[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation assay

Non Mutagenic

in vitro gene mutation study in mammalian cells

Non mutagenic

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

From 22 July 1994 to 24 January 1995

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, Annex (November 18, 1987) Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (hprt)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F 10 medium supplemented with 10% foetal calf serum and antibiotics
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

CYTOXICITY TEST
Range with metabolic activation:
500.0000 µg/ml
250.0000 µg/ml
125.0000 µg/ml
62.5000 µg/ml
31.2500 µg/ml
15.6250 µg/ml
7.8125 µg/ml
3.9063 µg/ml
1. 9531 µg/ml
0.9766 µg/ml
0.4883 µg/ml
0.2441 µg/ml

Range without metabolic activation:
500.0000 µg/ml
250.0000 µg/ml
125.0000 µg/ml
62.5000 µg/ml
31.2500 µg/ml
15.6250 µg/ml
7.8125 µg/ml
3.9063 µg/ml
1. 9531 µg/ml
0.9766 µg/ml
0.4883 µg/ml
0.2441 µg/ml

MUTAGENICITY TEST
Original experiment:
Range with metabolic activation:l
250.0000 µg/ml
83.3333 µg/ml
27.7778 µg/ml
9.2593 µg/ml

Range without metabolic activation:
100.0000 µg/ml
33.3333 µg/ml
11.1111 µg/ml
3.7037 µg/ml

Confirmatory experiment:
Range with metabolic activation:
300.0000 µg/ml
100.0000 µg/ml
33.3333 µg/ml
11.1111 µg/ml

Range without metabolic activation:
150.0000 µg/ml
50.0000 µg/ml
16.6667 µg/ml
5.5556 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

N-dimethylnitrosamine with metabolic activation; ethylmethanesulphonate without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours
- Expression time (cells in growth medium): from 7 to 8 days

SELECTION AGENT (mutation assays):
- 6-thioguanine

NUMBER OF REPLICATIONS:
- cultures were treated in duplicate with four test chemical concentrations, a positive and a negative control

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines: ARLETT, C.F., D.M. SMITH, M.H.L. Green, D.B. McGREGOR, G.M. CLARKE, J. COLE, J.C. ASQUITH (1990) Mammalian cell gene mutation assays based upon colony formation. In: Statistical Evaluation of Mutagenicity Test Data (ed Kirkland, D.J.) Cambridge University press, pp 67-101.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test with and without metabolic activation 12 concentrations of substance were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated by 2-fold intervals.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was seen during original experiment performed with and without metabolic activation.
No toxicity was seen during confirmatory experiment performed with and without metabolic activation.

Remarks on result:

other: strain/cell type: Chinese Hamster cell line V79

Remarks:

Migrated from field 'Test system'.

Conclusions:

Non mutagenic

Executive summary:

Method

The experiment was conducted in agreement to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test), EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture) and EEC Directive 87/302, Annex (November 18, 1987) Part B ( Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test).

The test was performed following the GLP.

The test article was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in DMSO.

A preliminary range finding test was run assessing cytotoxicity. The substance was tested at concentrations up to 500.0 µg/ml. Higher concentration could not be applied due to solubility limitations in the vehicle. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested. Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment.

Depending on the toxicity of the test compound 2.5-5.0x10E6 cells of passage 27 (original experiment) and passage 31 (confirmatory experiment) were plated in 30 ml growth medium into flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone.

In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 µl/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 µl/ml.

Cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).

The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype. At the end of the expression period the cultures were trypsinised pelleted, resuspended in fresh growth medium and counted.

The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 µg/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control).

After seven to eight days incubation at 37°C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.

In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The number of colonies which developed within these cultures reflected the viability at the end ofthe expression period (viability values).

Results

According to the acceptance criteria outlined in the above mentioned guidelines, in the presence and absence of metabolic activation, no relevant increase in mutant frequency was observed at any concentration level of substance tested in the original or the confirmatory experiment in comparison with the negative control.

In the two experiments performed in the presence of metabolic activation, statistically significant differences were obtained at two concentrations each. However, at none of these concentrations the number of normalized mutant clones differed by more than 20 compared to the respective control cultures.

Moreover, no concentration dependency was obvious and the effects were not reproducible (appeared at different concentrations in the two experiments). They are therefore considered to be purely fortuitous and not related to treatment with the test compound.

The positive controls induced a clear increase in mutant frequency.

