Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Hatcol 5236

Ames test: Negative

Chromosome aberration: Negative

Hatcol 3344

Ames test: Negative

Chromosome aberration: Negative

Hatcol 3331

Ames test: Negative

Hatcol 1189

Gene mutation test: Negative

Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Ames test: Negative

Chromosome aberration: Negative

Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids

Ames test: Negative

Gene mutation test: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March to 3 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Horse serum
Horse serum (Invitrogen Corporation) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heatinactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heatinactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse
serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 40 - 97%), containing 5.0 ± 0.5% CO2 in air, at a
temperature of 37.0 ± 1.0°C (actual range 25.4 - 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each
working day. Temporary deviations from the temperature, humidity and CO2 percentage that occurred
were caused by opening and closing of the incubator door. Based on laboratory historical data these
deviations are considered not to affect the study integrity.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Vehicle / solvent:

No correction was made for the purity/composition of the test substance.

The test substance was dissolved in ethanol (Merck, Darmstadt, Germany). Hatcol HXL 8865 concentrations were used within 2 hours after preparation.

The final concentration of the solvent in the exposure medium was 0.2 – 0.4% (v/v).

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Mutation experiment

No toxicity was observed up to and including the precipitating dose levels of 100 and 333 μg/ml in the dose range finding test. Therefore, the highest concentration to be tested was determined by the solubility in the culture medium. The following dose range was selected for both mutagenicity tests in the absence and presence of S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 μg/ml exposure medium. Further investigation showed that at a concentration of 33 μg/ml Hatcol HXL 8865 already precipitated in the exposure medium. Hatcol HXL 8865 was tested beyond the limit of the solubility to obtain adequate mutagenicity data. The results of the two mutation experiments were within the validity criteria.

In the second mutation experiment in the presence of S9-mix, one of the mutation frequencies of the solvent controls was above the limit of the historical control data range. In addition the test substance treated groups showed mutation frequencies above the historical control data range. Therefore this part of the experiment was repeated with a decreased amount of ethanol. In mutation experiment 2A the spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Conclusions:

Negative

In the absence of S9-mix, Hatcol HXL 8865 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, Hatcol HXL 8865 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.
In conclusion, Hatcol HXL 8865 is not mutagenic in the TK mutation test system under the experimental conditions.

Executive summary:

Evaluation of the mutagenic activity of Hatcol HXL 8865 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat). The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures were based on the most recent OECD and EC guidelines.

The test substance was dissolved in ethanol, prepared directly prior to use. In the first experiment, Hatcol HXL 8865 was tested up to a concentration of 333 μg/ml in the absence and presence of 4% (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, Hatcol HXL 8865 was again tested up to a concentration of 333 μg/ml, but in the absence and presence of 8% (v/v) S9-mix. The incubation times were 24 hours and 3 hours, respectively.

In the absence of S9-mix, Hatcol HXL 8865 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Hatcol HXL 8865 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that Hatcol HXL 8865 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Reference 2

Endpoint:

genetic toxicity in vitro, other

Type of information:

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

Remarks:

7 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the attached justification in section 13 for the full details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

lymphocytes: human

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Cultured peripheral human

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: cultured peripheral human 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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

not examined

Positive controls validity:

valid

Remarks on result:

other: Ames test

Remarks:

Hatcol 5236

Conclusions:

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. Based on the available information from the read across substances, the substance is not expected to be mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2003 to 16 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Mitosis

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Source: Healthy adult male volunteers.
Dose range finding study: 24 h fixation: age 37, AGT = 16.5 h (Dec. 2002); 48 h fixation: age 37, AGT = 17.5 h (Dec. 2002)
First cytogenetic assay: age 29, AGT = 16.4 h (Dec. 2002)
Second cytogenetic assay: age 39, AGT = 16.6 h (Dec. 2002)
(AGT= Average Generation Time of the cells).

Cell culture
Blood samples: Blood samples were taken from healthy adult male volunteers by venapuncture 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.
F10 complete culture medium: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/I) and 30 U/ml heparin.
Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml F10 complete culture medium (in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Murex) was added.
Environmental conditions: All Incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% CO2 in air in the dark, at 37 ± 1°C. The temperature, humidity and CO2-percentage-were monitored throughout the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 3331/ml culture medium with and without S9-mix.
First cytogenetic assay: Without and with S9-mix: 10, 33 and 100 µg Hatcol 3331/ml culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay: Without S9-mix 10, 33 and 100 µg Hatcol 3331/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time); With S9-mix: 10, 33 and 100 µg Hatcol 3331/ml culture medium (3 h exposure time, 48 h fixation time)

Vehicle / solvent:

Halcol 3331 was dissolved in ethanol absolute (Lichrosolv, Merck). Hatcol 3331 concentrations were prepared directly prior to use. The final concentration of the solvent in the culture medium amounted 1.0% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Metabolic activation system: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River (Sulzfeld, Germany).
Preparation of S9-fraction: The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures (SOP's), and allowed to acclimatise for at least 5 days. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days after injection, the rats were killed by decapitation. Rats were denied access to food for at least 12 hours preceding sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. The livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).
Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.63 mg MgCI2.6H20; 2.46 mg KCI; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mlx components 0.5 ml S9-fraction (batches 03-3 and 03-5) was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin); The concentration of the S9-fraction in the-exposure-medium was 1.8% (v/v).

Study design
Dose range finding test: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Hatcol 3331 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Hatcol 3331 for 3 h, 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was determined by the solubility of Hatcol 3331 in the culture medium.
After 3 h exposure to Hatcol 3331 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.
Cytogenetic assay: The cytogenetic assay was carried out with minor modifications as described by Evans, 1984. Hatcol 3331 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.
First cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3331 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.
Second cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3331 for 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Hatcol 3331 in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time).
The cells that were treated for 24 h and 48 h in the absence of S9-mixwere not rinsed after exposure but were fixed immediately after 24 hand 48h (24 h-and 48 h fixation time).

Chromosome preparation: During the last 2.5 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. 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 tap water.
Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring of the cytogenetic assay: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically and biologically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Chi-square test.

Key result

Species / strain:

lymphocytes: Cultured peripheral human 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:

not examined

Positive controls validity:

valid

Additional information on results:

Dose-range-finding test: At a concentration of 100 µg/ml Hatcol 3331 precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of Hatcol 3331.
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 3331/ml culture medium with and without S9-mix.

