Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

KGD 1409 shows mutagenic effects in two Salmonella/microsome tests (strains TA 98, TA 100, TA 102, TA 1535 and TA 1537; +/- S9 mix) (Herbold, 2002; Meyer, 2015). In a HPRT test (V79 cells; +/- S9 mix) a negative result was observed after exposure to KGD 1409 (Wollny, 2016). A micronucleus test in vitro (V79 cells) revealed a clastogenic effect in the presence of S9 mix after 4h treatment to high concentrations of KGD 1409 (Naumann, 2016).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar to May 2015

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-06-12

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 50, 160, 500, 1600, 5000 µg/plate (without and with S9 mix)

Vehicle / solvent:

Solvents used: demineralized water (test substance), deionized water (mitomycin C), DMSO (sodium azide, cumene hydroperoxide, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100, TA 1535), 2-nitrofluorene (TA 98), 4-nitro-1,2-phenylene diamine (TA 1537), mitomycin C (TA 102, plate incorp.), cumene hyd (TA 102, preincub. trials), 2-aminoanthracene (all strains)

Remarks:

The positive controls sodium azide, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

METHOD: Standard plate test and preincubation test

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: plate incorporation test

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test) with KGD 1409 (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	13	110	5	16	205
50	10	104	4	16	210
160	10	91	6	12	218
500	9	97	5	14	200
1600	10	92	6	15	201
5000	15	111	4	18	203
Positive control	660	1395	39	1167	782
Dose (µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	12	154	9	28	296
50	11	139	9	29	329
160	11	164	10	26	321
500	13	134	8	31	301
1600	15	148	8	25	300
5000	20	144	8	26	272
Positive control	132	2980	279	2791	1158

Table 2: Summary of results from the Salmonella mutagenicity assay (independent preincubation test) with KGD 1409 (mean values of revertants per plate)

 

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	10	114	6	16	240
50	10	114	7	21	245
160	8	111	7	20	261
500	10	105	6	13	253
1600	8	109	7	16	259
5000	20	118	8	17	265
Positive control	656	842	37	1101	550
Dose (µg per plate)	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	10	156	10	27	334
50	10	150	8	28	343
160	11	162	9	27	341
500	9	164	9	28	339
1600	15	162	8	26	359
5000	25	178	8	28	337
Positive control	239	2204	382	2251	1013

 

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects or precipitations.

Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535. The Salmonella/microsome test thus showed the test item to have a mutagenic effect.

In both experiments the positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluoren, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Conclusions:

positive

Executive summary:

The mutagenic potential of KGD 1409 was evaluated in a Salmonella/microsome test (plate incorporation test and preincubation methood) with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects or precipitations.

Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535.

Therefore, the test item was considered to be mutagenic without and with S9 mix in the preincubation modification of the Salmonella/microsome test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug to Sep 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

screening version (only one plate per dose and strain investigated, purity of test item not specified)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 38
- Purity: not specified
- Expiration date of the lot/batch: 2002-10-05

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 induced rat livers

Test concentrations with justification for top dose:

plate incorporation test and preincubation test: 16, 50, 160, 500, 1600, 5000 µg/plate (all strains);
preincubation test: 900, 1300, 1800, 2600, 3600, 5200 µg/tube (TA 1535 only)

Vehicle / solvent:

Solvents used: deionized water (test substance), DMSO (positive controls)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, nitrofurantoin and 4-nitro-1,2-phenylene diamine were only used without S9 mix and 2-aminoanthracene was only used with S9 mix

Details on test system and experimental conditions:

METHOD: Standard plate test and preincubation test

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537 at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

other: weakly positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: plate incorporation test

Doses up to and including 5200 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed.

In the plate incorporation trial no evidence of mutagenic activity of KGD 1409 was seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. However, in the preincubation trials evidence of mutagenic activity of KGD 1409 was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest effective dose was 1800 µg per tube. Due to the weakness of the effect it cannot be excluded, that this effect is based on an impurity and not on KGD 1409 itself.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Conclusions:

weakly positive

Executive summary:

KGD 1409 was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on the five Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C using doses of up to and including 5200 µg per tube.

Doses up to and including 5200 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed.

