Glycerides, C16-18 and C18-unsatd. mono-, di- and tri-, citrates, potassium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative in a reliable in vitro bacterial Reverse Mutation Assay conducted according to OECD 471, employing Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 exposed to the substance at concentrations up to 5000 µg/plate (plate incorporation method) in the presence and absence of mammalian metabolic activation (Aroclor 1254 induced liver S9 mix). Structural analogues of the substance were also found to be negative in an in vitro bacterial gene mutation assay and in an in vitro mammalian cell point mutation assay. The in vitro cytogenetics assay has been waived on the basis that a reliable in vivo study is available on a structural analogue.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to internationally accepted testing guideline, well documented.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98 his mutation: D3052; other markers: rfa uvrB; plasmid: pKM101 / TA 100 his mutation: G46; other markers: rfa uvrB; plasmid: pKM101 / TA 1535 his mutation: G46; other markers: rfa uvrB; / TA 1537 his mutation: C3076; other markers: rfa uvrB.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S9 mix

Test concentrations with justification for top dose:

50 / 160 / 500 / 1600 / 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofurane
- Justification for choice of solvent/vehicle: not mentioned

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and tetrahydrofurane

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Nitrofluorene (2.5 µg/plate) for the strains TA 98; Sodium azide (5.0 µg/plate) for TA 100; Sodium azide (2.5 µg/plate) for TA 1535; 9-Aminoacridine (5.0 µg/plate) for TA 1537.

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2.5 µg/plate) with all strains

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation, performed in two independent tests


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 96 hours


NUMBER OF REPLICATIONS: 3 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: not mentioned


OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures


OTHER: none

Evaluation criteria:

According to Ames a test article which caused no mutagenic effects at a concentration of 5000 µg/plate will be called non-mutagenic.

Statistics:

no statistics performed

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: not performed

COMPARISON WITH HISTORICAL CONTROL DATA: not performed

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Remarks on result:

other: strain/cell type: Salmonella typhimurium

Remarks:

Migrated from field 'Test system'.

Table #1: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 98]			[Strain TA 100]			[Strain TA 1535]
Conc. [unit]	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	16 ± 3	48 ± 10	no	146 ± 16	168 ± 10	no	8 ± 3	12 ± 3	no
50	14 ± 2	39 ± 8	no	166 ± 1	163 ± 8	no	6 ± 1	9 ± 2	no
160	18 ± 1	50 ± 5	no	136 ± 16	168 ± 3	no	7 ± 1	15 ± 4	no
500	15 ± 7	49 ± 8	no	182 ± 13	172 ± 9	no	10 ± 3	12 ± 4	no
1600	18 ± 7	59 ± 9	no	175 ± 11	163 ± 15	no	7 ± 1	12 ± 5	no
5000	21 ± 4	55 ± 4	no	166 ± 18	179 ± 8	no	7 ± 2	18 ± 4	no
Positive control	8.4	7.3	no	499 ± 38	2028 ± 39	no	242 ± 34	215 ± 14	no

*solvent control with THF  

Table #2: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 1537]
Conc. [unit]	- MA	+ MA	Cytotoxic (yes/no)
0*	7 ± 1	13 ± 3	no
50	10 ± 3	16 ± 5	no
160	11 ± 2	15 ± 4	no
500	11 ± 1	21 ± 2	no
1600	15 ± 3	21 ± 2	no
5000	13 ± 1 B,P	20 ± 4	no
Positive control	49 ± 14	154 ± 13	no

*solvent control with THF; B = background lawn reduced; Precipitation 

Table #3: Preincubation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 98]			[Strain TA 100]			[Strain TA 1535]
Conc. [unit]	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	21 ± 5	22 ± 6	no	81 ± 8	84 ± 9	no	9 ± 2	6 ± 3	no
50	24 ± 4	23 ± 6	no	101 ± 16	77 ± 11	no	7 ± 3	9 ± 2	no
160	19 ± 4	17 ± 6	no	88 ± 10	81 ± 11	no	6 ± 3	8 ± 2	no
500	24 ± 4	20 ± 3	no	66 ± 2	82 ± 8	no	4 ± 2	9 ± 2	no
1600	24 ±3	17 ± 4	no	82 ± 4	93 ± 15	no	7 ± 3	9 ± 3	no
5000	23 ± 9	19 ± 3	no	80 ± 10	104 ± 20	no	10 ± 6	11 ± 1	no
Positive control	103 ± 17	1059 ± 330	no	539 ± 17	1033 ± 163	no	327 ± 46	220 ± 49	no

