Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity:

in vitro: Bacterial reverse mutation test, S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535, up to 5000 µg/plate, plate incorporation & preincubation method: negative ± S9 (OECD 471, GLP)

in vitro: Bacterial reverse mutation test, S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538, up to 5000 µg/plate, plate incorporation method: negative ± S9 (read-across from PPS, OECD 471, GLP)

in vitro: mammalian cell micronucleus test, human lymphocytes, 657.3 µg/mL, 1150.3 µg/mL and 2013 µg/mL: negative ± S9 (read-across from PPS, OECD 487, GLP)

in vitro: Mammalian cell forward mutation test, mouse lymphoma L5178Y cells, 2000, 1000, 500, 250 and 125 µg/ml: negative ± S9 (OECD 490, GLP)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2007 - 11 January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000-05-19
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
other: TA 1535, TA 97a, TA 98, TA 100, TA 102
Details on mammalian cell type (if applicable):
TA97a: hisD6610, TA 98: hisD3052 TA 100 und TA 1535 hisG46, TA102:hisG428, TA97, TA98 and TA100 contain uvrB; TA97, TA98, TA100 and TA102 contain pKM 101 and rfa.
Metabolic activation:
with and without
Metabolic activation system:
S9 [obtained by TRINOVA Biochem GmbH, Gießen, Germany (Batch nos: 2081, 2113, 2118)] Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254 per kg body weight intraperitoneally.
Test concentrations with justification for top dose:
First experiment : 5010, 1503, 501, 150 and 50 µg/plate (Plate incorporation method)
Second experiment : 5002, 2501 and 1251 µg/plate (pre-incubation method)
Vehicle / solvent:
- Solvent used: deionised water
- Justification for choice of solvent : deionised water was chosen as solvent, because the test item was completely soluble, and this solvent doesn’t have any effects on the viability of the bacteria or the number of spontaneous revertants.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine ; 2-Aminoanthracene
Remarks:
See -Any other information on materials and methods incl. tables- for details
Details on test system and experimental conditions:
Bacteria :
12 hours before the start of each experiment, a stock culture of each strain was thawed and an aliquot was placed into a culture flask containing 70 ml nutrient broth. The flasks were incubated at 37°C for 12 hours.

Description of the Method
Preparations :
In the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilized and the first batches poured. On the day before the test, the remaining plates were poured. On the day of the test, the overnight cultures were checked for growth. The incubation chamber was heated to 37°C. The water bath and the heating block were turned to 43°C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0°C.

Experimental Parameters
First Experiment :
Date of treatment : 12 December 2007
Concentrations tested : 5010 / 1503 / 501 / 150 / 50 µg/plate
Incubation time : 48 hours
Incubation temperature : 37°C
Tester strains : TA97a, TA98, TA100, TA102, TA1535
Method : Plate incorporation method

Second Experiment
Date of treatment : 09 January 2008
Concentrations tested : 5002 / 2501 / 1251 µg/plate
Incubation time : 48 hours
Incubation temperature : 37°C
Tester strains : TA97a, TA98, TA100, TA102, TA1535
Method : pre-incubation method

Description of the Method
Plate incorporation method :
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidin-biotin-solution 0,5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45°C. 0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mixture was gently vortexed, then poured an a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

Pre-incubation method
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C. 0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 mL top agar were added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

References
Genotype Confirmation
-Histidine requirement : Each strain was streaked on a biotin and a histidin-biotin-plate, using a sterilized wire loop.
-Ampicilline-Resistance (pKM 101) resp. ampicilline-tetracycline-resistance (pAQ1) The strains were streaked on ampicilline agar, TA102 on ampicilline-tetracycline agar. TA1535 was taking the function of control strain, since it is not ampicilline resistant.
-UV-sensitivity (uvrB) : Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 33 cm. Incubation over night at 37°C followed.
-Crystal violet sensitivity (deep rough) : For each strain, two plates were used. 0,1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (9 mm diameter), each soaked with 10 μL of crystal violet solution (0,1%) were placed into the middle of each plate, followed by incubation over night.
-Spontaneous revertants : Four replicates, with/without S9, for each solvent which was used in the test.
-Determination of Titre : The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0,1 mL on maximal-soft agar. It should give a density of 10E9 cells/mL (at the least).
-Toxicity Control : Performed analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar.
-Sterility Control : Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar.
-Positive Controls : Using diagnostic mutagens, four replicates were prepared. The stock solutions of the substances were diluted to effect an application volume of 0,1 mL/plate.
Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Key result
Species / strain:
other: TA 1535, TA 97a, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The study is regarded as a valid guideline study with certificated GLP compliance. The test item didn't show mutagenic effects in both experiments. The number of revertant colonies was not significantly increased in comparison with the spontaneous revertants (solvent only), as the increase factor never reached 2.0. In the first experiment, a weak dose-response correlation could be observed in TA97, whereas in the second experiment, this relation was not verified.
Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.
Therefore it can be stated, that under the conditions of the test, the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
Executive summary:

This study was performed in order to evaluate the mutagenic potential of 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain.

