Dehydratase, carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A read-across to xanthan lyase was done as the two enzymes belong to the same sub-class and therefore similar results are expected. 

It was concluded that Xanthan lyase, batch PPE55581 showed no evidence of mutagenic activity in the bacterial system in the presence and absence of S9.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

31 October 2018 - 15 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the similarity between the two enzymes that belong to the same enzyme sub-class, similar results are expected for carbonic anhydrase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan locus in the genome of five strains of bacteria.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver.

Test concentrations with justification for top dose:

The OECD Guideline 471 recommends a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). The test item was tested as 5000 µg total organic solids (TOS)/mL maximum dose, equivalent to 7881 µg enzyme concentrate dry matter/mL, fulfilling the recommendation.

Test 1: 7.9, 23.6, 78.8, 236.4, 788.1, 2364, 7881 μg enzyme concentrate dry matter/mL
Five concentrations of the test item in the final test: (78.8, 236.4, 788.1, 2364 and 7881 enzyme concentrate dry matter/mL).

Vehicle / solvent:

Vehicle for enzyme: Purified water.
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 1x 10^9 cells/mL

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 1 °C for 3 hours (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count and observation of the bacterial background lawn. 0.1 ml aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates in duplicates. The plates were inverted and incubated at 37 ± 2°c for about 72 hours and scored.

Evaluation criteria:

The test substance was considered as positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appertaining solvent control in one or more of the strains, in the presence or absence of S9 mix, if this response is dose related (at least 3 doses) and reproducible. In case of a dose related and reproducible numerical increase, which is below a doubling but at least 50% higher than the solvent control, the result is considered as equivocal and needs further clarification.

Statistics:

Not performed.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but no issues were reported in this study.
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

No evidence of toxicity was obtained following exposure to Xanthan lyase, batch PPE55581. No precipitate was observed on any of the plates containing Xanthan lyase, batch PPE55581.

The results with strain TA98 in the presence of S9 mix were obtained from a repeated test. Some increases in revertant colony numbers compared to the concurrent vehicle control counts were observed in strain TA1535 in the absence of S9 mix. The response was flat with no real concentration-response relationship and did not reach 3-fold increase. Hence, the acceptance criteria for a positive response was not fulfilled. The Sponsor indicated that the increases may have arisen from the test item containing various nutrients composing a rich growth medium to the bacterial cultures and therefore stimulating their growth. No evidence of mutagenic activity was seen with any other strains at any concentration of Xanthan lyase, batch PPE55581 in either the presence or absence of S9 mix.

Conclusions:

It was concluded that Xanthan lyase, batch PPE55581 showed no evidence of mutagenic activity in the presence and absence of S9, in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of Xanthan lyase, batch PPE55581, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to Xanthan lyase, batch PPE55581 diluted in water. Water was also used as a vehicle control.

The mutation tests were performed according to the treat-and-wash method. They were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Two independent mutation tests were performed. Concentrations of Xanthan lyase, batch PPE55581 up to 7881 μg enzyme concentrate dry matter/mL were tested. Other concentrations used were a series of about half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to Xanthan lyase, batch PPE55581 in either test. No precipitate was observed on any of the plates containing Xanthan lyase, batch PPE55581. Some increases in revertant colony numbers compared to the concurrent vehicle controls were observed in strain TA1535 in the absence and presence of S9 mix in the first test, and in the absence of S9 mix in the second test. The response was flat with no real concentration response relationship and did not reach 3-fold increase. Hence, the acceptance criteria for a positive response was not fulfilled. The increases may have arisen from the test item containing various nutrients composing a rich growth medium to the bacterial cultures and therefore stimulating their growth. No evidence of mutagenic activity was seen with any other strains at any concentration of Xanthan lyase, batch PPE55581 in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory, and within the acceptable ranges defined in Gatehouse et al (1990). At present, there is a limited number of data available for the treat-and-wash method of the Ames test. For this reason, as well as to provide a robust set of current laboratory records, historical data for the standard Ames test are also reported.

It was concluded that Xanthan lyase, batch PPE55581 showed no evidence of mutagenic activity in the presence and absence of S9, in this bacterial system under the test conditions employed.

Reference:

Gatehouse, D.G. et al (1990) Bacterial mutation assays in: Kirkland, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part I. Basic Mutagenicity Tests. Cambridge University Press, Cambridge. pp.13-61.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)