4,4'-((4-(phenylamino)naphthalen-1-yl)methylene)bis(N,N-dimethylbenzenamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic in Ames test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from similar substance

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Detailed read across justification is attached in section 13. Source study has reliability 4, due to the lack of data on composition of test substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix, 10 µg in DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9-mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S9-mix

Details on test system and experimental conditions:

Tissue preparation
S9 fraction
5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20 % solution in peanut oil - w/v) per kg bw 5 days before sacrifice.
During this time the animais are housed in Makrolon cages in air-conditioned rooms. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles are available ad libitum.
5 days after administration the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of + 40 °C).
The livers are weighed and washed in an equivalent volume of a 150 mM KCl solution (1 ml ~ 1 g wet liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 × g for 10 minutes at + 40 °C, 5 ml portions of the supernatant (so-called S9 fraction) are quickly deep-frozen in dry ice and stored at - 70 °C to - 80 °C for 2 months at the most.

S9-mix
The S9-mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 3 volumes of S9 fraction are mixed with 7 volumes of S9 supplement (cofactors). This preparation, the so-called S9-mix, is kept an ice until used. The concentrations of the cofactors in the S9-mix are:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 100 mM.
The phosphate buffer is prepared by mixing an NS2HPO4 solution with an NaH2PO4 solution in a ratio of about 4 : 1.

Bacteria
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy to histidine prototrophy is determined. The indicator organisms TA 1535, TA 1537, TA 98 and TA 100 selected by Ames especially far this purpose are derivatives of Salmonella typhimurium. 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 cansiderably 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 bis C 3076 in the case of TA 1537 and the +2 type his 0 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 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.
For testing, deep-frozen (- 70 °C to - 80 °C) bacterial cultures (1 ml in 15 ml glass tubes) are thawed at room temperature, 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37 °C for 16 hours. As a rule, a germ density of above 10^8 bacteria/ml is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

Standard plate test
Test tubes containing 2 ml portions af soft agar which consists af 100 ml agar (0.6% agar 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination af mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S9 mix (in tests with metabolic activation) or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, samples are poured onto Vogel-Banner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
0.1 ml. test solution, 0.1 ml bacterial suspension and 0.5 ml S9 mix are incubated at 37 °C for the duratian of 20 minutes. Subsequently, 2 ml. of soft agar is added and, after mixing, the samples are poured onta the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Titer determination
In general, the titer is determined only in the experiments with S9-mix both without test substance (solvent only) and after adding the two highest amounts of substance.
For this purpose, 0.1 ml of the overnight cultures is diluted to 10^-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining
components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 10^-6)
0.5 ml S9-mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

Checking out the tester strains
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (∆ uvrB); ampicillin resistance (R factor plasmid). Histidine auxotrophy is automatically checked in each experiment via the spontaneous rate.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed depending an the strain, experiment and test conditions in the standard plate test fram about 100 µg - 500 µg/plate onward. In the preincubation study, bacteritoxicity was found without S9-mix from about 4 µg - 20 µg/plate onward or with metabolic activation at doses ≥ 20 µg/plate (TA 1535), ≥ 100 µg/plate (TA 100, TA 98) or ≥ 500 µg/plate (TA 1537).

Solubility
Incomplete solubility of test substance in DMSO from about 500 µg/plate onward.

Remarks on result:

other: standard plate test and preincubation test; with and without S9-mix

Conclusions:

Non mutagenic in Ames test.

Executive summary:

Method

Ames test using TA 1535, TA 100, TA 1537, TA 98 strains in the dose range 0.16 µg - 5000 µg/plate for the standard plate test and 0.16 - 750 µg/plate for the plate incorporation test. Both types of test were conducted with and without metabolic activation by rat liver S9-mix. Positive and solvent controls were used.

Results

Solubility: incomplete solubility of test substance in DMSO from about 500 µg/plate onward.

Toxicity: a bacteriotoxic effect was observed in the standard plate test from about 100 - 500 µg/plate onward and in the preincubation test from about 1 - 20 µg/plate onward without S9-mix or at doses ≥ 20 µg (TA 1535), ≥ 100 µg (TA 100, TA 98) or ≥ 500 µg (TA 1537) with metabolic activation.

Mutagenicity: an increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S9-mix or after the addition of a metabolising system.

Overall, test substance resulted as non mutagenic in the Ames test under these experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from similar substance

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only four strains were tested instead of five as currently required (OECD guideline 471, 1997). Reliablity 4 has been assigned due to the lack of information on composition of test substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

1st: 0, 20, 100, 500, 2500 and 5000 µg/plate
2nd: 0, 4, 20, 100, 500 and 1000 µg/plate
3rd: 0, 10, 30, 80, 250 and 750 µg/plate
4th: 0, 4, 20, 100, 500 and 1000 µg/plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanhracene

Remarks:

all strains, with S9-mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537, without S9-mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

TA 98, without S9-mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitroso-guanidine

Remarks:

TA 100 and TA 1535, without S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate incorporation and preincubation.

