Ethanamine, N-ethyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate (>1 <6.5 mol EO and >1 < 6.5 mol PO)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: neg. (BASF 2017)

HPRT: neg. (BASF 2018)

MNT in vitro: inconclusive (BASF 2018)

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:

31-03-2017 - 29-08-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The analyses of the test item (= test substance) was carried out at Competence Center Analytics (see analytical report, study code 16L00510), BASF SE, Ludwigshafen, Germany.
Name of test substance: Laromer LR 8889
Test substance No.: 16/0422-1
Batch identification: 160005P040
CAS No.: 173046-61-2
Identity: confirmed
Purity: 98.2 area-% (HPLC, 201 nm)
98.4 area-% (HPLC, 231 nm)
Content: > 99 g/100 g
water content: 0.04 g/100 g
UVCB susbtance
(UVCB = Substances of Unknown or Variable composition)
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions is guaranteed until 16 Oct 2017 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor
division.
Date of production: 21 Oct 2016
Physical state, appearance: liquid, yellowish, clear
Storage conditions: room temperature
More detailed information may be requested from the sponsor (BASF SE).

Species / strain / cell type:

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

Remarks:

without TA 98

Details on mammalian cell type (if applicable):

The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined (2, 3, 4). The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions (5). The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was 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 clarification/substantiation.

Vehicle / solvent:

Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 109 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay. The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

Checking the tester strains:
The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA was checked for UV sensitivity. Histidine and tryptophan auxotrophy was checked in each experiment via the spontaneous rate.

Standard plate test:
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1, 2).
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test:
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.. 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were 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.
• Fresh bacterial culture containing approximately 109 cells per mL were used.
Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system. A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test using the tester strain TA 1535 and TA 98 with S9 mix at 5000 μg/plate.

Vehicle 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:

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test using the tester strain TA 1535 and TA 98 with S9 mix at 5000 μg/plate.

Vehicle 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:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No test substance precipitation was found with and without S9 mix.

Conclusions:

Under the experimental conditions chosen here, it is concluded that Laromer LR 8889 is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-01-2017 - 20-02-2018

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Name of test substance: Laromer LR 8889
Test substance No.: 16/0422-1
Batch identification: 160005P040
CAS No.: 173046-61-2
Identity: Confirmed
Content/composition: > 99 g/100 g [UVCB substance (UVCB = Substances of Unknown or Variable composition) Water content: 0.04 g/100 g]
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 16 Oct 2017 as indicated by the sponsor, and the sponsor holds this responsibility.
The test facility is organizationally independent from the BASF SE sponsor division.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line (1, 2) is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested:
1st Experiment
without S9 mix
0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL
with S9 mix
0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00; 400.00 μg/mL
2nd Experiment
without S9 mix
0; 2.50; 5.00; 10.00; 20.00; 40.00; 50.00 μg/mL (invalid; data not shown)
with S9 mix
0; 9.38; 18.75; 37.50; 75.00; 100.00; 150.00; 200.00 μg/mL
3rd Experiment
without S9 mix
0; 2.50; 5.00; 10.00; 15.00; 20.00; 25.00; 40.00 μg/mL
with S9 mix
0; 10.00; 20.00; 40.00; 80.00; 100.00; 120.00; 140.00 μg/mL
4th Experiment
without S9 mix
0; 2.50; 5.00; 10.00; 12.50; 15.00; 17.50; 20.00; 25.00 μg/mL

Vehicle / solvent:

Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Culture medium:
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No.
P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).

Treatment medium (without S9 mix):
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%
(v/v) FCS.
Treatment medium (with S9 mix):
Ham's F12 medium containing stable glutamine and hypoxanthine.

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) FCS

Selection medium ("TG" medium):
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with:
- 6-thioguanine (10 μg/mL)
- 10% (v/v) FCS

For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks).

Routine passage (preparation of a single cell suspension):
- Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree).
- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks.
- This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS.
- Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
- Cells were counted in a counting chamber or using a cell counter.
- Cell suspensions were diluted with complete culture medium to the desired cell count.

