Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- genetic toxicity in vitro: negative; 4 studies negative (Sire (2012) Genetic toxicity in bacteria - Chromosome aberration -Mammalian gene mutation), (Marzin (1980) Genetic toxicity in bacteria),
- genetic toxicity in vivo: no data available

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 30 March 2012 to 04 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EU / OECD guidelines and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

19/07/2011

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

The dose-levels selected for the preliminary tests were 10, 100, 500, 1000, 2500 and 5000 µg/plate (expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 13.3, 133, 665, 1330, 3325 and 6650 µg/plate in terms of registered substance (100% UVCB)).
The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 415.6, 831.3, 1662.5, 3325 and 6650 µg/plate in terms of registered substance (100% UVCB)) for the five strains in both mutagenicity experiments, with and without S9 mix.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: water for injection
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the vehicles usually used for in vitro tests.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Anthramine

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method

DURATION
- Preincubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATES: three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHER: SCORING METHOD: automated
Positive controls:
Without S9 mix:
- sodium azide (NAN3) for strains TA 1535 and TA 100 (1 µg/plate),
- 9-Aminoacridine (9AA) for strain TA 1537 (50 µg/plate),
- 2-Nitrofluorene (2NF) for strain TA 98 (0.5 µg/plate),
- Mitomycin C (MMC) for strain TA 102 (0.5 µg/plate).

With S9 mix:
- 2-Anthramine (2AM) for strains TA 1535, TA 1537, TA 98 (2 µg/plate) and TA102 (10 µg/plate),
- Benzo(a)pyrene (BAP) for strain TA 100 (5 µg/plate).

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 7.6.1/1 and 7.6.1/2

Cytotoxicity / choice of top concentrations:

other: In the second experiment with S9 mix (pre-incubation method), a moderate to strong toxicity was noted in the five strains used at the dose-level of 5000 µg/plate (See table 7.6.1/2).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: In the second experiment with S9 mix (pre-incubation method), a moderate to strong toxicity was noted in the five strains used at the dose-level of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the three strains used, either with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The control data reported in these report are in the range of the historical control data observed in the laboratory. The study was therefore considered valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1:First experiment (direct plate incorporation) - Mean revertant colony counts

	TA 1535			TA 1537			TA 98			TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	10	14	No	7	7	No	20	29	No	115	140	No	294	468	No
312.5	14	16	No	6	6	No	26	28	No	130	141	No	280	559	No
625	10	11	No	4	7	No	15	31	No	144	164	No	348	465	No
1250	12	14	No	5	10	No	18	29	No	125	136	No	318	508	No
2500	14	10	No	6	6	No	19	27	No	132	162	No	312	466	No
5000	7	12	No	8	7	No	18	23	No	122	154	No	276	403	No
NAN3	718	-	-	-	-	-	-	-	-	513	-	-	-	-	-
2AM	-	215	-	-	108	-	-	828	-	-	-	-	-	3415	-
9AA	-	-	-	277	-	-	-	-	-	-	-	-	-	-	-
2NF	-	-	-	-	-	-	107	-	-	-	-	-	-	-	-
BAP	-	-	-	-	-	-	-	-	-	-	776	-	-	-	-
MMC	-	-	-	-	-	-	-	-	-	-	-	-	2554	-	-

 

 

*solvent control with water for injection

MA: metabolic activation

NaN3: sodium azide

9AA :9-Aminoacridine

2NF:2-Nitrofluorene

MMC:Mitomycin C

2AM:2-Anthramine

BAP:Benzo(a)pyrene

 

Table 7.6.1/2:Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) - Mean revertant colony count

 

