Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenicity of the test item was observed in a reliable in vitro gene mutation test in bacteria.

The test item did also not induce gene mutations in a reliable study using CHO cells in vitro with and without metabolic activation.

It was also negative in a reliable clastogenicity test in human lymphocytes in vitro with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The histidine dependent strains are derived from S. typhimurium strain LT2 throught a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.
When summarised the mutations on the TA strains and the E. coli strain, used in this study can be described as follows:

SALMONELLA TYPHIMURIUM:
-Strain TA 1537; Genotype: his C 3076; rfa-; uvrB-: Type of mutations indicated: frame shift mutations
-Strain TA 98; Genotype: his D 3052; rfa-; uvrB-; R-factor Type of mutations indicated: frame shift mutations
-Strain TA 1535; Genotype: his G 46; rfa-; uvrB-: Type of mutations indicated: base-pair substitutions
-Strain TA 100; Genotype: his G 46; rfa-; uvrB-; R-factor Type of mutations indicated: base-pair substitutions

ESCHERICHIA COLI
-Strain WP2 uvrA; Genotype: trp-; uvrA-: Escherichia coli: base-pair substitutions and others

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Test concentrations with justification for top dose:

In the pre-experiment the concentration rnge of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
The test item precipitated in the overlay agar in experiment I with and without metabolic activation at 5000, except in strain TA 1537 (without metabolic activation) and WP2 uvrA with metabolic activation precipitation was observed at 2500 - 5000 µg/plate.
The undissolved particles had no influence on the data recording.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Negative Controls: Concurrent untreated and solvent controls were performed.

S9 (Preparation by RCC-CCR): Phenobarbital/beta-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i. p. (Desitin; D-22335 Hamburg) and beta-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution followed by centrifugation at 9000 g. Aliquotes of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week.
The protein concentration in the S9 preparation was 34.5 mg/mL (lot no. R 300404).

S9 Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Rationale for test conditions:

The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.

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:

cytotoxicity

Remarks:

at 2500 - 5000 µg/plate with and without S9 mix in Experiment I and at 33 - 1000 µg/plate without S9 mix and 2500 µg/plate with S 9 mix in Experiment II

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:

cytotoxicity

Remarks:

at 5000 µg/plate with S9 mix in Experiment I and II

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:

cytotoxicity

Remarks:

at 1000 - 5000 µg/plate without S9 mix and at 2500 - 5000 µg/plate with S9 mix in Experiment I, at 333 - 5000 µg/plate without S9 mix and 2500 - 5000 µg/plate with S9 mix in Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix in Experiment I

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test item was assessed for its poetntail to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in three independent experiments two with and without liver microsomal activation. A third experiment had to be performed, since in experiment I with metabolic activation in strain TA 1537 no bacteria were applicated onto the plates (reported as part of experiment I). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	5000	/	/
TA 1537	2500 - 5000	/	1000	2500 - 5000
TA 98	5000	2500 - 5000	5000	2500 - 5000
TA 100	333 - 5000	1000 - 5000	1000 - 5000	/
WP2 uvrA	5000	1000 - 5000	/	/

/ = no reduced background growth

Toxic effects, evident as a reduction in the number of revertants, were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	2500 - 5000	2500 - 5000	33 - 1000	2500
TA 98	/	5000	/	5000
TA 100	1000 - 5000	2500 - 5000	333 - 5000	2500 - 5000
WP2 uvrA	5000	5000	/	/

/ = no toxic effects evident as a reduction in the number of revertants

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In strain WP2 uvrA of the second experiment without metabolic activation the historical range of positive controls was just not reached. This minor effect was judged to represent fluctuations. The threshold of two times the corresponding solvent control was exceeded by far (factor of 6.5), so the test was considered valid.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in three independent experiments two with and without liver microsomal activation. A third experiment had to be performed, since in experiment I with metabolic activation in strain TA 1537 no bacteria were applicated onto the plates (reported as part of experiment I). Each concetration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	5000	/	/
TA 1537	2500 - 5000	/	1000	2500 - 5000
TA 98	5000	2500 - 5000	5000	2500 - 5000
TA 100	333 - 5000	1000 - 5000	1000 - 5000	/
WP2 uvrA	5000	1000 - 5000	/	/

