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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in bacteria, as determined in an OECD 471 study.

The test substance is not cytogenic, as determined in an OECD 487 study.

The test substance is not mutagenic in mammalian cells, as determined in an OECD 490 study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Apr 2017 to 01 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 03 January 2018 (expiry date)
- Solubility in vehicle: Water: >770 g/L
- Stability in vehicle: Water: Stable
- Appearance: Clear colourless liquid (determined by Charles River Den Bosch)

OTHER
- Purity/Composition correction factor: Yes, according to the purity: 1.28
Target gene:
- S. typhimurium: his
- E. coli: trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 (i.p.) induced rats livers
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST
Strains TA100 and the WP2uvrA, with and without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

EXPERIMENT 1 (DIRECT PLATE ASSAY)
- Strains TA98, TA1535 and TA1537, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate

EXPERIMENT 2 (PRE-INCUBATION ASSAY)
- Strains TA98, TA100, TA1535, TA1537 and WP2 uvrA, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Milli-Q water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION
- DOSE-RANGE FINDING TEST and EXPERIMENT 1: in agar (plate incorporation)
- EXPERIMENT 2: preincubation

DURATION
- Preincubation period (EXPERIMENT 2 only): 30 minutes by 70 rpm at 37°C
- Exposure duration: 48 ± 4 h in the dark at 37.0 ± 1.0°C

NUMBER OF REPLICATIONS
3 plates/dose

COLONY COUNTING
Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
see section "Any other information on materials and methods incl. tables"
Species / strain:
S. typhimurium, other: TA1535, TA1537 and TA98
Remarks:
Dose range Experiment and Experiment 1: Direct Plate Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Dose range Experiment: Direct Plate Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98
Remarks:
Experiment 2: Pre-Incubation Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Experiment 2: Pre-Incubation Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

HISTORICAL CONTROL DATA
- Positive historical control data: see Table 1 in ‘Any other information on results inc. tables’.
- Negative (solvent) historical control data: see Table 2 in ‘Any other information on results inc. tables’.

Table 1 Negative (solvent) historical control data

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

5 - 36

3 - 32

3 – 23

3 – 23

8 - 41

9 - 52

66 - 156

65 - 154

10 – 56

9 - 69

Mean

12

12

6

8

16

23

100

100

25

31

SD

5

4

3

4

5

7

15

16

6

7

n

1865

1862

1740

1715

1852

1912

1853

1877

1571

1583

Table 2 Positive historical control data

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

125 - 1381

78 - 1058

55 – 1311

55 – 1051

410 – 1995

250 - 1907

554 – 1848

408 - 2651

112 – 1951

85 - 1359

Mean

828

218

686

376

1270

883

892

1352

1165

388

SD

151

109

320

142

338

340

174

342

488

152

n

1875

1829

1560

1716

1766

1851

1820

1857

1506

1557

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2018 - 19 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
A correction factor of 1.32 was used to correct for the water content.
Appearance: Turbid yellowish liquid
Test item storage: At room temperature
Batch: SEALS 2014-63-2-001
Purity/Composition: UVCB
-75,90% Tris(2-hydroxyethyl)ammonium 2-hydroxypropane-1,2,3-tricarboxylate
-24,10% water
Stable under storage conditions until: 31 December 2018 (expiry date)
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
Type and identity of media:
- Sex, age and number of blood donors if applicable:
Blood was collected from healthy adult, non-smoking volunteers
The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016 and 2017) are presented below:
Dose-range finding study: age 35, AGT = 15.8 h
First cytogenetic assay: age 23, AGT = 14.2 h
Second cytogenetic assay: age 23, AGT = 13.9 h


Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
Cytokinesis block (if used):
Cytochalasine B (5 μg/mL) for 24 hours (1.5 times normal cell cycle)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3 hr exposure; 27 hr harvest: 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
Without S9-mix, 24 hr exposure, 24 hr harvest 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
With S9-mix, 3 hr exposure; 27 hr harvest: 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL

First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr harvest: 0, 1250, 2500 and 5000 μg/mL
With S9-mix, 3hr exposure; 27 hr harvest: 0, 1250, 2500 and 5000 μg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure, 24 hr harvest: 0,10, 100, 500, 1000, 2500 and 5000 μg/mL

