Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames test / OECD 471): negative with and without metabolic activation

In vitro chromosome aberration test in Chinese hamster ovary (CA / OECD 473): negative with and without metabolic activation

Gene mutation potential, mammalian cells - (Q)SAR predictions are negative for three types of studies and using 3 different (Q)SAR models (Leadscope Enterprise model, MultiCASE CASE Ultra model and SciMatics SciQSAR model)

- Mutations in the thymidine kinase (TK) locus in mouse lymphoma cells in vitro: negative

- Mutations in the hypoxanthine‐guanine phosphoribosol transferase (HGPRT) locus in Chinese Hamster Ovary (CHO) cells in vitro: negative

- Comet assay in Mouse in vivo: negative

Based on the available information, the substance is not considered a genetic toxicant.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October-November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1977

Qualifier:

according to guideline

Guideline:

other: Standard Operation Procedure of Level Biotechnolocy Inc. (SOP: MP007-04)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

The test article will be dissolved to the stock concentration of 100 mg/mL by ethanol. 5mg/plate of the test item will be used as the highest dose in "dose range finding test". In the groups with metabolic activation, 0.5 mL of metabolic mixture will be used instead of 0.2M phosphate buffer stated in 6.4B of the study report.
According to the result of the dose range finding test, 5 mg/plate was chosen as the highest dose and the other four doses (2.5, 1.25, 0.625, 0.313 mg/plate) were then determined for five Salmonella typhimurium strains.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The vehicle should not be suspended of chemical reaction with the test article and should be compatible with the survival of the bacteria and the S9 activity.

Negative solvent / vehicle controls:

yes

Remarks:

water/ethanol

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene, SA

Details on test system and experimental conditions:

BACTERIAL SYSTEM:
- Salmonella typhimurium strain TA98, TA100, TA102, TA1535, TA1537
- Bacterial source: Moltox Inc., USA
- The genotype confirmation of bacterial strains was performed according to SOP MP007-04 before testing.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate (all groups including positive, negative, vehicle and five dosage testing groups)
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:

A. The doses used were:
without S9 mix:
TA 98: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-nitrofluorene, 1µg/plate)
TA100: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (sodium azide, 1µg/plate)
TA102: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (mitomycin C, 0.2 µg/plate)
TA1535: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (sodium azide, 1µg/plate)
TA1537: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (9-aminoacridine, 50 µg/plate)

without S9 mix:
TA 98: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-nitrofluorene, 1µg/plate)
TA100: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (benzo(a)pyrene, 1µg/plate)
TA102: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-aminoanthracene, 5 µg/plate)
TA1535: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-aminoanthracene, 5 µg/plate)
TA1537: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-aminoanthracene, 5 µg/plate)

B. Plate incorporaton method was employed and performed as following:
(a) the components were added sequentially according to SOP MP007-04: (1) 0.5 mL of 0.2M Phosphate buffer, pH=7.4 (without S9 metabolic activation), (2) 0.05 mL of each testing concentration of test article, negative, vehicle, or positive control solution, 83) 0.1 mL of overnight culture of the Salmonella typhimurium strains (containing aproximately 1-2x10E9 cells/mL), (4) 2 mL of molten top agar (with 0.5 mM histidine/biotin)
(b) the contents of each tube were mixed and poured onto the surface of minimal glucose agar plates.
(c) when the top agar has been solidified, the plates were inverted and placed in a 37+-1ºC incubator for 48-72 hours. The colonies were then counted.

In the groups with metabolic activation, 0.5 ml of S9 metabolic mixture was used instead of 0.5 mL of 0.2M Phosphate buffer stated in B(a)

Evaluation criteria:

1. The raw data of revertant colony values were represented with Mean+-SD.
2. Cell toxicity determination: (A) A cytotoxic effect was concluded when a decrease in revertant colonies over the negative/vehicle control was lower than 0.5-fold, loss of bacterial lawn, or pin colony appeared. (B) Plates would be labeled and excluded from statistics while cytotoxic effect occured.
3. An increase in revertants over the negative control would be as the cut-off between a mutagenic and non-mutagenic response: (A) TA98, TA100 and TA102: more than two-fold increase in revertants over the negative/vehicle control, then the test article would be considered as a potential mutagen. (B) TA1535 and TA1537: more than three-fold increase in revertants over the negative/vehicle control, then the test article would be considered as a potential mutagen.
4. If the test aritlce was considered as a potential mutagen, the raw data would be further analyzed by ANOVA to evaluate the difference between negative/vehicle control group and the test article groups, and p<0.05 indicates the significant difference.
5. If the data showed statistically significant, then to evaluate the dose-related response in the numbers of revertant colonies on the test article group as compared with the vehicle control group. Once dose-related response was confirmed, the test articles would be considered as a mutagen.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Table 1. Result of genotyping of Salmonella typhimurium strains.