First cytogenetic assay
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg Hatcol3331/ml culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Second cytogenetic assay
To obtain more information about the possible clastogenicity of Hatcol 3331, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Hatcol 3331 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Hatcol 3331. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 10, 33 and 100 µg Hatcol 3331/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix: 10, 33 and 100 µg Hatcol3331/ml culture medium (3 h exposure time, 48 h fixation time)
All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Both in the absence and presence of S9-mix Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

Remarks on result:

other: all strains/cell types tested

APPENDIX I DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASES PORTRAITS

Aberration	Abbreviation	Description
Chromatid gap	g'	An achromatic lesion which appears as an unstained region in the chromatid arm, the size of which is equal to or smaller than the width of the chromatid and the apparently “broken” segments of the chromatid arm are in alignment.
Chromosome gap	g"	An achromatic lesion which appears as an unstained region in both chromatids at the same position, the size of which is equal to or smaller than the width of the chromatid and the apparently “broken” segments of the chromatids are in alignment.
Chromatid break	b'	An achromatic lesion in a chromatid arm, the size of which is larger than the width of the chromatid. The brown segments of the chromatid arm are aligned or unaligned.
Chromosome break	b"	An achromatic lesion in both chromatids at the same position, the size of which is larger than the width of the chromatid. The broken segments of the chromatids are aligned or unaligned.
Chromatid deletion	d'	Deleted material at the end of a chromatid arm.
Minute	m'	A single, usually circular, part of a chromatid lacking a centromere.
Double minutes	m"	Two, usually circular, parts of a chromatid lacking a centromere.
Dicentric chromosome	dic.	A chromosome containing two centromeres.
Tricentric chromosome	tric.	A chromosome containing three conctromeres.
Ring chromosome	r	A ring structure with a distinct lumen.
Exchange figure	exch.	An exchange(s) between two or more chromosomes resulting in the formation of a tri- or more-armed configuration.
Chromosome intrachange	intra.	A chromosome intrachange is scored after rejoining of a lesion within one chromosome.
Pulverized chromosome	p	A fragmented or pulverized chromosome.
Multiple aberrations	ma	A metaphase spread containing ten or more of the above mentioned aberrations (chromatid and chromosome gaps not included) ma is counted as 10 aberrations.
Polyploidy	poly	A chromosome number that is a multiple of the normal diploid number.
Endoreduplication	endo	A form of polyploidy in which each centromere connects two or four pairs of chromatids instead of the normal one pair.

 

Conclusions:

Interpretation of results:
negative with and without metabolic activation

It is concluded that this test is valid and that Hatcol 3331 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

Evaluation of the ability of Hatcol 3331 to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of Hatcol 3331 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 3331 was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997).

- European Economic Community (EEC). Directive 2000/321EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome AberrationTest".

Batch D21287 of Hatcol 3331 was a clear colourless liquid with a purity of 97.3%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3331 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction, Hatcol 3331 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, intwo independently repeated experiments.

Finally, it is concluded that this test is valid and that Hatcol 3331 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2003 to 14 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in accordance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100,333, 1000, 3330 and 5000 µg/plate
Mutation assay: 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

The test substance was dissolved in ethanol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin (DM); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METABOLIC ACTIVATION SYSTEM
Preparation of S9-frction: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.
The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were injected intraperitoneally with a solution (20% (w/v)) of Arcelor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C) and identified by the day of preparation.
Before Use, all S9-batches were characterized with the metabolic activation requiring positive control; benzo(a)pyrene (Sigma) in tester strain TA98 at the concentration of 5 µg/plate.
Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 ml Mill-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgC2 solution; 1 ml 0.33 M KCI solution. The above solution was filter (0.22µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-frction was added (10% (v/v) S9-frction) to complete the S9-mix in the second experiment.
The S9 batches used were no. 03-2 and 03-3.

EXPERIMENTAL PROCEDURE
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with the tester strain TA100 in the absence and presence of S9-mix. Eight concentrations of HATCOL 3344, 3, 10, 33, 100,333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of HATCOL 3344 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10E+09cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1°C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST: HATCOL 3344 was tested in the tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first experiment of the mutation test.
Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of HATCOL 3344 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.
Toxicity: To determine the toxicity of HATCOL 3344 the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTATION ASSAY: Based on the results of the dose range finding test, HATCOL 3344 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537, TA98 and WP2uvrA and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: HATCOL 3344 precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of HATCOL 3344 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.
Toxicity: 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.
Number of revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

TABLE 1 – MUTAGENIC RESPONSE OF HATCOL 3344 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plate (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	718± 49	446± 65	351± 40	978± 78	577± 24
Solvent control	8± 1	5± 2	17± 3	132± 14	21± 5
3				105± 13
10	9± 1	7± 3	17± 2	98± 12	14± 2
33	7± 5	5± 2	19± 3	101± 11	15± 3
100	6± 3	4± 2	15± 2	112± 14	13± 3
333	6± 1	6± 1	18± 1	113± 8	16± 4
1000^SP	7± 4	5± 2	17± 3	111± 1	18± 7
3330^SP				104± 2
5000^SP				121± 7
With S9-mix^1
Positive control	213± 9	851± 55	1154± 58	1403± 125	86± 12
Solvent control	7± 4	8± 3	22± 2	115± 20	20± 4
3				109± 12
10	8± 2	5± 2	24± 3	112± 14	21± 3
33	11± 3	4± 2	19± 4	114± 8	16± 9
100	5± 2	7± 2	22± 5	110± 6	14± 1
333	7± 1	6± 2	15± 2	102± 6	17± 6
1000^SP	10± 3	7± 3	22± 4	113± 8	17± 2
3330^SP				116± 6
5000^SP				120± 10

Solvent control: 0.1ml ethanol;¹The S9-mix contained 5% (v/v) S9 fraction;^SPSlight Precipitate

 

TABLE 2 – MUTAGENIC RESPONSE OF HATCOL 3344 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	259± 8	242± 16	346± 15	1002± 30	585± 7
Solvent control	9± 2	5± 2	17± 3	128± 10	8± 3
10	11± 4	6± 1	18± 4	117± 14	10± 3
33	10± 3	6± 3	19± 4	124± 8	8± 3
100	10± 5	6± 2	16± 6	119± 8	9± 2
333	12± 1	9± 4	15± 1	138± 7	8± 4
1000^SP	11± 4	6± 2	15± 3	126± 5	12± 2
With S9-mix^1
Positive control	99± 3	251± 3	469± 18	463± 34	240± 3
Solvent control	8± 3	5± 1	25± 3	115± 7	13± 4
10	10± 2	7± 4	25± 9	112± 5	13± 3
33	8± 2	5± 3	27± 10	123± 7	12± 3
100	9± 2	5± 1	31± 4	115± 5	11± 3
333	9± 2	6± 3	23± 2	125± 6	14± 3
1000^SP	11± 3	4± 1	25± 5	124± 9	12± 1

Solvent control: 0.1 ml ethanol;¹The S9-mix contained 10% (v/v) S9 fraction;^SPSlight Precipitate

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Based on the results of the study it is concluded that HATCOL 3344 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

HATCOL 3344 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 3344 was tested up to concentrations of 5000 µg/platein the absence and presence of S9-mix in the tester strain TA100. HATCOL 3344 precipitatedon the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in thenumber of revertants was observed.

