Phosphorothioic triamide, N-propyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: not mutagenic (BASF, 2008)
in vitro Micronucelus assay: negative (BASF, 2010)
HPRT: negative (BASF, 2011)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 8712 / 056
- Purity: 99.6 g / 100 g
- Date of production: 01 Oct 2007
- Physical state and appearance: solid, white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator - 20 °C
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle water over a period of 4 hours was verified analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in water. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. All test substance formulations were prepared immediately before administration.

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 fraction

Test concentrations with justification for top dose:

1st Experiment (Standard Plate Test): 0; 20; 100; 500; 2500 and 5000 µg/plate
2nd Experiment (Preincubation Test): 0; 312.5; 625; 1250; 2500 and 5000 µg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further calculation / substantiation.

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: - 2-aminoanthracene (2-AA): with S9 mix, 2.5 µg/plate dissolved in DMSO (TA 1535, TA 100, TA 1537, TA 98) or 60 µg/plate dissolved in DMSO (E.coli WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: 48 - 72 hours in the dark at 37 °C

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer

Rationale for test conditions:

Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substances and are used to screen for possible mammalian mutagens and carcinogens.

Evaluation criteria:

- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
- Titer: The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
- Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
- Solubility: Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interefere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification / substantiation.

Statistics:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was >= 10^8 / mL.

Assessment criteria
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in preincubation test only at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

E. coli WP2 uvr A

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
- Precipitation: No test substance precipitation was found.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see tables below
- Negative (solvent/vehicle) historical control data: see tables below

Table 1: Historical Negative Control Data TA 1535

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	01.00 – 02.07	400	10	24	17	2
SPT	-	DMSO	11.04 – 02.07	400	10	25	17	2
SPT	-	Acetone	01.96 – 11.06	259	11	25	18	2
SPT	1 : 9	Water	01.00 – 01.07	400	12	24	18	2
SPT	1 : 9	DMSO	11.04 – 03.07	400	10	24	17	2
SPT	1 : 9	Acetone	01.96 – 11.06	258	11	25	19	2
PIT	-	Water	04.98 – 02.07	400	10	25	18	2
PIT	-	DMSO	07.03 – 03.07	400	10	25	17	2
PIT	-	Acetone	01.96 – 11.06	216	10	25	18	3
PIT	1 : 9	Water	11.97 – 02.07	400	11	25	18	2
PIT	1 : 9	DMSO	06.03 – 03.07	400	10	24	16	2
PIT	1 : 9	Acetone	01.96 – 11.06	201	10	24	18	2

*: revertants/plate

Table 2: Historical Negative Control Data TA 100

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	12.99 – 02.07	400	82	150	110	9
SPT	-	DMSO	11.04 – 02.07	400	80	135	109	9
SPT	-	Acetone	01.96 – 11.06	306	91	160	117	14
SPT	1 : 9	Water	12.99 – 01.07	400	88	152	115	2
SPT	1 : 9	DMSO	11.04 – 03.07	400	81	154	111	11
SPT	1 : 9	Acetone	01.96 – 11.06	301	85	160	119	15
PIT	-	Water	04.98 – 02.07	400	90	160	115	13
PIT	-	DMSO	09.03 – 03.07	400	81	153	109	10
PIT	-	Acetone	01.96 – 11.06	223	91	160	116	14
PIT	1 : 9	Water	03.98 – 02.07	400	92	159	120	15
PIT	1 : 9	DMSO	07.03 – 03.07	400	81	157	110	10
PIT	1 : 9	Acetone	05.96 – 11.06	198	96	160	118	15

*: revertants/plate

Table 3: Historical Negative Control Data TA 1537

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	11.99 – 02.07	400	5	16	10	2
SPT	-	DMSO	10.04 – 03.07	400	5	16	10	2
SPT	-	Acetone	01.96 – 11.06	261	5	18	10	2
SPT	1 : 9	Water	10.99 – 01.07	400	6	17	11	2
SPT	1 : 9	DMSO	09.04 – 03.07	400	5	16	10	2
SPT	1 : 9	Acetone	01.96 – 11.06	261	5	20	11	2
PIT	-	Water	04.98 – 02.07	400	5	16	10	2
PIT	-	DMSO	07.03 – 03.07	400	5	17	10	2
PIT	-	Acetone	01.96 – 11.06	213	6	17	10	2
PIT	1 : 9	Water	11.97 – 02.07	400	6	20	11	2
PIT	1 : 9	DMSO	07.03 – 03.07	400	5	17	10	2
PIT	1 : 9	Acetone	01.96 – 11.06	204	5	20	11	2

