Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 17, 2011 to June 27, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2A Genotoxicity: Specific Aspects of Regulatory Tests, Step 5

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 1591ZG-075; Appearance: white powder

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Main Assay I (plate incorporation method): 5000, 1580, 500, 158, 50.0 15.8, 5.00 and 1.58 ug/plate,
Main Assay II (pre-incubation assay): 5000, 2500, 1250, 625, 313, 156 and 78.1 ug/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Solutions of the test substance, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test substance. Concentrations were expressed in terms of substance as received.

The following growth media were used:
Nutrient Broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5 % in distilled water and autoclaved prior to use.
This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar: Oxoid Nutrient Broth No 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 L) and autoclaved.
The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar: Minimal medium agar was prepared as 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar: "Top Agar" (overlay agar) was prepared as 0.6 % Difco Bacto-agar + 0.5 % NaCl in distilled water and autoclaved. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL.

One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study.

Bacterial strains:

TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.

Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

Main experiments:
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test substance solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.

Experiment I (plate-incorporation method):
The components of the assay (the tester strain bacteria, the test substance and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows: overlay agar (held at 45 °C) 2 ml, test or control substance solution 0.1 ml, S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml, bacterial suspension 0.1 ml.

Experiment II (pre-incubation method):
The components were added in turn to an empty test-tube: bacterial suspension 0.1 ml, test or control substance solution 0.05 ml, S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml.

The solutions were vortexed and incubated at 37 °C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate.

Rationale for test conditions:

Solubility of the test substance was evaluated in a preliminary trial using DMSO, sterile distilled water and ethanol. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. The test substance was found to be soluble in DMSO at 100 mg/ml after 20 minute sonication. This result permitted a maximum concentration of 5000 ug/plate to be used in the Main Assay I.

Evaluation criteria:

Results shows that mean plate counts for untreated and positive control plates fell within the normal historical range. In the plate incorporation assay for TA1537, the positive control value in the absence of S9 metabolism slightly exceeded the maximum acceptable value based on the historical control data. However, the revertant number was lower than the maximum value observed in the historical control data.
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. The study was accepted as valid.
The test substance did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main Assay I (plate incorporation method):

- No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.
- Dose related precipitation of the test substance, that did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations.

Main Assay II (pre-incubation method):
- Toxicity, as indicated by thinning of the background lawn, was observed at the two or three highest dose levels with all tester strains in the absence and presence of S9 metabolism.
- Dose related precipitation of the test substance, that did not interfere with the scoring, was observed at the end of the incubation period at the three highest concentrations.
- Slight increases in revertant numbers, without any dose-effect relationship, were observed with TA1537 tester strain in the presence of S9 metabolism. These increases did not reach the two fold increases and were considered to be attributable to a chance event not related to the action of the test substance.

- The sterility of the S9 mix and of the test substance solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions.
- Marked increases in revertant numbers were obtained in these tests, following treatment with the positive control items, indicating that the assay system was functioning correctly.

Conclusions:

Under the study conditions, the test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay at any dose level and in any tester strain, in the absence or presence of S9 metabolism. It was concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Executive summary:

A study was conducted to determine the test substance ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9-mix. The test substance was used as a solution in DMSO. In Main Assay I, using the plate incorporation method, the substance was tested at the maximum dose level of 5000 µg/plate and at seven lower dose levels spaced at approximately half-log intervals: 1580, 500, 158, 50.0 15.8, 5.00 and 1.58 µg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Dose-related precipitation of the test substance that did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations. As no increases in revertant numbers were observed, a pre-incubation step was included for all treatments of Main Assay II. The test substance was assayed at the maximum dose level of 5000 µg/plate and at six lower dose levels spaced by a factor of two: 2500, 1250, 625, 313, 156 and 78.1 µg/plate. Toxicity, as indicated by thinning of the background lawn, was observed at the two or three highest dose levels with all tester strains in the absence and presence of S9 metabolism. Dose related precipitation of the test substance, did not interfere with the scoring, that was observed at the end of the incubation period at the three highest concentrations. Under the study conditions, the test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay at any dose level and in any tester strain, in the absence or presence of S9 metabolism. It was concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism (Ciliutti, 2011).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 25, 2017 to March 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

CAS No.162492-07-1; EINECS No.: 500-740-9; Batch No.: 1591ZG-101; Purity >98 %; Appearance: homogeneous white powder

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Test concentrations were based on the results from the pre-test.
Experiment I:
With and without metabolic activation: 500, 250, 125, 62.5, 31.25, 15.63 ug/mL
Experiment II:
Without metabolic activation: 500, 250, 125, 62.5, 31.25, 15.63 ug/mL

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMEM and DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Cultures:
In order to reduce the amount of spontaneous mutants, the cells are treated with DMEM-HAT (hypoxanthine-aminopterin-thymidine) medium (15 % horse serum (HS)) for 24 h at 37.0 ± 1.5 °C and 5.0 ± 0.5 % CO2. After a recovery period of 48 h at 37.0 ± 1.5 °C and 5.0 ± 0.5 % CO2, the cells are treated a second time in DMEM-TH (thymidine-hypoxanthine-) medium until they are transferred into DMEM complete culture medium. This procedure is referred to as “Cleansing”.
Cleansed and for mycoplasma contamination screened stocks of cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guar-antees similar parameters of the experiment and reproducible characteristics of the cells. In accordance to the corresponding SOP the V79 cells are thawed 5 - 10 d prior to treat-ment and cultivated in DMEM complete culture medium in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Metabolic Activation System:
S9 (liver enzyme mixture from male rats used for the test with metabolic activation) is not produced by LAUS GmbH, but is obtained from Trinova Biochem GmbH, Gießen. Upon arrival in the test facility, it is stored at < -80 °C. Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.

Chemicals and Media:

- Complete Culture medium DMEM with 5 % HS: Horse Serum 5 %, Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O 1 %.

-Selection medium DMEM with –TG: Horse Serum 5 %, Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O 1 %, 6-Thioguanine (1 mg/mL) 2 μg/mL.

Solutions:
- Phosphate buffer for S9-Mix (1 M): NaH2PO4 * H2O 0.4612 g, Na2HPO4 * 2H2O. 4.318 g, H2O demin. Ad 250 mL, pH was adjusted with HCl (1 M) to 7.4.
- NADP-solution for S9-Mix (0.1M): NADP-Disodiumsalt (M 787.4 g/mol) 1 g, H2O demin. 12.7 mL.
- Glucose-6-phosphate-solution for S9-Mix: Glucose-6-phosphat disodium salt * 2H2O (M 340.13 g/mol) 3401.3 mg, H2O demin. ad 10 mL.
- Salt solution for S9-Mix: KCl 1.23 g, MgCl2 * 6H2O 0.814 g, H2O demin. ad 10 mL.

Test Vessels: all vessels used were made of glass or sterile plastic.

Rationale for test conditions:

Solubility Test:
In a non-GLP pre-test, the solubility of the test substance was determined in DMEM, H2O demin., ethanol, DMSO, acetone, tetrahydrofuran and ethylene glycol. In none of the tested solvents the test substance was soluble at the concentration of 200 mg/mL. Further solubility tests with DMSO were performed. At the concentrations of 200 mg/mL and 100 mg/mL the test substance transformed into a hard insoluble lump after a while. At the concentrations of 50 mg/mL, 40 mg/mL, 30 mg/mL, 20 mg/mL and 10 mg/mL small precipitates were visible. In order to maximize the homogeneity of the suspensions, 30 min ultrasonic treatment was done, but the precipitates were still clearly visible at all these concentrations. Therefore, in addition all suspensions were shaken at 37 °C for 16 h. After this treatment only some transparent thread-like structures were left over. Those were hardly visible with the unaided eye and it was considered that the test substance was nearly dissolved. No notable difference in solubility was seen and therefore the concentration of 50 mg/mL was selected to prepare the series of dilutions.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Turbidity was visible at the concentrations 500 μg/mL and 250 μg/mL in experiment I (+S9 and -S9) as well as experiment II (S9). However no precipitates could be detected.

No cytotoxic effects were observed in any of the test substance concentrations in experiment I. Therefore, all concentrations were analysable for mutagenicity. In experiment II, a slight reduction of growth was detected at the test substane concentrations 500 μg/mL (replicate A: 42.2 %, replicate B: 41.4 %) and 250 μg/mL (replicate A: 60.5 %, replicate B: 58.8 %).
However both values are still far above the threshold of 20 % relative survival. Therefore, all concentrations were analysable for the evaluation of the mutagenicity.
No relevant and reproducible increase in mutant colony was observed in the main experiments up to the maximum concentration. All mutant frequencies remained well within the historical range of solvent controls.
No statistically significant increase in mutant frequency in comparison to the solvent control was detected. Also a concentration dependent effect was not observed.

Acceptability:

The gene mutation assay is considered acceptable if it meets the following criteria:

1. the mutant frequency found in the solvent controls falls within the laboratory historical

95% control limit.

2. the positive control substances must produce a significant increase (p < 0.05) in mutant frequency and lies in the range of the laboratory historical 95% control limit.

3. two experimental conditions (+S9 and -S9) are tested unless one resulted in positive results.

4. adequate number of cells (spontaneous MF is 5 – 20+E6) and concentrations (minimum of 4) are analysable.

5. the criteria for the selection of top concentration are met.

All acceptability criteria were met. Therefore, the study is considered as valid.

Conclusions:

Under the study conditions, the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test substance was considered to be non-mutagenic under the conditions of the HPRT assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce mutations at the HPRT locus on chromosome X in Chinese Hamster V79 cells according to In Vitro Mammalian Cell Gene Mutation Test (OECD 476), in compliance with GLP. The assay comprised a pre-test and two independent experiments (experiment I and II), using two parallel cultures each (replicates). The pre-test was done to detect a potential cytotoxic effect of the test substance. Based on the results, the concentrations for the main experiments were determined. In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation. The exposure time was 4 h. The following nominal concentrations were investigated: 500, 250, 125, 62.5, 31.25 and 15.63 μg/mL. Experiment II was performed with a treatment period of 24 h without metabolic activation. The same concentrations of the test substance were investigated. Cytotoxicity was almost not observed and therefore not a limiting factor for the dose selection. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in comparison to the solvent control in both experiments up to the maximal concentration of the test substance. Under the study conditions, the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test substance was considered to be non-mutagenic under the conditions of the HPRT assay (Fruhmesser, 2017).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 26, 2002 to November 03, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

CAS No.: 162492-07-1; Batch No.: lab; Purity: unknown by the sponsor, treated as 100 % pure; Appearance: white to off-white solid

Species / strain / cell type:

lymphocytes: cultured peripheral human

Metabolic activation:

with and without

Test concentrations with justification for top dose:

First cytogenetic assay:
Without and with S9-mix: 10, 33 and 100 ug test substance/ml culture medium

Second cytogenetic assay:

Without S9-mix:
10, 33, 66 and 100 ug test substance/ml culture medium (24 h exposure time, 24 h fixation time)
10, 20, 33, 66 and 100 ug test substance/ml culture medium (48 h exposure time, 48 h fixation time)
With S9-mix:
10, 33 and100 ug test substance/ml culture medium (3 h exposure lime, 48 h fixation time)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Hanks' Balanced Salt So/ution (HBSS) without calcium and magnesium

Details on test system and experimental conditions:

Test system: cultured peripheral human Iymphocytes,
Source: Healthy adult male volunteers,
Dose range finding study: age 36, AGT = 17.6 h,
First cytogenetic assay: age 38, AGT = 16.7 h,
Second cytogenetic assay: age 37, AGT = 16.6 h,
(AGT= Average Generation Time of the cells).

Cell culture:

Blood sample:
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. The blood samples were stored at a temperature between 4 and 25 °C. Within 4 h after blood collection, Iymphocyte 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) heatinactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), peniciilin/streptomycin (50 U/ml and 50 ug/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.

Lymphocyte cultures:
WhoIe 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 was added.

Environmental conditions:
All incubations were carried out in a humid atmosphere (80-100 %) containing 5 ±0.5 % C02 in air in the dark at 37 ± 1 °C. The temperature, humidity and CO2-percentage were monitored throughout the experiment.

Evaluation criteria:

A chromosome aberration test was considered acceptable if it met the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range {min=O, max=5 (mean=1 .0, standard deviation=1.1) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=O, max=4 (mean=0.8, standard deviation=0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded; for n=134 and 90 respectively).
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The number of cells with chromosome aberrations found In the solvent control cultures were within the laboratory historical control data range {min=0, max=6 (mean=1.1, standard deviation=1.1) aberrant cells per 100 metaphases In the absence of S9-mlx; gaps excluded and min=0, max=5 (mean=0.8, standard deviation=1.0) aberrant cells per 100 metaphases In the presence of S9-mix; gaps excluded; for n=652 and 433 respectively).
The positive control substances (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 the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two Independent experiments.

Conclusions:

Under the study conditions, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence or presence of S9-mix, in two independently repealed experiments. The test substance was therefore considered not to be clastogenic in human lymphocytes.

Executive summary:

A study was conducted to determine the effect of the test substance on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix) according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The test substance was tested up to 100 µg/ml for a 3 h exposure time with 24 h fixation time, in the absence and presence of S9-mix. The substance precipitated in the culture medium at this dose level. In the second cytogenetic assay, the substance was tested up to 100 µg/ml for a 24 h and 48 h continuous exposure time with a 24 and 48 h fixation time, in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction, the substance was also tested up 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 functioned properly. Under the study conditions, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence or presence of S9-mix, in two independently repealed experiments. The test substance was therefore considered not to be clastogenic in human lymphocytes (Buskens, 2002).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria (Ames test)

A study was conducted to determine the test substance ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9-mix. The test substance was used as a solution in DMSO. In Main Assay I, using the plate incorporation method, the substance was tested at the maximum dose level of 5000 µg/plate and at seven lower dose levels spaced at approximately half-log intervals: 1580, 500, 158, 50.0 15.8, 5.00 and 1.58 µg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Dose-related precipitation of the test substance that did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations. As no increases in revertant numbers were observed, a pre-incubation step was included for all treatments of Main Assay II. The test substance was assayed at the maximum dose level of 5000 µg/plate and at six lower dose levels spaced by a factor of two: 2500, 1250, 625, 313, 156 and 78.1 µg/plate. Toxicity, as indicated by thinning of the background lawn, was observed at the two or three highest dose levels with all tester strains in the absence and presence of S9 metabolism. Dose related precipitation of the test substance, did not interfere with the scoring, that was observed at the end of the incubation period at the three highest concentrations. Under the study conditions, the test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay at any dose level and in any tester strain, in the absence or presence of S9 metabolism. It was concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism (Ciliutti, 2011).

Gene mutation in mammalian cells

A study was conducted to investigate the potential of the test substance to induce mutations at the HPRT locus on chromosome X in Chinese Hamster V79 cells according to In Vitro Mammalian Cell Gene Mutation Test (OECD 476), in compliance with GLP. The assay comprised a pre-test and two independent experiments (experiment I and II), using two parallel cultures each (replicates). The pre-test was done to detect a potential cytotoxic effect of the test substance. Based on the results, the concentrations for the main experiments were determined. In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation. The exposure time was 4 h. The following nominal concentrations were investigated: 500, 250, 125, 62.5, 31.25 and 15.63 μg/mL. Experiment II was performed with a treatment period of 24 h without metabolic activation. The same concentrations of the test substance were investigated. Cytotoxicity was almost not observed and therefore not a limiting factor for the dose selection. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in comparison to the solvent control in both experiments up to the maximal concentration of the test substance. Under the study conditions, the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test substance was considered to be non-mutagenic under the conditions of the HPRT assay (Fruhmesser, 2017).

Chromosomal aberration in human lymphocytes

A study was conducted to determine the effect of the test substance on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix) according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The test substance was tested up to 100 µg/ml for a 3 h exposure time with 24 h fixation time, in the absence and presence of S9-mix. The substance precipitated in the culture medium at this dose level. In the second cytogenetic assay, the substance was tested up to 100 µg/ml for a 24 h and 48 h continuous exposure time with a 24 and 48 h fixation time, in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction, the substance was also tested up 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 functioned properly. Under the study conditions, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence or presence of S9-mix, in two independently repealed experiments. The test substance was therefore considered not to be clastogenic in human lymphocytes (Buskens, 2002).

Justification for classification or non-classification

Based on the results of in vitro assays, the test substance does not need to be classified for genotoxicity according to CLP (EC 1272/2008) criteria.