Conclusion

Non mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Non clastogenic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

From November 28 to January 23, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male animals-mean = 181.4 g (= 100 %) (min = 173 g, max = 191 g); female animals- mean = 146.5 g (=100 %) (min = 140 g, max = 154 g)
- Assigned to test groups randomly: Yes
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-H (V 1534), ad libitum, ssniff® GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From December 03, 2002 to December 05, 2002

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SUSPENSION: On the day of first administration the test substance was dissolved in deionized water at an appropriate concentration.
A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Stability and homogeneity in the vehicle : guaranteed for 96 hours in deionized water by the sponsor (archived with the raw data)

Duration of treatment / exposure:

2 d

Frequency of treatment:

twice at an interval of 24 h

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 per sex per group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Tissues and cell types examined:

- 2,000 polychromatic erythrocytes were counted for each animal.
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No preliminary experiments were performed, as corresponding toxicological information was available. Since 5000 mg per kg body weight resulted in no lethality in acute oral toxicity testing a limit test with 2000 mg per kg body weight was performed.
.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.

-Staining procedure: The slides were stained as follows:-
-5 minutes in methanol
-5 minutes in May-Grunwald's solution
-brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-rinsing in distilled water
-drying
-coating with Entellan

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered as positive if there is a significant dose- related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.

Statistics:

Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Exposure was assured by the presence of light grey coloured tissues at necropsy

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment. No signs of toxicity were detected. All dosed animals showed black discoloured faeces and Body surface showed grey discolorations which were noted on hairless areas 24 hours after the first administration up to the end of study.
The dissection of the animals revealed a nearly black coloured content of the gastro-intestinal tract and light grey coloured tissues.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the test tem-treated groups was within the normal range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7 — 4.9). No statistically significant increase in micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Conclusions:

Not clastogenic

Executive summary:

Method

The study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley). At study start the animals were 6 weeks of age and had mean body weights of 181.4 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

Observations

The number of polychromatic erythrocytes containing micronuclei was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20 % from the control value.

Endoxan®induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Conclusions

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus testin vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The assessment of mutagenicity potential of the substance is based on one in vitro study, carried out on Similar Substance 01, and one in vivo study, carried aout on Similar Substance 02.

Further information are reported in the Read Across justification attached to section 13.

IN VITRO BACTERIAL REVERSE MUTATION TEST

An ames test is in progress and expceted to be negative.

IN VITRO MAMMALIAN CELL GENE MUTATION TEST

The substance was tested on chinese hamster cells V79 according to OECD 476. Test article was a greenblack powder with purity of 54.62%. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours.

A preliminary range finding test was run assessing cytotoxicity. The substance was tested at concentrations up to 500.0 µg/ml. Higher concentration could not be applied due to solubility limitations in the vehicle. In the part with metabolic activation, at the highest concentration a 100 % growth inhibiting effect could be seen, while the next lower concentration of 250.0 µg/ml inhibited 90.92 %. Without metabolic activation treatment with substance proved growth inhibiting down to the concentration of 125 µg/ml. The next lower concentration of 62.5 µg/ml revealed an acute inhibition of growth of 63.99 %. Accordingly, 250.0 µg/ml with and 100.0 µg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation: the original experiment was performed at the following concentrations: 9.26, 27.78, 83.33 and 250.0 µg/ml. The mean growth inhibiting value found at the highest concentration after treatment was 71.35 %. No toxicity was seen after expression.

In the confirmatory experiment the concentrations applied were 11.11, 33.33, 100.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 93.76%. The mean growth inhibitory effect after the expression period was 12.28 %.

Mutagenicity test without metabolic activation: the original experiment was performed at the following concentrations: 3.70, 11.11, 33.33 and 100.0 µg/ml. The mean growth inhibition value found at the highest concentration after treatment was 38.33 %. No toxicity was seen after expression.

In the confirmatory experiment the concentrations applied were 5.56, 16.67, 50.0 and 150.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 71.47 %. No toxicity was seen after expression.

For both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the substance and its metabolites did not show any mutagenic activity in this forward mutation system.

IN VIVO MAMMALIAN SOMATIC CELL STUDY: CYTOGENICITY/ERYTHROCYTE MICRONUCLEUS

The study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley). At study start the animals were 6 weeks of age and had mean body weights of 181.4 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20 % from the control value.

Endoxan®induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus test in vivo.

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

— mammalian bone marrow chromosome aberration test;

— mouse spot test;

— mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

An in vitro mammalian cell gene mutation test is available and the test substance does not show any mutagenic effect.

As per REACH Regulation requirements, a further in vivo study is considered in which the test substance didn’t induce increase the number of polychromatic cells with micronuclei.

Therefore, as reported in the Flow chart R.7.7 -1, no further test are required and it is possible to conclude that the test substance is Not Classified for Mutagenic Toxicity.