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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

Chromosome aberration test (OECD 473): negative in human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD 476): negative in mouse lymphoma cells (L5178Y) with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept. 18 - Nov. 10, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure was a GLP study performed similar to OECD guideline 471 (but with 2-aminoanthracene as sole indicator of efficacy of S9-Mix)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Principles of method if other than guideline:
Crude enzyme preparations, like the present batch of Subtilisin contain the free amino acid histidine and tryptophan, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay.
To overcome this problem, all strains were exposed to Subtilisin in liquid culture (“treat and plate assay”).
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon, trp operon
Species / strain / cell type:
bacteria, other: Salmonella typhimurium TA1537, TA98, TA1535, TA100, Escherichia coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced Spraque Dawley rats obtained from MP Biomedicals, LLC. 29525 Fountain Parkway Solon, Ohio 44139.
Test concentrations with justification for top dose:
5000 µg test substance (dry matter) per ml and 2-fold dilutions (6 doses), with and without the metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile deionised water
- Justification for choice of solvent/vehicle: substance is water-soluble and any human exposure will be in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, 2-nitrofluorene, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium before plating, i.e a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3 hours
- Incubation time (selective incubation) : 64 hours

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:
A test substance is regarded as positive when it has induced at least a doubling in the mean number of revertants per plate compared to the appropriate solvent control in one or more of the strains, in the presence or absence of S9, if this response is dose related and reproducible.
Statistics:
No statistics performed.
Key result
Species / strain:
bacteria, other: Salmonella typhimurium TA1537, TA98, TA1535, TA100, Escherichia coli WP2uvrA
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:
The test material is a fluid enzyme preparation. It contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. This means, that comparison of viable counts between exposed cultures and control culture in a “treat and plate” assay reflects growth stimulation/inhibition as well as cell killing. It is our experience, that in a treat and plate assay, where bacteria are exposed to different doses of such a test substance in separate liquid cultures for a certain time, the spontaneous revertant levels fluctuate more than in the direct "plate incorporation assay."
No significant toxicity was evident in the majority of the tests with and without metabolic activation. Reduced viabilities are evident at the three highest doses in tests with TA100 and TA1535 without the presence of S-9, mainly in the first experiment. These observations have no significant influence on the overall evaluation of the results.
No treatments of any of the Salmonella and E.coli strains with subtilisin resulted in any increases in revertant numbers that meets these criteria for a positive or equivocal response.
The results of the controls in the study were within the historical ranges.
Remarks on result:
other: all strains/cell types tested
Executive summary:

Subtilisin (Batch Number: PPA 28009) was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrA.

Crude enzyme preparations, like the present batch contain the free amino acid histidine and tryptophan, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all strains were exposed to PPA 28009 in liquid culture (“treat and plate assay”).

Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg (dry matter) per mL as highest concentration. After incubation the test substance was removed by centrifugation prior to plating.

The study was conducted with and without the metabolic activation system S9 - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix).

Two identical and independent experiments were conducted.

The treatment of the Salmonella and E.coli strains with Batch PPA 28009, in the presence or absence of S9 mix, did not result in any increases in revertant numbers. Batch number PPA 28009 was found not mutagenic.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug. 23 - Oct. 31, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was a GLP study conducted in compliance with OECD guideline 473 (July 1997).
Remarks:
/ less metaphases scored (200 per concentration instead of 300)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
/ less metaphases scored (200 per concentration instead of 300)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL and broad range of dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 4-Nitroquinoline 1-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: 3 hours (first experiment, in absence or presence S9); 3 hours (second experiment, in presence S9); 20 hours (second experiment, in absence S9)
- Treatment plus recovery time: 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

NUMBER OF CELLS EVALUATED: a total of 200 cells per dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Hyperdiploid cells

Evaluation criteria:
A test article is considered as positive in this assay if:
1) the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicates, and
2) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.
Statistics:
Fisher's exact test, p≤0.05 significant.
Heterogeneity between replicates evaluated by means of a binomial dispersion test, p≤0.05 significant.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Subtilisin is a proteolytic enzyme which implies, that the test substance has the potential of breaking down the metabolising system S9. In a preliminary study it was demonstrated, that the test material inactivated S9 significantly. Therefore, the main study was conducteds with heat-inactivated test substance.

