complex mixture from biological origin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No substance-specific data on genetic toxicity is available. Data from a suitable read-across partner was used to assess the genotoxic properties. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

An in vitro genotoxicity test battery (OECD 471, 487, 490 GLP) is available for the read-across substance. It was tested negative all three tests. The test item is considered to not induce genotoxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

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:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item did not induce two-fold increases in the number of revertant colonies, in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

The test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, S. typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA were exposed to the test substance (47.2% solid content) in purified water at concentrations of 5000, 2500, 1250, 625, 313 µg/plate (TA1535, WP2 uvrA, TA98), 5000, 2500, 1250, 625, 313, 156µg/plate (TA1537) and 2500, 1250, 625, 313, 156, 78.1 µg/plate (TA100) in the presence and absence of mammalian metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

For both experiments, the following dose levels were selected for scoring:

Experiment No.:	S9	Treatment time (hours)	Harvest time (hours)	Concentration (µg/mL)
1	-	3	31-32	212, 147 and 102
1	+	3	31-32	570, 396 and 275
2	-	31	31	133, 92.1 and 64.0

Conclusions:

The test material did not induce structural and/or numerical chromosomal damage in human lymphocytes. The test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), human lymphocytes cultured in vitro were exposed to the test item HH-2015-623 (47.2% solid content) in cell culture medium.Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. A second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours. Solutions of the test item were prepared in culture medium.

Dose levels for treatment were selected on the basis of the cytotoxicity observed in previous experiments where unacceptable values of frequency of micronucleated cells were observed or the required cytotoxicity was not achieved to select dose levels for scoring micronuclei.

Dose levels of 528, 440, 367, 306, 255, 212, 177, 147, 123, 102 and 85.3 µg/mL were used for the three hour treatment in the absence of S9 metabolism.

Dose levels of 684, 570, 475, 396, 330, 275, 229 and 191 µg/mL were used for the three hour treatment in the presence of S9 metabolism.

Dose levels of 191, 159, 133, 111, 92.1, 76.7, 64.0, 53.3 and 44.4 µg/mL were used for the continuous treatment in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI).

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All results were within the distribution of historical negative control data. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that HH-2015-623 does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487 for in vitro mutagenicity data.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity
In the first experiment, in the absence of S9 metabolic activation, no cell survived treatment at the highest dose level, marked toxicity reducing relative total growth (RTG) to 20% of the concurrent negative control was noted at 125 µg/mL, slight toxicity was observed at 104 µg/mL, while no relevant toxicity was noted over the remaining concentrations tested.
In the presence of S9 metabolism, no cell survived treatment at the two highest dose levels; treatment with the test item at 384 µg/mL yielded mild toxicity reducing RTG to 57% of the concurrent negative control value, while no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the presence of S9 metabolic activation, no cell survived after treatment at the highest dose level; the next concentration (440 µg/mL) yielded moderate toxicity reducing RTG to 26% of the concurrent negative control value. Mild toxicity was observed at 400 µg/mL (RTG = 54%), while no relevant toxicity was seen over the remaining dose levels tested.
Mutation results
No statistically significant increase in mutant frequency was observed at any concentration, in any experiment, in the absence or presence of S9 metabolism.
A very small percentage of chemicals are uniquely positive using the 24 hour treatment; moreover the longer treatment becomes necessary when a chemical’s insolubility precludes testing at adequate level of cytotoxicity. Since no precipitation of the test item in the treatment mixture was noted and adequate levels of cytotoxicity were achieved both in the absence and presence of S9 metabolism, data obtained were considered sufficient to provide evidence of negative results.
For the negative and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive control.

Conclusions:

The test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 490, mouse lymphoma L5178Y cells cultured in vitro were exposed 4 hours to the test item (47.2% solid content) at following concentrations:

Main Assay I (-S9, 3 hour treatment): 150, 125, 104, 86.8, 72.3 and 60.3 μg/mL.

Main Assay I (+S9, 3 hour treatment): 600, 480, 384, 307, 246 and 197 μg/mL.

Main Assay II (+S9, 3 hour treatment): 480, 440, 400, 367, 333 and 305 μg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to little or no cytotoxicity, were observed in the absence of S9 metabolism. In its presence, test item treatments did not meet appropriate cytotoxicity levels reducing relative total growth (RTG) to 57% at the highest analysable concentration. Based on these results, a second experiment (Main Assay 2) was performed in the presence of S9 metabolism.

The selection of the concentrations used in the main experiment were based on data from a pre-experiment. No precipitation of the test item was noted any experiment and growth inhibition was observed in the main experiment without and with metabolic activation.

The relative total growth (RTG) was 20% (without metabolic activation) and 57% and 26% (with metabolic activation) for the highest concentrations evaluated. The positive controls showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. Furthermore, in the main experiment without and with metabolic activation, all validity criteria were met.

No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. It is concluded that HH-2015-623 does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity data.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The genotoxic potential of the test item was assayed in three OECD guideline studies (471, 487 and 490) using a read-across approach. The substance did not show genotoxicity in any of the studies, and classification is not warranted.