S9 fraction:
The S9 fraction was prepared according to Ames et al. (3) at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 30 - 70%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

Time schedule:
Day 1: Seeding of the cells pretreated with "HAT" medium: in 300 cm² flasks (20x106 cells in 40 mL)
Day 2: Test substance incubation (approx. 20 – 24 hours after seeding); exposure period (4 hours); removal of test substance by intense washing; 1st passage of the treated cells in 175 cm2 flasks (2x106 cells in 20 mL medium) and seeding of the cloning efficiency 1 (survival) in 60 mm petri dishes (200 cells in 5 mL medium).
Day 5: 2nd passage of the treated cells (seeding of 2x106 cells in 20mL medium)
Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1. 3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability)
From day 16: Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2

Cell stocks (1.0-mL portions) stored in liquid nitrogen were thawed at 37°C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). After 24 hours, the medium was replaced to remove any dead cells. At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.

Pre-treatment of cells with "HAT" medium:
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pre-treatment with "HAT" medium. 0.8 - 1x106 cells were seeded per flask (175 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10% (v/v) FCS was incubated for a further 3 - 4 days.

Attachment period:
For each test group, about 20x106 logarithmically growing cells per flask (300 cm²) were seeded into about 40 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours.

Exposure period:
After the attachment period, the medium was removed from the flasks and the treatment medium was added (see table below). The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity (see Table 2, 3, 4 and 5 page 21, 23 and 24).

Expression period:
The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2x106 cells were seeded in 20 mL medium (in 175 cm2 flasks). The flasks were left to stand in the incubator for about 3 days at 37°C, relative humidity of ≥ 90% and 5% (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm2 flasks with 2x106 cells. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (3rd passage).

Selection period:
For selection of the mutants, two 175 cm2 flasks with 2x106 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 – 7 days. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

S9 mix
The S9 mix was prepared freshly prior to each experiment (3). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used.
The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (4) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

Evaluation criteria:

