Sophorolipids: fermentation products of glucose and rapeseed-oil fatty acids methyl esters with yeast Candida Bombicola

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genotoxicity studies were conducted with the test substance: OECD 471, OECD 476 and OECD 487, all performed under GLP conditions.

These studies were all negative for genotoxicity (mutagenicity and clastogenicity).

Link to relevant study records

Reference

Reference 1

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:

2003

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella thiphymurium TA1535 --> His G 46 --> additional mutations: rfa uvrB
Salmonella thiphymurium TA100 --> His G 46 --> additional mutations: rfa uvrB pKM 101
Salmonella thiphymurium TA102 --> His G 428 (pAQ1) --> additional mutations: rfa pKM 101
Salmonella thiphymurium TA1537 --> His C 3076 --> additional mutations: rfa uvrB
Salmonella thiphymurium TA98 --> His D 3052 --> additional mutations: rfa uvrB pKM 101

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix - liver microsomal fraction (S9) of rats

Test concentrations with justification for top dose:

Based on the toxicity showed in the preliminary studies the doses were chosen as follows:
1st experiment with and without S9 mix: from 312.5 to 5000µg/plate, idem for TA102 in the 2nd one
2nd experiment: from 156.25 to 2500µg/plate

Vehicle / solvent:

The tested substance sopholiance was dissolved in DMSO (dimethylsulfoxide).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Remarks:

5 known mutagens tested to check sensitivity

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-anthramine

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The tested substance sopholiance does not show any mutagenic activity in the bacterial reverse mutation test with Salmonella Typhimurium

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Mouse lymphoma

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Lyophilized S-9 mix (MutazymeTM) reconstituted with
purified water to provide a 10% S-9 mix.
Added to the cell cultures at 10% v/v.

Test concentrations with justification for top dose:

Due to the potentially cytotoxic nature of the
test article, eight concentrations separated by two fold intervals ranging down from
the solubility limit, 10 mM or 2000 μg/mL will be used in the cytotoxicity
Range-Finder.

Vehicle / solvent:

Vehicle controls will comprise treatments with the chosen
vehicle diluted to the same extent as the test article
solutions.
The preferred vehicles are water or dimethyl sulphoxide
(DMSO). If other vehicles or volumes of the organic
vehicles exceeding 1% (v/v) need to be used, the effects on
mutant frequencies may need to be checked. The choice of
vehicle will be confirmed in the raw data.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

Untreated Controls Cultures treated with culture medium alone will act as untreated controls (UTC). UTC will be included in this study only if the chosen vehicle is not commonly used in this laboratory (at the discretion of the Study Director).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The substance tested did not show any mutagenic result in the test conditions

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

human peripheral blood lymphocytes

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Cytokinesis block (if used):

Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 μg/mL concentration to block cytokinesis.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay (A1), the doses tested ranged from 0.5 to 5000 μg/mL, which was the limit dose for a substance of unknown or variable composition. Cytotoxicity [≥ 55 ± 5% reduction in cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 500 μg/mL in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at 5000 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 92.4 to 425 μg/mL for the non-activated 4 and 24-hour exposure groups; and from 132 to 500 μg/mL for the S9-activated 4-hour exposure group.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

other: water

Evaluation criteria:

Evaluation of Test Results
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
BioReliance Study No. AG19AR.348REACH.BTL 15
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

These results indicate Sophorolipids was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Executive summary:

The test substance, Sophorolipids, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay (A1), the doses tested ranged from 0.5 to 5000 μg/mL, which was the limit dose for a substance of unknown or variable composition. Cytotoxicity [≥ 55 ± 5% reduction in cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 500 μg/mL in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at 5000 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 92.4 to 425 μg/mL for the non-activated 4 and 24-hour exposure groups; and from 132 to 500 μg/mL for the S9-activated 4-hour exposure group.

In the initial micronucleus assay (B1), cytotoxicity (≥ 55 ± 5% reduction in CBPI relative to the vehicle control) was observed at doses ≥ 222 μg/mL in the non-activated 4-hour exposure group and at doses ≥ 361 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose in any of the exposure groups. In the non-activated 24-hour exposure group, due to lack of requisite cytotoxicity, the micronucleus assay was repeated at doses ranging from 59 to 200 μg/mL.

In the repeat assay (B2), cytotoxicity (≥ 55 ± 5% reduction in CBPI relative to the vehicle control) was observed at doses ≥ 162 μg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose.

The doses selected for evaluation of micronuclei were 92.4, 132, and 261 μg/mL for the non-activated 4-hour exposure group; 132, 261, and 425 μg/mL for the S9-activated 4-hour exposure group; and 59, 131, and 162 μg/mL for the non-activated 24-hour exposure group.

In the non-activated 4 and 24-hour exposure groups, neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.

In the S9-activated 4-hour exposure group, no statistically significant increases in micronuclei induction were observed at any dose (p > 0.05; Fisher’s Exact test). The results were within the 95% control limit of the historical negative control data. However, the Cochran-Armitage test was positive for a dose response increase (p ≤ 0.05). Since the increased induction of micronuclei was not significant and was within the 95% control limit of the historical negative control data, it was considered not biologically relevant.

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

Given the lack of genotoxic effects of the test substance in the three in vitro assays, no classification for mutagenicity is warranted.