*: revertants/plate

Table 4: Historical Negative Control Data TA 98

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	01.00 – 02.07	400	17	44	29	5
SPT	-	DMSO	10.04 – 03.07	400	15	40	29	4
SPT	-	Acetone	01.96 – 11.06	263	19	50	29	5
SPT	1 : 9	Water	01.00 – 01.07	400	20	49	36	5
SPT	1 : 9	DMSO	10.04 – 03.07	400	21	50	35	5
SPT	1 : 9	Acetone	01.96 – 11.06	262	25	50	38	5
PIT	-	Water	01.98 – 02.07	400	18	44	28	4
PIT	-	DMSO	07.03 – 03.07	400	17	40	29	4
PIT	-	Acetone	01.96 – 11.06	216	19	45	28	5
PIT	1 : 9	Water	11.97 – 02.07	400	17	49	35	2
PIT	1 : 9	DMSO	07.03 – 03.07	400	20	50	33	5
PIT	1 : 9	Acetone	01.96 – 11.06	203	20	50	36	6

*: revertants/plate

Table 5: Historical Negative Control Data E.coli WP2 uvrA

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	09.99 – 01.07	400	25	55	33	5
SPT	-	DMSO	09.04 – 03.07	400	25	59	34	5
SPT	-	Acetone	04.96 – 11.06	234	25	55	34	5
SPT	1 : 9	Water	09.99 – 01.07	400	25	56	36	6
SPT	1 : 9	DMSO	09.04 – 03.07	400	25	60	38	6
SPT	1 : 9	Acetone	04.96 – 11.06	242	25	59	37	6
PIT	-	Water	10.97 – 02.07	400	25	52	32	5
PIT	-	DMSO	06.03 – 03.07	400	25	54	33	5
PIT	-	Acetone	01.96 – 11.06	191	25	54	32	5
PIT	1 : 9	Water	11.97 – 02.07	400	25	57	37	6
PIT	1 : 9	DMSO	07.03 – 03.07	400	25	56	36	5
PIT	1 : 9	Acetone	01.96 – 11.06	197	25	52	36	6

*: revertants/plate

Table 6: Historical Positive Control Data TA 1535

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	MNNG 5.0	03.05 – 02.07	400	509	1602	752	187
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	77	342	132	28
PIT	-	MNNG 5.0	06.04 – 03.07	400	512	1317	708	150
PIT	1 : 9	2-AA 2.5	03.04 – 03.07	400	83	398	130	33

*: revertants/plate

Table 7: Historical Positive Control Data TA 100

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	MNNG 5.0	02.05 – 03.07	400	500	1574	823	201
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	521	1878	876	205
PIT	-	MNNG 5.0	06.04 – 03.07	400	520	1302	797	141
PIT	1 : 9	2-AA 2.5	04.04 – 03.07	400	517	1497	829	174

*: revertants/plate

Table 8: Historical Positive Control Data TA 1537

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	AAC 100	02.05 – 03.07	400	212	1172	441	109
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	72	200	129	22
PIT	-	AAC 100	05.04 – 03.07	400	221	1028	429	100
PIT	1 : 9	2-AA 2.5	03.04 – 03.07	400	80	198	123	19

*: revertants/plate

Table 9: Historical Positive Control Data TA 98

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	NOPD 10	02.05 – 03.07	400	332	1316	616	145
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	502	1414	697	160
PIT	-	NOPD 10	06.04 – 03.07	400	388	1430	630	169
PIT	1 : 9	2-AA 2.5	03.04 – 03.07	400	510	1223	663	120