	TA 1535			TA 1537			TA 98			TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	18	15	No	8	14	No	24	29	No	134	142	No	332	585	No
312.5	9	13	No	6	16	No	25	31	No	130	168	No	328	654	No
625	17	9	No	10	18	No	25	35	No	148	145	No	392	609	No
1250	13	13	No	6	8	No	29	30	No	138	141	No	379	465	No
2500	19	10	No	12	6	No	19	36	No	135	124	No	357	348	No
5000	11	19	Yes (Mt)	7	15	Yes (Mt)	19	39	Yes (Mt)	112	101	Yes (Mt)	368	180	Yes (St)
NAN3	610	-	-	-	-	-	-	-	-	485	-	-	-	-	-
2AM	-	83	-	-	186	-	-	859	-	-	-	-	-	3394	-
9AA	-	-	-	353	-	-	-	-	-	-	-	-	-	-	-
2NF	-	-	-	-	-	-	103	-	-	-	-	-	-	-	-
BAP	-	-	-	-	-	-	-	-	-	-	510	-	-	-	-
MMC	-	-	-	-	-	-	-	-	-	-	-	-	2766	-	-

 

 

*solvent control with water for injection

MA: metabolic activation

NaN3: sodium azide

9AA :9-Aminoacridine

2NF:2-Nitrofluorene

MMC:Mitomycin C

2AM:2-Anthramine

BAP:Benzo(a)pyrene

Mt: Moderate toxicity

St: Strong toxicity

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under these experimental conditions, the test item Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

This study was performed to investigate the potential of the test item, reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, to induce reverse mutation in Salmonella typhimurium. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the dose-levels of reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes,37°C).

Five strains of bacteria Salmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in water for injections.

 

Since the test item was freely soluble and non-cytotoxic in the preliminary test, the highest dose‑level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (doses expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 415.6, 831.3, 1662.5, 3325 and 6650 µg/plate in terms of registered substance (100% UVCB)) for the five strains in both mutagenicity experiments, with and without S9 mix. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. No noteworthy toxicity was noted at any dose-levels in the absence of S9 mix (in either experiments) or in the presence of S9 mix when using the direct plate incorporation method (first experiment). In the second experiment with S9 mix (pre-incubation method), a moderate to strong toxicity was noted in the five strains used at the dose-level of 5000 µg/plate.The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 30 March 2012 to 05 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EU / OECD guidelines and GLP

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

19/07/2011

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human, primary culture

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/ml (doses expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 51.9, 103.9, 207.9, 415.6, 831.25, 1662.5, 3325 and 6650 µg/mL in terms of registered substance (100% UVCB)),, with and without metabolic activation (no precipitate was observed in the culture medium either at the beginning or at the end of the treatment period).
Experiment 2: 31.3, 62.5, 125, 250, 500 and 1000 µg/ml (i.e. 41.6, 83.1, 166.3, 332.5, 665 and 1330 µg/l in terms of registered substance), without S9 mix; 62.5, 125, 250, 375, 500 and 750 µg/ml (i.e.83.1, 166.3, 332.5, 498.75, 665 and 997.5 µg/ml in terms of registered substance), with S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injection , batch Nos. 1F1731, 2F0284 and 2F0703 (CDM Lavoisier, France).
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the vehicles usually used for in vitro tests. Therefore, a suspension in water for injection was used for treatment.
- volume of vehicle/solvent in the medium : 110 µL/ 5.5 mL culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Type and identity of media:
Human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours. Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinized sterile tubes.


METABOLIC ACTIVATION SYSTEM : (details)
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
The S9 fraction was preserved in sterile tubes at -80°C, until use.
The S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to the culture medium.
The composition of S9 mix was as follows:
Glucose-6-phosphate: 5 mM
NADP: 4 mM
KCl: 33 mM
MgCl2: 8 mM
Sodium phosphate buffer pH 7.4: 100 mM
S9 fraction, batch No. 2861, protein concentration: 36.4 mg/mL: 10% (v/v)
water: to volume

The final concentration of S9 fraction in the culture medium was 1.5% (i.e. 15% S9 mix).

POSITIVE CONTROL (S) SUBSTANCE
- without S9 mix: Mitomycin C (MMC), used at a final concentrations of 2 and 3 µg/mL (3 hours of treatment) and at 0.2 and 0.3 µg/mL (continuous treatment),
- with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 12.5 and 25 µg/mL (the dose-level which gave a satisfactory response in terms of quality and quantity of metaphases and extent of chromosomal damage was selected for analysis).

