Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11- 30 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

link to target

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

11- 30 Jul 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read-across

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The present analogue approach contemplates Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine (CAS 98679-19-7) as target substance for read across from the source substance Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5). The read-across approach is used to evaluate the hazardous potential of the target substance with respect to ecotoxicological endpoints and human health for REACH Annex VII.
Based on similar chemical structures, read-across based on different compounds having the same type of effect(s) as described in scenario 2 of the Read-Across Assessment Framework document can be used as a basis for assessment.
The target substance (Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine) and the source substance (Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates) are both organic UVCB substances.
In consequence, read-across can be justified due to the high structural similarities as well as common properties, which will be outlined in detail below.

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

Source: Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (EC 306-227-4, CAS 96690-34-5)
The substance is manufactured from the educts phosphorus pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) and Amines, C12-14-tert-alkyl (CAS 68955-53-3, EC 273-279-1).

Target: Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine (EC 308-859-6, CAS 98679-19-7)
The substance is manufactured from the educts phosphor pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) and bis(2-ethylhexyl)amine (CAS 106-20-7, EC 203-372-4)

Both substances do not contain any impurities which may impact the feasibility of read-across.

3. ANALOGUE APPROACH JUSTIFICATION
Both substances are organic UVCBs and are manufactured from the educts phosphorus pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) in similar ratios, the only slight difference is the amine part. In the source chemical, Amines, C12-14-tert-alkyl is used, and in the target bis(2-ethylhexyl)amine.
So, the reaction products have a similar distribution pattern with regard to varied alkyl phosphate species (mainly dialkyl phosphates). Both used amines have similar molecular weights, i.e. 185.35-213.41 g/mol (CAS 68955-53-3) and 241.46 g/mol (CAS 106-20-7), which are hence not expected to alter their toxicokinetic behaviour essentially. The fact that one amine is mono-substituted and the other di-substituted, is also not considered to impact the suitability for read-across, as amines are considered to be rather stable functional groups, e.g. hydrolytically stable, and are not considered to be altered to a relevant extent in the body. Both amines are not covalently bound to the phosphates, so they are expected to dissociate into similar dissociation products, too.
Last but not least, both substances show similar physico-chemical properties. They are both liquid at all relevant handling temperatures, both are characterized by negative glass transition temperatures they do not boil but decompose, have similar densities and low vapour pressures indicating that they are no volatile liquids. They have both logPow values and water solubilities which, despite slight variations, are not expected to alter essentially their uptake and distribution through the body.
As there is not sufficient data on both source and target substance available, and the amine is considered to be the main difference and also not covalently bound to the phosphates, data on human health relevant endpoints is retrieved from publically available data sources, i.e. ACToR (https://actor.epa.gov/actor/searchidentifier.xhtml) and RTECS (http://ccinfoweb.ccohs.ca/rtecs/search.html). Further, ecotoxicological properties were estimated via US EPA EpiSuite ECOSAR Class Program v1.11.
Data indicate that both amines produce severe irritating reactions when applied into the eye or onto the skin. Further, the acute dermal toxicity LD50 values are nearly identical in the rabbit (acute tox. Cat. 4), and when applied orally, the available LD50 values similarly indicate that a classification as acute toxic Cat. 4 is triggered. With regard to ecotoxicological properties, the estimated values slightly differ. However, they consistently trigger the same classification, as both substances are not biodegradable; the source chemical is not, so the same can be applied to the amines.
So in consequence, taking into account the similar manufacturing process, i.e. identical and similar educts, and so similar reaction products, similar physico-chemical properties of the source and target chemical and similar (eco)toxicological properties of the source and target amines, the read-across from Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates to Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine is scientifically justified

4. DATA MATRIX

Table: Data Matrix, source and target chemical
Endpoint Source: CAS 96690-34-5 Target: CAS 98679-19-7
Physical state at 20°C, 1013 hPa liquid liquid
Glass transition temperature -44.95 °C -72.1 °C (onset)
Decomposition 243.36 °C 167.1°C (onset)
Density 0.97 g/cm³ 0.968 g/cm³
Vapour pressure ≤ 54 Pa at 20°C < 6.7 Pa at 20°C
logPow ≥ 0.69 - ≤ 5.6 (estimated) - 0.61 at 23°C
Water solubility 1322 mg/L at 25 °C and pH 3.57 17.14 g/l at 20°C
Surface tension 42.9 mN/m at 20°C and 0.66g/l 37.8 mN/m ≤ST ≤ 41.2 mN/m at 20°C and 1g/l
Flash point 1 35°C at 101.325 kPa 107.5°C at 101.3 kPa