Salmonella typhimurium strains	Histidine requirement		UV sensitivity	Crystal Violet sensitivity	Ampicilin resistance	Tetracycline resistance	Spontaneous revertants
	His+ Bio+ Plate	His- Bio+ Plate	UV irradiated	Zone of growth inhibition*	Ampicilin plate	Ampicilin tetracycline pate	without S9
TA98	+	-	-	+	+	-	32.7 +-3.8
TA100	+	-	-	+	+	-	158.0 +-4.6
TA102	+	-	+	+	+	+	463.3 +-15.5
TA1535	+	-	-	+	-	-	15.3 +-5.1
TA1537	+	-	-	+	-	-	12.7 +-2.1

* remark: +: a clear zone of inhibition appeared around the disc

Table 2. Result of the dose range finding test (in TA100).

dose (mg/plate)		number of revertants / plate (mean+-S.D., n=3)
		without S9 mixture
negative control (sterile water)		151.3 +-5.7
positive control (sodium azide, 1µ/plate)		478.0 +-10.6*
vehicle control (ethanol)		161.3 +-4.5
test article	5	156.7 +-3.1
test article	2.5	152.3 +-3.1
test article	1.25	157.7 +-14.0
test article	0.625	172.3 +-8.4
test article	0.313	157.0 +-5.0

* remark: more than two-fold increase in revertants over the negative control

Table 3. Result of Bacterial Reverse Mutation Test (5 strains)

group (mg/plate)		Number of revertants / plate (without S9 activator, Mean +- S.D., n=3)
		TA98	TA100	TA102	TA1535	TA1537
negative control		34.3 +-4.0	153.0 +-13.0	294.7 +-3.1	12.0 +-2.6	11.0 +-2.0
positive control		246.0 +-14.4	486.7 +-31.9	2832.0 +-57.7	498.0 +-31.0	192.3 +-64.5
vehicle control		31.3 +-9.0	138.0 +-13.2	263.3 +-20.0	11.7 +-1.5	12.3 +-4.0
test article	5	28.7 +-1.5	128.7 +-12.7	276.0 +-12.2	10.0 +-1.0	16.0 +-2.6
test article	2.5	37.7 +-2.5	135.3 +-6.5	277.3 +-20.0	11.0 +-2.0	18.3 +-4.5
test article	1.25	37.7 +-2.1	126.7 +-17.0	302.7 +-17.5	10.7 +-3.1	12.7 +-2.5
test article	0.625	40.7 +-7.2	124.3 +-2.5	298.0 +-28.2	9.0 +-1.0	13.3 +-4.2
test article	0.313	43.0 +-4.0	153.7 +-12.9	312.7 +-10.1	9.7 +-2.3	14.7 +-1.5
group (mg/plate)		Number of revertants / plate (with S9 activator, Mean +- S.D., n=3)
negative control		38.0 +-2.6	111.3 +-6.0	323.0 +-43.8^a	13.0 +-1.0	12.0 +-2.6
positive control		806.0 +-24.6*	291.3 +-33.0*	670.7 +-7.0*	213.0 +-67.8*	337.3 +-36.9*
vehicle control		41.7 +-1.2	129.7 +-2.1	283.3 +-34.9	16.0 +-1.0	8.7 +-2.9
test article	5	42.7 +-5.0	130.0 +-13.7	296.0 +-19.1	11.3 +-1.5	12.3 +-4.2
test article	2.5	39.3 +-10.3	110.0 +-5.6	283.0 +-15.6^a	13.7 +-2.5	13.0 +-1.0
test article	1.25	35.7 +-7.6	113.7 +-13.6	288.7 +-37.2	13.0 +-1.0	10.3 +-2.5
test article	0.625	34.0 +-9.5	121.0 +-19.2	273.3 +-20.1	11.3 +-1.2	13.0 +-3.5
test article	0.313	41.0 +-8.2	120.3 +-5.8	301.3 +-14.2	9.7 +-1.5	12.0 +-3.5

Remark:

1. The use of positive control substance for each strain is listed above

2. *: more than two or three-fold increase in revertants over the negative control

3. a: n=2

Conclusions:

According to the numbers of revertant colonies at all the tesing conditions of the five strains used in this study (TA98, TA100, TA102, TA1535, TA1537), the test article did not present genotoxic effect.