A concentration of 5000 µg/mL showed no precipitation in the culture medium and was used as the OECD guideline recommended highest dose level.
Vehicle / solvent:
- Vehicle: RPMI 1640 medium (Culture medium)
- Justification for choice of vehicle: The test item formed a clear yellow solution or a yellow homogenous suspension in RPMI 1640 medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hanks’ Balanced Salt Solution without calcium and magnesium.
True negative controls:
no
Positive controls:
yes
Remarks:
Without Metabolic Activation
Positive control substance:
mitomycin C
Remarks:
Mitomycin C was used as a direct acting clastogen at a final concentration of 0.25 and 0.38 µg/mL for a 3 hour exposure period and 0.15 and 0.23 µg/mL for a 24 hour exposure period.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hanks’ Balanced Salt Solution without calcium and magnesium.
True negative controls:
no
Positive controls:
yes
Remarks:
Without Metabolic Activation
Positive control substance:
other: Colchicine
Remarks:
Colchicine was used as a direct acting aneugen at a final concentration of 0.1 µg/mL for a 3 hour exposure period and 0.05 µg/mL for a 24 hour exposure period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hanks’ Balanced Salt Solution without calcium and magnesium.
True negative controls:
no
Positive controls:
yes
Remarks:
With Metabolic Activation
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide was used as an indirect acting clastogen, requiring metabolic activation, at a final concentration of 15 and 17,5 µg/mL for a 3 hour exposure period.
Details on test system and experimental conditions:
Environmental conditions:
Humidity: 38 - 88%
CO2: 5.0 ± 0.5%
Temperature: 34.0 - 37.0°C

TEST ITEM PREPARATION:
The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

METHOD OF APPLICATION:
in medium

DURATION
Without and with S9-mix: 3 hour treatment, 27 hour harvest time
The exposure period was followed by centrifugation, rinsing, a second centrifugation and incubation for another 24 hours with cytokinase block.

Without S9-mix: 24 hour treatment, 24 hour harvest time.
The exposure period was followed by immediate fixation without rinsing.


ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B

STAIN: 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol:acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover.

NUMBER OF CELLS EVALUATED: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
Evaluation criteria:
A test was considered acceptable if it met the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
To evaluate the statistical significane of induction, the Chi-square test is performed ( Chi-square test, onesided, p < 0.05).
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: no, The pH and osmolarity of a concentration of 5000 μg/mL were 7.5 and 310 mOsm/kg respectively, the pH of a concentration of 2500 μg/mL was also 7.5 (compared to 8.0 and 294 mOsm/kg in the solvent control respectively).

- Definition of acceptable cells for analysis:
Cells could be scored when they had seperate main nuclei of approximately equal size, when the nuclear boundaries were able to be distinguished or when the main nuclei were linked by nucleoplasmic bridges. Trinucleated, quadranucleated, or multinucleated cells or cells where main nuclei were undergoing apoptosis were not scored.

RANGE-FINDING/SCREENING STUDIES:

A concentration of 5000 μg/mL showed no precipitation in the culture medium and was used as the highest concentration


FIRST CYTOGENETIC ASSAY
All dose levels were selected for scoring
With S9-mix:
In the presence of S9-mix, The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

Without S9-mix:
In the absence of S9-mix, The test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the intermediate dose level. However, no significant trend was observed.


SECOND CYTOGENETIC ASSAY
All dose levels were selected for scoring:
Without S9-mix:
The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei


ACCEPTABILITY:
The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei.
The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei.
In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

There can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly.

Tables: See Attached Background material

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 2018 - 26 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
A correction factor of 1.32 was applied in this study to correct for the water content.
Appearance: Turbid yellowish liquid
Test item storage: At room temperature
Batch: SEALS 2014-63-2-001
Purity/Composition: UVCB
-75,90% Tris(2-hydroxyethyl)ammonium 2-hydroxypropane-1,2,3-tricarboxylate
-24,10% water
Stable under storage conditions until: 31 December 2018 (expiry date)
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 313, 625, 1250, 2500, 5000 μg/mL

Experiment 1
3 hour treatment (without and with S9): 39, 78, 156, 313, 625, 1250, 2500, 5000 μg/mL
Experiment 2
24 hour treatment (without S9): 500, 1000, 1250, 1500, 1750, 2000, 2200, 2400, 2600, 2800, 3000, 3200 and 3400 μg/mL


Vehicle / solvent:
- Vehicle used: RPMI 1640
- Justification of Vehicle: Based on a solubility test, exposure medium was selected as vehicle.