In the first and in the second mutation assay, HATCOL 3344 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 3344 precipitated on the platesat this dose level. The bacterial background lawn was not reduced at any ofthe concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 334 did not induce a dose-related, two-fold 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 an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 3344 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2003 to 26 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not specified

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were taken from healthy adult male volunteers by venapuncture 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.

Test System: Cultured peripheral human lymphocytes.
Source: Healthy adult male volunteers.
Dose range finding study: age 29, AGT = 16.4 h (Dec. 2002)
First cytogenetic assay: age 39, AGT = 16.6 h (Dec. 2002)
Second cytogenetic assay: Without S9-mix (24 h fixation time) and with S9-mix: age 33, AGT = 16.8 h (Dec. 2002). Without S9-mix (48 h fixation time): age 29, AGT = 16.4 h (Dec. 2002)
(AGT= Average Generation Time of the cells).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1, 3, 10, 33 and 100 µg/ml – Dose range finding study
10, 33 and 100 µg/ml – 1st and 2nd cytogenetic assays

Vehicle / solvent:

Hatcol 3344 was dissolved in ethanol absolute (Lichrosolv, Merck). Hatcol 3344 concentrations were prepared directly prior to use. The final concentration of the solvent in the culture medium amounted 1.0% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Metabolic activation system: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River (Sulzfeld, Germany).
Preparation of S9-fraction: The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures (SOP's), and allowed to acclimatise for at least 5 days. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five (batch 03M5 and 03-6) or six (batch 03-7) days after injection, the rats were killed by decapitation. Rats were denied access to food for at least 12 hours preceding sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. The livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Patter homogeniser. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).
Preparation of S9-Mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.63 mg MgCI2.6H20; 2.46 mg KCI; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mix-components 0.5 ml S9-fraction (batches 03-5, 03-6 and 03-7) was added (50% (v/v) 59-frction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-frction in the exposure medium was 1.8% (v/v).

Dose range finding test: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Hatcol 3344 was tested in the absence and in the presence of 1.8% (v/v) S9-frction.
Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Hatcol 3344 for 3 h, 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was determined by the solubility of Hatcol 3344 in the culture medium.
After 3 h exposure to Hatcol 3344 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.

Cytogenetic assay: The cytogenetic assay was carried out with minor modifications as described by Evans, 1984. Hatcol 3344 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.
First cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3344 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.
Second cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 3344 for 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Hatcol 3344 in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time).
The cells that were treated for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 hand 48 h (24 hand 48 h fixation time).

Chromosome preparation: During the last 2.5 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. 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 tap water.
Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring of the cytogenetic assay: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and "the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if
a) It induced a dose-related statistically significant (Chi-square test, P< 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics

Key result

Species / strain:

lymphocytes: human

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:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding tes: At a concentration of 100 µg/ml Hatcol 3344 precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of Hatcol 3344.
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 3344/ml culture medium with and without S9-mix.

First cytogenetic assay: Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg HatcoI 3344/ml culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Second cytogenetic assay: To obtain more information about the possible clastogenicity of Hatcol 3344, a second cytogenetic assay was performed In which human lymphocytes were continuously exposed to Hatcol 3344 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Hatcol 3344. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 10, 33 and 100 µg Hatcol 334/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
With S9-mix: 10, 33 and 100 µg Hatcol 334/ml culture medium (3 h exposure time, 48 h fixation time)
All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Remarks on result:

other: all strains/cell types tested

The ability of Hatcol 3344 to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

It is concluded that this test is valid and that Hatcol 3344 is not clastogenic in human lymphocytes under the experimental conditons described in the study report.

Executive summary:

Evaluation of the ability of Hatcol 3344 to induce chromosome aberrations in cultured peripheral human lymphocytes.

The report describes the effect of Hatcol 3344 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 3344 was tested In two independent experiments.

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997).

- European Economic Community (EEC). Directive 2000/321EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

Batch H102-01-28 of Hatcol 3344 was a clear colourless liquid with a purity of 96.9%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 3344was tested up to 100 µg/ml for a 3 h exposure timewith a 24 h fixation time in the absence and presence of S9 -mix. Hatcol 3344 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In thepresence of 1.8% (v/v) S9-fraction Hatcol 3344was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

Finally, it is concluded that this test is valid and that Hatcol 3344 is not clastogenic in human lymphocytes under the experimental conditions described in the report

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2002 to 06 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Test System: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R –factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chI : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The strain was regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST: 3, 10, 33,100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
MUTATION ASSAY: 10, 33, 100, 333 and 1000 µg/plate in the absence and presence of S9-mix.

Vehicle / solvent:

The test substance was dissolved in ethanol. Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin (DM); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

CELL CULTURE
Preparation of Bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilzation of the Eschenchia coli strain: WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCL buffer pH 8.0. then gently resuspended in 0.2 vol. 0.12 M Tris-Hei, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates: Agar plates (ø 9 em) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code l28) in Vogel-Bonner Medium E, 20 g glucose. N.B. The agar plates for the test with the Salmonella typhimurium strins also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar: Top agar medium, containing 0.6% (w/v) agar and 0.5% (w/v) NaCI, was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1°C.
Environmental conditions: All incubations were carried out in the dark at 37 ± 1°C. The temperature was monitored during the experiment.

METABOLIC ACTIVATION SYSTEM
Preparation of S9-frction: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.
The animals were housed at NOTOX in a special room under standard laboratory conditions, as described In the Standard Operating Procedures. The rats were injected intraperitoneally with a solution (20% (w/v)) of Aroclor 1254 (500 mg/kg bodyweight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (O°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Nai-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate bufer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored In liquid nitrogen (-196°C) and identified by the day of preparation.
Before use, all S9-batches were characterized with the metabolic activation requiring positive control; benzo[a]pyrene (Sigma) in tester strain TA98 at the concentration of 5 µg/plate.
Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 ml Mi1i-Q water* (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0,08 M MgCl2 solution; 1 ml 0.33 M KCI solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml 59-frction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-frction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
The S9-batches used were no. 02-8 and 02-9.