Acceptance criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.
Assessment criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit). Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction (7, 8). The calculation was performed using R (9). If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TREATMENT CONDITIONS
The pH value of the test substance preparation (stock solution) was adjusted by adding small amounts of HCl. Osmolality and pH values were not influenced by test substance treatment. In this study, in the absence of S9 mix, no test substance precipitation was observed in culture medium at the end of treatment up to the highest applied concentration of 50μg/mL. In the presence of S9 mix precipitation was observed in culture medium at the end of treatment at 100.00μg/mL and above in the 1st, 2nd and 3rd Experiment.
CELL MORPHOLOGY
After 4 hours of treatment the morphology and attachment of the cells treated with at least the highest applied concentration was adversely influenced (grade > 2) in all experimental parts scored for gene mutations. This occurred in samples regardless of the presence or absence of metabolic activation.
CYTOTOXICITY
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in all four experiments in the presence of S9 mix, at least at the highest applied concentrations. A decrease in the number of colonies was observed at 200.00 μg/mL (RS: 2.0%) in the 1st Experiment and from about 120.00 μg/mL (RS: 13.9%) onward in the 3rd Experiment. The cell densities were distinctly reduced. Without S9 mix, there was a decrease in the number of colonies from about 50.00μg/mL in the 1st Experiment. The cell densities were distinctly reduced after the 2nd passage. In the 3rd Experiment there was a decrease in the number of colonies from about 20.0 μg/mL (RS: 3.4%) onward after an exposure period of 4 hours and from about 17.50 μg/mL (RS: 10.9%) onward after an exposure period of 4 hours in the 4th Experiment. The cell densities were distinctly reduced. In the 2nd Experiment after 4 hours treatment in the absence of metabolic activation the cloning efficiency 1 showed strong cytotoxicity from 20.00 μg/mL onward. Thus, with only three remaining test groups this experimental part of the study did not fulfill the recommendations of the current OECD Guideline 476 and was discontinued.
MUTANT FREQUENCY
In the absence of metabolic activation, no relevant increase in the number of mutant colonies was observed in the 1st Experiment. The corrected mutation frequency of the test substance treated groups ranged between 0.68 - 2.95 per 10^6 cells. All values were within the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 5.97 per 10^6 cells) and statistically not significant as compared to the concurrent negative control value (MFcorr.: 1.99 per 106 cells). A dose related response trend was also not observed. In the 3rd Experiment after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 4.27 – 14.29 per 10^6 cells) were close to the respective vehicle control values (MFcorr.: 4.24 per 10^6 cells), except the cultures treated with the highest evaluated concentration (15.00 μg/mL). The mutation frequency observed in these cultures (MFcorr.: 14.29 per 106 cells) were, in contrast to the lower concentration within this experiment, above the range of the 95% control limit of our historical negative control data and statistically significant. Furthermore, this value led to a positive trend in the dose response analysis. In order to address the relevance of the obtained results a fourth experiment was performed. In the 4th Experiment without S9 mix the test substance did not cause a biologically relevant increase in the mutant frequencies. The obtained corrected mutation frequencies from the test substance treated cultures ranged between 2.82 - 6.70 per 106 cells. All values were statistically not significant and within the 95% control limit of our historical negative control data, except the value obtained at 15.00 μg/mL (MFcorr.: 6.70 per 10^6 cells). This increase is considered as not relevant, since at the next higher tested concentration (17.5 μg/mL) the corrected mutant frequency was lower (2.82 per 10^6 cells) and not statically significant. In the presence of metabolic activation, no relevant increase in the number of mutant colonies was observed in the 1st Experiment. The corrected mutation frequency of the test substance treated groups ranged between 0.27 - 1.98 per 10^6 cells. All values were within the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 7.91 per 10^6 cells) and statistically not significant as compared to the concurrent negative control value (MFcorr.: 3.67 per 10^6 cells). In the 2nd Experiment there was a statistically significant, dose related increase in the mutant frequencies. The values for the corrected mutation frequencies in the test groups 75.0 and 100.0 μg/mL (MFcorr.: 7.69 and 10.71 per 10^6 cells, respectively) were statistically significant compared to the respective vehicle control (MFcorr.: 2.13 per 10^6 cells). They were above the 95% upper control limit (6.84 mutants per 10^6 cells) and at 100.00 μg/mL exceeded the maximum value of our historical negative control data range (MFcorr.: 0.00 – 9.93 per 10^6 cells). In order to address the relevance of the obtained results a third experiment was performed. In the 3rd Experiment with S9 mix the test substance did not cause a biologically relevant increase in the mutant frequencies. The obtained corrected mutation frequencies from the test substance treated cultures ranged between 1.16 - 7.80 per 106 cells. All values were statistically not significant and within the 95% control limit of our historical negative control data. The respective negative control value for the corrected mutation frequency was 4.84 per 10^6 cells. The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 143.01 – 241.36 per 10^6 cells; with S9 mix: MFcorr.: 105.47 – 162.2 per 10^6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 10^6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 10^6 cells).

Conclusions:

In the absence and the presence of metabolic activation, Laromer LR 8889 is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

Experimental completion date: 2017-08-11

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Results of repeat experiments in the presence of S9 were not reproducible, 2 out of 3 experiements did not show a dose response relationship with regard to cytotoxicity or micronuclei induction, results for positive control substances partly within the range of the historical negative control values, used statistical trend test inappropriate for the dataset.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 160005P040
- Expiration date of the lot/batch: Oct., 16th, 2017
- Purity test date: Feb. 15th, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

TK6 cells were supplied by the European Collection of Cell Cultures (ECACC), Salisbury, UK.

Cultures are maintained at Covance Laboratories Ltd. in tissue culture flasks containing HEPES-buffered RPMI 1640 medium with GlutaMAXTM-1 including 10% (v/v) heat inactivated foetal calf serum, 100 Units/mL/100 µg/mL penicillin / streptomycin at 37±1ºC, 5% (v/v) CO2 in air, in a humidified environment. They were subcultured regularly at low density, and before overgrowth occured, to maintain low aberration frequencies. Stocks of cells preserved in liquid nitrogen were reconstituted for each experiment so as to maintain karyotypic stability. The cells are routinely screened for mycoplasma contamination.

The final volume of culture medium in each tube (following completion of treatment) was 5 mL. Cells were maintained at 37±1°C, 5% CO2 in air, in a humidified environment prior to treatment. All cultures were incubated on a slope.

A check on culture cell growth from time of culture establishment to time of treatment (calculation of population doubling) confirmed cultures in the Range-Finder and all three Micronucleus Experiments were in exponential growth at the time of treatment.

Cytokinesis block (if used):

Cyto-B

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Preliminary solubility data indicated that Laromer LR 8889 was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 558.92 mg/mL. The solubility limit in culture medium was in the range of 174.66 to 349.33 µg/mL, as indicated by precipitation at the higher concentration which persisted for at least 28 hours after test article addition. A maximum concentration of 5000 µg/mL was selected for the Range-Finder Experiment, as recommended by OECD test guideline 487 for UVCBs (OECD, 2016). Concentrations for Micronucleus Experiment 1 were selected based on the results of this cytotoxicity Range-Finder Experiment.

The test article solutions were protected from light and used within approximately 2 hours of initial formulation. The following concentration ranges were tested:

Experiment Treatment Concentration Range (µg/ml) Final Concentration Range (µg/mL)
Range-Finder3+27 hour, -/-S-9 1.814 to 500.0 18.14 to 5000
30+0 hour, -S-9 1.814 to 500.0 18.14 to 5000
Experiment 1 3+27 hour, -S-9 0.005 to 2.000 0.050 to 20.00
3+27 hour, +S-9 1.000 to 10.00 10.00 to 100.0
30+0 hour, -S-9 0.005 to 2.000 0.050 to 20.00
Experiment 2 3+27 hour, -S-9 0.200 to 10.00 2.000 to 100.0
3+27 hour, +S-9 1.500 to 12.00 15.00 to 120.0
30+0 hour, -S-9 0.100 to 2.000 1.000 to 20.00
Experiment 3 3+27 hour, +S-9 1.500 to 20.00 15.00 to 200.0

Appropriate concentrations for micronucleus analysis could not be selected from treatments in the absence of S-9 in Micronucleus Experiment 1. Therefore, these treatments were performed again in Micronucleus Experiment 2 as stated in the table above.

Vehicle / solvent:

0.05 ml DMSO were used as vehicle control, due to the lipohilicity of the test substance

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Noscapine (NOS)

Details on test system and experimental conditions:

DURATION
- Exposure duration: 3h (w/ and w/out S9) or 30h (w/out S9 only)
- Expression time (cells in growth medium): 30h (including exposure time)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:
Treatment S-9 Cytotoxicity Range-Finder Micronucleus Experiment
3+27* 30+0 * 3+27* 30+0 *
Vehicle control - 2 2 4 4
+ 2 4
Test article - 1 1 2 2
+ 1 2
Positive controls - 2 2
+ 2
* Hours treatment + hours recovery

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cells were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides labelled with appropriate study details. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes, then washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature prior to analysis.

NUMBER OF CELLS EVALUATED:
2000 (1000 per culture)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The micronucleus had the same staining characteristics and a similar morphology to the main nuclei
- Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus
- Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Replication index (RI)
RI = (number binucleate cells + 2 (number multinucleate cells))/ total number of cells in treated cultures
Relative RI (expressed in terms of percentage): Relative RI (%) = (RI of treated cultures / RI of vehicle controls) x100
Cytotoxicity (%) is expressed as (100 – Relative RI).

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Osmolality and pH measurements on post-treatment incubation medium were taken from the highest three concentrations tested per treatment regime in the Range-Finder Experiment.

Evaluation criteria:

Evaluation Criteria
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:

1.A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2.An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3.A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by- case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account (Thybaud et al., 2007).

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No marked changes in pH of greater than one pH unit was observed at the three concentration assessed (1800, 3000 and 5000 µg/mL) as compared to the concurrent vehicle controls (individual data not reported) in the Range-Finder Experiment.
A marked decrease in osmolality was observed at the highest concentration assessed (5000 µg/mL) as compared to the concurrent vehicle controls. However, no marked changes in osmolality of greater than 50 mOsm/kg was observed at the other two concentrations assessed (1800 and 3000 µg/mL, individual data not reported). As concentrations tested in the Micronucleus Experiments were much lower than even 1800 µg/mL, it was considered the marked decrease at 5000 µg/mL did not affect the outcome of the study.
The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for Micronucleus Experiment 1.

Conclusions:

In cultured human lymphoblastoid TK6 cells, Laromer LR 8889 did not induce micronuclei or induced micronuclei of questionable biological relevance when tested up to cytotoxic concentrations following 3+27 hour treatment or 30+0 hour treatment in the absence of S-9, respectively. In the same test system, following 3+27 hour treatment, none of the three experiments delivered conclusive and reproducible data. A significantly higher amount of micronuclei compared to vehicle control observed in experiment one, was only present in halfway of the concentration range tested and not in the respective high doses. Furthermore the result was not reproducible in experiment two and three. Therefore there's no clear indication for a clastogenic potential of Laromer LR 8889.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MNT in vivo: neg. (BASF 2013, RA to CAS 195008 -76 -5)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

adopted August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 35.6g (SD +/- 1.5g)
- Assigned to test groups randomly: yes
- Housing: single in Makrolon Type II/III cages with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: relative non-toxicity
- Amount administered: 10ml/kg b.w.
- Lot/batch no. (if required): MKBF8603V

Duration of treatment / exposure:

24h and 48h (only highest dose tested)

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
500, 1000, 2000mg/kg b.w.
Basis:
actual ingested

No. of animals per sex per dose:

7 (5 for vehicle and positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral, gavage in sterile water
- Doses / concentrations: 40mg/kg b.w.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: the maximum recommended dose of 2000mg/kg b.w. was tolerated without causing toxic reactions

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Gruenwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.

A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

only clinical signs evaluated

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): not altered
- Statistical evaluation: no significant increase detected

Conclusions:

The test substance is considered to be non-mutagenic in this micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both substances are reaction products of the same alcohol (propylidynetrimethanol, TMP) either ethoxylated or ethoxylated and propoxylated, acrylic acid, and a secondary amine, which differ only in the length of the side chaine (butyl or ethyl). Consequently, the reactive groups as well as the distribution of comparable components. These components contain reaction products of the ethoxylated or ethoxylated and propoxylated TMP with 1-3 acrylic acid groups. These molecules can then react with usually one, but potentially up to three secondary amines. The number of EO or PO groups is comparable for both substances (1-6.5 units).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: CAS 195008-76-5, TMPeo/poTA, reaction products with 1-Butanamine, N-Butyl
Target: CAS 173046-61-2, TMPeoTA, reaction products with Diethylamine

No significant impurities have been detected in either reaction mass.
For the source substance, TMPeo/poTA, reaction products with 1-Butanamine, N-Butyl content ranges from 10-30%, while the major component is TMPeo/poTA (70-90%). Added stabilisers, water, and unreacted acrylic acid are only present at a concentration of 0.3% or less.
The same was observed for the target substance, i.e., the major component is TMPeoTA, followed by TMPeoTA, reaction products with diethylamine. The largest impurity is unreacted diethylamine with a concentration of <0.2%. Further components are stabilisers, water, and unreacted acrylic acid and TMP, all below the concentration of DEA.

3. ANALOGUE APPROACH JUSTIFICATION
Both, target and source substance, did not cause gene mutations in bacteria or mammalian cells. Tests on clastogenicity in vitro were positive for the source substance in three repeat experiments without S9 and 1/3 experiment with S9 at the highest concentration only, and only if cytotoxicity was observed. No higher concentration could be scored due to too severe cytotoxicity. For the target substance, in a study of limited validity, one out of three experiments in the presence of S9 yielded a positive result in combination with cytotoxicty at the upper acceptance level of the current OECD guideline. In summary, both substances cause cytotoxicity with a steep dose response in mammalian cell cultures. At cytotoxicity levels that should already be interpreted with caution, an increase in the number of micronuclei was observed in some cultures. Though this finding was believed to be an in vitro artefact caused by cytotoxicity, an in vivo MNT has been performed with the source substance to unequivocally exclude the relevance of this finding in vivo. No increase in the number of micronuclei was observed in this study.
Because of the very comparable results in in vitro studies with source and target substance, the same result would also be expected for the in vivo situation. Consequently, read across for the in vivo MNT is considered valid, and the result of this study can also be used in the assessment of the target substance.

4. DATA MATRIX
Source Target
Ames neg. neg.
HPRT neg. neg.
MNT in vitro pos. at cytotox. hints for MNT increase
concentrations at cytotoxic concentrations
but study of limited validity
MNT in vivo neg. RA performed

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

only clinical signs evaluated

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

The test substance Laromer LR 8889 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

DOSES: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT) with and without S9

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

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 (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance Laromer LR 8889 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT Test

The substance Laromer LR 8889 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. Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were evaluated for gene mutations:

- without S9 mix

0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00 μg/mL (Exp. 1)

0; 2.50; 5.00; 10.00; 15.00 μg/mL (Exp. 3)

0; 12.50; 15.00; 17.50; 20.00 μg/mL (Exp. 4)

- with S9 mix

0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL (Exp. 1)

0; 9.38; 18.75; 37.50; 75.00; 100.00 μg/mL (Exp. 2)

0; 20.00; 40.00; 80.00; 100.00 μg/m (Exp. 3)

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In this study, in all experimental parts, at least the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in four experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance Laromer LR 8889 is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

MNT in vitro

Laromer LR 8889 was tested in an in vitro micronucleus assay using duplicate cultures of human lymphoblastoid TK6 cells in three independent experiments. Treatment of cells for 3h (expression time of 27h) or 30h in the absence of S-9 were similar to those observed in concurrent vehicle controls and within the historical control data for all concentration analyzed.

Treatment of cells for 3h (27h expression time) in the presence of S-9 in experiment 1 resulted in frequencies of MNBN cells (2.85%, 2.80% and 2.96%), which were significantly higher than those observed in concurrent vehicle controls (0.8%) at 65, 70 and 75 µg/mL, which induced 47, 57 and 67% cytotoxicity, respectively. These highest value exceeds the acceptable cytotoxicity range according to the OECD guideline, while results from cultures leading to cytotoxicity above 55% should be interpreted with extreme caution. Contrary to that, the MNBN cell frequency at 80µg/ml and 85 µg/ml were similar to the vehicle control.Furthermore 80 and 85 µg/ml treatment only induced 21 and 19 % cytotoxicity respectively. Due to the missing dose response, this experiment does not provide clear evidence of clastogenicity according to the interpretation of the registrant.

In the repeat experiment, no increase in micronuclei was observed, but cytotoxicity did not exceed 36%. Thus, this experiment does not meet the requirements of the OECD 487 test guideline (55±5%).

The second repeat experiment resulted in frequencies of MNBN cells (3.1%), which were significantly higher (p≤0.001) than those observed in concurrent vehicle controls (1%) at 60 µg/mL. However, this increase was observed in conjunction with 59% cytotoxicity and according to the respective OECD Guidline, care should be taken in interpreting positive results only found in the higher end of this 55 ± 5% cytotoxicity range. The two lower concentrations analysed (30 and 50 µg/mL), which induced 7 and 18% cytotoxicity, did not induce an increase in micronuclei.

Overall, the positive results obtained were not dose-dependent in exp. 1 and occured only at highly cytotoxic concentrations. In addition, results were not reproducible between trials, the positive control did not always respond properly, and the trend test is unappropriate for these data. No clear conclusions on the clastogenig potential can be drawn from this study, and it was diregarded for the assessment of the registered substance.

Instead, to assess the clastogenic potential, data from the related substance TMPeoTA, reaction products with 1 -Butanamine, N-butyl- CAS 195008 -76 -5) were employed.

Gene mutation in bacteria

In a gene mutation assay in bacteria (Ames) according to OECD471 and GLP (BASF 1992) TMPeoTA, reaction products with 1-Butanamine, N-butyl- did not lead to an increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Concentrations up to 5000µg/plate were tested in Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli 1412 uvrA. S9 fraction was prepared from aroclor induced rat liver.

Gene mutation in mammalian cells

The read across substance was also not mutagenic in an HPRT assay with mammalian cells according to OECD 476 and GLP (BASF 2012). Two independent experiments were carried out, both with and without the addition of phenobarbital and β-naphthoflavone induced rat liver S9 mix. The maximum doses assessed were limited by the cytotoxic properties of the test substance: without S9 mix (4h and 24h exposure) 0.16; 0.31; 0.63; 1.25; 2.50μg/mL with S9 mix (4h exposure) 6.25; 12.5; 25; 50μg/mL (1st experiment); 4.38; 8.75; 17.5; 35; 70μg/mL (2nd experiment).

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.

Micronucleus test in vitro

An in vitro study according to OECD 487 and GLP was performed to assess the potential of TMPeoTA, reaction products with 1-Butanamine, N-butyl- to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). The cells were incubated with the test substance at varying concentrations in DMSO for 4h in the presence or absence of phenobarbital and β-naphthoflavone induced rat liver S9 and harvested after 24h after the start of the experiment. Due to ambigous results and low cell quality (slides not scorable), three independent experiments were performed with the following doses (concentrations used were limited by the high cytotoxicity of the test substance):

1st Experiment

• 4 hours exposure; 24 hours harvest time; without S9 mix: not scorable
• 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 and 100.00 μg/mL

2nd Experiment

• 4 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.13; 0.25; 0.50; 1.00; 2.00 and 4.00 μg/mL
• 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 7.50; 15.00; 30.00; 60.00 and 100.00 μg/mL

3rd Experiment

• 4 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.16; 0.31; 0.63; 1.25; 2.50 and 5.00 μg/mL
• 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 5.00; 10.00; 20.00; 40.00 and 80.00 μg/mL

At least 1 000 cells of 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 the expected increase in the number of cells containing micronuclei. PH and osmolarity were not affected and no precipitation occured at the concentrations used.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) or low cell quality was observed at least at the highest applied test substance concentration in all experimental parts at all test conditions. Without metabolic activation, the test substance induced a slight, but significant increase in 2 independent experiments only in the highest dose scorable due to cytotoxicity. With metabolic activation, only one out of three experiments led to an increase in micronuclei in the highest dose above historical control values. Thus the test substance is considered to have a clastogenic effect in vitro at or close to cytotoxic concentrations without metabolic activation. Results obtained after addition of S9 remain ambiguous.

Micronucleus test in vivo

7 male NMRI mice were orally exposed via gavage to a single dose of 500, 1000, and 2000mg/kg b.w. of TMPeoTA, reaction products with 1-Butanamine, N-butyl- in corn oil. At least 2000 bone marrow polychromatic erythrocytes were evaluated for micronuclei 24 and 48h (only 2000mg/kg b.w.) after exposure. No increase in micronuclei was observed, the ratio of polychromatic to normochromatic erythrocytes was unaltered, and the number of polychromatic erythrocytes was not decreased compared to vehicle control animals. Animals exposed to the positive control of 40mg/kg bw.w cyclophosphamide showed a substantial increase in micronuclei.

Justification for classification or non-classification

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

The registered substance did not cause gene mutation in bacteria or mammalian cells. In an unreliable in vitro micronucleus assay, an increase in micronuclei was observed in one experiment at cytotoxic concentrations and with S9 only. This result could not be repeated in two further experiments. Due to irregularities in the obtained dose responses also for cytotoxicity as well as a faulty statistical analysis, the data are not considered reliable. Conseqently, data for the structurally related CAS 195008 -76 -5 were employed to assess the endpoint of clastogenicity. This substance also did not cause point mutations in bacteria and mammalian cells. In vitro, a clastogenic effects (without S9) was observed at cytotoxic concentrations, which were not confirmed in an in vivo MNT. As a result, both substances are considered as not genotoxic and no classification is required.