/ = no reduced background level

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations in strains TA 1537, TA 98, 100 and WP2 uvrA.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, meither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2016 to 25 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

In accordance with the OECD guidelines for testing of chemicals, No. 473, “In Vitro Mammalian Chromosome Aberration Test” adopted on 26 September 2014.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: ESD0021912
- Expiration date of the batch: 17.03.2020
- Purity test date: 100% [w/w]

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29 °C)

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colchicine

Metabolic activation:

with

Metabolic activation system:

Rat

Test concentrations with justification for top dose:

Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosome aberration test. Top dose: 0.25 mg/mL

Vehicle / solvent:

Dimethylsulfoxide (DMSO)- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item soluble in DMSO at 200 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3-6 hours and 20-24 hours

NUMBER OF REPLICATIONS: Three concentratrion of test item, each concentrations in Duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellet was mixed with 5 mL of freshly prepared 0.56% warm Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later centrifuged at 1800 to 2000 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 2 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cells were incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 2 mL of cold acetic acid:methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator before use.
Slides were stained using 5% Giemsa stain for 20 minutes.


NUMBER OF CELLS EVALUATED: 150 metaphase plates for each replicate

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 metaphase

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:

Rationale for test conditions:

OECD guidelines for testing of chemicals, No. 473, “In Vitro Mammalian Chromosome Aberration Test”

Evaluation criteria:

• All slides including vehicle control, treatment and positive controls of chromosome aberration test were coded before evaluation.
• The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate.
• Cytotoxicity was determined by calculating percentage reduction in mitotic index (%) for all treated and vehicle control cultures were determined.
• Gaps were recorded separately and reported but generally not included in the total aberration frequency.
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control. The increase is dose-related when evaluated with an appropriate trend test

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p < 0.05).

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Water solubility: No
- Precipitation: precipitation was observed at 0.5, 1.0 and 2.0 mg/mL

Remarks on result:

other: non-clastogenic

TABLE 1.           SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST

 

Set No.	Treatment	Dose (mg/mL)	Mitotic Index		Mean Mitotic Index	Mean Percentage Mitotic Index	Percentage Reduction in Mitotic Index
			Replicate 1	Replicate 2
Set 1 (+S9)  (3-6 hours)	Vehicle control	-	0.0533	0.0499	0.0516	5.16	-
	Test item [Hostastat FE 20 LIQ]	0.0625	0.0455	0.0431	0.0443	4.43	14.03
		0.125	0.0431	0.0406	0.0419	4.19	18.81
		0.25	0.0394	0.0374	0.0384	3.84	25.50
Set2 (-S9) (3-6 hours)	Vehicle control	-	0.0544	0.0518	0.0531	5.31	-
	Test item [Hostastat FE 20 LIQ]	0.0625	0.0438	0.0439	0.0439	4.39	17.36
		0.125	0.0446	0.0418	0.0432	4.32	18.61
		0.25	0.0400	0.0415	0.0408	4.08	23.23
Set 3 (-S9) (20-24 hours)	Vehicle control	-	0.0504	0.0524	0.0514	5.14	-
	Test item [Hostastat FE 20 LIQ]	0.0625	0.0438	0.0456	0.0447	4.47	12.99
		0.125	0.0411	0.0423	0.0417	4.17	18.88
		0.25	0.0394	0.0375	0.0385	3.85	25.11

+S9: With metabolic activation; -S9: Without metabolic activation  

TABLE 2.     SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

                                                                                              