*: revertants/plate

Table 10: Historical Positive Control Data E.coli WP2 uvrA

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	4-NQO 5.0	01.05 – 03.07	400	500	1587	653	188
SPT	1 : 9	2-AA 60.0	01.05 – 03.07	400	150	383	231	30
PIT	-	4-NQO 5.0	05.04 – 03.07	400	500	1339	593	102
PIT	1 : 9	2-AA 60.0	05.04 – 03.07	400	150	307	226	25

*: revertants/plate

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose Range: 20 µg - 5000 µg/plate (SPT), 312.5 µg - 5000 µg/plate (PIT)

Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

Solubility: No precipitation of the test substance was found.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 487 (DRAFT)

Version / remarks:

Version 5, 02 Nov 2009

Deviations:

no

GLP compliance:

yes

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

N/A

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source
supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
During exposure to the test substance for 4 hours only, MEM medium was used without FCS
supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1st Experiment
4 hours exposure; 24 hours harvest time; without S9 mix:
0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix:
0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 250.0; 500.0; 1 000.0; 1 500.0; 2 000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 250.0; 500.0; 1 000.0; 2 000.0; 3 000.0 μg/mL

Following the requirements of the current draft guideline a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 µL/mL or 10 mM, which is the lowest. In case of toxicity, the top concentration should produce 55 % +/- 5 % cytotoxicity (cell number and / or proliferation index) compared to the respective vehicle control. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of exposure period.
In the pretest the parameters pH value and osmolarity were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
In addition, no test substance precipitation in the vehicle acetone was observed up to the highest applied concentration of 5800 µg/mL (stock solution). In culture medium test substance precipitation was observed at the end of treatment at 5800 µg/mL after 4 and 24 hours treatment in the absence and the presence of S9 mix.
After 4 hours treatment in the absence and the presence of S9 mix cytotoxicity indicated by reduced cell numbers of about or below 55 % +/- 5 % of control was observed at the highest applied concentration of 5800 µg/mL. Besides, in the pretest with 24 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 1450 µg/mL and above.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, acetone was selected as vehicle,
which had been demonstrated to be suitable in the in vitro mircronucleus test and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hours
- Recovery time: 0 or 20 hours
- Harverst time: 24 hours

NUMBER OF REPLICATIONS: The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected
culture conditions (last measurement based on the BrdU method of Speit et al. [ 7]: 12 hours; Feb 2009). Thus, the selected harvest time of 24 hours is about 2 times the normal cell cycle length.

NUMBER OF CELLS EVALUATED: A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least
2 000 cells for each test group.

DETERMINATION OF CYTOTOXICITY
- Method: cell count

Evaluation criteria:

A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent negative/vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data.

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative/vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
If the results of this test were statistically significant compared with the respective negative/vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been be printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In this study, clearly reduced cell counts of about or below 55% ± 5% of control were observed at all experimental parts at least at the highest concentration scored for the occurrence of micronuclei.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

RESULTS

MICRONUCLEUS ANALYSIS

1st Experiment

In the 1st Experiment after 4 hours exposure with and without metabolic activation no

relevant increase in the number of micronucleated cells was found. In the experimental part

without S9 mix a single, statistically significant increase in the number of micronucleated cells

(0.9%) exceeding the concurrent vehicle control value (0.2% micronucleated cells) was

observed at 725 μg/mL. However, in the 1st Experiment all values after treatment with the

test substance (0.2 – 1.3% micronucleated cells) were close to the concurrent vehicle control

values (0.2% and 0.8% micronucleated cells without and with S9 mix, respectively) and

clearly within our historical negative control data range (0.3 - 1.8% micronucleated cells).

2nd Experiment

In the 2nd Experiment after 24 hours exposure without S9 mix no relevant increase in the

number of micronucleated cells was found. The values after treatment with the test

substance (0.1 – 0.6% micronucleated cells) were close to the concurrent vehicle control

value (0.5% micronucleated cells) and clearly within our historical negative control data range

(0.3 - 1.8% micronucleated cells).

