2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 Feb 2021 to 15 Jun 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

EC Number:

620-539-0

Cas Number:

1218787-30-4

Molecular formula:

No molecular formula

IUPAC Name:

2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

Test material form:

solid

Details on test material:

- Chemical names: N,N-bis-2-(hydroxyethyl)-alkyl-(C16-C18)-amine
2,2'-(C16-18 (evennumbered) alkyl imino) diethanol
- CAS number: 1218787-30-4
- EC number: 620-539-0
- slightly yellow solid

Based on the qualitative and quantitative information on the composition, the sample used is representative of the boundary composition shared and agreed by each registrant.

Method

Target gene:

Point mutations which involve substitution, addition or deletion of one or a few DNA base pairs.
All Salmonella: rfa, uvrB genes
E. coli: trp, uvrA

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: The strain is deficient in the DNA nucleotide excision repair system.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: liver, rat
- method of preparation of S9 mix: according to Ames et al.
- final culture medium final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay
using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

Tester strains of TA98 and TA100 were subjected to the test item in the pre-experiment at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.

The test item concentrations to be applied in the main experiments were chosen according to the
results of the pre-experiment. 5000 µg/plate was selected as the
maximum concentration. The concentration range covered two logarithmic decades. Two independent
experiments were performed at the following concentrations:
Experiment I:
3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA1535 and TA1537 (without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100 and E. coli WP2 uvrA (pKM101), TA1535 and TA1537
(with metabolic activation))
Experiment II:
0.316, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(with metabolic activation))

Vehicle / solvent:

Negative controls (A. dest., Eurofins Munich, Lot No. 210219, 210319, 210409) and solvent controls (DMSO, AppliChem Lot No. 0001926479) were treated in the same way as all dose groups.

The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic
activation (-S9).
Phosphate-buffer (0.2 M) contains per litre of purified water:
0.2 M NaH2PO4 x H2O 120 mL
0.2 M Na2HPO4 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min
at 121 °C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
0.2 M phosphate-buffer 9.5 parts
0.15 M KCl solution 0.5 parts

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

2.5 µg/plate for TA98, TA100, TA1535 and TA1537; 10 µg/plate for E. coli WP2 uvrA (pKM101); with metabolic activation for all

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA (pKM101); without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

TA98, TA1537; Without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535; Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Replicatoins: In triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 109 cells/mL
- Test substance dissolved in DMSO and diluted prior to treatment

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: For the pre-incubation method 100 µL of the test item-preparation is pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration/duration of treatment: After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark

MUTAGENICITY:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.


- CRITERIA OF VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020)):
- S9 + S9
STRAIN|- S9 min | - S9 max | + S9 min | + S9 max
TA98 14 61 15 60
TA100 44 143 60 154
TA1535 5 35 4 37
TA1537 3 35 5 41
E. coli WP2 uvrA (pKM101) 110 315 142 381
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation criteria:

MUTAGENICITY:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

- CRITERIA OF VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020)):
- S9 + S9
STRAIN|- S9 min | - S9 max | + S9 min | + S9 max
TA98 14 61 15 60
TA100 44 143 60 154
TA1535 5 35 4 37
TA1537 3 35 5 41
E. coli WP2 uvrA (pKM101) 110 315 142 381
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

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:

TEST-SPECIFIC CONFOUNDING FACTORS
No known confounding factors were present in the test substance.

RANGE-FINDING/SCREENING STUDIES (if applicable):
pre-experiment at the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data
All controls resulted in values within acceptable limits.
For all test methods and criteria for data analysis and interpretation:
- No concentration-response relationship is presented
- Statistical analysis; According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Ames test:
- Signs of toxicity: cyto toxicity observed with increasing concentration both with and without S9 mix
- Mean number of revertant colonies per plate and standard deviation: Both values are presented in the study for all strains used. An additional Mutation Factor has been calculated by dividing the mean revertants of the test item with the mean revertants of the vehicle control. Cytotoxicity may result in decrease in revertants seen with increasing concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)


Positive Historical Control without S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Substance 4-NOPD NaN3 NaN3 4-NOPD MMS
Conc./plate 10 µg 10 µg 10 µg 40 µg 1 µL
Mean 443.2 614.2 826.2 123.3 1702.7
SD 168.7 208.2 362.2 49.4 551.4
Min 89 166 28 33 831
Max 2013 2493 1863 570 3391
RSD [%] 38.1 33.9 43.8 40 32.4
n 985 1034 935 934 89

Negative Historical Control without S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Mean 28.4 95.4 15.6 15.6 181.5
SD 8 16.3 5.9 6.1 42.7
Min 14 44 5 3 110
Max 61 143 35 35 315
RSD [%] 28.2 17 37.8 39.2 23.5
n 976 1023 926 927 87

Positive Historical Control with S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Substance 2-AA 2-AA 2-AA 2-AA 2-AA
Conc./plate 2.5 µg 2.5 µg 2.5 µg 2.5 µg 10 µg
Mean 1305.9 1054.5 185.7 187.3 708.4
SD 792.7 611 130.1 104 153.5
Min 70 119 19 23 424
Max 3609 2920 1856 1476 1138
RSD [%] 60.7 57.9 70.1 55.5 21.7
n 976 1026 929 929 89

Negative Historical Control with S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Mean 29.9 92.7 14.1 16.2 219.4
SD 7.3 14 5.3 6.3 48.6
Min 15 60 4 5 142
Max 60 154 37 41 381
RSD [%] 24.5 15.1 37.5 38.8 22.2
n 974 1020 923 924 87

Any other information on results incl. tables

Precipitation of the test item was observed in all tester strains used in experiment I and II. In experiment I, precipitation was noted at concentrations of 316 µg/plate and higher (with and without metabolic activation). In experiment II, precipitation was observed at concentrations of 1000 µg/plate (without and with metabolic activation). The observed precipitation did not interfere with the scoring and thus it did not impact the results.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, CAS 1218787-30-4 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, CAS 1218787-30-4 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

 

The test item, CAS 1218787-30-4, was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

 

Experiment I:
3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA1535 and TA1537 (without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100 and E. coli WP2 uvrA (pKM101), TA1535 and TA1537
(with metabolic activation))

 

Experiment II:
0.316, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(with metabolic activation))

 

Precipitation of the test item was observed in all tester strains used in experiment I and II. In experiment I, precipitation was noted at concentrations of 316 µg/plate and higher (with and without metabolic activation). In experiment II, precipitation was observed at concentrations of 1000 µg/plate (without and with metabolic activation). The observed precipitation did not interfere with the scoring; thus it did not impact the results.

 

The microbial contamination observed in a few plates in experiment I at concentrations of
5000 µg/plate did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella revertants and thus did not affect the evaluation.

 

Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.

 

In experiment I toxic effects of the test item were observed in tester strains TA98 and TA100 at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). Toxic effects of the test item were also noted in tester strain TA1535 concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA1537, toxic effects of the test item were noted at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate (with metabolic activation). Toxic effects of the test item were also observed in tester strain E. coli WP2 uvrA (pKM101) at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

 

In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations of 3.16 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA100, toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of1000 µg/plate and higher (with metabolic activation). Toxic effects of the test item were also observed in tester strain TA1535 at concentrations of 10.0 µg/plate and higher (without metabolic activation)
and at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA1537, toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation).

 

Toxic effects of the test item were also observed in tester strain E. coli WP2 uvrA (pKM101) at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA1537 at a concentration of 3.16 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn.

 

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. In addition all validity crierion were met.