Set No.	Treatment	Dose (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 1 (+S9) (3-6 hours)	Vehicle control	-	3.89	-	1.0	1.0	1.0	0.7
	Positive Control (Cyclophosphamide)	10 µg/mL	3.17	22.80	13.0	11.0	10.5	7.0*
	Test item  [Hostastat FE 20 LIQ]	0.0625	3.44	14.39	2.5	1.5	1.5	1.0
		0.125	3.28	19.29	1.5	1.0	1.0	0.7
		0.25	3.08	25.73	3.5	2.5	2.5	1.7

MI: Mitotic Index; *: Statistically significant;+S9: With metabolic activation

 

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

                                                                                   

Set No.	Treatment	Dose (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 2 (-S9) (3-6 hours)	Vehicle control	-	4.05	-	1.0	1.0	1.0	0.7
	Positive Control (Mitomycin-C)	0.05 µg/mL	3.26	24.42	13.0	10.5	10.5	7.0*
	Test item [Hostastat FE 20 LIQ]	0.0625	3.48	17.66	2.5	1.5	1.5	1.0
		0.125	3.42	19.44	2.5	1.5	1.5	1.0
		0.25	3.26	24.39	2.5	2.0	2.0	1.3

MI: Mitotic Index; *: Statistically significant;-S9: Without metabolic activation

 

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

Set No.	Treatment	Dose (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 3 (-S9) (20-24 hours)	Vehicle control	-	4.26	-	1.0	1.0	1.0	0.7
	Positive Control (Mitomycin-C)	0.05 µg/mL	3.26	30.71	14.0	11.0	10.0	6.7*
	Test item [Hostastat FE 20 LIQ]	0.0625	3.69	17.67	2.0	1.5	1.5	1.0
		0.125	3.61	19.96	2.5	1.0	1.0	0.7
		0.25	3.39	26.82	2.5	2.0	2.0	1.3

MI: Mitotic Index; *: Statistically significant;-S9: Without metabolic activation

 

Conclusions:

Based on the results obtained, the test item, Hostastat FE 20 LIQ is considered as non-clastogenic at and up to the dose of 0.25 mg/mL, both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item,Hostastat FE 20 LIQ, wasevaluated for chromosomal aberration in human lymphocytes, as per the OECD guideline for the testing of chemicals,No. 473 “In vitroMammalian Chromosome AberrationTest” adopted on26^thSeptember2014.

Test item was soluble in DMSO at 200 mg/mL. Slight precipitation was observed at 0.25 mg/mL and thereafter precipitation was observed at the higher doses up to 2 mg/mL. No change in pH was observed at any of the concentrations tested up to 2 mg/mL. As there was slight precipitation at 0.25 mg/mL, the same was selected as the highest concentration for testing in the initial cytotoxicity test.

In the initial cytotoxicity test, the test item resulted in not greater than  25.50% reduction of Mitotic Index (MI) at the concentrations of0.0625, 0.125 and 0.25mg/mL both at short term (in the presence and absence of metabolic activation) and long term treatments (in the absence of metabolic activation). Hence, the above indicated concentrations were selected for the chromosome aberration test.

In the chromosomal aberration test, the cells were treated withthe test itemat the doses of0.0625, 0.125 and 0.25mg/mL as low, mid and high doses respectivelyusing DMSO as the vehicle. The treatment was carried out in duplicates for short term period (3 to 6 hours) both in the presence and absence of metabolic activation and long term (20 to 24 hours) in the absence of metabolic activation.

Concentration of 10 µg/mL ofCyclophosphamide Monohydrate (+S9) and 0.05µg/mLof Mitomycin-C (-S9 both for short term and long term) were used as positive controls. Cells were arrested at metaphase using 0.3 µg/mL of colchicine. The cells were harvested between 2 to 3 hours of cell cycle arrest and slides were prepared and stained using 5% Giemsa stain.