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: no
- Exposure duration: 3, 20 and 44 hours
- Expression time (cells in growth medium): 20 or 44 hours after beginning of exposure
- Selection time (if incubation with a selection agent) : not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 or 44 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid (3 h before harvesting)

STAIN (for cytogenetic assays): After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.

NUMBER OF REPLICATIONS : two

NUMBER OF CELLS EVALUATED : Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analyzed when at least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test item was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether an item induces mitotic inhibition. Mitotic index was determined without blind scoring.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


OTHER:
SCORING METHOD: Microscopic evaluation: The following structural aberrations were recorded for each metaphase : gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations).

Evaluation criteria:

Evaluation of a positive response: A test item is considered positive for inducing chromosomal aberrations if a reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration is observed at one or more dose-levels and at one or two harvest times.

Evaluation of a negative response: A test item is considered negative for inducing chromosomal aberrations if no significant increase is observed in the number of cells with chromosomal aberrations for any of the dose levels and at any harvest times.

Reference to historical data or other considerations of biological relevance were also taken into account in the evaluation of the findings.

Statistics:

For each experiment and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the χ2 test unless treated culture data were lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance.

Species / strain:

lymphocytes: primary human cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see table 7.6.1/1

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table 7.6.1/1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered to be valid.

With a treatment volume of 15 µL/5.5 mL of culture medium, the dose-levels used for treatment were as follows:
- 0.53, 1.07, 2.13, 4.26, 8.52, 17.05, 34.09 and 68.18 µg/mL for the first experiment, both with and without S9 mix,
- 2.13, 4.26, 8.52, 17.05, 34.09 and 68.18 µg/mL for the second experiment, both with and without S9 mix.

At the end of the 3 hour treatment periods both with and without S9 mix, a slight to moderate precipitate was observed in the culture medium at dose-levels >= 34.09 µg/mL and >= 17.05 µg/mL for the first and second experiments, respectively.
At the end of the 20- and 44- hour treatment periods without S9 mix, a slight to moderate precipitate was observed in the culture medium at dose-levels >= 17.05 µg/mL and >= 34.09 µg/mL, respectively.

EXPERIMENTS WITHOUT S9 MIX:
Cytotoxicity:
No noteworthy decrease in the mitotic index was noted at any of the tested dose-levels following the 3-, 20- or the 44-hour treatments.

Metaphase analysis:
The dose-levels selected for metaphase analysis were:
- 17.05, 34.09 and 68.18 µg/mL for the 3- and 20-hour treatments, the latter being the highest achieved dose-level,
- 68.18 µg/mL for the 44-hour treatment, which was the highest achieved dose-level.

Following the 3-hour treatment in the first experiment, a statistically significant increase (p < 0.05) in the frequency of cells with structural chromosomal aberrations was noted at 34.09 µg/mL (4.0%). This increase was not dose-related and the frequency of aberrant cells obtained at 34.09 µg/mL remained within the historical data range for the vehicle control (4.0% versus 0 to 5.0% for the historical data). Moreover, in a second independent experiment, no statistically significant increase in the frequency of cells with structural or numerical chromosomal aberrations was noted after either the 20 or the 44-hour treatments. Therefore, the increase in the frequency of aberrant cells noted in the first experiment was considered to be non-biologically significant.

EXPERIMENTS WITH S9 MIX:
Cytotoxicity:
No noteworthy decrease in the mitotic index was noted at any of the tested dose-levels in either experiment and at either harvest time.

Metaphase analysis:
The dose-levels selected for metaphase analysis were:
- 17.05, 34.09 and 68.18 µg/mL for the 20-hour harvest time in both experiments, the latter being the highest achieved dose-level,
- 68.18 µg/mL for the 44-hour harvest time, which was the highest achieved dose-level.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest time.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

At the dose-level of 5000 µg/mL, the pH of the culture medium was approximately 7.4 (7.1 for the vehicle control) and the osmolality was equal to 345 mOsm/kg H₂O (296 for the vehicle control).

