Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 19, 2012 to August 01, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP complaince with international guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Results obtained in a repeated experiment

Deviations:

yes

Remarks:

Results obtained in a repeated experiment

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Results obtained in a repeated experiment

Deviations:

yes

Remarks:

Results obtained in a repeated experiment

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes, regular intervals.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue homogenate

The rat S9 liver tissue fraction used for the present study had the following characteristics:

Lot Number : 2879

Inducing Agents: Phenobarbital – 5,6-Benzoflavone

Received from: MOLTOX, Molecular Toxicology, Inc.

Storage at RTC: Approximately -80°C

Strain : Sprague Dawley

Sex of donors: Male

Protein content: 42.73 mg/mL



S9 mix

The S9 liver tissue fraction will be prepared according to RTC standard procedures or will be obtained from an appropriate supplier (MOLTOX, Molecular Toxicology, Inc., USA). Induction of drug metabolising enzyme-levels is routinely performed using phenobarbitone and betanaphtoflavone (mixed induction). Induction with Aroclor 1254 will be performed if specifically requested by the Sponsor. Records pertaining to the preparation of the S9 tissue fraction are kept on file at RTC.

The mixture of S9 tissue fraction and cofactors (S9 mix) will be prepared in the following proportions:

S9 tissue fraction 3.0 ml

NADP (0.1M) 0.4 ml

G-6-P (0.1M) 0.5 ml

KCl (0.33M) 1.0 ml

MgCl2 (0.1M) 0.4 ml

Hepes (0.2M) 1.0 ml

H.B.S.S. 3.7 ml

10.0 ml



Treatment cultures:

Treatment medium without metabolic activation will be prepared for each test point in the following proportions:

Test item or control solution 0.05 ml

Supplemented Ham's F10 medium 4.95 ml

Treatment medium with metabolic activation will be prepared containing the test item and S9 mix in the following proportions:

Test item or control solution 0.05 ml

S9 mix 0.50 ml

Supplemented Ham's F10 medium 4.45 ml

Test concentrations with justification for top dose:

Experiment No. S9 Treatment time(hours) Harvest time (hours) Dose level(µg/mL)
1 ± 3 20 625, 313 ,156 ,78.1 and 39.1
2 - 20 20 80.0, 40.0 and 20.0

The dose levels of 0.30 and 0.10 µg/mL were selected for the scoring of cultures treated with Mitomycin-C (first and second experiment, respectively). The dose level of 15.0 µg/mL was selected for the scoring of cultures treated with Cyclophosphamide.

Vehicle / solvent:

distilled water, culture medium, DMSO, ethanol, acetone.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
The medium used for the growth of the cells has the following composition:
Ham's F.10 499.0 ml
Streptomycin sulphate 50,000 IU/ml
Penicillin G 50,000 IU/ml 1.0 ml
Newborn Calf Serum 88.2 ml

DURATION
- Expression time (cells in growth medium): Each 25 sq.cm. flask will be seeded with 300,000 cells in supplemented Ham's F10. These cells should be in exponential growth phase at the time of treatment.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1 x 10^6 cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled:
(i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control.
(ii) The increases must exceed the historical control values.
(iii) The increases are reproduced in both replicate cultures.
The evaluation is based on the set of results, which excludes gaps. A more detailed explanation of the criteria for evaluation of the results is given in the Study Protocol.

Statistics:

For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps. Following treatment in the absence of S9 metabolism, a statistically significant increase in the incidence of cells bearing structural aberrations including and excluding gaps was observed in the first main experiment, at the intermediate dose level selected for scoring. No other statistically significant increases were observed in any experiment, at any dose level, in the presence or absence of S9 metabolic activation. A statistically significant increase of endoreduplicated cells was observed at the highest dose level after treatment in the presence of S9 metabolic activation

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: and osmolality: Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism. These results are not presented in this report but are retained in the study file and archived as indicated in the Study Protocol.

Deviations from Study Protocol

Results presented for the first experiment in the presence of S9 metabolic activation were obtained in a repeated experiment. In the original one, a low cellular growth was observed, probably due to the toxicity of S9 metabolic fraction. Data concerning the previous experiment are not presented in the report but will be archived as indicated in the Study Protocol.

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce structural chromosomal aberrations in Chinese hamster ovary cells in vitro.However, treatment with the test item in the presence of S9 metabolic activation induced a biologically relevant increase in the number of cells with endoreduplicated chromosomes, indicating that the test item may have the potential to inhibit cell cycle progression. Statistically significant increases in aberrant cells, compared with the relevant control values, were seen in cultures treated with the positive controls Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Conclusions:

On the basis of these results, it is concluded that PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 does not induce chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions.
It is also concluded that the test item induces endoreduplication in the presence of S9 metabolic activation.

Executive summary:

The test item PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 was assayed for the ability to cause chromosomal damage in Chinese hamster ovary cells, following in vitro treatment in the absence and presence of S9 metabolic activation. Two main experiments for chromosomal damage were performed.

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 20 hours corresponding to approximately 1.5 cell cycle length was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using the same harvest time. A continuous treatment until harvest at 20 hours was used.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO). For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol and on the basis of the solubility of the test item. Dose levels of 1250, 625, 313, 156, 78.1, 39.1,19.5 and 9.77 μg/mL were used both in the absence and presence of S9 metabolism.

The dose range used in the second experiment was modified to take into account the results observed in the previous experiment.

Dose levels of 160, 80.0, 40.0, 20.0, 10.0, 5.00, 2.50 and 1.25 μg/mL were used. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

Dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of population doubling or relative cell count. Based on the obtained results, the following dose levels were selected for scoring:

Experiment No.	S9	Treatment time (hours)	Harvest time (hours)	Dose level (ug/mL)
1	+ -	3	20	625,313 and 156 156, 78.1 and 39.1
2	-	20	20	80.0, 40.0 and 20.0

One hundred metaphase spreads were scored for chromosomal aberrations from each culture, with the exception of one culture treated with Mitomycin-C from the first main experiment, where, due to the high incidence of aberrant cells (excluding gaps), scoring was terminated at 50 metaphases. Following treatment with test item, no statistically significant increase in the number of cells bearing structural aberrations, including or excluding gaps, was observed at any dose level or treatment time in the absence or presence of S9 metabolic activation. For the first main experiment, a statistically significant increase of endoreduplicated cells over the concurrent negative control was observed at the highest dose level selected for scoring (625 μg/mL) in the presence of S9 metabolism. Few endoreplicated cells were also observed at the intermediate dose level selected for scoring (313 μg/mL).

Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatment with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the test system. It is concluded that PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 does not induce structural chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions. However, treatment with the test item induces an increase in the number of cells with endoreduplicated chromosomes only in the presence of metabolic activation and at the highest dose level selected for scoring (625 μg/mL), indicating that the test item may have the potential to inhibit cell cycle progression.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The substance PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 did not induce reverse mutation in Salmonella typhimurium or Escherichia coli (Baslini 91050) , not induce mutation at the TK locus of L5178Y mouse lymphoma cells

(Salvador 91070) and not induce chromosomal aberrations in Chinese hamster ovary cells (Ciliutti 91060).

Based on results it is concluded that PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) is not genotoxic with or without metabolic activation.

Justification for selection of genetic toxicity endpoint
GLP compliant , OECD and EU guidelines, well described

Justification for classification or non-classification

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.

For the purpose of classification for germ cell mutagenicity, substances are classified if there is at least 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.

All the presented results from in vivo tests are negative, therefore it is concluded that PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) is not genotoxic with or without metabolic activation.