Besides after 4 hours exposure in the presence of metabolic activation a single, statistically

significant increase in the number of micronucleated cells was found at an intermediate

concentration of 1 000 μg/mL (2.2% micronucleated cells). Due to inhomogeneous data this

value was confirmed by scoring an increased sample of 2 000 micronucleated cells per test

group. This value was slightly above our historical negative control data range (0.3 - 1.8%

micronucleated cells). However, this increase of micronucleated cells occurred at an

intermediate concentration, means no dose-dependency was observed in this experiment.

PROLIFERATION INDEX

The proliferation index (PI) is based on the scoring of at least 1 000 cells per culture (2 000

cells per test group) for the different test groups without and with metabolic activation and

includes the measurement of colony size.

In this study, in the absence and the presence of S9 mix no cytotoxicity indicated by reduced

PI values was observed. In the 2nd Experiment after 4 hours exposure with S9 mix a sample

of 2 000 cells was scored at 1 000 μg/mL due to inhomogeneous genotoxicity data.

CELL COUNT

In this study, clearly reduced cell counts of about or below 55% ± 5% of control were

observed at all experimental parts at least at the highest concentration scored for the

occurrence of micronuclei.

In detail, in the absence of S9 mix in the 1st Experiment after 4 hours treatment clearly

reduced cell numbers were obtained at 1 450 μg/mL and above (51.9% of control). In the

2nd Experiment after 24 hours treatment cytotoxicity was observed at 1 000 μg/mL and

above (50.5% of control). In the presence of S9 mix after 4 hours in the 1st Experiment

growth inhibition indicated by reduced cell counts was observed at 2 900 μg/mL and above

(27.0% of control). In the 2nd Experiment in the presence of S9 mix reduced cell numbers

occurred at 2 000 μg/mL and above (60.6% of control).

CELL MORPHOLOGY

In this study, cell attachment and/or cell quality was influenced from about 3 000 μg/mL

onward after 4 hours treatment in the absence and presence of metabolic activation. After

24 hours test substance treatment without S9 mix cell attachment and/or cell quality was

strongly influenced from 1 500 μg/mL onward.

TREATMENT CONDITIONS

Osmolarity and pH values were not influenced by test substance treatment.

Test item precipitation in culture medium at the end of treatment period was observed from

about 2 900 μg/mL onward in the absence and presence of metabolic activation.

DISCUSSION

According to the results of the present in vitro micronucleus assay, the test substance

LIMUS-Sambaydestillation did not lead to a biologically relevant increase in the number of

micronucleated cells either without S9 mix or after the addition of a metabolizing system in

two experiments performed independently of each other. The frequencies of micronuclei after

test substance treatment were close to the range of the concurrent vehicle control values at

both exposure times and nearby the range of our historical negative control data.

In the 1st Experiment after 4 hours treatment in the absence of metabolic activation a single,

statistically significant increase in the number of micronucleated cells (0.9%) was obtained at

the lowest concentration scored for the occurrence of micronucleated cells (725 μg/mL).

However, no dose-dependency was obtained and the value was clearly within the range of

our historical negative control data. Therefore, this observation has to be regarded as

biologically irrelevant.

In the 2nd Experiment in the presence of metabolic activation a single, statistically significant

increase in the number of micronucleated cells (2.2%) was obtained after 4 hours exposure

with an intermediate concentration (1 000 μg/mL). Due to inhomogeneous data this value

was confirmed by scoring an increased sample of 2 000 micronucleated cells per test group.

This value slightly exceeded our historical negative control data range (0.3 - 1.8%

micronucleated cells). Due to missing dose-dependency and lacking confirmation in the

1st Experiment after 4 hours treatemnt in the presence of metabolic activation at a

comparable concentration range (725, 1 450 and 2 900 μg/mL) this single finding has to be

regarded as biologically irrelevant.

The number of micronucleated cells in the vehicle control groups were within our historical

negative control data range and, thus, fulfilled the acceptance criteria of this study .