Executive summary:

"Bacterial Reverse Mutation Test" was used in this study to evaluate the genotoxicity of the substance in accordance with OECD Guideline for the testing of chemicals #471 (1997): Bacterial reverse mutation test, and the operation was executed according to the standard operation procedure of Level Biotechnology Inc. (SOP: MP007 -04).

Salmonella typhimurium TA100 was chosen and 5 mg/plate of the test article was used as highest dose in "dose range finding test" to determine the testing dose for "Bacterial Reverse Mutation Test". The result of the "dose range finding test" indicated that the test article showed non-cytotoxic and non-mutagenic effects in Salmonella typhimurium TA100.

According to the result of the "dose range finding test", 5 mg/plate was chosen as the highest dose and the other four doses (2.5, 1.25, 0.625, 0.313 mg/plate) were then determined for five Salmonella typhimurium strains, "Bacterial Reverse Mutation Test". Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were used, and plate incorporation method in the presence and absence of S9 metabolic mixture were applied in this test.

Based on the results of this test, no significant increase in the number of revertant colonies was observed. The test article did not present genotoxic effect at al concentrations of testing strains in the condition of both presence and absence of S9 metabolic mixture. In conclusion, the test article was non-genotoxic in the testing system applied in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2011 - December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Lot number L108081
Storage condition: room temperature, desiccation and protected from light.
Fully miscible in ethanol at room temperature.
pH value: neutral

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cell line with epithelial-like morphology and a modal chromosome number 20 ±2.

Metabolic activation:

with and without

Metabolic activation system:

The S9 mixture consisted of S9 fraction (Aroclor 1254-induced; Moltox, BOONE, U.S.A.) and cofactor was used as the metabolic activation system.
The test item was exposed to hte metabolic activation system by preparing it in culture medium supplement with 1% (v/v) S9 mixture.
The S9 mixture was prepared freshly based on the Reagent Formula: M6063-02.

Type and composition of metabolic activation system:
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

The test item was prepared at 500 mg/mL in ethanol and 5 mg/mL were used as the highest dose for cytotoxicity test. The other testing dosages were serial diluents of 5 mg/mL in half interval with ethanol.

Vehicle / solvent:

Vehicle control:
For both With and Without metabolic activation (with and without S9 mixture): Ethanol
Source: Sigma USA
Lot number: 83220
Storage condition: room temperature
Expiration day: May 25, 2014

Test item is fully soluble in ethanol.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

Preparation of CHO-K1 cells for cytotoxiciy and chromosome aberration test
The CHO-K1 cell was propageted and exponentially growing cells were seeded in a 6-well culture plate at 4x10E5 cell/well and 2X10E5 cells/well for short-term and long-term treatment, respectively. Seeded cells were cultured in culture medium for 21 ±3 hours before treatment

Procedure of cytotoxicity study

Exposure concentrations
Testing dosages were designed with half intervals between test points. The treatment under different test schemes was designed as showed in the following table:
Short-term (3 hours): for both tests with and without metabolic activation, the vehicle was ethanol 1%, and the test item dosages were 5, 2.5, 1.25, 0.625, 0.313 mg/mL. The positive control for tests wtihout metabolic activation was Mitomycin C (0.5 µg/mL) and for tests with metabolic activation was Benzo(a)pyrene (25 µg/mL).
Long-term (18 hours): tests without metabolic activation using a vehicle testing dosage of 1 % (ethanol) and test item testing dosages of 5, 2.5, 1.25, 0.625, 0.313 mg/mL. The positive was Mitomycin C (0.5 µg/mL).

Treatment of target cells
Each test was performed in duplicate and incubated at 37 ±1 ºC and 5 ± 1% of CO2 for designated treatment duration. Dosing volume of test item was 10 µg/mL, which make the final concentration of ethanol be 1%.

Cell morphology
The morphology of the cells was observed and recorded by photographing under microscope at 100x magnitude after designated treatment duration.

Cytotoxicity assay
Cytotoxicity is determined by MTT assay. the same valume of DMSO is used as blank control for absorbance reading. THe absorbance is read at 570 nm (OD570) in a microprate spectrophotomerter. The cell viability (%) is calculated according to the following formula:
Cell viability (%) = 100 x (OD570nm (positive or test article) - OD570nm (blank) ) / (OD570 nm (Negative) - OD570nm (blank) )


Procedures of chromosome aberration test
The concentration for the chromosome aberration test is determined based on the result of the citotoxicity assay. At least three analyzable consentrations which cell viabilities are over 50% are performed. The culture medium is used as the negative control. The positive controls used in different treatment are dexribed here:
Short-term (3 hours) without metabolic activation S9.
Treatment: Negative control with no testing dosage
Treatment: Positive: Mitomycin C, 0.5 µg/mL
Treatment: Vehicle, Ethanol, 1%
Treatment with Test item: testing dosages of 5, 2.5, 1.25 mg/mL

Short-term (3hous) with metabolic activation S9
Treatment: Negative control with no testing dosage
Treatment: Positive: Benzo(a)pyrene C, 23 µg/mL
Treatment: Vehicle, Ethanol, 1%
Treatment with Test item: testing dosages of 5, 2.5, 1.25 mg/mL

Long-term (18 hours) without metabolic activation S9.
Treatment: Negative control with no testing dosage
Treatment: Positive: Mitomycin C, 0.5 µg/mL
Treatment: Vehicle, Ethanol, 1%
Treatment with Test item: testing dosages of 5, 2.5, 1.25 mg/mL

Treatment of target cells
Each test was performed in duplicate and incubated at 37 ±1 ºC and 5 ± 1% of CO2 for designated treatment duration. Dosing volume of test item was 10 µg/mL, to make the final concentration of ethanol be 1%.

Chromosome slide preparation
Clocemid is added into the culture medium at a final concentration of 0.1 µg/mL 2 hours before cell harvest. Collected cells were treated with hypotonic solution (0.075M KCl) and fixed with a mixture of methanol/acetic acid (3:1, v/v). Cells were dropped on clean slides and stained with Giemsa solution.

Chromosome aberration examination
a) At least 200 well-spread metaphase cells with a number of centromeres equal to the modal number (20 ± 2) is scored per concentration and control.
b) The structural choromsome aberrations, including chromosome brekage (csb) and exchange (cse), chromatid brekage (ctb) and exchange (cte), and other abnormalities, suh as polyploidy shall be scored and recorded by photographing.
c) Calculate the frequancy of cell with chromosome aberration in each tratment, including every testing dosages of testing article, positive and negative controls.
d) If the number of cells with chromosome aberration in short-term treatment of positive control is significantly increased copared to the negative control (p<0.05), the examination of chromosome aberration in long-term treatment of positive control can be skipped.

Evaluation criteria:

Cytotoxicity
The cell viability is greater than 50% will be considered that the test article is non-cytotoxic to CHO-K1 cells. Any changes in general morphology, vacuolization, detachment, lysis and mambrane integrity will be recorded.