The results indicatedno statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentrations tested.The mean percentage reduction in mitotic index observed at 0.25 mg/mL, the highest concentration tested was 25.73 in the presence of metabolic activation and 24.39 and 26.82 in the absence of metabolic activation for short and long term treatments, respectively.

The respective positive controls tested induced 6.7% to 7.0% of mean aberrated cells which was statistically significant. The mean percentage reduction in mitotic index was in the range of 22.80 to 30.71 when compared with the respective vehicle controls.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2016 to 01 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

OECD guidelines for testing of chemicals, No. 476, “In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes” adopted on 28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Gene mutation test in mammalian cells in vitro

Target gene:

HPRT

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

CHO AA8 cells

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 homogenate

Test concentrations with justification for top dose:

0.25 mg/mL, Based on the precipitation and pH test, 0.25 mg/mL was chosen as the highest dose for the initial cytotoxicity test and no cytotoxicity was observed in the initial cytotoxicity test up to 0.25 mg/mL tested, 0.25 mg/mL was chosen as the highest concentration for the gene mutation test.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

CHO AA8 cells, Batch No. 5000062 procured from American Type Culture Collection (ATCC)
and Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin)

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results.

Statistics:

Data of mutant frequencies was analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance.

Key result

Species / strain:

mammalian cell line, other: CHO AA8 cells

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:

The test item, Hostastat FE 20 LIQ resulted in mutant frequencies of 15.35 to 17.39 mutant colonies/106 cells in the presence of metabolic activation and 16.39 mutant colonies/106 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 15.12 to 16.91 mutant colonies/106 cells were observed with 15.89 mutant colonies/106 cells in the vehicle control. There was no concentrated related or statistically significant increase in the mutant frequencies when compared with vehicle control at any of the tested concentrations.

TABLE 1.           SUMMARY OF INITIAL CYTOTOXICITY TEST

Refer Appendix 1

Set No.		Treatment		Dose (mg/mL)		Average colony count		Adjusted Cloning Efficiency (ACE)		Relative Survival (%)
Set 1 +S9		Vehicle control		-		174.83		1.18		-
		Hostastat FE 20 LIQ		0.03125		164.83		1.08		91.21
			0.0625		163.00		1.02		86.30
			0.125		164.50		1.01		85.97
			0.25		164.17		0.98		83.00
Set 2 -S9	Vehicle control	-	171.83	1.13	-
	Hostastat FE 20 LIQ	0.03125	171.00	1.11	97.99
		0.0625	167.67	1.07	95.18
		0.125	168.83	1.05	92.84
		0.25	166.00	1.02	90.69

 +S9: with metabolic activation; -S9: without metabolic activation

TABLE 1.           SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST

                                                                                              Refer Appendix2

Set No.	Treatment	Dose (mg/mL)	Average colony count	Adjusted Cloning Efficiency (ACE)	Relative Survival (%)
Set 1 +S9	Vehicle control	-	179.67	1.14	-
	Hostastat FE 20 LIQ	0.03125	174.50	1.10	96.79
		0.0625	174.33	1.08	94.72
		0.125	168.00	1.02	89.70
		0.25	165.50	0.99	87.43
	Benzo(a)pyrene	0.003	148.17	0.79	69.35
Set 2 -S9	Vehicle control	-	176.83	1.12	-
	Hostastat FE 20 LIQ	0.03125	175.67	1.10	98.34
		0.0625	175.67	1.10	99.00
		0.125	166.50	1.00	89.67
		0.25	165.00	0.98	88.23
	4 Nitroquinoline 1-oxide	0.001	150.50	0.81	72.67

 +S9: with metabolic activation; -S9: without metabolic activation.

TABLE 1.           SUMMARY OF GENE MUTATION TEST

Refer Appendix 3

Set No.	Treatment	Dose (mg/mL)	Average colony count	Cloning Efficiency	Average Mutant Colonies/ 10^6cells	Mutant Frequency/ 10^6cells
Set 1 +S9	Vehicle control	-	183.00	0.92	15.0	16.39
	Hostastat FE 20 LIQ	0.03125	172.50	0.86	14.0	16.22
		0.0625	172.50	0.86	15.0	17.39
		0.125	169.17	0.85	13.0	15.35
		0.25	168.17	0.84	13.0	15.45
	Benzo(a)pyrene	0.003	165.33	0.83	115.5	140.27*
Set 2 -S9	Vehicle control	-	188.67	0.94	15.0	15.89
	Hostastat FE 20 LIQ	0.03125	182.83	0.91	14.0	15.30
		0.0625	171.83	0.86	13.0	15.12
		0.125	164.83	0.82	13.0	15.80
		0.25	165.67	0.83	14.0	16.91
	4 Nitroquinoline 1-oxide	0.001	160.00	0.80	114.0	142.49*

  +S9: with metabolic activation; -S9: without metabolic activation; *: Statistically significant  

APPENDIX 1.INDIVIDUAL DATA OF INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Dose (mg/mL)	Replicate	No. of Colonies
				Dish 1	Dish 2	Dish 3
Set 1 +S9	Vehicle control	-	1	177	169	180
			2	169	175	179
	Hostastat FE 20 LIQ	0.03125	1	168	159	165
			2	168	163	166
		0.0625	1	163	168	161
			2	162	165	159
		0.125	1	163	169	162
			2	164	166	163
		0.25	1	168	163	159
			2	166	167	162
Set 2 -S9	Vehicle control	-	1	176	173	168
			2	169	166	179
	Hostastat FE 20 LIQ	0.03125	1	171	176	170
			2	168	168	173
		0.0625	1	167	165	170
			2	171	164	169
		0.125	1	170	165	172
			2	166	171	169
		0.25	1	165	164	169
			2	168	167	163

+S9: with metabolic activation; -S9: without metabolic activation.   

(Contd..,). INDIVIDUAL DATA OF INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Dose (mg/mL)		Replicate	Cell count/ flask	Average Cell count
Set 1 +S9	End of treatment	Vehicle Control		1	4175000	4200000
				2	4225000
		Hostastat FE 20 LIQ	0.03125	1	3975000	4062500
				2	4150000
			0.0625	1	3900000	3887500
				2	3875000
			0.125	1	3825000	3837500
				2	3850000
			0.25	1	3750000	3712500
				2	3675000
Set 1 -S9	End of treatment	Vehicle Control		1	4125000	4087500
				2	4050000
		Hostastat FE 20 LIQ	0.03125	1	4025000	4025000
				2	4025000
			0.0625	1	4025000	3987500
				2	3950000
			0.125	1	3850000	3862500
				2	3875000
			0.25	1	3800000	3837500
				2	3875000

 

Beginning of treatment	Replicate	Cell count/ flask	Average Cell count
	1	3100000	3112500
	2	3125000

 

APPENDIX 1.  INDIVIDUAL DATA OF PARALLEL CYTOTOXICITY TEST

Set No.	Treatment	Dose (mg/mL)	Replicate	No. of Colonies
				Dish 1	Dish 2	Dish 3
Set 1 +S9	Vehicle control	-	1	180	182	178
			2	176	179	183
	Hostastat FE 20 LIQ	0.03125	1	177	169	181
			2	169	170	181
		0.0625	1	177	176	173
			2	176	174	170
		0.125	1	169	175	164
			2	165	169	166
		0.25	1	167	164	163
			2	170	165	164
	Benzo(a)pyrene	0.003	1	156	149	146
			2	147	143	148
Set 2 -S9	Vehicle control	-	1	177	174	180
			2	182	169	179
	Hostastat FE 20 LIQ	0.03125	1	175	178	172
			2	176	179	174
		0.0625	1	172	176	178
			2	176	173	179
		0.125	1	170	169	164
			2	168	166	162
		0.25	1	166	167	163
			2	168	164	162
	4 Nitroquinoline 1-oxide	0.001	1	150	142	154
			2	155	150	152

+S9: with metabolic activation; -S9: without metabolic activation.   