The increase in the frequencies of micronuclei induced by the positive control substances

EMS and CPP demonstrated the sensitivity of the test system and of the metabolic activity of

the S9 mix employed. Although the values in the 1st Experiment in the absence and

presence of metabolic activation (2.4% micronucleated cells, each) were slightly below the

range of the historical positive control data (without S9 mix: 2.6 – 8.3% micronucleated cells;

with S9 mix: 2.5 - 23.5% micronucleated cells; see APPENDIX 7) the rates of micronucleated

cells were clearly increased compared to the concurrent vehicle control groups (0.8% and

0.2%, respectively). Thus, the positive controls fulfilled the acceptance criteria of this study.

CONCLUSION

Thus, under the experimental conditions chosen here, the conclusion is drawn that LIMUSSambaydestillation

has not the potential to induce micronuclei (clastogenic and/or

aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of

metabolic activation.

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test substance has not the potential to induce micronuclei (clastogenic and / or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested and the test groups in bold type were evaluated:

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 362.5; 725.0; 1450.0; 2900.0; 4350.0; 5800.0 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 362.5; 725.0; 1450.0; 2900.0; 4350.0; 5800.0 µg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 250.0; 500.0; 1000.0; 1500.0; 200.0 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 250.0; 500.0; 1000.0; 2000.0; 3000.0 µg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. at least 2000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to a statistically significant and biologically relevant increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. The single statistically significant outlier value observed in the 2nd Experiment in the presence of metabolic activation after 4 hours treatment with 1000 µg/mL has to be considered biologically irrelevant due to missing dose-dependency and lacking confirmation in the 1st Experiment at a comparable concentration range.

Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2010 - February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Treatment medium (4-hour exposure period)
Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No.
FG 0815).
Culture medium and Treatment medium (24-hour exposure)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%
(v/v) fetal calf serum (FCS).
Pretreatment medium ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10-3 mg/mL)
- aminopterin (0.18 x 10-3 mg/mL)
- thymidine (3.88 x 10-3 mg/mL)
- 10% (v/v) fetal calf serum (FCS)
Selection medium ("TG" medium)
Hypoxanthine-free Ham's F12 medium supplemented with:
- 6-thioguanine (10 μg/mL)
- 1% (v/v) stable glutamine (200 mM)
- 10% (v/v) fetal calf serum (FCS)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S-9 mix

Test concentrations with justification for top dose:

1st Experiment (invalid due to technical error)
without S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)
0; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL
3rd Experiment
without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
4th Experiment
without S9 mix (24-hour exposure period)
0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0 μg/mL
5th Experiment
without S9 mix (4-hour exposure period)
0; 46.9; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test subtance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Methylcholanthrene (MCA): with S9 mix, 20 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20-24 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days

NUMBER OF CELLS EVALUATED: 10^5 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a doseresponse
relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient
evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see any other information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

RESULTS

Unfortunately, in the 1st Experiment a false test substance was used which had both, a

similar test substance name and a similar test substance number. Due to this technical error

the data obtained in the 1st Experiment in the absence and presence of S9 mix has to be

regarded as invalid and, thus, the data are neither reported nor discussed (for details see raw

data).

In addition, the 2nd and 3rd Experiment in the absence of metabolic activation did not fulfill

the recommendations of the current OECD Guideline 476 due to excessive cytotoxicity or

lacking cytotoxicity, respectively. Both experimental parts were discontinued after the

1st subcultivation.

MUTANT FREQUENCY

In this study, no relevant increase in the number of mutant colonies was observed either

without S9 mix or after the addition of a metabolizing system. In all experiments after 4 and

24 hours treatment with the test substance the values for the corrected mutation frequencies

(MFcorr.: 0.00 – 8.82 per 106 cells) were close to the respective vehicle control values (MFcorr.:

1.18 – 6.81 per 106 cells) and clearly within the range of our historical negative control data

(without S9 mix: MFcorr.: 0.00 – 15.95 per 106 cells; with S9 mix: MFcorr.: 0.00 – 12.62 per

106 cells).

The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix;

20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the

corrected mutant frequencies (without S9 mix: MFcorr.: 186.96 – 561.60 per 106 cells; with

S9 mix: MFcorr.: 61.36 – 79.80 per 106 cells) were clearly within our historical positive control

data range (without S9 mix: MFcorr.: 48.83 – 999.46 per 106 cells; with S9 mix: MFcorr.: 26.29 –

413.54 per 106 cells).