Table: Data Matrix, source and target amine
Endpoint Source amine: CAS 68955-53-3 Target: CAS 106-20-7
Acute oral toxicity LD50 = 552 mg/kg (m/f mice, 470 - 719 mg/kg) (ACToR) LD50 = 800µL/kg (mouse, intraperitoneal) (ACToR, RTECS)
LD50 = 320 mg/kg (male rats) (ACToR; RTECS) LD50 = 1640mg/kg (rat) (ACToR, RTECS)
LD50 = 300 mg/kg (rat) (RTECS)
Acute dermal toxicity LD50 = 251 mg/kg (m/f rats, 190 - 322 mg/kg) (ACToR) LD50 = 1190uL/kg (rabbit) (ACToR, RTECS)
LD50 = 1.12 g/kg (rabbits, 0.83 - 1.51) (ACToR, RTECS)
Acute toxicity inhalation LC50 = 157 ppm (female rats, 1.19 mg/L; 90 - 249 ppm) (ACToR, RTECS)
LC50 > 231 ppm (male rats, 1.75 mg/L) (ACToR, RTECS) No data
LC50 > 940 mg/m³/4h (rats) (RTECS)
Skin irritation Severe reaction (rabbit, Draize test) (RTECS) Severe reaction (rabbit, Draize test) (RTECS)
Severe reaction (rabbit, open irritation test) (RTECS)
Mild reaction (rabbit, open irritation test) (RTECS)
Eye irritation Severe reaction (rabbit, Draize test) (RTECS) Severe reaction (rabbit, Draize test) (RTECS)
Genetic Toxicity No data Negative ±S9 (Ames test) (ACToR)
Fish 96-hr LC50 0.644 mg/L* 0.047 mg/L*
0.632 mg/L** 0.016 mg/L**
Daphnid 48-hr LC50 0.104 mg/L* 0.010 mg/L*
0.458 mg/L** 0.014 mg/L**
Green Algae 96-hr EC50 0.047 mg/L* 0.003 mg/L*
0.939 mg/L** 0.059 mg/L**
Fish ChV 0.015 mg/L* 0.000546 mg/L*
0.082 mg/L** 0.003 mg/L**
Daphnid ChV 0.012 mg/L* 0.00131 mg/L*
0.088 mg/L** 0.004 mg/L**
Green Algae ChV 0.019 mg/L* 0.00134 mg/L*
0.424 mg/L** 0.039 mg/L**
* Aliphatic Amines
** Neutral Organic SAR (Baseline Toxicity)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

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:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment I: 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate with and without metabolic activation
Experiment II: 0.50, 1.58, 5.0, 15.8, 50, 158, 500 and 1580 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of bacteria and S9 activity

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Remarks:

aqua dest.

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (10 µg/plate) for TA100 and TA1535; 4-nitro-o-phenylene-diamine (10 or 40 µg/plate) for TA98 and TA1537; methylmethanesulfonate (1 µL/plate) for TA102; +S9: 2-aminoanthracene (2.5 or 10 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn or reduction in the number of revertants down to a mutation factor of ca. ≤ 0.5 compared to solvent control.

Evaluation criteria:

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values were used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occur and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with and without metabolic activation.
A biologically relevant increase is described as follows:
- if in the tester strains TA 98, TA 00 and TA 102 the number of reversions is at least twice as high than the reversion rate of the solvent control.
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

A statistical evaluation of the results was not regarded as necessary since the biological relevance is the criterion for interpretation of results according to OECD 471.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 50 µg/plate (-S9), ≥ 500 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 100 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 156 µg/plate (-S9), ≥ 1580 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 158 µg/plate (-S9), ≥ 1580 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 316 µg/plate (+S9); Exp. II: ≥ 50 µg/plate (-S9), ≥ 500 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 67 µg/plate (-S9), ≥ 1580 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: a preliminary cytotoxicity experiment in TA 98 and TA 100 was performed to determine appropriate concentrations for treatment in the main experiment. Cytotoxicity in TA 98 was observed at concentrations of ≥ 316 µg/plate in the absence of S9 mix and at concentrations of ≥ 1000 µg/plate in the presence of S9 mix. For TA 100, cytotoxic effects were observed at ≥ 100 µg/plate with and without S9 mix, respectively. In both strains, no bacterial background lawn was observed at ≥ 2500 µg/plate (with S9 mix) and at ≥ 5000 µg/plate (with and without S9 mix). Therefore, the highest concentration used in the main experiment was 2500 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: in experiment I in tester strain TA 102 (without metabolic activation), the mean value of revertant colony numbers of the negative control (aqua dest.) was below the lower limit of the historical control data for the negative control. Since the values fell only slightly below the lower limit of the control data, the data were considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in experiment I, in the absence of S9 mix, cytotoxicity was observed at concentrations ≥ 100 µg/plate in tester strain TA 1537 and at ≥ 316 µg/plate in the remaining tester strains. In the presence of S9 mix, cytotoxicity was evident at concentrations ≥ 1000 µg/plate in all tester strains, except for TA 100, in which cytotoxicity was already seen at ≥ 316 µg/plate. In experiment II, cytotoxic effects were already visible at concentrations ≥ 50 µg/plate in TA 100, TA 1535 and TA 1537 in the absence of S9 mix. In TA 98 and TA 102, cytotoxicity was observed without S9 mix at concentrations ≥ 158 and 500 µg/plate, respectively. In the presence of S9 mix, tester strains TA 100 and TA 1535 showed cytotoxicity at ≥ 500 µg/plate, whereas in the remaining tester strains cytotoxicity was seen at ≥ 1580 µg/plate.