 

(Contd..,). INDIVIDUAL DATA OF PARALLEL CYTOTOXICITY TEST

Set No.	Treatment	Dose (mg/mL)		Replicate	Cell count/ flask	Average Cell count
Set 1 +S9	End of treatment	Vehicle Control		1	3650000	3687500
				2	3725000
		Hostastat FE 20 LIQ	0.03125	1	3625000	3675000
				2	3725000
			0.0625	1	3550000	3600000
				2	3650000
			0.125	1	3500000	3537500
				2	3575000
			0.25	1	3550000	3500000
				2	3450000
		Positive Control		1	3175000	3100000
				2	3025000
Set 1 -S9	End of treatment	Vehicle Control		1	3675000	3675000
				2	3675000
		Hostastat FE 20 LIQ	0.03125	1	3550000	3637500
				2	3725000
			0.0625	1	3700000	3662500
				2	3625000
			0.125	1	3475000	3500000
				2	3525000
			0.25	1	3425000	3475000
				2	3525000
		Positive Control		1	3100000	3137500
				2	3175000

 

Beginning of the treatment	Replicate	Cell count/ flask	Average Cell count
	1	2950000	2912500
	2	2875000

APPENDIX 1.INDIVIDUAL DATA OF GENE MUTATION TEST

Set No.	Treatment	Dose (mg/mL)	No. of Colonies			No. of Mutant Colonies
			Replicate 1	Replicate 2	Replicate 3	Replicate 1	Replicate 2	Replicate 3	Replicate 4	Replicate 5
Set 1 +S9	Vehicle control	-	183	192	177	2	5	1	4	3
			169	185	192	3	0	5	5	2
	Hostastat FE 20 LIQ	0.03125	178	175	172	2	0	5	4	4
			169	169	172	1	2	2	3	5
		0.0625	175	175	168	3	2	2	5	3
			169	166	182	2	0	5	5	3
		0.125	175	162	175	0	5	2	4	4
			175	166	162	2	3	2	0	4
		0.25	159	175	166	3	0	3	2	4
			166	178	165	3	4	4	0	3
	Benzo(a)pyrene	0.003	149	158	166	24	28	25	21	22
			178	169	172	23	21	19	26	22
Set 2 -S9	Vehicle control	-	189	196	189	3	4	4	2	3
			178	192	188	3	2	1	4	4
	Hostastat FE 20 LIQ	0.03125	182	169	188	3	2	2	5	1
			179	187	192	3	2	4	1	5
		0.0625	166	178	178	2	3	5	0	4
			169	175	165	2	1	2	4	3
		0.125	162	159	168	2	2	2	5	4
			166	168	166	0	2	2	3	4
		0.25	163	163	169	4	4	2	2	3
			169	168	162	2	2	2	4	3
	4 Nitroquinoline 1-oxide	0.001	159	158	166	26	21	22	22	25
			166	158	153	23	21	18	22	28

+S9: with metabolic activation; -S9: without metabolic activation.   

 

 

 

                                                                           

                                                                           

APPENDIX 2.  TEST ITEM AND POSITIVE CONTROL DILUTION PREPARATION FORGENE MUTATION TEST

Test Item	Quantity of Test Item	Concentration of Test Item Stock (mg/mL)	Volume of Stock (µL)	Volume of DMSO (µL)	Concentration of Test Item Dilution (mg/mL)	Treatment dose level (mg/mL)
Hostastat FE 20 LIQ	25 mg	-	-	1000	25	0.25
	-	25	500	500	12.5	0.125
		12.5	500	500	6.25	0.0625
		6.25	500	500	3.125	0.03125
Positive Control	Quantity of Positive Control	Concentration of Stock (mg/mL)	Volume of Stock (µL)	Volume of DMSO (µL)	Concentration of positive control dilution (mg/mL)	Treatment dose level (mg/mL)
	2 mg	-	-	2000	1000	-
Benzo(a) Pyrene	-	1	300	700	300	3
	2 mg	-	-	2000	1000	-
4 Nitroquinoline 1-oxide	-	1	100	900	100	1

Note: 50 µL of each test item dilutions, positive and vehicle control were added to each flask to a final volume of 5 mL.