CYTOTOXICITY

Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control or

extremely low cell numbers at the 1st subcultivation were observed in the 2nd, 4th and

5th Experiment in the absence and presence of S9 mix in the highest applied concentrations.

In detail, in the 2nd Experiment in the absence of S9 mix, there was a decrease in the

number of colonies from 125.0 μg/mL onward after an exposure period of 24 hours. In the

presence of S9 mix there was a decrease in the number of colonies from 4 000.0 μg/mL

onward after an exposure period of 4 hours.

In contrary, in the 3rd Experiment in the absence of S9 mix when applying concentrations up

to 200 μg/mL no decrease in the number of colonies was obtained.

In the 5th Experiment in the absence of S9 mix there was a decrease in the number of

colonies from 1 500.0 μg/mL onward after an exposure period of 4 hours. In addition, in the

presence of S9 mix after 4 hours exposure there was a decrease in the number of colonies at

an intermediate concentration of 750.0 μg/mL: Test groups from 3 000.0 μg/mL onward could

nor be determined due to low cell numbers at 1st subcultivation.

It is remarkable that in all experimental parts assessed for gene mutations clearly reduced

colony counts in term of survival (CE1) were obtained at intermediate concentrations around

500.0 μg/mL. However, no adverse effects were obtained by means of viability (CE2) when

subcultivation of the test groups was feasible. These findings occurred far below the border

of saturation which started at about 4 000.0 μg/mL. Or described otherwise, just before

reaching total cytotoxicity an increase in colony numbers (only CE1) was observed.

CELL MORPHOLOGY

Details on the observations on cell morphology (cell attachment) are given in Appendix 3.

In all experimental parts with 4 and 24 hours treatment in the absence and presence of

S9 mix the morphology and attachment of the cells was adversely influenced at the highest

applied concentrations, except when using only 200.0 μg/mL as top concentration in the

3rd Experiment in the absence of S9 mix.

TREATMENT CONDITIONS

Osmolarity and pH values were not influenced by test substance treatment.

In all experimental parts of this study in the absence and presence of S9 mix test substance

precipitation in culture medium determined at the end of treatment period was observed from

about 4 000 μg/mL onward.

DISCUSSION

The 1st Experiment of this study was cancelled because of testing a false test substance.

However, the data are recorded and included in the raw data of this study.

According to the results of the present in vitro study, the test substance LIMUSSambaydestillation

did not lead to a relevant increase in the number of mutant colonies

either without S9 mix or after the addition of a metabolizing system in five experiments

performed independently of each other. The mutant frequencies at any concentration were

within the range of the concurrent vehicle control values and within the range of our historical

negative control data (see Appendix 4).

The mutation frequencies of the vehicle control groups were within our historical negative

control data range including all vehicles used in our laboratory and, thus, fulfilled the

acceptance criteria of this study (see Appendix 4)

The increase in the frequencies of mutant colonies induced by the positive control

substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the

metabolic activity of the S9 mix employed. The values were within the range of the historical

positive control data and, thus, fulfilled the acceptance criteria of this study (see Appendix 5).

CONCLUSION

Thus, under the experimental conditions chosen here, the conclusion is drawn that

LIMUS-Sambaydestillation is not a mutagenic substance in the HPRT locus assay using

CHO cells in the absence and the presence of metabolic activation.

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster Ovary (CHO) cells in vitro. Five independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

Based on the observations and the toxicity data of a previously performed pretest for an in vitro micronucleus assay and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated:

1st Experiment (invalid due to technical error)

without S9 mix (4 -hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL

with S9 mix (4 -hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL

2nd Experiment

without S9 mix (24 -hour exposure period) (failed recommendations of OECD 476)

0; 125.0; 250.0; 500.0; 1000.0; 2000.0; 4000.0; 6000.0 µg/mL

with S9 mix ((4 -hour exposure period)

0; 250.0; 500.0; 1000.0; 2000.0; 4000.0; 6000.0 µg/mL

3rd Experiment

without S9 mix (24 -hour exposure period) (failed recommendations of OECD 476)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

4th Experiment

without S9 mix (24 -hour exposure period)

0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1000.0; 2000.0; 4000.0 µg/mL

5th Experiment

without S9 mix (4 -hour exposure period)

0; 46.9; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL

with S9 mix (4 -hour exposure)

0; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

Due to a technical error in the 1st Experiment in the absence and presence of S9 mix the data obtained has to be regarded as invalid and, therefore, they are not reported.

In the 2nd and 3rd Experiment after 24 hours treatment in the absence of metabolic activation the recommendations of the current OECD Guideline 476 were not fulfilled due to excessive cytotoxicity or lacking cytotoxicity, respectively. Therefore, these experimental parts were discontinued.

In all experimental parts assessed as valid regarding the current OECD Guideline 476 and evaluated for gene mutations the highest concentrations applied were clearly cytotoxic.

On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in five experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro:

Ames test:

There is no Ames-Test for NPPT available but the following data is suitable for a read across:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose Range: 20 µg - 5000 µg/plate (SPT), 312.5 µg - 5000 µg/plate (PIT)

Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

Solubility: No precipitation of the test substance was found.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Micronucleus Assay:

There is no micronucleus assay for NPPT available but the following data is suitable for a read across:

The substance LIMUS-Sambaydestillation was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system based on OECD guideline 487 and GLP (BASF, 2010).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested and the test groups in bold type were evaluated:

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 250.0; 500.0; 1 000.0; 1 500.0; 2 000 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 250.0; 500.0; 1 000.0; 2 000.0; 3 000.0 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least 2 000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to a statistically significant and biologically relevant increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. The single statistically significant outlier value observed in the 2nd Experiment in the presence of metabolic activation after 4 hours treatment with 1 000 μg/mL has to be considered biologically irrelevant due to missing dose-dependency and lacking confirmation in the 1st Experiment at a comparable concentration range.

Thus, under the experimental conditions described, LIMUS-Sambaydestillation is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the

absence and the presence of metabolic activation.

HPRT:

There is no HPRT-Test for NPPT available but the following data is suitable for a read across:

The substance LIMUS-Sambaydestillation was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according ot OECD 476 guideline and GLP (BASF, 2011). Five independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the observations and the toxicity data of a previously performed pretest for an in

vitro micronucleus assay and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated:

1st Experiment (invalid due to technical error)

without S9 mix (4-hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)

0; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL

3rd Experiment

without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

4th Experiment

without S9 mix (24-hour exposure period)

0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0 μg/mL

5th Experiment

without S9 mix (4-hour exposure period)

0; 46.9; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

Due to a technical error in the 1st Experiment in the absence and presence of S9 mix the

data obtained has to be regarded as invalid and, therefore, they are not reported.

In the 2nd and 3rd Experiment after 24 hours treatment in the absence of metabolic activation the recommendations of the current OECD Guideline 476 were not fulfilled due to excessive cytotoxicity or lacking cytotoxicity, respectively. Therefore, these experimental parts were discontinued.

In all experimental parts assessed as valid regarding the current OECD Guideline 476 and evaluated for gene mutations the highest concentrations applied were clearly cytotoxic.

On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in five experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance LIMUSSambaydestillation is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Read Across justification:

LIMUS-Sambaydestillation is a reaction mass of phosphorothioic triamide, N-butyl- (NBPT) and phosphorothioic triamide, N-propyl- (NPPT), the subject of this registration. The relation of NBPT and NPPT in the reaction mass is ca. 3:1 and the overall total content of NBPT and NPPT in the reaction mass is up to 85 %. Thus, as the reaction mass contains a certain amount of NPPT (up to ca. 21 %) and based on the structural similarities between NBPT and NPPT (NPPT is one CH2 -group shorter) the reaction mass is suitable for a read across and thus for filling the data gaps of NPPT.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT test (OECD 476, GLP) and the in vitro Micronucleus test (OECD 487 (draft), GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.