Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 October 2015 - 25 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine

EC Number:

810-543-2

Cas Number:

1618093-67-6

Molecular formula:

not applicable

IUPAC Name:

Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine

Test material form:

solid: flakes

Details on test material:

Amidoamine 2 (UVCB based on hydrogenated tallow not hydrogenated palm oil):
- Name of test material (as cited in study report): FCM (DETA)M C16-18 TLW hydrogenated (Amidoamine 2 (UVCB based on hydrogenated tallow not hydrogenated palm oil))
- Substance type: Amidoamine
- Chemical name (old): Amides, from diethylenetriamine and hydrogenated tallow
- Chemical name (new): Triglycerides, C16-18 (even-numbered), reaction products with diethylenetriamine
- CAS 68920-82-1
- Physical state: pale yellowish solid at 20 °C
- Batch No.: PU22340011
- Expiry date of batch: 21 August 2014
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions

Specific details on test material used for the study:

Test item without emulsifier was investigated.

Source substance:

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
Former CAS No.: 1618093-67-6
New chemical name: Triglycerides, C16-18 (even-numbered), reaction products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target substance:

Amidoamine 2 (UVCB, low nitrogen):
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
CAS No.: 1618093-67-6
New chemical name: Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: PU50070067
Purity: 100 % (UVCB)
Storage condition of test material: Room temperature, protected from light
Stability: stable under test conditions

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Preliminary Toxicity Test: male, aged 35 years
Main Experiment: male, aged 29 years

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Based on the precipitate observations in the solubility test, the maximum dose level selected for the Preliminary Toxicity Test was 320 µg/mL. Since the OECD 473 guideline requires only one precipitating dose level to be tested, irrespective of toxicity it was decided to reduce the maximum dose for the Preliminary Toxicity Test to a level where the lowest precipitating dose level could more easily be identified and exclude the majority of dose levels where precipitate was seen.

The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable.

Vehicle / solvent:

Vehicle and positive controls were used in parallel with the test item.
The vehicle control used was Minimal Essential Medium (Sigma, Batch No. RNBD4261).

Controls

Untreated negative controls:

yes

Remarks:

The vehicle control used was Minimal Essential Medium.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1. The number of cells with structural chromosome aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.

3. There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is outside the range of the laboratory historical control data.

2. At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.

3. The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day,
orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames
test.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Any other information on results incl. tables

Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)

Treatment Group	Replicate	Mitotic Index (%)	Number of Cells Scored	Total Number of Aberrations		Frequency of Aberrant Cells (%)
				(+ Gaps)	(- Gaps)	(+ Gaps)	(- Gaps)
Vehicle Control (MEM)	A	3.40	150	2	1	2	1
	B	2.90	150	0	0	0	0
	Total	(100)	300	2	1	2 (0.7)	1 (0.3)
10 µg/ml	A	2.20	150	0	0	0	0
	B	2.30	150	0	0	0	0
	Total	(71)	300	0	0	0 (0.0)	0 (0.0)
20 µg/ml	A	3.40	150	2	2	2	2
	B	2.25	150	3	3	3	3
	Total	(90)	300	5	5	5 (1.7)	5 (1.7)
40 µg/ml	A	2.70	150	2	1	2	1
	B	1.45	150	1	1	1	1
	Total	(66)	300	3	2	3 (1.0)	2 (0.7)
80 µg/ml	A	1.50	150	1	1	1	1
	B	5.00	150	1	1	1	1
	Total	(103)	300	2	2	2 (0.7)	2 (0.7)
Mitomycin C 0.2 µg/ml	A	1.85	35*	21	18	18	15
	B	1.65	150	20	20	12	12
	Total	(56)	185	41	38	30 (16.2)	27 (14.6)**

* Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

** P < 0.001

Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (S9)

Treatment Group	Replicate	Mitotic Index (%)	Number of Cells Scored	Total Number of Aberrations		Frequency of Aberrant Cells (%)
				(+ Gaps)	(- Gaps)	(+ Gaps)	(- Gaps)
Vehicle Control (MEM)	A	5.45	150	1	1	1	1
	B	4.75	150	1	0	1	0
	Total	(100)	300	2	1	2 (0.7)	1 (0.3)
10 µg/ml	A	2.40	150	1	1	1	1
	B	3.00	150	1	1	1	1
	Total	(53)	300	2	2	2 (0.7)	2 (0.7)
20 µg/ml	A	4.60	150	1	1	1	1
	B	2.10	150	2	1	2	1
	Total	(66)	300	3	2	3 (1.0)	2 (0.7)
40 µg/ml	A	4.95	150	2	2	2	2
	B	2.75	150	0	0	0	0
	Total	(75)	300	2	2	2 (0 .7)	2 (0.7)
80 µg/ml	A	3.25	150	1	0	1	0
	B	5.35	150	1	0	1	0
	Total	(84)	300	2	0	2 (0.7)	0 (0.0)
Cyclophosphamide 2.0 µg/ml	A	3.75	67*	16	16	16	15
	B	0.80	69*	15	15	15	15
	Total	(45)	136	31	31	31 (22.1)	30 (22.1)**

* Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

** P < 0.001

Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure Without Metabolic Activation (S9)

Treatment Group	Replicate	Mitotic Index (%)	Number of Cells Scored	Total Number of Aberrations		Frequency of Aberrant Cells (%)
				(+ Gaps)	(- Gaps)	(+ Gaps)	(- Gaps)
Vehicle Control (MEM)	A	8.00	150	0	0	0	0
	B	7.40	150	0	0	0	0
	Total	(100)	300	0	0	0  (0.0)	0 (0.0)
10 µg/ml	A	4.40	150	4	2	4	2
	B	3.95	150	0	0	0	0
	Total	(100)	300	4	2	4 (1.3)	2 (0.7)
20 µg/ml	A	6.80	150	2	0	2	0
	B	3.60	150	1	1	1	1
	Total	(68)	300	3	1	3 (1.0)	1 (0.3)
40 µg/ml	A	6.70	150	0	0	0	0
	B	4.15	150	0	0	0	0
	Total	(70)	300	0	0	0 (0.0)	0 (0.0)
80 µg/ml	A	7.60	150	0	0	0	0
	B	6.25	150	0	0	0	0
	Total	(90)	300	0	0	0 (0.0)	0 (0.0)
Mitomycin C 0.2 µg/ml	A	3.25	82*	17	16	15	15
	B	2.45	150	16	14	15	13
	Total	(37)	232	33	30	30 (12.9)	28 (12.1)**

* Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

** P < 0.001

Frequency of polyploid cells was 0% in treatment groups ofthe test item.

Historical Aberration Ranges for Vehicle Control Cultures

1. 4(20)-hour exposure without S9

% cells with aberrations (-gaps): 0.48 (mean)

Standard Deviation: 0.56

% cells with polyploids: 0.05 (mean)

Standard Deviation: 0.17

2. 4(20)-hour exposure with S9 (1%)

% cells with aberrations (-gaps): 0.40 (mean)

Standard Deviation: 0.48

% cells with polyploids: 0.02 (mean)

Standard Deviation: 0.11

3. 24-hour exposure without S9

% cells with aberrations (-gaps): 0.44 (mean)

Standard Deviation: 0.52

% cells with polyploids: 0.07 (mean)

Standard Deviation: 0.22

4. 4(20)-hour exposure with S9 (2%)

% cells with aberrations (-gaps): 0.53 (mean)

Standard Deviation: 0.56

% cells with polyploids: 0.09 (mean)

Standard Deviation: 0.24

Historical Aberration Range for Positive Control Cultures

1. 4(20)-hour exposure without S9 (Mitomycin C)

% cells with aberrations (-gaps): 39.86 (mean)

Standard Deviation: 14.04

% cells with polyploids: 0.02 (mean)

Standard Deviation: 0.13

2. 4(20)-hour exposure with S9 (1%) (Cyclophosphamide)

% cells with aberrations (-gaps): 30.66 (mean)

Standard Deviation: 9.65

% cells with polyploids: 0.02 (mean)

Standard Deviation: 0.12

3. 24-hour exposure without S9 (Mitomycin C)

% cells with aberrations (-gaps): 39.09 (mean)

Standard Deviation: 14.57

% cells with polyploids: 0.02 (mean)

Standard Deviation: 0.013

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The test item was assayed in an in vitro chromosome aberration test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix).

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure.

The test item was completely dissolved in Minimal Essential Medium. Minimal Essential Medium served as the vehicle control. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 1.25 to 320 µg/mL medium were employed.

The maximum dose was reduced to 320 µg/mL based on the precipitate observations in the solubility test and the requirement to test the lowest precipitating dose level.

Hence, 80.0 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the absence of S9, mitomycin C (MMC) was used as positive control at 0.2 and 0.05 µg/mL for 4(20)-hour cultures and 24-hour continuous exposure cultures, respectively. It was dissolved in Minimal Essential Medium.

In the presence of S9, cyclophosphamide (CP) was used as positive control at 2 µg/mL. It was dissolved in dimethyl sulphoxide.

 

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells

with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item demonstrated no-marked toxicity in the preliminary toxicity test and the maximum mdose level selected for the main test was based on the lowest precipitating dose level. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose

level.