BACTERIA
For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures (1 ml in 15 ml giass tubes) are thawed at room temperature. 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/l) and incubated in the shaking water bath at 37 °C for 16 hours.
A germ density equal/greater than 10^8 bacteria/ml is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

STANDARD PLATE TEST
The experimental procedure is based an the method of Ames et al.
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino acid solution (minimal amino acid salution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C and the remaining components are added in the follawing order:
0.1 ml test solution
0.1 ml. bacterial suspension
0.5 ml S9 mix (in tests with metabolic activatian) or 0.5 ml phasphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

PREINCUBATION TEST - Experiment 4th
The experimental pracedure is based an the method described by Yahagi et al. and Matsushima et al.
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S9-mix are incubated at 37 °C for the duratian of 20 minutes. Subsequently, 2 ml af soft agar is added and after mixing, the samples are poured onto the Vogel-Banner agar plates within apprax. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

REPLICATES
- Experiments:
standard plate test: 1st and 2nd experiment performed on all the strains; the 3rd only on TA 98.
preincubation test: 4th experiment performed on all the strain.
- Number of plates: 3 test plates per dose or per control (in each experiment).

TITER DETERMINATION
In general, the titer is determined only in the experiments with S9-mix both without test substance (solvent only) and after adding the two highest amounts of substance.
For this purpose, 0.1 ml of the overnight cultures is diluted to 10^-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C and the remaining components are added in the following order:
0.1 ml solvent (withaut and with test substance)
0.1 ml bacterial suspension (dilution: 10^-6
0.5 ml S9-mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

S9 FRACTION - Preparation
The S9 fraction is prepared according to Ames et al. 5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20 % solution in peanut oil - w/v) per kg body weight 5 days before sacrifice.
During this time the animals are housed in Makrolon cages in air-conditioned rooms. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles are available ad libitum.
5 days after administration the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of +4 °C.
The livers are weighed and washed in an equivalent volume of a 150 mM KCl solution, then cut into small pieces 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 (so-called S9 fraction) are quickly deepfrozen in dry ice and stored at -70 °C to -80 °C for 2 months at the most.

S9 MIX - Preparation
The S9-mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of S9 supplement (cofactors). This preparation, the so-called S9-mix, is kept on ice until used. The concentrations of the cofactors in the S9-mix are:
8 mM of Mg Cl2
33mM of KCl
5mM of glucose-6-phosphate
4mM of NADP
100 mM of phosphate buffer (pH 7.4)
The phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4 : 1.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no increase in the number of his+ revertants

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriotoxic was observed in the standard plate test at doses > 500 µg/plate and in the preincubation test between 100 - 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S9-mix or after the addition of a metabolising system.

TOXICITY
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed depending on the strain and test canditions at dases > 500 µg/plate in the standard plate test.
In the preincubation assay bacteriotoxicity was observed from about 100 - 500 µg/plate onward.

SOLUBILITY
Incomplete solubility af test substance in DMSO from about 500 µg/plate onward.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance is not mutagenic in the Ames test under the experimental conditions assayed.

Executive summary:

Method

The substance was tested for mutagenicity in the Ames test. Strains TA 1535, TA 100, TA 1537, TA 98 were tested at doses in the range of 4 - 5000 µg/plate, in both the standard plate test and preincubation test. Experiments were performed with and without metabolic activation (rat liver S9 mix).

Results

Incomplete solubility of the test substance in DMSO was observed from about 500 µg/plate onward.

A bacteriotoxic effect was observed in the standard plate test at doses > 500 µg/plate and in the preincubation test from about 100 - 500 µg/plate onward.

An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S9 mix or after the addition of a metabolising system.

According to these results, the test substance is not mutagenic in the Ames test under the experimental conditions assayed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on available information, a weight of evidence approach has been followed for the assessment. Structural analogues have been tested for mutagenic potential in bacteria. A detailed description of the read across process is attached in section 13.

Genetic toxicity studies in bacteria (Ames test) were conducted following OECD guideline 471, using four Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Based on current version of this guideline, the use of a fifth strain is required. However, for both substances, no indications of mutagenicity were found in any tested strain, with or without metabolic activation, using standard plate test as well as preincubation test. Therefore, a mutagenic potential could be reasonably excluded.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008) a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- somatic cell mutagenicity tests in vivo, in mammals; or

- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays

Based on negative results in two available studies, relying on a weight of evidence approach, a mutagenic potential to bacteria was excluded for Basic Blue 026 leuco.

Accordingly, it is not classified under the CLP Regulation (EC 1272/2008).