The study was conducted in accordance with the following guidelines:

-OECD Guidelines for the Testing of Chemicals Part 471, adopted July 21st, 1997 „Bacterial Reverse Mutation Test"

-EU-Guideline 67/548 EWG, last changed by 29th amendment, Annex V, Method B.13/14 "Mutagenicity - Salmonella typhimurium, reverse mutation

assay", adopted May 19th, 2000

Two valid experiments were performed.

 

First Experiment:

Five concentrations of the test item, dissolved in deionised water (ranging from 5010 to 50 µg/plate) were used. Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

None of the concentrations caused an increase in the number of revertant colonies in the tested strains. The test item didn't show any mutagenic effects in the first experiment.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the

negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second experiment:

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging from 5002 to 1251 µg/plate) and a modification in study performance (pre-incubation method).

The test item didn't show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Therefore, no concentration-effect relationship could be determined.

The test item 1-(2-Hydroxy-3-sulfopropyI)-pyridinium-betain is considered as "not mutagenic under the conditions of the test".

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1983-01-20 - 1983-01-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
performed on read-across substance, TA1538 used instead of e.g. TA102 as fifth strain, but performed under GLP
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strains: Salmonella typhimurium TA1535, TA 1537, TA 98, TA100 and TA 1538 were used
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
strains: Salmonella typhimurium TA1535, TA 1537, TA 98, TA100 and TA 1538 were used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
other: TA 1538
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction S9-Mix (from specific pathogen free Wistar rats, prepared on the day of the experiment
Test concentrations with justification for top dose:
1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- On the day of the experiment, the test material was dissolved in distilled water at the highest investigated dose. The other doses were dilutions from this stock solution with the solvent in half-log intervals.
Untreated negative controls:
other: solvent control (distilled water)
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl methanesulfonate, 9-Aminoacridine, 2-Nitrofluorene, N-Ethyl-N-Nitro-N-Nitrosoguanidine, 2-Aminoanthracene, Benzo-(a)-pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The bacteria were grown overnight in a shaking water bath for 16 hours at 37°C in 2 . 5X Nutrient Broth No. 2. After centrifugation, the bacteria were resuspended to a concentration of approximately 1 x 10E8 to 2 x 10E9 cells per millilitre in 0.16% Nutrient Broth and 0.5% Sodium chloride. The concentration of germs was controlled photometrically and determined in an experimental test with Histidine-rich potassium chloride solution on selective agar plates.

DETERMINATION OF CYTOTOXICITY
- To estimate the toxicity of the test material prototrophic bacteria (spontaneous revertants of TA 1537 = RTA) were added as an internal standard of to the selective agar plates together with TA 1537. The difference in the number of colonies on plates with and without added prototrophic bacteria at each dose level of the test material was compared to the number of colonies obtained with the negative control. The ratio of the two values was expressed as relative survival rate with the negative control at a 100 % survival rate.
Additionally, the toxicity of the test material can be evidenced in the mutagenicity assay, by a reduction in the number of revertants or by a clearing of the background lawn
Evaluation criteria:
A material is identified as a mutagen in this test system if there is a reproducible demonstration of a dose effect relation with a 2-fold increase in the number of revertants over the controls in at least one strain. With the strain TA 100 a 1.5-told increase is the criterion for a positive result.
Statistics:
The difference in the number of colonies on plates with and without added prototrophic bacteria at each dose level of the test material was compared to the number of colonies obtained with the negative control. The ratio of the two values was expressed as relative survival rate with the negative control at a 100 % survival rate.
Additionally, the toxicity of the test material can be evidenced in the mutagenicity assay, by a reduction in the number of revertants or by a clearing of the background lawn
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No relevant increase of the revertant colony numbers was obtained in Salmonella typhimurium strain used at all dose levels tested in comparison with the corresponding controls. The presence of microsomal activation did not influence these findings.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
In the toxicity experiment with 1-3 Sulfopropyl-Pyridinium-Betain , neither quantitative nor qualitative evidence of a toxic effect was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

The study was performed according to the OECD Guideline 471 with deviations (strain TA 1538 instead of TA 102) according to the principles of the good laboratory practice and therefore considered to be of high quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material did not induce significant increases in the frequency of revertant colonies in any of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of the test.
Executive summary:

The test substance 1 -(3 -sulfopropyl)-pyridinium-betain (PPS) was investigated according to OECD TG471 for its potential to cause gene mutation in Salmonella typhimurium strains (TA98, TA100, TA1537, TA1535 and TA1538) (Guenard, 1983). Under the conditions of this experiment 1-(3-sulfopropyl)-pyridinium-betain (PPS) was found to cause no toxic effects. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain in comparison with the corresponding controls. The presence of microsomal activation did not influence these findings. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material caused neither base-pair substitutions, nor frameshift mutations. Therefore the test results with 1 -(3 -sulfopropyl)-pyridinium-betain (PPS) revealed no indication of gene mutagenic activity.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Klimisch 1 source record, but performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
expression phase and harvest time were slightly modified - this deviations are not considered to influence the outcome of the study
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (23 years old) for the first experiment and from a 33 year-old female donor for Experiment II. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats
Test concentrations with justification for top dose:
657.3 µg/mL, 1150.3 µg/mL and 2013 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water, the final concentration of deinonised water in the culture medium was 10 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. The solubility of the test item in the solvent is approximately 1500 g/L.
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent solvent controls (culture medium with 10 % deionised water) (local tap water deionised at Harlan CCR)).
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Remarks:
Dilutions of stock solutions were prepared on the day of the experiment. Stability of Demecolcin, Mitomycin C and CPA in solution is unknown but a mutagenic response in the expected range is sufficient biological evidence of chemical stability.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 40 hours
- Exposure duration: 4 and 20 hours

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: at least 1000 binuecleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: other: To describe a cytotoxic effect the CBPI (cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis (7).
Evaluation criteria:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data.
b) The positive control substances should produce significant increases in the number of cells with micronuclei.

Evaluation of Results

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:
Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
lymphocytes: human lymphocytes
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on osmolarity or pH value was observed.
- Effects of osmolality: No relevant influence on osmolarity or pH value was observed.
- Water solubility: 1500 g/L
- Precipitation: no precipitation of the test item in the pre-test nor in the actual experiments

RANGE-FINDING/SCREENING STUDIES:
With respect to the molecular weight of the test item, 2013.0 µg/mL of 1-(3-sulphonatopropyl) pyridinium (approx. 10 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 13.1 and 2013.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, no confounding factors observed
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

The study was performed according to the OECD Guideline 487 with deviations (expression phase and harvest time were slightly modified - these deviations are not considered to influence the outcome of the study) and is considered to be of the highest quality (reliability Klimisch 1). The vehicle water and the positive control substances fulfilled validity criteria of the test system. The positive controls mitomycin C, demecolcin and cyclophosphamide induced micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. None of the cultures treated with 1-(3-sulphonatopropyl)pyridinium in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of micronuclei. Based on this test, 1-(3-sulphonatopropyl)pyridinium is considered not to be non-mutagenic in human lymphocytes.
Executive summary:

The test item 1-(3-sulphonatopropyl)pyridinium, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro (Bohnenberger, 2012). Cultured human lymphcytes were used and the following study design was performed: experiment 1without S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 2 without S9 -mix (exposure period 20 hrs, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 1 and b with S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours). In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage on coded slides. The highest applied concentration in the pre-test on toxicity (2013.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, statistically significant increases were observed in Experiment I and II in the presence of S9 mix after treatment with 2013.0 µg/mL (0.70, 0.75 % micronucleated cells, respectively). The values are clearly within the range of the historical control data (0.20 - 1.70 % micronucleated cells) and therefore regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. The positive controls mitomycin C, demecolcin and cyclophosphamide induced statistically significant increases in cells with micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. .

