Reaction products of Hydrogen amino-4-hydroxynaphthalene-2-sulfonate, Potassium substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate and Sodium substituted-({4-[(2-chloroethyl)sulfonyl]butanoyl}amino)benzenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 40875/A TE was not mutagenic in the Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000µg/plate, under the conditions of testing employed.

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:

2018

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

Specific details on test material used for the study:

Test Item: FAT 40875/A TE
Physical Appearance: Dark red powder
Purity as per Certificate of Analysis (Content): 86.4% all organic components
Batch No.: BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured Date: December 20, 2017
Expiry Date: December 11, 2022
Recommended Storage Condition : Ambient (+2 to +8°C), Protect from light.
pH: 4.3 (aq. susp.(2% (w/w) at room temperature).

Target gene:

DNA base pairs

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Source: Health Protection Agency National Collection of Type Cultures (NCTC) 61, Colindale Avenue London NW9 5EQ Great Britain

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB) Ferguson Building Craibstone Estate, Bucksburn Aberdeen, AB21 9YA Scotland, U.K.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. The S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 °C. The details of S9 homogenate used in the study are furnished as a Certificate in Appendix I of the report. S9 homogenate was thawed immediately before use and mixed with the cofactor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 MgCl2 and 33 KCI in PBS.

S9 mix was prepared by mixing 1 and 5.5 mL of the S9 homogenate with 9 and 49.5 mL of the co-factors solution for the preliminary toxicity and
mutation assay respectively, kept in an ice bath and used within one hour.

Test concentrations with justification for top dose:

Concentration: 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate.

The test item did not cause precipitation on the basal agar plates at any of the tested doses.
Also, no toxicity to tester strains was observed at any test dose. The Intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates, both in the presence and absence of metabolic activation.
Based on these observations, it was decided to test the OECD 471 recommeded top dose of 5000 µg/plate in the mutation assay.

Vehicle / solvent:

Sterile Water (SW)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With Metabolic activation - TA98, TA100, TA1535, TA1537, WP@ uvrA (pKM101)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation - TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation - TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation - WP2 uvrA (pKM101)

Details on test system and experimental conditions:

Three replicate plates were maintained for the initial as well as the confirmatory mutation assays.
Three Independent test experiments were performed viz; preliminary toxicity test, initial mutation assay and confirmatory mutation assay.

Method and treatment/exposure:
The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas, the confirmatory mutation assay was carried out using the pre­ incubation mode of exposure.

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respectivethreshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prelimnary toxicity test: The test item did not cause precipitation on the basal agar plates at any of the tested doses. The test item did not show toxicity to the tester strain 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, both in the presence and absence of metabolic activation. Based on these observations, it was decided to test the OECD 471- recommended top dose of 5000 µg/plate in the mutation assay.

Genotypic Characterization: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains were demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility's historical control data.

Stability of Test Item in the Vehicle and Analytical Verification of Dosing Formulations: FAT 40875/A TE was found to be stable and re-suspendable in Milli-Q water for 24 hours at room temperature at the concentrations of 0.0255 and 252.20 mg/mL. The results of concentration analysis of FAT40875/ATE dose formulations from the initial mutation assay indicated that the mean of percentage agreement with claimed concentration were 105.2 and 101.8 % (with an RSD of 2.6 and 0.5%, respectively) of their respective claimed concentrations of 500 and 50000 µg/mL, confirming that the concentration of the test item was within the acceptable limit (±15% of claimed concentrations and an RSD of 10%). No test item was detected in the vehicle control.

The results of concentration analysis of FAT 40875/A TE dose formulations from the confirmatory mutation assay indicated that the mean of percentage agreement with claimed concentration were 96.7 and 85.6 % (with an RSD of 1.6 and 0.6%, respectively) of their respective claimed concentrations of 500 and 50000 µg/mL, confirming that the concentration of the test item was within the acceptable limits. No test item was detected in the vehicle control.

 

These results from the initial and confirmatory mutation assays indicate that the top dose of 5000 µg/plate level was achieved and support the validity of the study conclusion.

Conclusions:

The test item, FAT 40875/A TE, was not mutagenic in this bacterial reverse mutation test.

Executive summary:

The test item, FAT 40875/A TE was tested for its mutagenic potential in the bacterial reverse mutation assay. This test was conducted in accordance to OECD test guideline 471, in a GLP certified laboratory.

The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in 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).

FAT 40875/A TE formed a free flowing suspension in sterile water (SW) at 50 mg/mL. The test item was found stable and re-suspendable in Milli-Q water for 24 hours at room temperature, at the fortification levels of 0.025 and 250 mg/mL.

In a preliminary toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause toxicity to the tester strain 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 OECD 471 -recommended top dose of 5000 µg/plate was tested in the mutation assay.

In the mutation assay the bacterial tester strains were exposed to FAT 40875/A TE in triplicate at 50, 158, 500, 1581 and 5000 µg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas, the confirmatory mutation assay was carried out using the pre-incubation mode of exposure. The vehicle control (SW) 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 assays showed that, FAT 40875/A TE 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 a 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 results of the concentration analysis of the dose formulation samples of both the initial and confirmatory mutation assays confirmed that the top dose level of 5000 µg/plate was achieved in both assays and the results support the validity of the study conclusion. The study indicated that FAT 40875/A TE was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000 µg/plate, under the conditions of testing employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item, FAT 40875/A TE was tested for its mutagenic potential in the bacterial reverse mutation assay. This test was conducted in accordance to OECD test guideline 471, in a GLP certified laboratory.

The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in 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).

In a preliminary toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause toxicity to the tester strain 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 OECD 471 -recommended top dose of 5000 µg/plate was tested in the mutation assay.

In the mutation assay the bacterial tester strains were exposed to FAT 40875/A TE in triplicate at 50, 158, 500, 1581 and 5000 µg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas, the confirmatory mutation assay was carried out using the pre-incubation mode of exposure. The vehicle control (SW) 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 assays showed that, FAT 40875/A TE 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 a 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 results of the concentration analysis of the dose formulation samples of both the initial and confirmatory mutation assays confirmed that the top dose level of 5000µg/plate was achieved in both assays and the results support the validity of the study conclusion. The study indicated that FAT 40875/A TE was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000µg/plate, under the conditions of testing employed.

Justification for classification or non-classification

FAT 40875/A TE was not mutagenic in the Bacterial Reverse Mutation Assay, thus not classified for mutagenecity as per the Regulation (EC) N0. 1272/2008.