EXPERIMENTAL PROCEDURE
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without 59-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of HATCOL 5236 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1°C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not signiifcant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST: HATCOL 5236 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the first experiment of the mutation test.
Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards.
Precipitation of HATCOL 5236 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.
Toxicity: To determine the toxicity of HATCOL 5236, the reduction of the bacterial background lawn, the increase in the size of the micro colonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTATION ASSAY: Based on the results of the dose range finding test, HATCOL 5236 was tested up to concentrations of 1000 µg/plate In the absence and presence of S9-mix in two mutation assays.
The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: HATCOL 5236 precipitated in the top agar at concentrations of 333 and 1000 µg/plate.
Precipitation of HATCOL 5236 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.
Toxicity: 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.
Number of revertants: All bacterial strains showed negative responses over the entire dose range, I.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

TABLE 1: MUTAGENIC RESPONSE OF HATCOL 5236 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plate (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	966± 106	631± 188	465± 50	1066± 51	611± 49
Solvent control	15± 3	8± 5	23± 7	157± 9	14± 4
3				156± 12	15± 3
10	12± 3	7± 2	24± 4	142± 19	14± 2
33	9± 1	7± 3	13± 1	160± 7	13± 2
100	8± 1	10± 2	24± 2	158± 18	9± 2
333	8± 1	7± 3	19± 6	156± 20	12± 4
1000^SP	8± 3	5± 1	21± 3	149± 11	14± 5
3330^SP				157± 18	13± 6
5000^SP				149± 9	12± 2
With S9-mix^1
Positive control	138± 24	183± 81	591± 25	1003± 137	145± 16
Solvent control	12± 2	8± 4	19± 2	159± 15	14± 4
3				155± 17	15± 3
10	11± 4	12± 2	16± 1	153± 2	10± 1
33	12± 4	8± 1	21± 6	161± 8	15± 3
100	12± 4	8± 3	22± 5	149± 13	15± 2
333	10± 2	7± 4	19± 6	144± 18	15± 5
1000^SP	11± 2	7± 3	16± 1	146± 9	13± 5
3330^SP				148± 14	12± 3
5000^SP				143± 17	12± 5

Solvent control: 0.1 ml ethanol;¹The S9-mix contained 5% (v/v) S9 fraction;^SPSlight Precipitate

 

TABLE 2: MUTAGENIC RESPONSE OF HATCOL 5236 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plate (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	499± 14	370± 35	344± 35	676± 23	414± 100
Solvent control	6± 2	6± 1	14± 3	141± 27	20± 3
10	7± 2	7± 2	16± 3	120± 4	19± 4
33	9± 3	6± 2	19± 7	121± 15	19± 2
100	7± 2	6± 2	19± 3	125± 14	21± 4
333	7± 2	6± 1	19± 4	104± 9	21± 6
1000 SP	8± 4	6± 4	14± 1	121± 12	25± 3
With S9-mix^1
Positive control	103± 7	67± 5	350± 51	276± 20	199± 22
Solvent control	8± 2	10± 2	15± 2	117± 15	19± 5
10	8± 1	8± 4	27± 3	104± 9	26± 5
33	7± 2	9± 2	26± 2	98± 3	21± 6
100	10± 3	7± 3	27± 10	109± 15	18± 7
333	9± 3	6± 3	22± 1	96± 6	16± 7
1000^SP	10± 5	5± 1	21± 3	101± 11	16± 5

Solvent control: 0.1ml ethanol; ¹The S9-mix contained 10% (v/v); ^S9fraction; SP Slight Precipitate

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Based on the results of this study it is concluded that HATCOL 5236 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

HATCOL 5236 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

The study procedures described in this report were based on the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (Adopted July 21, 1997).

- European Economic Community (EEC). Adapting to technical progress for the twenty-sixth time Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination ofToxicity; B.13/14: "Mutagenicity: “Reverse Mutation Assay using bacteria". EEC Publication Commission Directive (Published June 8, 2000).

In the dose range finding test, HATCOL 5236 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. HATCOL 5236 precipitated on the plates at dose levels of 1000 µg/plate and upwards. 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.

In the first and in the second mutation assay, HATCOL 5236 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 5236 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 5236 did not induce a dose-related, two-fold 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 an independently repeated experiment.

Based on the results of this study it Is concluded that HATCOL 5236 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2003 to 06 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not specified in the study report.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Whole blood treated with an anti-coagulant (heparin) obtained from healthy male subjects was cultured in the presence of a mitogen (phytohaemagglutinin).
Test System: Cultured peripheral human lymphocyes.
Source: Healthy adult male volunteers.
Dose range finding study: age 37, AGT = 17.5 h (Dec. 2002)
First cyogenetic assay: age 36, AGT= 17.0 h (Dec. 2002)
Second cyogenetic assay: age 28. AGT = 16.2 h (Dec. 2002)
(AGT= Average Generation Time of the cells).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 5236/ml culture medium with and without S9-mix.
First cytogenetic assay: Without and with S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay: Without S9-mix 10, 33 and 100 µg Hatcol 5236/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time); With S9-mix 10,33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 48 h fixation time).

Vehicle / solvent:

Hatcol 5236 was dissolved in ethanol absolute.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cell culture
Blood samples: Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocye cultures were started.
F10 complete culture medium: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicilln/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.
Lvmphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml F10 complete culture medium (in the absence and presence of 59-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Murex) was added.
Environmental conditions: All incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% CO2 in air in the dark at 37 ± 1°C. The temperature, humidity and CO2-percentage were monitored throughout the experiment.

Negative control: The vehicle of the test article, being ethanol.
Positive controls
Without metabolic activation (-S9-mix): Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period.
With metabolic activation (+S9-mix): Cyclophosphamide (CP; CAS no. 50-18-0. Endoxan-Asta, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 15 µg/ml for a 3 h exposure period (24 h fixation time).
Solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium.
Solutions of reference substances were prepared immediately before use.

Metabolic activation system: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River (Sulzfeld, Germany).

Preparation of S9-fraction: The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures (SOP's), and allowed to acclimatise for at least 5 days. The rats were injected intrperitoneally with a solution (20% w/v) of Arclor 1254 (500 mg/kg body weight) in corn oil. Five (batches 03-5 and 03-6) or six (batch 03-7) days after injection, the rats were killed by decapitation. Rats were denied access to food for at least 12 hours preceding sacrifice. The livers of the rats were removed aseptically, and washed in cold (O°C), sterile 0.1 M sodium phosphate bufer (pH 7.4) containing 0.1 mM NaTEDTA. The livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C). .

Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.63 mg MgCI2.6H2O; 2.46 mg KGI; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPE5. The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction (batches 03-5, 03-6 and 03-7) was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-frction in the exposure medium was 1.8% (v/v).

Study design
Dose range finding test: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Hatcol 5236 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. Lymphocyte cultures (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/mI) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Hatcol 5236 for 3 h, 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. The highest tested concentration was determined by the solubility of Hatcol 5236 in the culture medium. After 3 h exposure to Hatcol 5236 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time). Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level was determined by the solubility.