No precipitate was observed in the culture medium either at the beginning or at the end of the treatment period.

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment, a moderate to severe toxicity was observed at dose-levels ≥ 625 µg/mL, as shown by a 48-100% decrease in the mitotic index .

Following the 20-hour treatment, a severe toxicity was observed at dose-levels ≥ 500 µg/mL, as shown by a 82-100% decrease in the mitotic index .

Following the 44-hour treatment, a severe toxicity was observed at dose-levels ≥ 500 µg/mL, as shown by a 99-100% decrease in the mitotic index .

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.            156.3, 312.5 and 625 µg/mL for the 3-hour treatment, the latter inducing a 48% decrease in themitotic index,

.            62.5, 125 and 250 µg/mL for the 20-hour treatment, higher dose-levels being too cytotoxic,

.            250 µg/mL for the 44-hour treatment, this dose inducing a 17% decrease in the mitotic index and higher dose-levels being too cytotoxic .

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments.

 

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a slight to severe toxicity was observed at dose-levels ≥ 156.3 µg/mL, as shown by a 32-100% decrease in the mitotic index.

At the 20-hour harvest time in the second experiment, a moderate to severe toxicity was observed at dose-levels ≥ 250 µg/mL, as shown by a 54-100% decrease in the mitotic index.

At the 44-hour harvest time (second experiment), a marked to severe toxicity was observed at dose-levels ≥ 375 µg/mL, as shown by a 69-100% decrease in the mitotic index.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.            78.13, 156.3 and 312.5 µg/mL for the 20-hour harvest time in the first experiment, the latter inducing a 36% decrease in themitotic index and higher dose-levels being too cytotoxic,

.            62.5, 125 and 250 µg/mL for the 20-hour harvest time in the second experiment, the latter inducing a 54% decrease in themitotic index ,

.            250 µg/mL for the 44-hour harvest time, this dose inducing a 15% decrease in themitotic index and higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is considered to be non-clastogenic in this in vitro chromosome aberration test, in the absence and presence of metabolic activation.

Executive summary:

This study was performed to investigate the potential of the test item, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, to induce structural chromosome aberrations in human lymphocytes in vitro. The study was performed according to OECD guideline no. 473 and EC guideline n° B10 and in compliance with the Principles of Good Laboratory Practice. 

 

The test item (dissolved in water for injections) was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment.

The second experiment was performed as follows:

-  without S9 mix, cells were exposed continuously to the test or control items until harvest,

- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment. Three hours before harvest, each culture was treated with a Colcemid® solution to block cells at the metaphase-stage of mitosis. After hypotonic treatment, the cells were fixed, spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

The negative control was the vehicle (Water for injections).

 

The dose-levels selected for the first experiment were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL (doses expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 51.9, 103.9, 207.9, 415.6, 831.25, 1662.5, 3325 and 6650 µg/mL in terms of registered substance), both with and without S9 mix.

Based on the cytotoxicity observed in the first experiment, the dose-levels selected for the second experiment were as follows:

.            31.3, 62.5, 125, 250, 500 and 1000 µg/mL ( i.e. 41.6, 83.1, 166.3, 332.5, 665 and 1330 µg/l in terms of registered substance), without S9 mix,

.            62.5, 125, 250, 375, 500 and 750 µg/mL (i.e.83.1, 166.3, 332.5, 498.75, 665 and 997.5 µg/ml in terms of registered substance), with S9 mix.

 

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment, a moderate to severe toxicity was observed at dose-levels ≥ 625 µg/mL, as shown by a 48-100% decrease in the mitotic index.

Following the 20-hour treatment, a severe toxicity was observed at dose-levels ≥ 500 µg/mL, as shown by a 82-100% decrease in the mitotic index.

Following the 44-hour treatment, a severe toxicity was observed at dose-levels ≥ 500 µg/mL, as shown by a 99-100% decrease in the mitotic index.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.            156.3, 312.5 and 625 µg/mL for the 3-hour treatment, the latter inducing a 48% decrease in the mitotic index,

.            62.5, 125 and 250 µg/mL for the 20-hour treatment, higher dose-levels being too cytotoxic,

.            250 µg/mL for the 44-hour treatment, this dose inducing a 17% decrease in the mitotic index and higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments.