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 1-(3-sulphonatopropyl)pyridinium is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-05 - 2017-12-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
OECD Guideline for the testing of chemicals No. 490 (adopted July 2015)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene forward mutation test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Target gene:
tk+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
L5178Y TK+/− (Clone 3.7.2C) mouse lymphoma cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518)
- Suitability of cells: Cells are recommended by the guideline. The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The following culture media were used:
Minimal medium A
RPMI1640 (1X) 516.1 mL
L-glutamine (200 mM) 5.4mL
Sodiumpyruvate (100 mM) 6.0mL
Non-essential amino acids (100X) 5.4mL
Streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL 1.1 mL
F 68 Pluronic 6.0mL

Minimal medium B
RPMI 1640 (1X) 522.1 mL
L-glutamine (200 mM) 5.4mL
Sodiumpyruvate (100 mM) 6.0mL
Non-essential amino acids (100X) 5.4mL
Streptomycin sulphate 50000 IU/mL +
Penicillin G 50000 IU/mL 1.1 mL

Complete medium (5%)
Minimal medium A 950 mL
Horse serum(heat-inactivated) 50mL

Complete medium (10%)
Minimal medium A 900 mL
Horse serum(heat-inactivated) 100mL

Complete medium A(20%)
Minimal medium A 800 mL
Horse serum(heat-inactivated) 200mL

Complete medium B (20%)
Minimal medium B 800 mL
Horse serum(heat-inactivated) 200mL

- Properly maintained: Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: The cells are checked at regular intervals for the absence of mycoplasmal contamination.
- Periodically 'cleansed' against high spontaneous background: The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. Prior to use, cells were cleansed of pre-existing mutants.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital – 5,6-Benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
2000, 1000, 500, 250 and 125 µg/ml (top dose as required by the guideline)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI minimal medium A
- Justification for choice of solvent/vehicle: Solubility of the test item was evaluated in a preliminary trial using DMSO and culture medium. These solvents were selected since they are compatible with the survival of the cells and the S9 metabolic activity. In addition, there are many historical control data demonstrating that no mutagenic effects are induced by these solvents. The test item was found to be soluble in culture medium RPMI minimal medium A at the maximum concentration of 20.0 mg/mL. On the basis of this result, a concentration of 2000 μg/mL (the upper limit to testing indicated in the test guideline OECD No. 490) was selected as the top dose level to be used in the cytotoxicity test.
Untreated negative controls:
yes
Remarks:
RPMI minimal medium A
Negative solvent / vehicle controls:
yes
Remarks:
RPMI minimal medium A
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
Cell suspensions in complete medium were prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes. The treatment media were prepared as follows:

Without S9 metabolism - 3 hour treatment time
Cell suspension (2 x 10exp6 cells/mL in complete medium 5%) 5.0 mL
Complete medium(5%) 13.0mL
Solvent control or Test item solution 2.0 mL
Total 20.0 mL

Without S9 metabolism - 24 hour treatment time
Cell suspension(1 x 10exp6 cells/mLincomplete medium 10%) 3.0mL
Complete medium(10%) 15.0mL
Solvent control or Test item solution 2.0 mL
Total 20.0 mL

With S9 metabolism - 3 hour treatment time
Cell suspension(2x 10exp6 cells/mLincomplete medium 5%) 5.0mL
S9 mix 9.8mL
Complete medium (5%) 3.2mL
Solvent control or Test item solution 2.0 mL
Total 20.0 mL

With S9 metabolism - 3 hour treatment time - Positive control
Cell suspension(1 x 10exp6 cells/mLincomplete medium 5%) 10.0mL
S9 mix 9.8mL
Positive control (B(a)P) solution 0.2mL
Total 20.0 mL

Without S9 metabolism - 24 hour treatment time - Positive control
Cell suspension(1 x 10exp6 cells/mL in complete medium 10%) 3.0mL
Complete medium(10%) 16.8mL
Positive control (MMS) solution 0.2 mL
Total 20.0 mL
The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

DURATION
- Preincubation period:
- Exposure duration: In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
- Expression time (cells in growth medium): During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10exp5 cells/mL.
- Selection time (if incubation with a selection agent): After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 μg/mL) and an estimated 2×103 cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for one to two weeks and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED / NUMBER OF CELLS EVALUATED:
Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 μg/mL) and an estimated 2×10exp3 cells were plated
in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for one to two weeks and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.
Plating for viability
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for one to two weeks and wells containing clones were identified as above and counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
1. The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
2. The untreated/solvent control suspension growth over 2 days fell within the range: 8-32 (3 hour treatment), 32-180 (24 hour treatment).
3. The mutant frequencies in the untreated/solvent control cultures fell within the range of 50−170×10exp(−6) viable cells.
Every assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
1. The positive control induced a clear increase above the spontaneous background (induced mutent frequency = IMF) of at least 300×10−6. At least 40% of the IMF was
reflected in the small colony MF.
2. The positive control induced a clear increase in the small colony IMF of at least 150×10exp(−6).