Cytogenetic assay: The cytogenetic assay was carried out with minor modifications as described by Evans, 1984. Hatcol 5236 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.
First cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 5236 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min. 150 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.
Second cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of Hatcol 5236 for 24 hand 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. After 3 h exposure, the cells exposed to Hatcol 5236 in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time). The cells that were treated for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 hand 48 h (24 hand 48 h fixation time).
Chromosome preparation: During the last 2.5 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining ceil pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).
Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. 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 tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.
Mitotic index/dose selection for scoring of the cytogenetic assay: The mitotic index of each culture was determined by counting the number of meta phases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.
Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was 2 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square test

Key result

Species / strain:

lymphocytes: human

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:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test: At a concentration of 100 µg/ml Hatcol 5236 precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of Hatcol 5236.
In the dose range finding test blood cultures were treated with 1, 3, 10, 33 and 100 µg Hatcol 5236/ml culture medium with and without S9-mix.

First cytogenetic assay: Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. In the absence of S9-mix, Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
In the presence of S9-mix, Hatcol 5236 induced a statistically significant increase in the number of cells with chromosome aberrAtions at the intermediate concentration of 33 µg/ml, only when gaps were included.

Second cytogenetic assay: To obtain more information about the possible clastogenicity of Hatcol 5236, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Hatcol 5236 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Hatcol 5236. The following dose levels were selected for the second cytogenetic assay:
With S9-mix: 10, 33 and 100 µg Hatcol 5236/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)
Without S9-mix: 10,33 and 100 µg Hatcol 5236/ml culture medium (3 h exposure time, 48 h fixation time)
All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Hatcol 5236 did not Induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Negative with and without metabolic activation

Finally, it is concluded that this test is valid and that Hatcol 5236 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

Evaluation of the ability of Hatcol 5236 to induce chromosome aberrations in cultured peripheralhuman lymphocytes.

This report describes the effect of Hatcol 5236 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 5236 was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines:

-OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro MammalianChromosomeAberration Test (adopted 21st July 1997).

-European Economic Community (EEC). Directive 2000132/EC, Part B: Methods for theDetermination ofToxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

Batch H20139 of Hatcol 5236 was a clear pale yellow liquid with a purity of 97.6%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 3 h exposure timewith a 24 h fixation time in the absence and presence of S9-mix. Hatcol 5236 precipitated in theculture medium at this dose level.

In the second cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In thepresence of 1.8% (v/v) S9-frction Hatcol 5236 was also tested up to 100 µg/ml for a 3 hexposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay:In the absence of S9-mix, Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the presence of S9-mix, Hatcol 5236 induced a statistically significant increase in the numberof cells with chromosome aberrations at the intermediate concentration of 33 µg/ml, only whengaps were included. Since the type of aberrations observed were only breaks and gaps, theincrease was not dose related, only observed at the intermediate concentration of 33 µg/ml, only when gaps were included and the number of cells with chromosome aberrations was well within our historical control data range, the increase was considered not to be biologically relevant.

Second cytogenetic assay: Hatcol 5236 did not induce a statistically significant or biologically relevant Increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

Finally, it is concluded that this test is valid and that Hatcol 5236 is not clastogentic in human lymphocytes under the experimental conditions described in this report.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 23 Jun 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

analytical purity not reported

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

analytical purity not reported

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon and trp operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1, -S9, + S9: 3, 10, 33, 100, 333, 1000, 3300 and 5000 µg/plate
Experiment 2, -S9, + S9: 10, 33, 100, 333, and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitroquinoline N-oxide, -S9, 10 µg in DMSO, WP2uvrA; daunomycin (DM) 4µg in saline, -S9, TA98

Remarks:

sodium azide (SA, 5 µg/plate in saline, -S9, TA1535); 9-aminoacridine (9-AC; 60 µg/plate in Milli-Q-water, -S9,TA1537); methylmethanesulfonate (MMS; 650 µg/plate in DMSO, -S9, TA100)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracence (2AA), + S9, 2.5-5 µg in DMSO, all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: three in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of bacterial background lawn, increase in the size of the microcolonies and reduction of the revertant colonies were examined

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (not mutagenic) in the test if:
c) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation
d) The positive response should be reproducible in the at least one independently repeated experiment.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3300 and 5000 µg/plate in the absence and presence of S9-mix.

Precipitate
The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of test substance on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards. This precipitate was judged as an emulsion of test substance in the incubation period.

Toxicity
To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertant were observed.

Number of revertants
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Mutation assay

Based on the results of the dose finding test, the test item was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutations assays. The first mutation experiment was performed with the test strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate

The test substance precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of the test substance on the plates was observed at the start of the incubation period at the concentration of 1000 µg/plate. This precipitate was judged as an emulsion of the test substance at the end of the incubation period.

Toxicity

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.

Number of reventants

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertans in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Tab.1: Experiment 1 (plate incorporation) without metabolicactivation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA1535	TA1537	TA98	TA100	WP2uvrA
Solvent control	11±4	5±2	24±3	136±2	19±5
3				140±10	15±2
10	8±4	4±1	17±4	137±20	21±3
33	10±1	4±2	17±2	140±12	14±3
100	8±0	5±1	20±4	139±20	16±1
333	9±2	4±2	19±1	129±17	19±5
1000*	6±1	5±2	21±4	140±11	13±2
3330*				135±3	18±6
5000*				133±9	17±4
Positive control	187±6	420±121	465±32	787±46	492±24

Solvent control: ethanol

Positive controls: sodium azide (SA) 5 µg/plate (TA1535); 9-aminoacridine (9-AC) 60 µg/plate in DMSO (TA1537); methylmethanesulfonate (MMS) 650 µg/plate in DMSO (TA100); 4-nitroquinoline-N-oxide (WP2uvrA) 10 µg/plate in DMSO; daunomycin (DM) 4 µg in saline (TA98)

Tab. 2: Experiment 1 (plate incorporation) with metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA1535	TA1537	TA98	TA100	WP2uvrA
Solvent control	10±6	5±1	25±4	155±7	19±4
3				138±12	20±3
10	12±1	7±1	27±2	153±10	20±2
33	9±1	7±4	20±6	151±3	20±3
100	9±3	6±2	28±2	149±7	18±4
333	10±2	5±3	22±1	141±15	17±2
1000*	9±4	7±3	27±2	136±5	17±2
3300*				139±8	12±5
5000*				141±12	13±4
Positive control	131±7	139±22	571±16	765±24	84±7

Solvent control: ethanol

Positive controls: 2-aminoanthracene (2-AA) 1 µg/plate (TA100), 2.5 µg/plate (TA1535, TA1537, and TA98) and 5 µg/plate (WP2uvrA) in DMSO