 

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a slight to severe toxicity was observed at dose-levels ≥ 156.3 µg/mL, as shown by a 32-100% decrease in the mitotic index.

At the 20-hour harvest time in the second experiment, a moderate to severe toxicity was observed at dose-levels ≥ 250 µg/mL, as shown by a 54-100% decrease in the mitotic index.

At the 44-hour harvest time (second experiment), a marked to severe toxicity was observed at dose-levels ≥ 375 µg/mL, as shown by a 69-100% decrease in the mitotic index.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.            78.13, 156.3 and 312.5 µg/mL for the 20-hour harvest time in the first experiment, the latter inducing a 36% decrease in themitotic index and higher dose-levels being too cytotoxic,

.            62.5, 125 and 250 µg/mL for the 20-hour harvest time in the second experiment, the latter inducing a 54% decrease in themitotic index,

.      250 µg/mL for the 44-hour harvest time, this dose inducing a 15% decrease in themitotic index and higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

 

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 

In conclusion, the test item did not induce chromosome aberrations in cultured human lymphocytes, in the absence or in the presence of a rat metabolising system, therefore Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 21 June 2011 to 20 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EU / OECD guidelines and GLP

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

19/07/2011

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase, TK +/-, locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK +/- phenotype

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiments without S9 mix
The selected dose-levels were as follows:
- 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL for the first experiment (3-hour treatment) (doses expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 8.3, 16.6, 33.3, 66.5, 133, 266 and 532 µg/mL in terms of registered substance (100% UVCB)),
- 25, 50, 100, 200, 266.7, 333.3 and 400 µg/mL for the second experiment (24 hour treatment) (i.e. 33.3, 66.5, 133, 266, 354.7, 443.3 and 532 µg/mL in terms of registered substance).

Experiments with S9 mix
The selected dose-levels were as follows:
- 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL for the first and second experiments (i.e. 8.3, 16.6, 33.3, 66.5, 133, 266 and 532 µg/mL in terms of registered substance),
- 25, 50, 100, 200, 250, 300 and 400 µg/mL for the third experiment (i.e. 33.3, 66.5, 133, 266, 332.5, 399 and 532 µg/mL in terms of registered substance).

Vehicle / solvent:

- Vehicle used: water for injections, batch Nos. 1F1731, 2F0284 and 2F0703 (CDM Lavoisier, France).
- Justification for choice of solvent: Water was chosen because the product was soluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days with TFT
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) at 4µg/mL

NUMBER OF REPLICATIONS: two cultures per dose level/Two independent experiments. A third experiment was performed with metabolic activation only.

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

OTHER: differentiation of small and large colonies:
.           size of small colonies: < 25% of the diameter of the well,
.           size of large colonies: > 25% of the diameter of the well.



OTHER:

Evaluation criteria:

IWGT recommendations (Moore et al., 2003; Moore et al., 2006; Moore et al., 2007) were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-related trend is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (Moore et al., 2002):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

According to the OECD guideline 476, a statistical analysis of the data is not mandatory.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see table 7.6.1/1

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table 7.6.1/1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
- Effects of pH and osmolality: At the dose-level of 400 μg/mL, the pH and osmolality values of the culture medium were equivalent to those of the vehicle control culture.


RANGE-FINDING/SCREENING STUDIES: Using a test item concentration of 250 mg/mL in the vehicle and a treatment-volume of 2% (i.e. 400 μL/20 mL culture medium for the 3-hour treatments and 1000 μL/50 mL culture medium for the 24-hour treatment), the highest recommended dose-level of 5000 μg/mL was achievable. Thus, the dose-levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 μg/mL. No precipitate was observed in the culture medium at the end of the treatment periods (3- and 24-hour treatments). Following the 3-hour treatments with and without S9 mix, as well as following the 24-hour treatment without S9 mix, a severe toxicity was observed at dose-levels ≥ 500 μg/mL, as
shown by 100% decreases in the Adj. RTG.