In the plate incorporation trial no evidence of mutagenic activity of KGD 1409 was seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. However, in the preincubation trials evidence of mutagenic activity of KGD 1409 was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest effective dose was 1800 µg per tube. Due to the weakness of the effect it cannot be excluded, that this effect is based on an impurity and not on KGD 1409 itself.

Therefore, KGD 1409 was considered to be weakly mutagenic without and with S9 mix in the preincubation screening.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June to Aug 2016

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2016-07-08

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

62.5, 125, 250, 500, 1000, 2000 µg/mL (-/+ S9 mix)

Vehicle / solvent:

Vehicle(s)/solvent(s) used: deionised water (test substance); nutrient medium (ethylmethane sulfonate); DMSO (7,12-dimethylbenzanthracene)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate (EMS; -S9 mix); 7,12-dimethylbenzanthracene (DMBA; +S9 mix)

Details on test system and experimental conditions:

- DOSE SELECTION: According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye and microscopically. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 15.6 μg/mL and 2000 μg/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476. In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed up to the highest concentration with and without metabolic activation. The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. No precipitation or phase separation occurred up to the maximum concentration after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations. The cultures at the lowest concentration with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

- CULTURE MEDIUM: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

- SEEDING: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×107 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

- TREATMENT: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G". Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium. Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below. Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II). The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range. A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits). The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together. A t-test was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value is below 0.05.

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The main experiment was evaluated at the following concentrations: 125, 250, 500, 1000, 2000 µg/mL (-/+ S9 mix).

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiment up to the maximum concentration.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

The 95% confidence interval was slightly exceeded at 125.0 μg/mL in the second culture of the main experiment with metabolic activation (28.9 versus an upper limit of 28.7 mutant colonies/10⁶ cells). This isolated increase was judged as irrelevant as it was not dose-dependent and not reproduced in the second culture. Even though the t-test turned out significant, the mean value of the mutation frequency of both parallel cultures remained well within the 95% confidence interval with 19.25 mutant colonies/10⁶ cells

In the main experiment with and without S9 mix the range of the solvent controls was from 5.5 up to 15.1 mutants per 10⁶ cells; the range of the groups treated with the test item was from 6.6 up to 28.9 mutants per 10⁶ cells.

EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

negative

Executive summary:

KGD 1409 was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 2000 µg/mL with and without metabolic activation. No relevant cytotoxic effect occurred up to the maximum concentration with and without S9 mix. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix.

In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, KGD 1409 is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

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:

SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2016-11-08

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium (MEM) with supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1 (4 h treatment): 13.0, 22.7, 39.8, 69.6, 122, 213, 373, 653, 1143, 2000 µg/mL (+/- S9 mix)

Vehicle / solvent:

Vehicle(s)/solvent(s) used: deionised water (test substance); deionised water (mitomycin C); 0.9 % NaCl (cyclophosphamide)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 10 % deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C (- S9 mix); cyclophosphamide (+ S9 mix)

Details on test system and experimental conditions:

- CULTURE CONDITIONS
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week. Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. Per culture approximately 5.0 – 6.0 x 105 cells will be seeded into 25 cm2 plastic flasks. All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

- DOSE SELECTION
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration should be 10 mM, 2 mg/mL or 2 μL/mL, whichever is the lowest. At least three test item concentrations should be evaluated for cytogenetic damage. 2000 μg/mL of KGD1409 were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.0 to 2000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Since the first experiment showed a positive result, no further experiment was performed.

- PRE-EXPERIMENT
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of 10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

- CYTOGENETIC EXPERIMENT
a. Pulse exposure:
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS in the presence of Cytochalasin B (1.5μg/mL) for the remaining culture time of 20 hours.

b. Preparation of micronuclei:
Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvest and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37°C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on a clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

- EVALUATION OF CYTOTOXICITY AND CYTOGENETIC DAMAGE
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Evaluation criteria:

- INTERPRETATION OF RESULTS
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data

When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. narrow concentration spacing, other metabolic activation conditions, i.e. S9 concentration or S9 origin) could be useful.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Only one experiment was performed, since the test item was considered to be mutagenic after the first experiment. The exposure period was 4 hours with and without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.