 

Tab. 3: Experiment 2 (plate incorporation) without metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA1535	TA1537	TA98	TA100	WP2uvrA
Solvent control	11±4	7±2	20±4	132±13	16±7
10	10±2	8±5	17±3	143±3	15±4
33	9±1	6±0	23±6	134±8	19±3
100	11±2	7±4	16±5	135±10	13±2
333	11±2	8±2	19±6	136±8	14±2
1000*	11±2	9±3	17±6	137±6	17±5
Positive control	325±26	236±17	356±30	873±7	710±126

Solvent control: ethanol

Positive controls: sodium azide (SA) 5 µg/plate (TA1535); 9-aminoacridine (9-AC) 60 µg/plate in DMSO (TA1537); methylmethanesulfonate (MMS) 650 µg/plate in DMSO (TA100); 4-nitroquinoline-N-oxide (WP2uvrA) 10 µg/plate in DMSO; daunomycin (DM) 4 µg in saline (TA98)

Tab. 4: Experiment 2 (plate incorporation) with metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA100	TA1535	TA102	TA98	TA1537
Solvent control	8±0	5±2	26±6	136±5	17±5
10	8±3	7±3	27±3	112±12	18±5
33	11±1	7±2	29±4	125±14	13±3
100	9±2	4±4	22±3	108±8	12±4
333	11±1	7±4	30±2	131±5	14±3
1000*	9±3	5±3	23±2	135±15	16±6
Positive control	130±2	137±32	317±26	649±44	87±13

Solvent control: ethanol

Positive controls: 2-aminoanthracene (2-AA) 1 µg/plate (TA100), 2.5 µg/plate (TA1535, TA1537, and TA98) and 5 µg/plate (WP2uvrA) in DMSO

Conclusions:

Interpretation of results:
negative

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Apr - 1 Sep 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

The department of health of the government of the United Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Eagle's minimal essential medium supplemented with sodium bicarbonate, H EPES buffer, L-glutamine, peniciIlin/streptomycin, amphotericin B and 15% foetal calf serum

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1, 4h: -S9: 39.06, 78.13, 156.25, 312.5, 625*, 1250*, 2500*, 5000* µg/mL
Experiment 1, 4h: +S9: 39.06, 78.13, 156.25*, 312.5, 625*, 1250*, 2500*, 5000* µg/mL
Experiment 2, 20h: -S9: 39, 78.1, 156.25, 312.5, 625*, 1250*, 2500*, 5000* µg/mL
Experiment 2, 4 h, +S9: 156.25, 312.5, 625, 1250*, 2500*, 5000* µg/mL

* dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Exp I and II, - S9: Ethyl methansulphonate (EMS) 750 and 500 µg/mL; Exp I and II, + S9: Cyclophosphanamide (CP) 25 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h
SPINDLE INHIBITOR (cytogenetic assays): demelcocine (Colcemid) 0.1 µg/mL
STAIN (for cytogenetic assays): Giemsa 5%
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Exp1: an oily precipitate at and above 625 µg/mL, +S9 and –S9; Exp. 2: oily precipitate was observed at and above 1250 µg/mL, -S9, and at and above 2500 µg/mL, +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH when the test material was dosed into media
- Effects of osmolality: there was no increase in osmolality by more than 50 mOSM

Remarks on result:

other: all strains/cell types tested

Experiment 1

The qualitative assessment of the slides determined that there were scorable metaphase up to the maximum recommended dose level of 5000 µg/mL in the without and with metabolic activation (S9) groups. An oily precipitate was observed at and above 625 µg/mL in both treatment groups.

With regard to the mitotic index data, these findings show that no toxicity was observed up to the maximum recommended dose level of 5000 µg/mL in the absence or presence of S9. No toxicity was observed at or below the precipitating dose level of 625 µg/mL. Therefore the maximum five dose levels were selected for metaphase analysis.

Both of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected.

The test material induced statistically significant increases in the frequency of cells with gap-type aberration at 2500 and 5000 µg/mL in the absence of S9 mix and at 5000 µg/mL in the presence of S9 mix. There were no statistically significant increases in the frequency of cells with aberrations excluding gaps and therefore the observation were considered to have no toxicological significance.

The test material did not induce a significant increase in the numbers of polyploidy cells at any dose level in either of the treatment cases.

Experiment 2

The qualitative assessment of the slides determined, as in Experiment 1, that were scorable metaphases up to the maximum recommended dose level, 5000 µg/mL.

An oily precipitate was observed at and above 1250 µg/mL in absence of S9 mix and at and above 2500 µg/mL in the presence of S9 mix.

The data on mitotic index confirm that qualitative observations in that no dose-related inhibition of mitotic index was observed.

The precipitation of the test material appeared to have no effect on toxicity. Therefore, doses below that precipitating dose level and up to 5000 µg/mL were selected for metaphase analysis.

Both the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either including or excluding gaps, in the presence of metabolic activation (at 2% concentration) or with continuous 20 h exposure in the absence of S9 mix. Therefore, the small increases observed in Experiment 1 were confirmed to be of no toxicological significance.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases.

Tab. 1: Experiment 1: 4 h treatment, 20 h harvest - Without Metabolic Activation

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.0	0.0	0.0
312.5 µg/mL	105	-	-	-
625 µg/mL	96	0.5	1.5	1.0
1250 µg/mL	110	1.0	1.0	1.0
2500 µg/mL	92	0.0	2.5	0.0
5000 µg/ml	94	0.0	2.5	0.5
EMS 750 µg/mL	75	0.0	18.0	10.0

EMS: ethyl methanesulphonate; solvent control: acetone

  Tab. 2: Experiment 1: 4 h treatment, 20 h harvest- With Metabolic Activation (S9-Mix)

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.5	0.5	0.0
156.25 µg/mL	-	-	2.0	1.0
312.5 µg/mL	92	-	-	-
625 µg/mL	102	0.0	2.0	1.5
1250 µg/mL	106	0.0	1.0	0.5
2500 µg/mL	92	0.0	0.5	0.0
5000 µg/mL	101	0.0	3.5	2.0
CP 25 µg/mL	31	0.0	15.5	6.5

CP: cyclophosphamide; solvent control: acetone

 

Tab. 3: Experiment 2: 20 h treatment, 20 h harvest - Without Metabolic Activation

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.0	1.5	0.5
625 µg/mL	70	0.0	0.5	0.0
1250 µg/mL	81	0.0	2.5	1.5
2500 µg/mL	116	0.0	0.5	0.0
5000 µg/mL	105	-	2.5	1.5
EMS 500 µg/mL	28	0.0	51.0	44.0

