acetylxylan esterase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic activity of acetylxylan esterase has not been determined but another enzyme (Lipase, IUBMB 3.1.1.3) of comparable genotoxic profile has been analyzed and used as a source substance for read-across.

Lipase did not show mutagenic activity in the Ames assay and did not induce chromosomal aberrations in the in vitro mammalian chromosome aberration test performed with human lymphocytes. These results are also supported by an in vitro gene mutation studies in L5178Y mouse lymphoma cells.

Therefore, acetylxylan esterase is considered to be non mutagenic.

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:

other information

Justification for type of information:

According to ECHA Guidance Chapter R 7a: Endpoint specific guidance (version 6.0, July 2017), the following studies on genetic toxicity are required: In vitro gene mutation study in bacteria and one of the following, in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study. In case these studies are both negative, an in vitro gene mutation study in mammalian cells is requested in addition.
The present target substance, Acetylxylan esterase IUBMB 3.1.1.72, has not been tested but another enzyme (Lipase, IUBMB 3.1.1.3) has been investigated in the Ames test and in the in vitro chromosome aberration test. Both tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. These results are also supported by read-across from an in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on lipase.
The conclusion is that the target substance Acetylxylan esterase IUBMB 3.1.1.72 is considered to be non-mutagenic.
Justification for read-across: see attached justification.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as Experiment I. Since no relevant toxic effects were observed, 5000 µg/plate was chosen as maximal concentration. Due to observed minor contamination at higher concentrations eight concentrations were tested in Experiment II.

The concentration range included two logarithmic decades. The following concentrations (based on the total protein for this test item) were tested in Experiment II: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate.

Vehicle / solvent:

Deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine: TA 1537 and TA 98 without S9 Aminoanthracene (2-AA): TA 98, TA 100, TA 1535, TA 1537, and E. coli

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10E8-10E9 cells/mL

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose, both with and without metabolic activation, and the same number of plates for the controls

DETERMINATION OF CYTOTOXICITY: Performed between 3 to 5000 µg/plate

Evaluation criteria:

The assay is considered to be valid if all of the following criteria are met:
• The negative and positive control data are consistent with the historical control data for the testing laboratory
• The positive control data show marked increases over the concurrent negative control values
• The evaluation of the test data is not restricted by the loss of plates (e.g., through contamination).

A test item is considered as a mutagen if all of the following criteria are met:
• A biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
• A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
• The increases are reproducible between replicate plates
• An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

In Experiment I, a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 at 5000 µg/plate with S9 mix.

Vehicle controls validity:

valid

Untreated 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertanst (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA98 at 5000 µg/plate with S9 mix.
No substantial increase in revertant colony numbers of any of the five testerr strains was observed following treatment with the test substance 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 experiment I with S09 mix, the data in the negative control of strian WP2 uvrA were slightly above the test facility historical control range. Since the deviation is rather small, the effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Table 1a - Summary of Results Pre-Experiment and Experiment I (Without activation)

 

                                                         Revertant Colony Counts (Mean ±SD)

 

Substance	µg/plate	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Vehicle		29 ± 2	155 ± 3	22 ± 3	12 ± 3	69 ± 7
Untreated		35 ± 6	138 ± 10	19 ± 8	11 ± 5	68 ± 4
TS	3	32 ± 3	155 ± 8	18 ± 10	13 ± 4	77 ± 5
TS	10	35 ± 7	145 ± 2	22 ± 3	14 ± 1	80 ± 5
TS	33	36 ± 1	139 ± 7	23 ± 4	12 ± 2	73 ± 4
TS	100	37 ± 7	155 ± 19	23 ± 6	13 ± 3	72 ± 9
TS	333	29 ± 1	150 ± 2	20 ± 3	15 ± 3	71 ± 6
TS	1000	38 ± 7	154 ± 8	21 ± 4	13 ± 6	65 ± 8
TS	2500	21 ± 4*	111 ± 8*	15 ± 4*	7 ± 2*	53 ± 5*
TS	5000	14 ± 1*	106 ± 5*	16 ± 2*	7 ± 3*	55 ± 6*
NaN3	10		2161 ± 7	2141 ± 41
4-NOPD	10	306 ± 13
4-NOPD	50				94 ± 3
MMS	3.0 µL					1541 ± 141