*solvent control with THF  

Table #4: Preincubation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 1537]
Conc. [unit]	- MA	+ MA	Cytoxic (yes/no)
0*	8 ±3	11 ± 4	no
50	7 ± 2	9 ± 3	no
160	6 ± 2	11 ± 3	no
500	10 ± 3	13 ± 1	no
1600	7 ± 1	14 ± 2	no
5000	6 ± 4	11 ± 3	no
Positive control	55 ± 22	143 ± 35	no

*solvent control with THF 

Conclusions:

The test substance is negative in the Bacterial Reverse Mutation Assay with and without metabolic activation.

Executive summary:

In a Bacterial Reverse Mutation Assay according to OECD 471, strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium were exposed to the test substance (CAS 91744-23-9) in solvent tetrahydrofurane at concentrations of 50 / 160 / 500 / 1600 / 5000 µg/plate (plate incorporation) in the presence and absence of mammalian metabolic activation (aroclor 1254 induced liver S9 mix). 

There was no evidence of induced mutant colonies over background.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-06 until 2012-09-19

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. .


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control with THF			-	100.0	100.0	100.0	12.3	1.0	100.0	100.0	100.0	13.8	1.0
Positive control (EMS)	150.0		-	84.1	132.6	113.8	70.9	5.8	80.4	76.7	63.8	114.8	8.3
Test item	4.7		-	97.9	culture was not continued^#				87.8	culture was not continued^#
Test item	9.4		-	84.6	104.9	118.8	13.4	1.1	79.6	106.1	55.7	24.3	1.8
Test item	18.8		-	88.6	142.2	100.7	9.8	0.8	98.8	105.7	31.0	47.0	3.4
Test item	37.5		-	80.4	155.1	139.6	12.3	1.0	83.9	76.0	39.0	25.3	1.8
Test item	75.0	P	-	68.5	70.8	113.2	8.3	0.7	82.0	96.4	42.7	25.1	1.8
Test item	150.0	P	-	80.7	90.3	126.2	7.4	0.6	69.8	80.6	35.6	84.9	6.1
Experiment I / 4 h treatment				culture I					culture II
Solvent control with THF			+	100.0	100.0	100.0	11.9	1.0	100.0	100.0	100.0	10.7	1.0
Positive control (DMBA)	1.1		+	58.3	70.4	70.2	802.4	67.3	85.0	48.2	119.1	274.5	25.5
Test item	4.7		+	81.4	culture was not continued^#				113.4	culture was not continued^#
Test item	9.4		+	77.8	120.4	92.1	28.2	2.4	96.0	60.9	109.9	13.3	1.2
Test item	18.8		+	80.5	105.1	100.7	10.3	0.9	119.3	87.5	92.3	17.5	1.6
Test item	37.5		+	78.5	130.0	100.3	11.5	1.0	119.0	97.9	105.2	8.5	0.8
Test item	75.0	P	+	72.4	100.9	119.0	9.5	0.8	128.4	77.6	109.0	7.4	0.7
Test item	150.0	P	+	72.6	98.2	107.7	6.7	0.6	131.9	76.2	121.7	15.1	1.4
Experiment II / 24 h treatment				culture I					culture II
Solvent control			-	100.0	100.0	100.0	20.4	1.0	100.0	100.0	100.0	9.7	1.0
Positive control (EMS)	150.0		-	108.1	79.6	86.7	381.1	18.7	105.1	126.4	103.5	422.4	43.7
Test item	4.7		-	109.1	culture was not continued^#				106.8	culture was not continued^#
Test item	9.4		-	98.6	108.0	84.6	26.2	1.3	105.6	95.6	117.2	23.1	2.4
Test item	18.8		-	98.8	92.3	92.4	34.1	1.7	109.3	75.7	98.4	20.2	2.1
Test item	37.5	P	-	102.2	93.8	85.5	14.7	0.7	106.1	93.8	99.1	9.9	1.0
Test item	75.0	P	-	97.2	104.0	84.7	24.0	1.2	105.8	144.5	113.3	13.2	1.4
Test item	150.0	P	-	99.0	115.4	79.0	30.3	1.5	103.9	94.1	95.9	2.6	0.3
Experiment II / 4 h treatment
Solvent control with THF			+	100.0	100.0	100.0	35.2	1.0	100.0	100.0	100.0	21.0	1.0
Positive control (DMBA)	1.1		+	103.0	120.7	67.8	732.9	20.8	100.0	105.0	93.9	348.7	16.6
Test item	4.7		+	104.4	culture was not continued^#				101.6	culture was not continued^#
Test item	9.4		+	123.9	109.0	98.6	14.8	0.4	108.7	107.6	95.2	4.5	0.2
Test item	18.8		+	133.2	89.6	93.2	29.6	0.8	112.2	109.0	98.7	10.8	0.5
Test item	37.5		+	94.0	127.6	79.8	26.9	0.8	100.9	93.4	101.3	16.4	0.8
Test item	75.0	P	+	97.8	95.3	74.5	23.4	0.7	105.7	123.3	107.4	15.7	0.7
Test item	150.0	P	+	106.3	104.3	74.7	15.7	0.4	105.3	99.5	94.7	14.2	0.7