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA:
Cells treated with Subtilisin, either in the absence of S-9, had similar numbers of abberrations to those observed in concurrent solvent controls. There were no reproducible increases in aberration frequency that were significantly higher than those observed in the negative controls.
The negative controls were within the negative control ranges.
Normal frequencies of cells with numerical aberrations were seen under all treatment conditions.
The positive controls induced satisfactory levels of aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No significant cytotoxicity, however 30% reduction of mitotix index at the highest dose level at 20 hours treatment.
Conclusions:
Under the conditions of the test, the test item did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 mg/mL in either the absence and presence of S-9.
Executive summary:

The clastogenic potential of Subtilisin, Batch No. PPA 6865 was evaluated by its effect on chromosomes of human peripheral blood lymphocytes according to OECD guideline 473 (July 1997). Subtilisin is a proteolytic enzyme which implies, that the test substance has the potential of breaking down the metabolising system S9. Therefore, the main study was conducted with heat-inactivated test substance. Heparinized whole blood cultures from three male donors were established, and division of the lymphocytes was stimulated by adding phytohaemagglutinin to the cultures.

Two independent experiments were performed both in the absence and presence of metabolic activation by a rat S-9 mix induced with Aroclor.

Sets of duplicate cultures were treated with the solvent (sterile purified water), test substance or positive controls (-S-9: 4-Nitroquinoline 1-oxide, +S-9: Cyclophosphamide). Treatments with subtilisin covered a broad range of doses, where the highest dose level used was 5000 µg/mL.

In the first experiment, the lymphocyte cultures were exposed to the test substance in the absence or presence of S-9 for three hours and cells were harvested 17 hours later. The second experiment included a continuous exposure for 20 hours in the absence of S-9. The test article dose levels for chromosome analysis were selected by evaluating the effect of Subtilisin on mitotic index.

Chromosome aberrations were analysed at three consecutive dose levels. Cells were arrested in metaphase by colchicine and after centrifugation and hypotonic treatment, metaphase spreads were prepared and stained with Giemsa. A total of 200 cells were scored per dose level (100 from each replicate culture) from subtilisin treatments and negative controls.

The proportion of cells with structural aberrations in all cultures of the solvent controls (purified water) was within the limits of the historical ranges. The positive controls induced statistically significant increases in the proportion of cells with structural aberrations, thus demonstrating the sensitivity of the test procedure and the metabolic activity of the S-9 mix employed.

Cells treated with subtilisin, either in the absence and presence of S-9, had similar numbers of aberrations to those observed in concurrent solvent controls.

Subtilisin, batch No. PPA 6865,did not induce chromosome aberrations in cultured human blood lymphocytes when tested up to a concentration of 5000 ug/mL in either the absence or presence of S-9.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec. 12 1990 - Feb. 12, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was a GLP study conducted in compliance with OECD guideline 476.
Remarks:
/ only 2 hrs of exposure
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
/ only 2 hrs of exposure
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (6-thioguanine resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's Medium 10 (10% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL and 4 dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide, benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7days
- Selection time (if incubation with a selection agent): At the end of the expression time, the cultures were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable


SELECTION AGENT : 6-TG

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as relative survival

Evaluation criteria:
A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.
Statistics:
The mutation frequency was evaluated statistically by using logaritthmic tranformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
TEST-SPECIFIC CONFOUNDING FACTORS
Subtilisin is a proteolytic enzyme which implies, that the test substance has the potential of breaking down the metabolising system S9. In a preliminary study it was demonstrated, that the test material inactivated S9 significantly. Therefore, the study was conducted with heat-inactivated test substance in the presence of S-9.
- Water solubility: yes

RANGE-FINDING/SCREENING STUDIES: Preliminary range finder performed

COMPARISON WITH HISTORICAL CONTROL DATA:
Cells treated with Subtilisin, either in the absence and presence of S-9, had similar mutation frequencies as those observed in concurrent solvent controls. The negative controls were within the historical negative control ranges.