Criteria for outcome of assay
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
see attachment
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The addition of the test item solution did not have any obvious effect on the pH of the treatment medium.
- Effects of osmolality: A slight increase in osmolatity was observed at higher dose levels, in the presence of S9 metabolism; however this increase was less than the threshold value (50 mOsm/kg) suggested to avoid artifactual positive results (Scott D. et al., 1991).
- Water solubility: not exceeded
- Precipitation: none observed
Solubility of the test item was evaluated in a preliminary trial using DMSO and culture medium. These solvents were selected since they are compatible with the survival of the cells and the S9 metabolic activity. In addition, there are many historical control data demonstrating that no mutagenic effects are induced by these solvents. The test item was found to be soluble in culture medium RPMI minimal medium A at the maximum concentration of 20.0mg/mL. On the basis of this result, a concentration of 2000 μg/mL (the upper limit to testing indicated in the test guideline OECD No. 490) was selected as the top dose level to be used in the cytotoxicity test.

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity test
Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000 μg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL. No precipitation of the test item was noted by the end of the treatment incubation period at any concentration tested in any treatment series. No relevant toxicity, as indicated by reduction in relative survival (RS), was observed in any experimental condition examined, at any concentration tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Mutation frequency = 446 (209-1103, -S9, 3h) / 728 (187-1424, +S9, 3h) / 611 (221-2325, -S9, 24h)
- Negative (solvent/vehicle) historical control data: Mutation frequency = 81.3 (41.9-234, -S9, 3h) / 84.7 (44.9-234, +S9, 3h) / 81.0 (50.4-217, -S9, 24h)
Conclusions:
The study was conducted under GLP according to OECD guideline 490 (mouse lymphoma assay) on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of PPSOH to induce gene mutations in mammalian cells. In all experimental conditions examined, no increase in mutation frequency above the concurrent negative control value exceeded the Global Evaluation Factor. No statistically significant dose-effect relationship was observed at any treatment time, in the absence or presence of S9 metabolism. Therefore, PPSOH is considered to be non-mutagenic in this mouse lymphoma assay.
Executive summary:

The test item PPSOH was examined in an OECD 490 study under GLP for mutagenic activity by assaying for the induction of trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 2000μg/mL (the upper limit to testing indicated in the test guideline OECD No. 490) using RPMI minimal medium A as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000μg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.81μg/mL.

The experiment was performed using a short treatment time (3 hours) both in the absence and presence of S9 metabolic activation and a long treatment time (24 hours) in its absence. Neither relevant cytotoxicity, nor precipitation of the test item by the end of treatment were observed in any treatment series, at any concentration tested.

Based on the results obtained in the preliminary cytotoxicity assay, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

 

Assay No

S9

Treatment (h)

Dose level (µg/ml)

1

±

3

2000, 1000, 500, 250 and 125

2

-

24

2000, 1000, 500, 250 and 125

 

No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that PPSOH does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

There are several data available, all assessed with at least Klimisch 2, sufficiently covering all required endpoints, i.e. gene mutation in both bacteria and mammalian cells, and chromosome aberrations (micronucleus induction) in mammalian cells. Those in vitro genetic toxicity tests are designed, i.a. via addition of additional enzymes (metabolic activation; induced rat liver S9), to mimic the exposure of humans to a potential genotoxic agent very close. They are accepted surrogates for human data, and no indication is given that the obtained results are not relevant for humans / risk assessment. Hence, the database is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from PPSOH.

All available tests gave consistently negative results, which makes a mode of action analysis impossible, as no conclusions from any events can be drawn.

Additional information

Justification for classification or non-classification

All available test results for gene mutation and chromosome aberrations (micronucleus test) in vitro are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified. Gene and chromosome mutations are considered initial steps in rather complex carcinogenesis. As the substance does not induce those, no need to consider the substance as carcinogen is evident.

According to Regulation 1272/2008 and amendments, regarding “3.5. 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. Further, 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. Last but not least, to arrive at a classification, test results are considered from experiments determining mutagenic and/or genotoxic effects in germ and/or somatic cells of exposed animals. Mutagenic and/or genotoxic effects determined in in vitro tests shall also be considered.

In consequence, consistent negative results in three different in vitro test systems addressing three different events related to genetic damage allow to conclude that the substance does not need to be classified as germ cell mutagen.