 

TS = Test substance

* = Contaminated and Manual count

 

 

Table 1b - Summary of Results Pre-Experiment and Experiment I (With activation)

 

                                         Revertant Colony Counts (Mean ±SD)

 

Substance	µg/plate	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Vehicle	0	39 ± 6	170 ± 11	26 ± 7	12 ± 3	79 ± 6
Untreated		31 ± 5	172 ± 2	22 ± 6	16 ± 6	87 ± 6
TS	3	39 ± 6	164 ± 8	23 ± 9	15 ± 2	84 ± 11
TS	10	38 ± 3	146 ± 17	17 ± 4	13 ± 1	76 ± 5
TS	33	40 ± 3	167 ± 4	20 ± 1	13 ± 3	87 ± 7
TS	100	40 ± 2	161 ± 2	22 ± 3	14 ± 2	72 ± 8
TS	333	34 ± 2	161 ± 17	25 ± 8	11 ± 4	77 ± 8
TS	1000	38 ± 5	149 ± 9	21 ± 7	16 ± 3	76 ± 4
TS	2500	23 ± 4*	140 ± 7*	16 ± 5*	11 ± 1*	57 ± 7*
TS	5000	14 ± 4*	117 ± 6*	18 ± 4*	13 ± 2*	71 ± 7*
2-AA	2.5	1363 ± 198	1368 ± 100	189 ± 22	155 ± 12
2-AA	10					219 ± 3

 

TS = Test substance

* = Contaminated and Manual count

 

Table 2a - Summary of Experiment II (Without activation)

 

                                                              Revertant Colony Counts (Mean ±SD)

 

Substance	µg/plate	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Vehicle	0	34 ± 9	127 ± 13	21 ± 2	13 ± 4	46 ± 9
Untreated		33 ± 8	142 ± 10	28 ± 9	16 ± 2	52 ± 5
TS	3	30 ± 7	145 ± 15	29 ± 6	13 ± 3	52 ± 7
TS	10	34 ± 5	143 ± 15	20 ± 5	13 ± 8	48 ± 8
TS	33	36 ± 10	155 ± 32	23 ± 5	19 ± 7	55 ± 9
TS	100	44 ± 11	140 ± 8	24 ± 4	16 ± 6	53 ± 3
TS	333	29 ± 11	154 ± 33	29 ± 12	14 ± 1	48 ± 4
TS	1000	34 ± 5	145 ± 14	24 ± 3	11 ± 2	51 ± 2
TS	2500	31 ± 6	151 ± 17	31 ± 8	13 ± 4	67 ± 17
TS	5000	39 ± 9	158 ± 18	29 ± 1	17 ± 1	59 ± 7
NaN3	10		1766 ± 85	1615 ± 160
4-NOPD	10	354 ± 17
4-NOPD	50				114 ± 23
MMS	3.0µL					1453 ± 79

 

TS = Test substance

 

Table 2b - Summary of Experiment II (With activation)

 

                                       Revertant Colony Counts (Mean ±SD)

 

Substance	µg/plate	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Vehicle		40 ± 4	166 ± 8	38 ± 6	17 ± 5	63 ± 14
Untreated		37 ± 4	156 ± 4	30 ± 11	18 ± 7	68 ± 11
TS	3	42 ± 9	185 ± 27	38 ± 6	22 ± 4	67 ± 9
TS	10	34 ± 14	161 ± 17	31 ± 4	22 ± 2	62 ± 6
TS	33	43 ± 11	171 ± 1	28 ± 8	20 ± 3	68 ± 1
TS	100	37 ± 8	160 ± 2	39 ± 15	23 ± 4	71 ± 6
TS	333	45 ± 9	170 ± 15	42 ± 5	22 ± 10	61 ± 7
TS	1000	43 ± 6	167 ± 21	30 ± 6	24 ± 3	72 ± 14
TS	2500	46 ± 4	194 ± 26	43 ± 8	24 ± 3	83 ± 31
TS	5000	50 ± 8	169 ± 17	43 ± 1	24 ± 1	70 ± 3
2-AA	2.5	482 ± 20	828 ± 35	748 ± 95	127 ± 6
2-AA	10					314 ± 21

 

TS = Test substance

Conclusions:

The test 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 in accordance with OECD Test Guideline 471. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations (based on the total protein for this test item): Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 at 5000 µg/plate with S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance 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 and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosome aberration test in human lymphocytes

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a male donor (46 years old). Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hours after blood collection. If necessary, the blood was stored before use at 4°C.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9

Test concentrations with justification for top dose:

Experiment I (with and without S9): 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000 µg/mL
Experiment II (without S9): 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000 µg/mL
Experiment II (with S9): 533.1, 932.9, 1632.7, 2857.1, 5000 µg/mL

With respect to the current OECD Guideline 473 and protein content of the test item 5000 µg/mL was applied as the top concentration for the pre-test. No cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, the highest required concentration, 5000 µg/mL, was chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionized water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: In medium
DURATION
Exposure time 4 hours: About 72 hours after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm2 culture flasks. The culture medium was replaced with serum-free medium containing the test substance. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test substance was discarded and the cells were re-suspended in “saline G.” The washing procedure was repeated once. After washing the cells were re-suspended in complete culture medium and cultured until preparation.

Exposure time 22 hours (without S9 mix): About 73 hours after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm2 cell culture flasks. The culture medium was replaced with complete medium (with 10% FCS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.

All cultures were incubated at 37°C in a humidified atmosphere with 5.5% CO2 (94.5% air).

- Culture harvesting: Three hours before harvesting, colcemid was added to the cultured (final concentration of 0.2 μg/mL). The cultures were harvested by centrifugation 22 hours after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL of 0.0375 M KCl. The cell suspension was then allowed to stand at 37°C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least 2 slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa.

NUMBER OF REPLICATIONS: 2
SLIDE ANALYSIS: The slides were evaluated using NIKON microscopes with 100x oil emersion objectives. Breaks, fragments, deletions, exchanges, and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of aberration rates. 100 well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive controls in Experiment I with metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46±1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

The chromosomal aberration assay is considered acceptable if it met the following criteria:
The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range. The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

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:

No precipitation of the test substance in the culture medium was observed. No relevant increase in the osmolarity or pH value was observed (e.g. Exp. I: solvent control: 233 mOsm, pH 7.4 versus 278 mOsm and pH 7.4 at 5000 µg/mL. At both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carry structural chromosomal aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0-2.0% aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5-2.0% aberrant cells, excluding gaps) and within the range of the laboratory’s historical solvent control data (0.0-4.0% aberrant cells, excluding gaps). No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. In both experiments, either EMA or CPA were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table 1a: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment I; Without S9 mix)

 

Concentration (µg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent controls
Solvent control	4 hours	22 hours	12.4	100
32.5	4 hours	22 hours	Not determined	Not determined
56.8	4 hours	22 hours	Not determined	Not determined
99.5	4 hours	22 hours	Not determined	Not determined
174.1	4 hours	22 hours	Not determined	Not determined
304.6	4 hours	22 hours	Not determined	Not determined
533.1	4 hours	22 hours	14.7	118.1
932.9	4 hours	22 hours	13.8	111.3
1632.7	4 hours	22 hours	13.1	105.6
2857.1	4 hours	22 hours	13.4	108.1
5000	4 hours	22 hours	13.9	112.1

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

 

Table 1b: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment I; With S9 mix)

 

Concentration (µg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent controls
Solvent control	4 hours	22 hours	13.7	100
32.5	4 hours	22 hours	Not determined	Not determined
56.8	4 hours	22 hours	Not determined	Not determined
99.5	4 hours	22 hours	Not determined	Not determined
174.1	4 hours	22 hours	Not determined	Not determined
304.6	4 hours	22 hours	Not determined	Not determined
533.1	4 hours	22 hours	13.3	97.1
932.9	4 hours	22 hours	12.3	90.1
1632.7	4 hours	22 hours	14.2	104.0
2857.1	4 hours	22 hours	14.0	102.6
5000	4 hours	22 hours	15.4	112.5

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

 

 

Table 2a: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment II; Without S9 mix)

 