Conclusions:
Under the conditions of the test, the test substance had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 µg/mL in either the absence or presence of S-9.
Executive summary:

Subtilisin batch PPA 3366 was assayed for its ability to induce mutation at the HPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation (S-9 mix). A preliminary experiment established that Subtilisin (a protease) inactivated the enzymes of the S-9 mix. The positive control substance benzo(a)pyrene did not give a positive mutagenic response in the presence of Subtilisin and S-9. Therefore Subtilisin was inactivated for all treatments in the presence of S-9. Following a wide range of treatments, separated by half-log intervals and reaching 5000 ug/mL, cells survived all doses of Subtilisin showing 91% and 128% relative survival in the absence and presence of S-9 respectively, at the top dose. This dose together with the next four lower doses, was plated for viability and 6-thioguanine resistance 7 days after treatment. In the second experiment a narrower dose range was used to maximise the chance of detecting any dose related effects. The top dose plated in this experiment was again 5000 ug/mL in the absence and presence of S-9, which yielded 86% and 99% survival, respectively. Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutation frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid. No Subtilisin treatment, either in the absence or presence of S-9, resulted in a statistically significant increase in mutation frequency. Therefore when tested up to 5000 ug/mL, Subtilisin failed to induce mutation at the HPRT locus of L5178Y mouse lymphoma cells, both in the absence and presence of S-9 (active enzyme tested in the absence of S-9 and inactivated enzyme in the presence of S-9). It was concluded that Subtilisin, under the conditions employed in this study, had no mutagenic activity in this test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for read-across

There are no measured/experimental data for genetic toxicity available for thermomycolin (CAS 52233-31-5) in regard to genetic toxicity in bacteria, clastogenicity or mutagenicity in mammalian cells. To fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.4, read-across from an appropriate substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met”. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”.

For each specific endpoint the source substance(s) structurally closest to the target substance is/ are chosen for read across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read- across approach is provided in the technical dossier (see IUCLID Section 13).

As no reliable measured/experimental data are available on clastogenicity and genetic toxicity in mammalian cells for thermomycolin, read-across to reliable data on the analogue substance subtilisin (CAS 9014-01-1) was apllied.

Genetic toxicity in bacteria (Ames)

The in vitro genetic toxicity of the test material was assessed in a bacterial reverse mutation assay (Ames test) according to GLP criteria and OECD 471 (Pedersen, 2008). The mutagenic potential of the test material was assessed in S. typhimurium tester strains including TA 98, 100, 1535 and 1537 and in E.coli WP2 uvr A bacterial cells at concentrations up to 5000 μg/plate in 2 independent experiments. The test substance did not exhibit mutagenic properties in the absence or presence of metabolic activation. Reduced viabilities were evident at the three highest doses in tests with TA100 and TA1535 without the presence of S9 mix, mainly in the first experiment. Based on the results of the conducted study, the test material is not considered to exhibit mutagenic properties in bacterial cells.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was performed with the test material in primary human lymphocytes according to OECD Guideline 473 and GLP (Whitwell, 2001). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (S9-mix). In the first experiment cells were incubated with test material concentrations up to 5000 μg/mL for 3 hours with and without metabolic activation. The second experiment included a continuous exposure for 20 hours in the absence of the S9 -mix. 4-Nitroquinoline 1-oxide and Cyclophosphamide were used as positive control substances without and with S9-mix, respectively. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level in comparison to the negative controls. Vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. Positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system. As the test material is a proteolytic enzyme which implies, that the test material has the potential of breaking down the metabolising system S9. In a preliminary study it was demonstrated, that the test material inactivated S9 significantly. Therefore, the study was conducted with heat-inactivated test materiali n the presence of S9. The test material is therefore considered to be non-clastogenic to human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro Mammalian Cell Gene Mutation Assay according to OECD Guideline 476 and under GLP was performed with subtilisin in mouse lymphoma L5178Y cells (Clements, 1991). Two independent experiments (with 2 hours of exposure) were performed in the absence and presence of S9-mix with test material concentrations up to 5000 μg/mL. 4-nitroquinoline-1-oxide and benzo(a)pyrene were used as positive controls with and without S9 mix, respectively. Positive and negative controls were valid and in range of historical control data. As the test material is a proteolytic enzyme which implies, that the test material has the potential of breaking down the metabolising system S9. In a preliminary study it was demonstrated, that the test material inactivated S9 significantly. Therefore, the study was conducted with heat-inactivated test substance in the presence of S-9. No significant increase in mutation frequency occurred in any of the test conditions, indicating that the test material is not mutagenic in the mammalian cells in vitro.

Conclusion on genetic toxicity

The available data do not provide evidence that the structural analogue substance subtilisin (CAS 9014-01-1) exhibit mutagenic or clastogenic properties in bacteria or mammalian cells. Therefore, no potential for genetic toxicity is expected for thermomycolin (CAS 52233-31-5).

Justification for classification or non-classification

Based on the available data on the genetic properties of the structural analogue, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.