Fatty acids, C16 and C18-unsatd., polymers with bisphenol A, Bu glycidyl ether, epichlorohydrin and triethylenetetramine

Genetic toxicity: in vitro

Administrative data

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:

21 August 2012 to 4 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and in accordance with current test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

other: yellow/brown liquid

Details on test material:

Name: MonoFA_TETA_PAA_BADGE_BGE_Adduct
Synonyms: Aradur 460 J90 BD, Aradur 460J90
Batch number: WA 521
Date of receipt: 24 August 2012
Storage details: stored at 15 - 25°C protected from light
Purity: 100%
Expiry date: February 2014

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 10EO8 to 10EO9 cells/mL, based on cell count data from testing of each strain batch.

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Positive controls
2-nitrofluorene: Stock concentration; 50 µg/mL, final concentration; 5 µg/mL, Strain; TA 98, without metabolic activation
Sodium azide: Stock concentration; 20 µg/mL, final concentration; 2 µg/mL, Strain; TA100 and TA1535 without metabolic activation
9-aminoacridine: Stock concentration; 500 µg/mL, final concentration; 50 µg/mL, Strain; TA1537 without metabolic activation
Mitomycin C: Stock concentration; 2 µg/mL, final concentration; 0.2 µg/mL, Strain; TA102 without metabolic activation
Benzo[a]pyrene: Stock concentration; 100 µg/mL, final concentration; 10 µg/mL, Strain; TA98 with metabolic activation
2-aminoanthracene: Stock concentration; 50 µg/mL. final concentration; 5 µg/mL, Strain; TA100, TA1535 and TA1537 with metabolic activation
2-aminoanthracene: Stock concentration; 200 µg/mL, final concentration; 20 µg/mL, Strain; TA102 with metabolic activation

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S‑9, using final concentrations at 5, 15.81, 50, 158.1, 500, 1581 and 5000 mg/plate, plus negative (vehicle) and positive controls.
Experiment 2: treatments of all the tester strains were performed in the absence and in the presence of S-9. For strain TA98 in the absence of S-9 and strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9, the maximum test concentration was reduced to 200 µg/plate and for strain TA98 in the presence of S-9, the maximum test concentration was reduced to 500 µg/plate based on toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 0.8192 – 200 µg/plate or 2.048 – 500 µg/plate

Vehicle / solvent:

Dimethylformamide (DMF)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylformamide (DMF)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Remarks:

For concentrations see test concentrations section

Details on test system and experimental conditions:

Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 10EO8 to 10EO9 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
When the plates were set they were inverted and incubated at 37±1°C in the dark for 3 days.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p < 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Dunnett’s test used accordingly

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Following treatments of all the test strains in the absence and presence of S-9, only Experiment 2 treatments of strain TA100 in the presence of S-9 at 0.8192 µg/plate resulted in an increase in revertant numbers that was statistically significant when the data were analysed at the 1% level using Dunnett’s test. The increase was small in magnitude and occurred at a single low concentration with no indication of a concentration relationship. Accordingly this increase was considered to have been due to normal biological variability and not evidence of mutagenic activity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that MonoFA_TETA_PAA_BADGE_BGE_Adduct did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

MonoFA_TETA_PAA_BADGE_BGE_Adduct was assayed for mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254‑induced rat liver post-mitochondrial fraction (S‑9), in two separate experiments.

All MonoFA_TETA_PAA_BADGE_BGE_Adduct treatments in this study were performed using formulations prepared in Dimethylformamide (DMF).

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S‑9, using final concentrations of MonoFA_TETA_PAA_BADGE_BGE_Adduct at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed at 158.1 and/or 500 µg/plate and above in all strains in the absence and presence of S-9. In addition, evidence of toxicity was also observed at 50 µg/plate in strain TA102 in the absence of S-9 only.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For strain TA98 in the absence of S-9 and strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9, the maximum test concentration was reduced to 200 µg/plate and for strain TA98 in the presence of S-9, the maximum test concentration was reduced to 500 µg/plate based on toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 0.8192 – 200 µg/plate or 2.048 – 500 µg/plate, in order to examine more closely those concentrations of MonoFA_TETA_PAA_BADGE_BGE_Adduct approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. This modification to the standard plate incorporation methodology has been known to identify mutagenic chemicals that could not be detected using the standard plate incorporation assay system. Following these treatments, evidence of was observed at 80 and/or 200 µg/plate in strain TA98 in the absence of S-9 and strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9, and at 500 µg/plate in strain TA98 in the presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies were comparable with acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Although a statistically significant increase in revertant numbers was observed following MonoFA_TETA_PAA_BADGE_BGE_Adduct treatments of strain TA100 in the presence of S‑9 (Dunnett's Test, 1% level), this occurred at the lowest concentration tested, was not concentration-related or reproducible and was of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system.

It was concluded that MonoFA_TETA_PAA_BADGE_BGE_Adduct did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S‑9).