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.25 mg/mL, both in the presence and absence of metabolic activation under the laboratory conditions tested.

Executive summary:

The test item was evaluated for gene mutation in CHO AA8 cells, as per the OECD guideline for the testing of chemicals, No. 476 “In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes” adopted on 28 July 2015. Based on the results of solubility, pH and precipitation test, the test concentrations of 0.03125, 0.0625, 0.125 and 0.25 mg/mL were tested in the initial cytotoxicity test using DMSO as the vehicle both in the presence and absence of metabolic activation (3 to 6 hours). In the initial cytotoxicity test, the Relative Survival of the tested concentrations up to 2 mg/mL was more than 20%, when compared with the respective vehicle controls, both in the presence and absence of metabolic activation. Hence, concentrations of 0.03125, 0.0625, 0.125 and 0.25 mg/mL were selected for the gene mutation test. In the gene mutation test, the cells were treated with test item, at four concentrations using DMSO as a vehicle in duplicates in the presence and absence of metabolic activation (3 to 6 hours). Positive controls, 3 µg/mL of Benzo(a)pyrene (+S9) and 1 µg/mL of 4 Nitroquinoline 1- oxide (-S9) were used for the gene mutation test. Cytotoxicity of the test item and positive controls was assessed by the determining the Adjusted Cloning Efficiency (ACE) and Relative Survival (RS). The test item, Hostastat FE 20 LIQ resulted in mutant frequencies of 15.35 to 17.39 mutant colonies/106 cells in the presence of metabolic activation at the different test concentrations tested and 16.39 mutant colonies/106 cells in the vehicle control. In the absence of metabolic activation, test item resulted in mutant frequencies of 15.12 to 16.91 mutant colonies/106 cells and 15.89 mutant colonies/106 cells in the vehicle control. There was no concentration related or statistically significant increase in the mutant frequencies at any of the test item concentrations when compared with vehicle control. Positive controls resulted in mutant frequencies of 140.27 to 142.49 mutant colonies/106 cells in the presence and absence of metabolic activation, respectively and were statistically significant when compared with the respective vehicle controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the possibly observed effects are regarded as relevant for humans.

Additional information

In the bacterial test, a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA in concentrations up to 5000 µg/plate with and without metabolic activation. No increase in revertants was observed. The study is GLP-compliant and was performed according to OECD guideline 471.

The test item was evaluated for gene mutation in CHO AA8 cells. Based on initial cytotoxicity testing, concentrations of 0.03125, 0.0625, 0.125 and 0.25 mg/mL were selected for the main test.

There was no concentration related or statistically significant increase in the mutant frequencies at any of the test item concentrations with and without metabolic activation. The study is GLP-compliant and was performed according to OECD guideline 476.

Clastogenicity in vitro was examined in a chromosomal aberration test with human lymphocytes. Based on a initial cyctotoxicity test, concentrations of 0.0625, 0.125 and 0.25mg/mL were tested for a short term period (3 to 6 hours) both in the presence and absence of metabolic activation and a long term period (20 to 24 hours) in the absence of metabolic activation. The results indicated no statistically significant increase in the number of aberrated cells at any of the concentrations tested. The study is GLP-compliant and was performed according to OECD guideline 473.

Justification for classification or non-classification

As no effects were observed in three reliable in vitro genotoxicity tests, no classification is required according to Regulation (EC) No. 1272/2008 (CLP).