Chromosome aberration
-The statistical analysis shall be performed by validated computer system.
-The statistical evaluation will be analyzed by Poisson distribution. The p value of less than 0.05 (p <0.05) will be considered statistically significant. The number of cells with chromosome aberration in positive control should be significantly increased (p<0.05) comparing with that in negative control.
-If more than two testing dosages show significant increas in number of cells with chromosome aberration, it will be considered that the test article induce structural chromosome aberratin in CHO-K1 cells.
-If only one testing dosage show significant increased in number of cells with chromosome aberration, the Cochran-Armitage tred test (C-A test) will be performed for dose-dependent analysis. Only if there is a dose-dependent tred in number of cells with chromosome aberration, the test article will be considered genotoxic to CHO-K1 cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Result of cytotoxicity
The result of cell viability is summarized in Table 1. In 3-hour treatment in absence of S9 metabolic activation, the cell viability of the test item at 5, 2.5, 1.25, 0.625, 0.313 mg/mL were 106.03 ±0.57%, 97.75 ±2.67%, 94.96 ± 5.38%, 101.94 ± 2.42% and 93.00 ± 1.77% respetively. In 3-hour treatment in presence of S9 metabolic activaiton, the cell viability of the test item at 5, 2.5, 1.25, 0.625, 0.313 mg/mL were 111.08 ±4.32%, 11.08 ± 0.72%, 112.16 ± 2.17%, 106.66 ± 1.45% and 92.84 ±2.51% respectively. In 18.hour treatment in abssence of S9 metabolic activation, the cell viability of the test item at 5, 2.5, 1.25, 0.625, 0.313 mg/mL were 80.94 ±040%, 75.90 ± 3.23%, 76.40 ± 7.15%, 77.60 ± 4.27% and 70.65 ± 1.82% respectively.
Accordingly, the testing dosages which were busjected to chromosome aberratin test were 2.5, 1.25 and 0.625 mg/mL i nboht 3-hour treatment groups (with or without S9 metabolic activation) and 18.hour treatment group.

Result of chromosome aberration
The result of cell viability is summarized in Table 2. The number of cells with chromosome aberration observed in negative control under different test scheme ranged within hte historical data of chromosome aberration test. Compared with negative control, the number of cells with chromosome aberration in vehicle controls were not significantkyt different in all tst schemes (3-hour treatment with or without S9, 18-hour treatment without S9).
The number of cells with chromosome aberration in 200 observed metaphase cells in positive controls was 11 and 12 under 3-hour treatment with or without S9 metabolic activation respectively, which was significantly increased than that in negative controls (both p value <0.05).
The number of cells with chromosome aberration in 200 observed metaphase cells was 2, 3 and 2 at 5, 2.5 and 1.25 mg/mL of test item respectively under 3-hour treatment in the absernce of S9 metabolic activation. The number of cells with chromosome aberration in 200 ob The number of cells with chromosome aberration in 200 observed metphase cells was 5, 0 and 3 at 5, 2.5 and 1.25 mg/mL of test substance respectively under 18-hour treatment in the absence of S9 metabolic activation.
Compared with vehicle contorl, there were no significant difference in the number of cells with chromosome aberration in all testing dosages of the substance excep 5 mg/mL under 3-hour treatment in the presence of S9 metabolic activaiton (p value=0.0404).
Due to only testing dosage showed significatn difference in one test scheme, the Cochran-Armitage trend test was performed for dose-depeendent analysis. The result of C-A test indicated that there was no dose-dependent trend in 3-hour treatment with S9 metabolic activation. In other words, the test item would be considered not genotoxic to CHO-K1 cells.

Test item	Short-term (3-hour treatment)		Long-term (18 -hour treatment)
Dosage (mg/mL)	-S9	+S9	-S9
5
2.5
1.25
0.625
0.313
Negative control (culture medium)
Vehicle control (Ethanol)			85.32 ±1.24
Positive control (Mitomycin C9)	96.87 ±2.27	--	93.32 ±1.26
Positive control Benzo(a)pyerene	--	64.49 ± 1.51	--

 

 

Conclusions:

The genotoxic potential of the substance was evaluated in a chromosome aberration test in Chinese hamster ovary (CHO-K1) cells. The test was performed in accordance with OECD Guideline 473: In vitro Mammalian Chromosome Aberration Test. The results showed the test substance was not genotoxic to CHO-K1 cells under the testing conditions.

Executive summary:

The genotoxic potential of the substance was evaluated in a chromosome aberration test  in Chinese hamster ovary (CHO-K1) cells. The test was performed in accordance with OECD Guideline 473: In vitro Mammalian Chromosome Aberration Test.

The highest testing dosage of the substance in the citotoxicity study was 5 mg/mL The other testing dosages were diluted from 5 mg/mL with a dilution factor of 2. Five dosages of the test item ( 5, 2.5, 1.25, 0.625, 0.313 mg/mL) were tested for citotoxicity using MTT assay under three test schemes: 3 -hour treatment with S9 or without metabolic activation and 18 -hour treatment without S9 metabolic activation.

The testing dosages with over 50% cell viability, which were subjected to chromosome aberration test were 5, 2.5 and 1.25 mg/mL in both 3 -hour treatment groups (with or without S9 metabolic activation) and 18 -hour treatment group (without S9 metabolic activation). The result of chromosome aberration and Cochran-Armitage trend test demonstrated that the test item would not be considered positive in causing structural chromosome aberration to CHO-K1 cells.

In conclusion, the substance was not genotoxic to CHO-K1 cells under the testing conditions employed.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

QSAR prediction

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Danish (Q)SAR database / (Q)SAR Models, Division of Diet, Disease Prevention and Toxicology, National Food Institute, Technical University of Denmark, https://qsarmodels.food.dtu.dk.

Leadscope Predictive Data Miner, a component of Leadscope Enterprise version 3.1.1‐10.
MultiCASE CASE Ultra 1.4.6.6 64‐bit.
SciQSAR version 3.1.00.

2. MODEL (incl. version number)
Three types of studies have been considered to assess the potential for mutations in mammalian cells of the substance Dipenaertythritol hexaesters with 3,5,5,-trimethylhexanoic and n-heptanoic acids:
- Mutations in the thymidine kinase (TK) locus in mouse lymphoma cells in vitro
- Mutations in the hypoxanthine‐guanine phosphoribosol transferase (HGPRT) locus in Chinese Hamster Ovary (CHO) cells in vitro
- Comet assay in Mouse in vivo

For each one, predictions with three (Q)SAR models were performed:
- Leadscope Enterprise model
- MultiCASE CASE Ultra model
- SciMatics SciQSAR model

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
DPE777777 (constituent #1):
O=C(CCCCCC)OCC(COC(=O)CCCCCC)(COC(=O)CCCCCC)COCC(COC(=O)CCCCCC)(COC(=O)CCCCCC)COC(=O)CCCCCC

DPE777779 (constituent #2):
O=C(CCCCCC)OCC(COC(=O)CCCCCC)(COC(=O)CC(C)CC(C)(C)C)COCC(COC(=O)CCCCCC)(COC(=O)CCCCCC)COC(=O)CCCCCC

DPE777799 (constituent #3):
O=C(CCCCCC)OCC(COC(=O)CCCCCC)(COC(=O)CC(C)CC(C)(C)C)COCC(COC(=O)CCCCCC)(COC(=O)CC(C)CC(C)(C)C)COC(=O)CCCCCC

DPE777999 (constituen #4):
O=C(CCCCCC)OCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COCC(COC(=O)CCCCCC)(COC(=O)CC(C)CC(C)(C)C)COC(=O)CCCCCC

DPE779999 (constituent #5):
O=C(CCCCCC)OCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COC(=O)CCCCCC

DPE799999 (constituent #6):
O=C(CCCCCC)OCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COC(=O)CC(C)CC(C)(C)C

DPE999999 (constituent #7):
O=C(CC(C)CC(C)(C)C)OCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COCC(COC(=O)CC(C)CC(C)(C)C)(COC(=O)CC(C)CC(C)(C)C)COC(=O)CC(C)CC(C)(C)(C)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF for each model.

5. APPLICABILITY DOMAIN
The tool checks and indicates in the outcome report if the substance falls within the applicability domain of the corresponding model. Further details on the definition of the applicability domain and the calculations to determine whether the substance falls within it can be found in the corresponding QMRF attached in this section of the IUCLID dossier.
The results for the 7 constituents indicate that the substance falls within the applicability domain of each one of the applied models.

6. ADEQUACY OF THE RESULT
The applied models are valid and recognized (Q)SAR models and the substance falls within the applicability domains, therefore the predictions are considered reliable and adequate for the assessment of the potential for mutagenicity in mammalian cells.

Principles of method if other than guideline:

Danish (Q)SAR database / (Q)SAR Models, Division of Diet, Disease Prevention and Toxicology, National Food Institute, Technical University of Denmark, https://qsarmodels.food.dtu.dk.

Species / strain:

other: not applicable

Metabolic activation:

not applicable

Genotoxicity:

other: prediction for mutagenicity: no alert found

Cytotoxicity / choice of top concentrations:

other: not applicable

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The test substance is not predicted to have mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of Dipentaerythritol hexaesters of 3,5,5 -trimethylhexanoic and n-heptanoic acids show the substance does not meet the classification criteria for genetic toxicity according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.