COMPARISON WITH HISTORICAL CONTROL DATA: The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. The mutation frequencies of the positive controls also met the acceptance criteria of a study. The study was therefore considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment without S9 mix:
Following the 3-hour treatment, a moderate to severe toxicity was observed at dose-levels ≥ 200 μg/mL, as shown by a 59 - 100% decrease in the Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels ≥ 200 μg/mL, as shown by a 68 - 100% decrease in the Adj. RTG.
Experiment with S9 mix:
A moderate to severe toxicity was observed at dose-levels ≥ 200 μg/mL in the first and second experiments, as shown by a 39-100% decrease in the Adj. RTG. In the third experiment, a moderate to severe toxicity was induced at dose-levels ≥ 250 μg/mL, as shown by a 59-100% decrease in the Adj. RTG.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Summary of mutagenicity results

 

Test item concentration in µg/mL	Mean values of cultures 1 and 2
	Adjusted RTG%	CE2	MF x 10^-6	IMF
Experiment I without S9 mix, 3-hour treatment
Vehicle control^#	100	0.85	117
6.25	87	0.77	114	none
12.5	97	0.64	112	none
25	102	0.84	117	0
50	108	0.88	114	none
100	87	0.91	124	8
200	41	0.87	157	40
400	-	-	-	-
MMS 25 µg/mL	42	0.47	1206	1089
Experiment II without S9 mix, 24-hour treatment
Vehicle control^#	100	0.75	95
25	91	0.86	60	none
50	88	0.85	82	none
100	80	0.77	71	none
200	32	0.73	73	none
266.7	1	0.74	71	none
333.3	-	-	-	-
400	-	-	-	-
MMS 5 µg/mL	41	0.54	532	437
Experiment I with S9 mix, 3-hour treatment
Vehicle control^#	100	0.88	146
6.25	100	0.77	144	none
12.5	95	0.83	147	1
25	123	0.93	89	none
50	124	0.83	141	none
100	137	0.93	108	none
200	44	1.03	161	15
400	-	-	-	-
CPA 3 µg/mL	66	0.53	1351	1205
Experiment II with S9 mix, 3-hour treatment
Vehicle control^#	100	0.75	92
6.25	92	0.78	84	none
12.5	98	0.69	107	15
25	108	0.81	93	0
50	99	0.64	107	15
100	90	0.61	97	5
200	61	0.87	131	39
400	-	-	-	-
CPA 3 µg/mL	48	0.47	588	496
Experiment III with S9 mix, 3-hour treatment
Vehicle control^#	100	0.76	76
25	95	0.75	70	none
50	91	0.68	79	3
100	97	0.73	80	4
200	84	0.74	66	none
250	41	0.76	62	none
300	23	0.93	70	none
400	-	-	-	-
CPA 3 µg/mL	55	0.64	648	571

 

# Water for injections

Adj RTG%: Adjusted relative total growth

CE2: cloning efficiency

MF: mutation frequency

IMF: Induced Mutation Frequency

MMS:methylmethane sulfonate

CPA: Cyclophosphamide

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is considered to be non-mutagenic in this in vitro mouse lymphoma assay, in the absence and presence of metabolic activation.

Executive summary:

This study was performed to investigate the potential of the test item, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells. The study was performed according to OECD guideline no. 476 and EC guideline n° B17 and in compliance with the Principles of Good Laboratory Practice.

 

The test item, dissolved in water for injections, was tested in three independent experiments with a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, and two independent experiments without S9 mix.

Cultures of 20 mL at 5 x 10⁵cells/mL (3-hour treatment) or cultures of 50 mL at 2x 10⁵cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10 % (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. The negative control was the vehicle (Water for injections).

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

Since the test item was toxic in the preliminary test, the highest dose-level selected for the main test was 400 µg/mL, on the basis of the level of toxicity, according to the criteria specified in theinternational guidelines (decrease in the Adj. RTG).