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9, in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: 7.5 µg/mL in Hanks’ balanced salt solution without calcium and magnesium.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

TEST ITEM PREPARATION
The test item was dissolved in RPMI 1640. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test item concentrations were used within 3 hours of preparation.

DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth rate (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 x 10^-6. in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126 x 10^-6.

Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 x 10^-6.

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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: the test item did not precipitate in the exposure medium up to and including the concentration of 5000 μg/mL.
-pH and osmolarity: The pH and osmolarity at a concentration of 5000 μg/mL were 7.49 and 0.310 Osm/kg respectively (compared to 8.04 and 0.294 Osm/kg in the solvent control)

RANGE-FINDING/SCREENING STUDIES
The 3 hour exposure showed no significant cytotoxicity in the absence and presence of S9-mix at all dose levels.
The 24 hour exposure showed a relative suspension growth of 9% at the test item concentration of 2500 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 5000 μg/mL.

EXPERIMENT 1
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls. No significant cytotoxicity was observed.

EXPERIMENT 2
The dose levels of 500 to 1500 μg/mL, 1750 to 2200 μg/mL, 2600 and 2800 μg/mL, and 3200 and 3400 μg/mL showed similar cell growth delay. Therefore, the dose levels of 1250, 1750, 2200, 2800 and 3200 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were: 500, 1000, 1500, 2000, 2400, 2600, 3000 and 3400 μg/mL exposure medium.

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls. The relative total growth of the highest test item concentration (3400 μg/mL) was 13% compared to the total growth of the solvent controls.

ACCEPTABILITY
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (See table 1 and 2). It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for solvent control cultures treated was between 11 and 19 (3 hour treatment) and 79 and 83 (24 hour treatment).

Table 1 :Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 10^6survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

96

92

96

SD

29

30

29

n

268

248

285

Upper control limit (95% control limits)

160

152

160

Lower control limit (95% control limits)

32

31

32

SD = Standard deviation n = Number of observations

 Table 2: Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 10^6survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

808

684

1669

SD

239

206

843

n

136

124

146

Upper control limit (95% control limits)

1465

1222

4196

Lower control limit (95% control limits)

152

146

-859

SD = Standard deviation n = Number of observations

Distribution historical positive control data from experiments performed between November 2014 and November 2017.  

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutations in bacteria

The potential of the test substance (CAS no. 29340-81-6) and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA was determined in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both based on the most recent OECD and EC guidelines. The batch of CAS no. 29340-81-6 was a clear colourless liquid. A correction factor of 1.28 was used to correct for the purity. The test item was dissolved in Milli-Q water. In the dose-range finding study, CAS no. 29340-81-6 was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. In each of these tests, the test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In conclusion, based on the results of this study it is concluded that CAS no. 29340-81-6 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

 

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles. The dose levels that were tested in the dose finding study and experiment 1 were up to and including 5000 μg/mL in the absence and presence S9-mix. No toxicity was observed during the experiments. In experiment 2 the test item was tested up to concentrations of 3400 μg/mL in the absence of S9-mix. The relative total growth (RTG) was reduced to 13%.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence and presence of S9 -mix, the test item did not induce a significant increase in the mutation frequency in the first and second experiment. In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

In vitro cytogenicity

An in vitro micronucleus assay in cultured peripheral human lymphocytes with the substance was performed according to OECD 487 and GLP principles.Batch SEALS 2014-63-2-001 of the test item was a turbid yellowish liquid. A correction factor of 1.32 was used to correct for the water content (24.10%). The vehicle of the test item was culture medium. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. In the first cytogenetic assay, the test item was tested up to the recommended dose level of 5000μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, the test item was tested again up to 5000μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9- mix) functioned properly. The test substance did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. In conclusion, this test is valid and the substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this

report.

 

Short description of key information: The substance and its metabolites are not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the available studies, classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.