No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH was observed.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration of 2000 µg/mL.

In the absence of S9 mix, no relevant increase in the number of micronucleate cells was observed after treatment with the test item. In the presence of S9 mix, statistically significant increases in the number of micronucleate cells were observed after treatment with 653 and 2000 μg/mL (2.05 and 2.78 %). The second value clearly exceeded the range of the laboratory historical control data (0.0 - 2.62 % micronucleate cells).

Mitomycin C (0.3 μg/mL) and cyclophosphamide (2.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:

positive

Executive summary:

KGD 1409 was examined for chromosome breakage (clastogenic effects) and misdistribution of chromosomes (aneugenic effects) in the in vitro micronucleus test using Chinese hamster V79 cells according to OECD TG 487. Solvent controls and appropriate positive controls with known mutagens demonstrated the suitability and sensitivity of the test system. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration of 2000 µg/mL. In the absence of S9 mix, no relevant increase in the number of micronucleated cells was observed after 4h treatment with the test item. In the presence of S9 mix, statistically significant increases in the number of micronucleated cells were observed after 4h treatment with 653 and 2000 μg/mL (2.05 and 2.78 %). The second value clearly exceeded the range of the laboratory historical control data (0.0 - 2.62 % micronucleated cells).

Therefore, KGD1409 is considered to be mutagenic in the in vitro micronucleus test, when tested up to the highest required concentration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

KGD 1409 was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on the five Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and TA 102 (Herbold, 2002). The independent repeat was performed as preincubation for 20 minutes at 37°C using doses of up to and including 5200 µg per tube. Doses up to and including 5200 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. In the plate incorporation trial no evidence of mutagenic activity of KGD 1409 was seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. However, in the preincubation trials evidence of mutagenic activity of KGD 1409 was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest effective dose was 1800 µg per tube. Due to the weakness of the effect it cannot be excluded, that this effect is based on an impurity and not on KGD 1409 itself. Therefore, KGD 1409 was considered to be weakly mutagenic without and with S9 mix in the preincubation screening.

To verify the mutagenic potential of KGD 1409 a further Salmonella/microsome test (plate incorporation test and preincubation methood) with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 was performed in the presence and absence of S9 mix according to OECD TG 471 (Meyer, 2015). Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects or precipitations. Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535. Therefore, the test item was considered to be mutagenic without and with S9 mix in the preincubation modification of the Salmonella/microsome test.

KGD 1409 was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476 (Wollny, 2016). The cells were exposed to the test item for 4 hours in concentrations up to and including 2000 µg/mL with and without metabolic activation. No relevant cytotoxic effect occurred up to the maximum concentration with and without S9 mix. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, KGD 1409 is considered to be non-mutagenic in this HPRT assay.

In addition, KGD 1409 was examined for chromosome breakage (clastogenic effects) and misdistribution of chromosomes (aneugenic effects) in the in vitro micronucleus test using Chinese hamster V79 cells according to OECD TG 487 (Naumann, 2016). Solvent controls and appropriate positive controls with known mutagens demonstrated the suitability and sensitivity of the test system. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration of 2000 µg/mL. In the absence of S9 mix, no relevant increase in the number of micronucleated cells was observed after 4h treatment with the test item. In the presence of S9 mix, statistically significant increases in the number of micronucleated cells were observed after 4h treatment with 653 and 2000 μg/mL (2.05 and 2.78 %). The second value clearly exceeded the range of the laboratory historical control data (0.0 - 2.62 % micronucleated cells). Therefore, KGD 1409 is considered to be genotoxic in the in vitro micronucleus test, when tested up to the highest required concentration.

In summary, in vitro testing of KGD 1409 revealed evidence for a possible genotoxic potential of the test substance based on the positive results of two bacterial reverse mutation tests and an in vitro mammalian cell micronucleus test at high concentrations.

Justification for classification or non-classification

Even if the classification criteria are not fully met, a classification according to Regulation (EC) No.1272/2008 (CLP) with Muta. 2 ( H341: Suspected of causing genetic defects) is proposed based on the positive results of two bacterial reverse mutation tests and an in vitro mammalian cell micronucleus test.