^#   culture was not continued since a minimum of only four analysable concentrations is required

P  precipitation observed at the end of treatment

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item (Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read-across to K1 study therefore K2 is the maximum Klimisch value that can be assigned

Justification for type of information:

Please see the read-across report attached in section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. .


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control with THF			-	100.0	100.0	100.0	12.3	1.0	100.0	100.0	100.0	13.8	1.0
Positive control (EMS)	150.0		-	84.1	132.6	113.8	70.9	5.8	80.4	76.7	63.8	114.8	8.3
Test item	4.7		-	97.9	culture was not continued^#				87.8	culture was not continued^#
Test item	9.4		-	84.6	104.9	118.8	13.4	1.1	79.6	106.1	55.7	24.3	1.8
Test item	18.8		-	88.6	142.2	100.7	9.8	0.8	98.8	105.7	31.0	47.0	3.4
Test item	37.5		-	80.4	155.1	139.6	12.3	1.0	83.9	76.0	39.0	25.3	1.8
Test item	75.0	P	-	68.5	70.8	113.2	8.3	0.7	82.0	96.4	42.7	25.1	1.8
Test item	150.0	P	-	80.7	90.3	126.2	7.4	0.6	69.8	80.6	35.6	84.9	6.1
Experiment I / 4 h treatment				culture I					culture II
Solvent control with THF			+	100.0	100.0	100.0	11.9	1.0	100.0	100.0	100.0	10.7	1.0
Positive control (DMBA)	1.1		+	58.3	70.4	70.2	802.4	67.3	85.0	48.2	119.1	274.5	25.5
Test item	4.7		+	81.4	culture was not continued^#				113.4	culture was not continued^#
Test item	9.4		+	77.8	120.4	92.1	28.2	2.4	96.0	60.9	109.9	13.3	1.2
Test item	18.8		+	80.5	105.1	100.7	10.3	0.9	119.3	87.5	92.3	17.5	1.6
Test item	37.5		+	78.5	130.0	100.3	11.5	1.0	119.0	97.9	105.2	8.5	0.8
Test item	75.0	P	+	72.4	100.9	119.0	9.5	0.8	128.4	77.6	109.0	7.4	0.7
Test item	150.0	P	+	72.6	98.2	107.7	6.7	0.6	131.9	76.2	121.7	15.1	1.4
Experiment II / 24 h treatment				culture I					culture II
Solvent control			-	100.0	100.0	100.0	20.4	1.0	100.0	100.0	100.0	9.7	1.0
Positive control (EMS)	150.0		-	108.1	79.6	86.7	381.1	18.7	105.1	126.4	103.5	422.4	43.7
Test item	4.7		-	109.1	culture was not continued^#				106.8	culture was not continued^#
Test item	9.4		-	98.6	108.0	84.6	26.2	1.3	105.6	95.6	117.2	23.1	2.4
Test item	18.8		-	98.8	92.3	92.4	34.1	1.7	109.3	75.7	98.4	20.2	2.1
Test item	37.5	P	-	102.2	93.8	85.5	14.7	0.7	106.1	93.8	99.1	9.9	1.0
Test item	75.0	P	-	97.2	104.0	84.7	24.0	1.2	105.8	144.5	113.3	13.2	1.4
Test item	150.0	P	-	99.0	115.4	79.0	30.3	1.5	103.9	94.1	95.9	2.6	0.3
Experiment II / 4 h treatment
Solvent control with THF			+	100.0	100.0	100.0	35.2	1.0	100.0	100.0	100.0	21.0	1.0
Positive control (DMBA)	1.1		+	103.0	120.7	67.8	732.9	20.8	100.0	105.0	93.9	348.7	16.6
Test item	4.7		+	104.4	culture was not continued^#				101.6	culture was not continued^#
Test item	9.4		+	123.9	109.0	98.6	14.8	0.4	108.7	107.6	95.2	4.5	0.2
Test item	18.8		+	133.2	89.6	93.2	29.6	0.8	112.2	109.0	98.7	10.8	0.5
Test item	37.5		+	94.0	127.6	79.8	26.9	0.8	100.9	93.4	101.3	16.4	0.8
Test item	75.0	P	+	97.8	95.3	74.5	23.4	0.7	105.7	123.3	107.4	15.7	0.7
Test item	150.0	P	+	106.3	104.3	74.7	15.7	0.4	105.3	99.5	94.7	14.2	0.7

^#   culture was not continued since a minimum of only four analysable concentrations is required

P  precipitation observed at the end of treatment

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item (Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

B6C3F1 mice (10/sex/dose) received diets containing 0, 0.62%, 1.25%, 2.5%, 5.0% or 10% castor oil, continuously for 13 weeks. Ten additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters. At days 5 and 21, these animals were anesthetized with carbon dioxide, and blood was collected from the orbital sinus. These animals were killed following the blood collection on day 21.Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. Slides were stained with Hoechst 33258/pyronin Y. At least 2000 PCE and 10000 NCE from each animal were scored for micronuclei. Castor oil was found to be negative for cytogenicity as evidenced by a lack of micronuclei in peripheral blood erythrocytes of B6C3F1 mice exposed in dosed feeding for 13 weeks.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Mice (10/sex/dose) received diets containing 0, 0.62%, 1.25%, 2.5%, 5.0% or 10% castor oil, continuously for 13 weeks. Ten additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters. At days 5 and 21, these animals were anesthetized with carbon dioxide, and blood was collected from the orbital sinus. These animals were killed following the blood collection on day 21.Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. Slides were stained with Hoechst 33258/pyronin Y. At least 2000 PCE and 10000 NCE from each animal were scored for micronuclei.

- Short description of test conditions: Mice were housed individually. Polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets were used; the cages were stored on stainless steel racks equipped with an automatic watering system. Temperature in the animal room was maintained within 68-76°F; relative humidity ranged from 42% to 72%. Incoming air was filtered to remove particulates, and a flow rate was maintained to ensure complete exchange at least 10 times per hour. A controlled light cycle of 12 hours of daylight and 12 hours of darkness was maintained. Control feed or diet formulations of castor oil were available ad libitum; feeders were changed twice per week throughout the study.

- Parameters analysed / observed: Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. Slides were stained with Hoechst 33258/pyronin Y. At least 2000 PCE and 10000 NCE from each animal were scored for micronuclei.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

USP AA grade castor oil was obtained in one lot (#L-5G30-01) from Cas Chemical, Inc. (Bayonne, NJ). Purity and identity analyses were conducted by Midwest Research Institute (MRI) (Kansas City, MO). MRI reports on the analyses performed in support of the castor oil studies are on file at the National Institute of Environmental Health Sciences (Research Triangle Park, NC).