 

In the experiments without S9 mix, the selected dose-levels were: 6.25,12.5, 25, 50, 100, 200 and 400 µg/mL(i.e. 8.3, 16.6, 33.3, 66.5, 133, 266 and 532 µg/mL in terms of registered substance) for the first experiment (3-hour treatment), and 25, 50, 100, 200, 266.7, 333.3 and 400 µg/mL (i.e. 33.3, 66.5, 133, 266, 354.7, 443.3 and 532 µg/mL in terms of registered substance) for the second experiment (24‑hour treatment).

In the experiments with S9 mix, the selected dose-levels were: 6.25,12.5, 25, 50, 100, 200 and 400 µg/mL (i.e. 8.3, 16.6, 33.3, 66.5, 133, 266 and 532 µg/mL in terms of registered substance) for the first and second experiments, and 25, 50, 100, 200, 250, 300 and 400 µg/mL (i.e. 33.3, 66.5, 133, 266, 332.5, 399 and 532 µg/mL in terms of registered substance) for the third experiment.

 

Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no relevant increase in the mutation frequency was noted up to the dose-level of 200 µg/ml or 300 µg/ml in comparison to the vehicle control.

 

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. The mutation frequencies of the positive controls also met the acceptance criteria of a study. The study was therefore considered as valid.

 

In conclusion, the test item did not show any mutagenic activity in the mouse lymphoma assay, in the absence or in the presence of a rat metabolising system, therefore Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 01 July 1980 to 26 August 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is sufficiently documented and the test conditions are similar to current test guidelines. The study was conducted before GLP implementation.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

: S. typhimurium TA102 or E. coli WP2 uvrA was not used in this test, only one experiment was performed, the highest tested concentration in the main test did not induce any cytotoxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: S. typhimurium TA102 or E. coli WP2 uvrA was not used in this test, only one experiment was performed, the highest tested concentration in the main test did not induce any cytotoxicity.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

8, 40, 200, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
Vehicle controls tested: medium with solvent or vehicle alone
- Justification for choice of solvent/vehicle: data not available
- volume of vehicle/solvent in the medium: 0.1 mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Beta-propiolactone (50 µg/plate) in TA 1535; Hycanthone (50 µg/plate) in TA 1537; Niridazole (0.1 µg/plate) in TA 98 and TA 100; Ethidium Bromure (60 µg/plate) in TA98 with S9 mix; acetyl amino fluorene (50 µg/plate) in TA 1538 with S9 mix

Details on test system and experimental conditions:

- Duration:
* Preincubation period: none
* Exposure duration: 48 hrs
- Number of replicate: 3

Evaluation criteria:

The test substance is judged positive if there is a 3-fold increase in the number of mutant in one of the strains, when compared to the control plates.

Statistics:

no data

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See table 7.6.1/1

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

See table 7.6.1/1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:a preliminary study was conducted in order to determine the bacterial cytotoxicity of the test item. The test item was tested up to 0.05 mg/plate. 5000 µg/plate induced a severe toxicity (70-90% cytotoxicity) in all strains (iut is not precised if it was with and/or without metabolic activation). As no cytotoxicity ocurred in strains except TA1538 (50% toxicity) at the concentration of 1000 µg/plate, this concentration was designed as the top concentration in the main test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Number of revertants per plate (mean of 3 plates)

	TA 1535			TA 1537			TA 1538			TA 98			TA 100
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	24	33	No	7	9	No	19	24	No	24	39	No	197	150	No
8	13	30	No	8	8	No	34	28	No	25	47	No	194	179	No
40	21	33	No	6	9	No	37	32	No	21	31	No	193	141	No
200	22	27	No	8	13	No	26	28	No	22	34	No	199	144	No
1000	25	23	No	10	8	No	30	21	No	22	32	No	182	158	No
Beta-propiolactone	989	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Hycanthone	-	-	-	676	-	-	-	-	-	-	-	-	-	-	-
Niridazole	-	-	-	-	-	-	-	-	-	69	-	-	512	-	-
Ethidium Bromure	-	-	-	-	-	-	-	-	-	-	2080	-	-	-	-
acetyl amino fluorene	-	-	-	-	-	-	-	1523	-	-	-	-	-	-	-