Concentration (µg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent controls
Solvent control	4 hours	22 hours	16.0	100
32.5	4 hours	22 hours	Not determined	Not determined
56.8	4 hours	22 hours	Not determined	Not determined
99.5	4 hours	22 hours	Not determined	Not determined
174.1	4 hours	22 hours	Not determined	Not determined
304.6	4 hours	22 hours	Not determined	Not determined
533.1	4 hours	22 hours	16.4	102.5
932.9	4 hours	22 hours	15.1	94.4
1632.7	4 hours	22 hours	15.1	94.4
2857.1	4 hours	22 hours	11.4	70.0
5000	4 hours	22 hours	11.4	71.3

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

 

Table 2b: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment II; With S9 mix)

 

Concentration (µg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent controls
Solvent control	4 hours	22 hours	14.4	100
533.1	4 hours	22 hours	13.9	96.5
932.9	4 hours	22 hours	15.0	104.2
1632.7	4 hours	22 hours	12.5	86.8
2857.1	4 hours	22 hours	14.4	100.3
5000	4 hours	22 hours	14.6	101.4

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

 

Table 3: Structural chromosome aberrations Experiment I; preparation interval 22 hours with S9 mix: exposure period 4 hours

 

                                                                                                   Aberrations

                                     % Aberrant cells            Gaps   Chromatid type Chromosome type Other

Slide #	Cells scored	Incl. gaps*	Excl. gaps*	With exchanges	g ig	b f d ex	ib if id cx	ma cd
Solvent control 1 2 1+2	100 100 200	2.0	1.5	0.0	1 0 0 0 1 0	1 0 0 0 1 0 0 0 2 0 0 0	0 0 0 0 0 1 0 0 0 1 0 0	0 0 0 0 0 0
Positive control: CPA 22.5 µg/mL 1 2 1+2	50** 50** 100	39.0	35.0	3.0	7 2 1 0 8 2	26 3 0 3 15 0 0 0 41 3 0 3	2 0 0 0 0 0 0 0 2 0 0 0	1 0 0 0 0 0
TS: 1632.7 µg/mL 1 2 1+2	100 100 200	1.5	1.5	0.0	0 0 0 0 0 0	2 0 0 0 1 0 0 0 3 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 2857.1 µg/mL 1 2 1+2	100 100 200	1.0	1.0	0.0	0 0 0 0 0 0	0 1 0 0 1 0 0 0 1 1 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 5000 µg/mL 1 2 1+2	100 100 200	1.0	0.5	0.0	0 0 1 0 1 0	1 0 0 0 0 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0

 

*  Inclusive cells carrying exchanges

** 50 metaphase plates per culture were evaluated due to strong clastogenic effects

TS = Test substance

 

Abbreviations

 

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso- fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

 

Table 4: Structural chromosome aberrations Experiment I; preparation interval 22 hours without S9 mix: exposure period 4 hours

                                                                                                                    Aberrations

                                  % Aberrant cells               Gaps   Chromatid type Chromosome type  Other

Slide #	Cells scored	Incl. gaps*	Excl. gaps*	With exchanges	g ig	b f d ex	ib if id cx	ma cd
Solvent control 1 2 1+2	100 100 200	2.5	2.0	0.0	0 0 1 0 1 0	1 1 0 0 2 0 0 0 3 1 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
Positive control (EMS 825 µg/mL) 1 2 1+2	100 100 200	12.0	11.5	1.0	1 0 1 0 2 0	9 2 0 2 8 0 0 0 17 2 0 2	0 3 0 0 1 0 0 0 1 3 0 0	0 0 0 0 0 0
TS: 1632.7 µg/mL 1 2 1+2	100 100 200	3.0	1.5	0.0	2 0 1 0 3 0	0 0 0 0 3 0 0 0 3 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 2857.1 µg/mL 1 2 1+2	100 100 200	0.0	0.0	0.0	0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 5000 µg/mL 1 2 1+2	100 100 200	0.5	0.5	0.0	0 0 0 0 0 0	1 0 0 0 0 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0

 

*  Inclusive cells carrying exchanges

 

Table 5: Structural chromosome aberrations Experiment II; preparation interval 22 hours without S9 mix: exposure period 22 hours

 