EMS: ethyl methanesulphonate; solvent control: acetone

 

Tab. 4: Experiment 2: 4 h treatment, 20 h Harvest- With Metabolic Activation (S9-Mix)

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.0	1.0	1.0
1250 µg/mL	101	0.0	1.0	0.5
2500 µg/mL	91	0.0	0.0	0.0
5000 µg/mL	105	-	1.0	1.0
CP 25 µg/mL	27	0.5	22.0	10.0

CP: cyclophosphamide; solvent control: acetone

Conclusions:

Interpretation of results:
negative

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 Jan - 10 March 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon and trp operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (2-AA) 1, 2, 10, 0.5, 2 µg/plate (+S9 mix, TA100, TA1535, WP2uvrA,TA98 and TA1537, respectively)

Remarks:

-S9 mix: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) 3µg/plate (TA100), 5 µg/plate (TA1535) and 2 µg/plate (WP2uvrA-); 9-aminoacridine (9-AA) 80 µg/plate (TA1537); 4-nitroquinoline-N-oxide (4NQO) 0.2µg/plate (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in the revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels.

Statistics:

All of the data was analysed using the statistical methods recommended by the UKEMS with Dunnett's method of linear regression used to evaluate the result. To be considered negative, the number of revertants at each dose level should be less than twofold that of the vehicle control frequency.

Key result

Species / strain:

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

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: the dose range of the test material used in the preliminary toxicity study was 5, 50, 150, 500 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

Remarks on result:

other: all strains/cell types tested

Tab.1: Experiment 1 (plate incorporation) without metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA100	TA1535	WP2uvrA-	TA98	TA1537
Solvent control	114±8.0	26±6.9	23±1.5	37±9.2	12±1.2
50	118±5.3	28±6.1	24±5.5	39±7.1	10±2.0
150	113±13.5	27±3.6	22±7.5	42±3.0	11±5.3
500	113±7.5	25±5.7	20±1.5	33±3.1	12±5.5
1500	99±5.8	21±4.0	20±4.2	29±6.0	10±1.7
5000	108±11.3	22±7.8	18±1.0	35±4.0	11±1.7
Positive control	500±12.8	242±18.6	902±57.6	160±17.6	982±208.7

Solvent control: acetone

Positive controls: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) 3 µg/plate (TA100), 5 µg/plate (TA1535) and 2 µg/plate (WP2uvrA-); 9-aminoacridine (9-AA) 80 µg/plate (TA1537); 4-nitroquinoline-N-oxide (4NQO) 0.2 µg/plate (TA98)

 

Tab. 2: Experiment 1 (plate incorporation) with metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA100	TA1535	WP2uvrA-	TA98	TA1537
Solvent control	128±7.0	18±1.2	19±5.0	38±1.7	15±3.5
50	120±11.0	17±7.0	26±5.9	43±9.3	14±2.0
150	119±17.0	14±1.5	20±4.0	40±8.4	16±5.2
500	109±2.5	17±4.4	24±2.3	47±6.0	13±1.2
1500	117±8.5	13±2.3	22±6.0	43±4.0	13±3.2
5000	108±3.1	15±7.0	20±1.7	42±4.2	13±2.3
Positive control	1021±63.6	276±54.2	906±43.8	301±13.1	329±41.4

Solvent control: acetone

Positive controls: 2-aminoanthracene (2-AA) 1, 2, 0.5, and 10 µg/plate (TA100, TA1535, TA1537, TA98, and WP2uvrA)

Tab. 3: Experiment 2 (plate incorporation) without metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA100	TA1535	WP2uvrA-	TA98	TA1537
Solvent control	132±14.5	30±3.5	20±5.0	30±6.0	11±1.2
50	140±3.2	25±5.3	25±5.7	35±3.1	11±3.2
150	131±15.6	30±1.5	21±4.0	33±5.7	8±1.2
500	130±9.6	28±2.0	22±4.7	36±6.6	12±0.6
1500	133±80	29±3.0	21±3.5	35±3.8	11±1.0
5000	126±1.5	28±3.8	22±4.6	31±5.3	10±3.0
Positive control	508±18.0	261±81.0	966±66.5	157±36.5	1204±104.6

Solvent control: acetone

Positive controls: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) 3 µg/plate (TA100), 5 µg/plate (TA1535), and 2 µg/plate (WP2uvrA), 4-nitroquinoline-N-oxide (4NQO) 0.2 µg/plate (TA98), and 9-aminoacridine (9AA) 80 µg/plate

Tab. 4: Experiment 2 (plate incorporation) with metabolic activation: Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA100	TA1535	WP2uvrA-	TA98	TA1537
Solvent control	112±15.0	24±5.9	27±4.4	38±2.5	17±2.5
50	119±11.0	23±1.7	31±1.5	35±5.5	16±5.1
150	127±17.0	18±2.9	30±4.5	39±6.1	18±1.2
500	127±5.0	22±6.7	26±2.6	32±1.0	17±1.0
1500	121±16.2	23±5.6	29±6.1	37±3.1	15±1.0
5000	102±12.9	21±3.5	30±6.0	38±5.8	14±1.5
Positive control	1185±77.3	601±69.8	1088±53.7	246±22.7	179±10.1

Solvent control: acetone

Positive controls: 2-aminoanthracene (2-AA) 1 µg/plate (TA100), 2 µg/plate (TA1535, TA1537), 10 µg/plate (WP2uvrA), and 0.5 µg/plate (TA98)

Mutation study

Prior to use, the master strains were checked for characteristics, viability and spontaneous revision rate (all were found satisfactory).

No toxicity was exhibited to any strains of bacteria used. An oily precipitate was observed at 5000 µg/plate this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

Conclusions:

Interpretation of results:
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

HATCOL 5236

In Vitro Ames Assay: HATCOL 5236 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 5236 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. HATCOL 5236 precipitated on the plates at dose levels of 1000 µg/plate and upwards. 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. In the first and in the second mutation assay, HATCOL 5236 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 5236 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 5236 did not induce a dose-related, two-fold 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 an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 5236 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In Vitro Chromosome Aberration Assay: Evaluation of the ability of Hatcol 5236 to induce chromosome aberrations in cultured peripheral human lymphocytes. The report describes the effect of Hatcol 5236 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 5236 was tested in two independent experiments.