Conclusions:

Interpretation of results: negative

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

EC Number:

306-227-4

EC Name:

Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Cas Number:

96690-34-5

Molecular formula:

C4H11O4P to C8H20O7P2 as representative molecular formula of the composition as specified in section 1.2

IUPAC Name:

Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Specific details on test material used for the study:

Physical state at 20°C, 1013 hPa: liquid
Glass transition temperature: -44.95 °C
Decomposition: 243.36 °C
Density: 0.97 g/cm³
Vapour pressure: ≤ 54 Pa at 20°C
log Pow: ≥ 0.69 - ≤ 5.6 (estimated)
Water solubility: 1322 mg/L at 25 °C and pH 3.57
Surface tension: 42.9 mN/m at 20°C and 0.66g/l
Flash point: 135°C

Method

Target gene:

his operon

Species / strain

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:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment I: 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate with and without metabolic activation
Experiment II: 0.50, 1.58, 5.0, 15.8, 50, 158, 500 and 1580 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of bacteria and S9 activity

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Remarks:

aqua dest.

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (10 µg/plate) for TA100 and TA1535; 4-nitro-o-phenylene-diamine (10 or 40 µg/plate) for TA98 and TA1537; methylmethanesulfonate (1 µL/plate) for TA102; +S9: 2-aminoanthracene (2.5 or 10 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn or reduction in the number of revertants down to a mutation factor of ca. ≤ 0.5 compared to solvent control.

Evaluation criteria:

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values were used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occur and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with and without metabolic activation.
A biologically relevant increase is described as follows:
- if in the tester strains TA 98, TA 00 and TA 102 the number of reversions is at least twice as high than the reversion rate of the solvent control.
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

A statistical evaluation of the results was not regarded as necessary since the biological relevance is the criterion for interpretation of results according to OECD 471.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 50 µg/plate (-S9), ≥ 500 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 100 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 156 µg/plate (-S9), ≥ 1580 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 158 µg/plate (-S9), ≥ 1580 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 316 µg/plate (+S9); Exp. II: ≥ 50 µg/plate (-S9), ≥ 500 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: ≥ 316 µg/plate (-S9); ≥ 1000 µg/plate (+S9); Exp. II: ≥ 67 µg/plate (-S9), ≥ 1580 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: a preliminary cytotoxicity experiment in TA 98 and TA 100 was performed to determine appropriate concentrations for treatment in the main experiment. Cytotoxicity in TA 98 was observed at concentrations of ≥ 316 µg/plate in the absence of S9 mix and at concentrations of ≥ 1000 µg/plate in the presence of S9 mix. For TA 100, cytotoxic effects were observed at ≥ 100 µg/plate with and without S9 mix, respectively. In both strains, no bacterial background lawn was observed at ≥ 2500 µg/plate (with S9 mix) and at ≥ 5000 µg/plate (with and without S9 mix). Therefore, the highest concentration used in the main experiment was 2500 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: in experiment I in tester strain TA 102 (without metabolic activation), the mean value of revertant colony numbers of the negative control (aqua dest.) was below the lower limit of the historical control data for the negative control. Since the values fell only slightly below the lower limit of the control data, the data were considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in experiment I, in the absence of S9 mix, cytotoxicity was observed at concentrations ≥ 100 µg/plate in tester strain TA 1537 and at ≥ 316 µg/plate in the remaining tester strains. In the presence of S9 mix, cytotoxicity was evident at concentrations ≥ 1000 µg/plate in all tester strains, except for TA 100, in which cytotoxicity was already seen at ≥ 316 µg/plate. In experiment II, cytotoxic effects were already visible at concentrations ≥ 50 µg/plate in TA 100, TA 1535 and TA 1537 in the absence of S9 mix. In TA 98 and TA 102, cytotoxicity was observed without S9 mix at concentrations ≥ 158 and 500 µg/plate, respectively. In the presence of S9 mix, tester strains TA 100 and TA 1535 showed cytotoxicity at ≥ 500 µg/plate, whereas in the remaining tester strains cytotoxicity was seen at ≥ 1580 µg/plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative