c	14.11^c	766^c	22^c	6.86^c	155^c	9^c	9.32^c	161^c	13^c	11.00^c	568^d	18^d	3.87^d
^aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number   ^bRatio of treated/vehicle control (mean revertants per plate).The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)               ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                                                                                                         SD: Standard deviation

 

 

 

 

 

 

 

Table 6. Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	27	2	NA	108	2	NA	16	2	NA	14	3	NA	148	3	NA
50	25	2	0.95	107	4	0.99	15	2	0.92	12	2	0.85	147	3	0.99
158	27	2	1.00	106	3	0.98	15	1	0.90	12	1	0.85	146	4	0.99
500	25	3	0.94	108	3	1.00	16	3	1.00	11	2	0.78	146	4	0.99
1581	26	2	0.97	107	4	0.99	15	3	0.90	12	2	0.88	145	3	0.98
5000	25	2	0.94	103	3	0.96	14	2	0.88	12	2	0.90	145	4	0.98
Positive controls	276^c	15^c	10.36^c	567^d	19^d	5.25^d	158^d	11^d	9.65^d	166^e	11^e	12.12^e	564^f	8^f	3.80^f
^aValues are means of three replicates calculated from Appendix 6 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98: 2-Nitrofluorene (2 µg/plate),                     ^dTA100, TA1535: Sodium azide (1 µg/plate)                         ^eTA1537: 9-Aminoacridine (50 µg/plate),              ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                       NA: Not applicable                                                         SD: Standard deviation

 

 

 

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the study plan. It is concluded that the test item, Licocare RBW 300 FL TP, was not mutagenic in this bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

The test item, Licocare RBW 300 FL TP was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains ofSalmonella typhimuriumand WP2uvrA (pKM101) strain ofEscherichia coliin three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

Licocare RBW 300 FL TP formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at 50 mg/mL.

 

In a preliminary toxicity test for the selection of test doses for the mutation assay, the test itemdid not precipitate on the basal agar plates at any of the tested doses.The test item did not show toxicity to the tester strain TA100 at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the test item was tested up to the OECD 471-recommended top dose of 5000 µg/plate in the mutation assay.

 

In the mutation assay, the bacterial tester strains were exposed to                       Licocare RBW 300 FL TPin triplicate at 50, 158, 500, 1581 and 5000mg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure and the confirmatory mutation assay was conducted using the pre-incubation mode of exposure. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

 

The results of the study from both the initial and confirmatory mutation assay showed that, Licocare RBW 300 FL TP did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

 

The study indicated that Licocare RBW 300 FL TP was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000mg/plate, under the conditions of testing employed.