Batch H20139 of Hatcol 5236 was a clear pale yellow liquid with a purity of 97.6%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 5236 precipitated in the culture medium at this dose level. In the second cytogenetic assay, Hatcol 5236 was tested up to 100 µg/ml for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9 -mix. In the presence of 1.8% (v/v) S9-fraction Hatcol 5236 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay: In the absence of S9-mix, Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. In the presence of S9-mix, Hatcol 5236 induced a statistically significant increase in the numberof cells with chromosome aberrations at the intermediate concentration of 33 µg/ml, only when gaps were included. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related, only observed at the intermediate concentration of 33 µg/ml, only when gaps were included and the number of cells with chromosome aberrations was well within our historical control data range, the increase was considered not to be biologically relevant. Second cytogenetic assay: Hatcol 5236 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

Finally, it is concluded that this test is valid and that Hatcol 5236 is not clastogentic in human lymphocytes under the experimental conditions described in this report.

HATCOL 3344

In Vitro Ames Assay: HATCOL 3344 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the dose range finding test, HATCOL 3344 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the tester strain TA100. HATCOL 3344 precipitated on the plates at dose levels of 1000 µg/plate and upwards. 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. In the first and in the second mutation assay, HATCOL 3344 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. HATCOL 3344 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

HATCOL 334 did not induce a dose-related, two-fold 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 an independently repeated experiment.

Based on the results of this study it is concluded that HATCOL 3344 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In Vitro Chromosome Aberration Assay: The report describes the effect of Hatcol 3344 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 3344 was tested in two independent experiments.

Batch H102-01-28 of Hatcol 3344 was a clear colourless liquid with a purity of 96.9%. The test substance was soluble in ethanol.

In the first cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3344 precipitated in the culture medium at this dose level. In the second cytogenetic assay, Hatcol 3344 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9 -fraction Hatcol 3344 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hatcol 3344 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

Finally, it is concluded that this test is valid and that Hatcol 334 is not clastogenic in human lymphocytes under the experimental conditions described in the report.

HATCOL 3331:

In Vitro Chromosome Aberration Assay: Evaluation of the ability of Hatcol 3331 to induce chromosome aberrations in cultured peripheral human lymphocytes. The report describes the effect of Hatcol 3331 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Hatcol 3331 was tested in two independent experiments.

Batch D21287 of Hatcol 3331 was a clear colourless liquid with a purity of 97.3%. The test substance was soluble in ethanol. In the first cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Hatcol 3331 precipitated in the culture medium at this dose level. In the second cytogenetic assay, Hatcol 3331 was tested up to 100 µg/ml for a 24 hand 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9 -mix. In the presence of 1.8% (v/v) S9-fraction, Hatcol 3331 was also tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

Hatcol 3331 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, intwo independently repeated experiments. It is concluded that this test is valid and that Hatcol 3331 is not clastogenic in human lymphocytes underthe experimental conditions described in this report.

Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids

In vitro gene mutation in bacteria

The mutagenic potential of Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids was tested in a reverse mutation assay according to OECD Guideline 471 and under GLP conditions (Verspeek-Rip, 2003). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. In the first experiment (plate incorporation) tester strains were incubated with test material dissolved in ethanol at concentrations of 3, 10, 33, 100, 333, 1000, 3300 and 5000 µg/plate with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). In the second experiment, tester strains were incubated with the test substance dissolved in vehicle at concentrations of 10, 33, 100, 333, and 1000 µg/plate.

Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids (EC 451-190-0) did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

An in vitro Mammalian Cell Gene Mutation Assay according to OECD Guideline 476 and GLP was performed with Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids in mouse lymphoma L5178Y cells (Verspeek-Rip, 2014). In the first and second experiment, the cells were treated for 3 and 24 hours, respectively with 0.1, 0.3, 1, 3, 10, 33, 100, and 333 µg/mL of the test material in the presence or absence of S9-mix. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester

The mutagenic potential of the structural related PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Thompson, 1998). The following Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli WP2 uvr A were used. Tester strains were incubated with the test material dissolved in acetone at concentrations of 50, 150, 500, 1500, 5000 µg/plate in the first experiment and second experiment with and without the addition of a metabolic activation system (Arochlor 1254 induced rat liver S9 mix). The vehicle control (acetone) control plates gave counts of revertant colonies within the normal range. Appropriate positive controls induced marked increases in the frequency of revertant colonies, both with and without metabolic activation indicating the satisfactory performance and the sensitivity of the test and the efficacy of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. No cytotoxicity (visible reduction in the growth of the bacterial lawn at any dose level) was observed. An oily precipitate was observed at 5000 µg/plate, which did not prevent the scoring of revertant colonies. Thus, PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested and was considered non mutagenic under the conditions of this test.

An in vitro mammalian chromosome aberration test was performed with PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester in human lymphocytes comparable to OECD Guideline 473 and under GLP conditions (Wright, 1998). Duplicate cultures of human lymphocytes, treated with test material, were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). Four treatment conditions were used for the study: Experiment 1: 4 h exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16 h expression period and a 4 hour exposure in the absence of activation with a 16 h expression period. In Experiment 2 the 4-h exposure with addition of S9 was repeated (except that final S9 concentration was increased from 1% to 2%), whilst in the absence of activation the exposure time-was increased to 20 h. The following substance concentrations were used: Experiment 1, 4h, -S9: 39.06, 78.13, 156.25, 312.5, 625*, 1250*, 2500*, 5000* µg/mL; Experiment 1, 4h, +S9: 39.06, 78.13, 156.25*, 312.5, 625*, 1250*, 2500*, 5000* µg/mL; Experiment 2, 20h, -S9: 39, 78.1, 156.25, 312.5, 625*, 1250*, 2500*, 5000* µg/mL; Experiment 2, 4 h, +S9: 156.25, 312.5, 625, 1250*, 2500*, 5000* µg/mL (* dose levels selected for metaphase analysis).

The test substance did not induce cytotoxicity but an oily precipitate was visible in the first and second experiment at 625 µg/mL and above after 4 h incubation with without metabolic activation, and in the second experiment at and above 1250 µg/mL, -S9, and at and above 2500 µg/mL, +S9. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Ethyl methanesulphonate and cyclophosphamide were used as positive control materials inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

Gene Mutation Assay

Evaluation of the mutagenic activity of Hatcol HXL 8865 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat). The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures were based on the most recent OECD and EC guidelines.

The test substance was dissolved in ethanol, prepared directly prior to use. In the first experiment, Hatcol HXL 8865 was tested up to a concentration of 333 μg/ml in the absence and presence of 4% (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, Hatcol HXL 8865 was again tested up to a concentration of 333 μg/ml, but in the absence and presence of 8% (v/v) S9-mix. The incubation times were 24 hours and 3 hours, respectively.

In the absence of S9-mix, Hatcol HXL 8865 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Hatcol HXL 8865 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that Hatcol HXL 8865 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Justification for classification or non-classification

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. According to Regulation (EC) 1272/2008, the read across substances were not considered to be mutagenic, therefore the substance subject to registrations is not considered to be mutagenic.