 

 *solvent control with water

MA: metabolic activation

Beta-propiolactone (50 µg/plate) in TA 1535; Hycanthone (50 µg/plate) in TA 1537; Niridazole (0.1 µg/plate) in TA 98 and TA 100; Ethidium Bromure (60 µg/plate) in TA98 with S9 mix; acetyl amino fluorene (50 µg/plate) in TA 1538 with S9 mix

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under these experimental conditions, the test item Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

In a reverse gene mutation assay in bacteria (Marzin, 1980), strains TA 1537, TA 1535, TA 1538, TA 98 and TA100 of S. typhimurium were exposed to Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, in water at concentrations of 8, 40, 200 and 1000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation). 
 
Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

 

 
This study is classified as acceptable. This study satisfies the requirement for Test Guideline EU B.13/14 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

Bacterial gene mutation:

Two bacterial reverse mutation assays (Ames test) are available. A GLP-compliant Ames test conducted according to OECD TG 471 (of reliability 1 according to Klimisch cotation criteria) was selected as the key study (CitoxLab, 2012). A second Ames test (of reliability 2 according to Klimisch cotation criteria) was used as a supporting study (DREBS, 1980). In both Ames tests, the test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

Therefore, reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is considered not to be mutagenic in this Salmonella typhimurium assay.

 

Mammalian gene mutation:

One fully reliable study is available (CitoxLab, 2012), conducted according to OECD TG 476 and GLP (mouse lymphoma assay, 6.25 – 400 µg/mL (active ingredient i.e. sodium mono, di and triisopropylnapthalenesulphonate), with and without liver microsomal activation, 3 and 24 h of treatment).

The test item did not induce mutations in the thymidine kinase locus assay using the mouse lymphoma cell line L5178Y up to the highest tested concentrations.

Therefore, reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is considered not to be mutagenic in this mouse lymphoma assay.

 

Mammalian chromosome aberration:

One fully reliable study is available (CitoxLab, 2012) conducted according to OECD TG 473 and GLP (mouse lymphoma assay, with and without liver microsomal activation, 3-, 20- and 44-hour treatments).

The dose-levels selected for the first experiment were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL (active ingredient), both with and without S9 mix.

Based on the cytotoxicity observed in the first experiment, the dose-levels selected for the second experiment were as follows:

.            31.3, 62.5, 125, 250, 500 and 1000 µg/mL, without S9 mix,

.            62.5, 125, 250, 375, 500 and 750 µg/mL, with S9 mix.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments, at both harvest times and in both the absence and presence of S9 mix.

Therefore, reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is considered not to be clastogenic in this in vitro mammalian chromosome aberration assay.

 

In vivo

No data available. Based on REGULATION (EC) No 1907/2006 as at July 2011 and the absence of positive results in the three above mentioned in vitro tests no additional testing for genetic toxicity in vivo is necessary.  

 

Conclusion: 

For each endpoint (bacterial gene mutation, mammalian gene mutation, & mammalian chromosome aberration) reliable and GLP compliant in vitro studies are available that all gave negative results.

Therefore it can be concluded that reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

is neither clastogenic or aneugenic nor mutagenic.

Accordingly it can be concluded that reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

is not genotoxic.

Justification for selection of genetic toxicity endpoint
No study was selected since all available in vitro studies were negative.

Short description of key information:
- genetic toxicity in vitro: negative; 4 studies negative (Sire (2012) Genetic toxicity in bacteria - Chromosome aberration -Mammalian gene mutation), (Marzin (1980) Genetic toxicity in bacteria),
- genetic toxicity in vivo: no data available

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

Four in vitro genotoxicity tests are available. None of the tests showed evidence of genotoxicity. The substance is therefore not regarded to have genotoxic effects and does not require classification for genetic toxicity according to the Regulation (EC) 1272/2008 and GHS UN..