                                                                                                 Aberrations

                                 % Aberrant cells               Gaps     Chromatid type Chromosome type  Other

Slide #	Cells scored	Incl. gaps*	Excl. gaps*	With exchanges	g ig	b f d ex	ib if id cx	ma cd
Solvent control 1 2 1+2	100 100 200	0.5	0.5	0.0	0 0 0 0 0 0	0 0 0 0 1 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
Positive control (EMS 660.0 µg/mL) 1 2 1+2	100 100 200	13.0	10.0	2.0	3 0 8 0 11 0	10 0 0 1  9 0 0 3 19 0 0 4	0 1 0 0 0 1 0 0 0 2 0 0	0 0 0 0 0 0
TS: 1632.7 µg/mL 1 2 1+2	100 100 200	1.5	1.5	0.0	0 0 0 0 0 0	1 0 0 0 1 0 0 0 2 0 0 0	0 0 0 0 0 1 0 0 0 1 0 0	0 0 0 0 0 0
TS: 2857.1 µg/mL 1 2 1+2	100 100 200	2.5	2.0	0.0	0 0 1 0 1 0	0 0 0 0 4 0 0 0 4 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 5000 µg/mL 1 2 1+2	100 100 200	1.5	0.5	0.0	1 0 1 0 2 0	0 0 0 0 1 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0

 

*  Inclusive cells carrying exchanges

 

Table 6: Structural chromosome aberrations Experiment II; preparation interval 22 hours with S9 mix: exposure period 4 hours

 

                                                                                               Aberrations

                                  % Aberrant cells               Gaps   Chromatid type Chromosome type  Other

Slide #	Cells scored	Incl. gaps*	Excl. gaps*	With exchanges	g ig	b f d ex	ib if id cx	ma cd
Solvent control 1 2 1+2	100 100 200	1.0	1.0	0.0	0 0 0 0 0 0	1 0 0 0 1 0 0 0 2 0 0 0	0 0 0 0 0 1 0 0 0 1 0 0	0 0 0 0 0 0
Positive control (CPA 22.5 µg/mL) 1 2 1+2	100 100 200	14.0	13.5	2.5	3 0 1 0 4 0	10 2 0 6 18 2 0 0 28 4 0 6	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 1632.7 µg/mL 1 2 1+2	100 100 200	0.5	0.5	0.0	0 0 0 0 0 0	0 0 0 0 1 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 2857.1 µg/mL 1 2 1+2	100 100 200	0.0	0.0	0.0	0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0
TS: 5000 µg/mL 1 2 1+2	100 100 200	1.0	0.0	0.0	1 0 1 0 2 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0

 

*  Inclusive cells carrying exchanges

Conclusions:

The test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.

Executive summary:

The test substance, dissolved in deionized water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix in accordance with OECD Test Guideline 473. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test substance. In each experimental group two parallel cultures were analyzed. 100 metaphase plates per culture were scored for structural chromosomal aberrations, except for the positive controls in Experiment I, in the presence of S9 mix, where 50 metaphases were scored. 1000 cells were counted per culture for determination of the mitotic index. With respect to the current OECD Guideline 473 and protein content of the test item 5000 µg/mL were applied as top concentration in this study.

In this study, no precipitation of the test item in the culture medium was observed. No relevant increase in the osmolarity or pH value was observed (e.g. Exp. I: solvent control: 233 mOsm, pH 7.4 versus 278 mOsm and pH 7.4 at 5000 µg/mL). At both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices was observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0 - 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 – 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps). No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. In both experiments, either EMS (660 and 825 µg/mL) or CPA (22.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 June 1989 - 11 October 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fishers medium (10% and 20% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: 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:

The highest concentration tested was 5000 µg/mL, tested as supplied.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure is in aqueous solutions.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%)

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 logarithmic transformation 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

Conclusions:

It was concluded that Lipase had no mutagenic activity in the present test system.

Executive summary:

Lipase was assayed for its ability to induce mutation at the HGPRT 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).

 

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/mL, cells survived this dose of 5000 µg/mL (93% survival) in the absence and 500 µg/mL (11% survival) in the presence of S-9. This, together with the next three lower doses without S-9 and the next five lower doses with S-9, were plated for viability and 6-thioguanine resistance seven days after treatment (with the exception of experiment 1 treatments in the absence of S-9, plated after eight days). In the second experiment a narrower dose range was used to maximise the chance of detecting any lose related effects. The top doses plated in this experiment were 5000 µg/mL in the absence and 500 µg/mL in the presence of S-9, which yielded 104% and 5% 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 Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

 

It was concluded that Lipase had no mutagenic activity in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)