Cumulative data indicated a purity consistent with the USP specifications and the reported composition for castor oil.

The stability of the study material during the toxicology studies was monitored by determination of peroxide content and by high performance liquid chromatography. No deterioration of the castor oil study material was observed over the course of the studies.

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Obtained from Simonsen Laboratories (Gilroy, CA, USA).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mice were housed individually. Polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets were used; the cages were stored on stainless steel racks equipped with an automatic watering system. Temperature in the animal room was maintained within 68-76°F; relative humidity ranged from 42% to 72%. Incoming air was filtered to remove particulates, and a flow rate was maintained to ensure complete exchange at least 10 times per hour. A controlled light cycle of 12 hours of daylight and 12 hours of darkness was maintained. Control feed or diet formulations of castor oil were available ad libitum; feeders were changed twice per week throughout the study.

Route of administration:

oral: feed

Details on exposure:

Ad libitum.

Duration of treatment / exposure:

13 continuous weeks.

Frequency of treatment:

Daily

Dose / conc.:

0 other: %

Dose / conc.:

0.62 other: %

Dose / conc.:

1.25 other: %

Dose / conc.:

2.5 other: %

Dose / conc.:

5 other: %

Dose / conc.:

10 other: %

No. of animals per sex per dose:

10/sex/concentration.

Control animals:

yes, plain diet

Positive control(s):

Male mice treated for 4 weeks with urethane in the drinking water (0.2%). These animals were not part of the 13-week study, but were added as a measure of quality control for the assay.

Tissues and cell types examined:

Complete histopathology examinations were conducted on all mice from the control and 10% dose groups.

Details of tissue and slide preparation:

Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. Slides were stained with Hoechst 33258/pyronin Y. At least 2000 PCE and 10000 NCE from each animal were scored for micronuclei.

Statistics:

Shirley's test was used to assess any significance that were different from control groups by , p <0.05.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Frequency of Micronuclei in Peripheral Blood Erythrocytes of B6C3F1 Mice Exposed to Castor Oil in Dosed Feed for 13 Weeks

Percent in Feed	% Normachromatic erythrocytes with micronuclei	% Polychromatic erythrocytes with micronuclei	Number of mice
Male mice:
0	0.11 ± 0.02	1.20 ± 0.08	10
0.6	0.13 ± 0.02	1.18 ± 0.10	10
1.3	0.11 ± 0.01	1.16 ± 0.09	10
2.5	0.13 ± 0.01	1.24 ± 0.10	10
5.0	0.09 ± 0.02	1.40 ± 0.11	9
10	0.09 ± 0.01	1.21 ± 0.08	10
Female mice
0	0.10 ± 0.01	1.18 ± 0.07	10
0.6	0.09 ± 0.01	1.21 ± 0.10	10
1.3	0.07 ± 0.01	1.11 ± 0.08	9
2.5	0.09 ± 0.02	1.11 ± 0.08	10
5.0	0.09 ± 0.01	1.49 ± 0.18	10
10.0	0.06 ± 0.01	1.00 ± 0.10	10
Urethane
0.2%	1.68 ± 0.25	1.710 ± 0.25	3

Conclusions:

Castor oil was found to be negative for cytogenicity as evidenced by a lack of micronuclei in peripheral blood erythrocytes of B6C3F1 mice exposed in dosed feeding for 13 weeks.

Executive summary:

B6C3F1 mice (10/sex/dose) received diets containing 0, 0.62%, 1.25%, 2.5%, 5.0% or 10% castor oil, continuously for 13 weeks. Ten additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters. At days 5 and 21, these animals were anesthetized with carbon dioxide, and blood was collected from the orbital sinus. These animals were killed following the blood collection on day 21.Smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. Slides were stained with Hoechst 33258/pyronin Y. At least 2000 PCE and 10000 NCE from each animal were scored for micronuclei. Castor oil was found to be negative for cytogenicity as evidenced by a lack of micronuclei in peripheral blood erythrocytes of B6C3F1 mice exposed in dosed feeding for 13 weeks.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the findings of a number of different reliable in vitro and in vivo mutagenicity studies conducted on the substance and structural analogues, classification of the substance is not justified.