Castor oil, ester with glycerol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the absence of genetic toxicity data on target substance Castor oil, ester with glycerol an analogue read-across approach was conducted on suitable source substances.

In vitro

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and TA 102, and in E. coli WP2 uvrA.

Chromosome aberration (OECD 473): negative in human lymphocytes and Chinese hamster ovary cells (CHO) with and without metabolic activation.

Gene mutation in mammalian cells (OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation.

In vivo

Micronucleus test (OECD 474): negative in mammalian erythrocytes of B6C3F1 mouse

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix:
4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA98)
156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535, WP2 uvrA)
0.153, 0.305, 0.610, 1.22, 2.44, 4.88, 9.77 μg/ plate (TA1537)

+S9 mix:
9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA100)
156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535, WP2 uvrA, TA98)
9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA1537)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was not soluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF2; 0.01 or 0.1 µg/plate, TA100, TA98, E. coli); sodium azide (SA; 0.5 µg/plate, TA1535); 9-aminoacridine (9AA; 80 µg/plate, TA1537); +S9: 2-Aminoanthracene (2AA; 0.5-10 µg/plate, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation method

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:

Revertant colonies were counted for 3 plates per bacterial strain. The test result was considered positive, if the mean value out of 3 plates was at least twice as high as the negative vehicle control and a dose-dependency was found.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: at 3.13 (TA1537), 78.1 (TA98) and 100 (TA100) µg/plate and above; +S9: at 128 (TA1537) and 156 (TA100) µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was not soluble in water.
- Precipitation: in the dose-finding studies and the main study, precipitation was observed at the end of the exposure period at ≥ 625 and ≥ 1250 µg/plate in the presence and absence of metabolic activation, respectively.

RANGE-FINDING/SCREENING STUDIES:
The highest concentrations analysed in the main study were selected based on the solubility and/or cytotoxicity of the test substance in the cell culture medium. Three dose-finding studies were performed:

[Dose-finding study]
-S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
+S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
[Dose-finding restudy]
-S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
+S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
[Dose-finding additional study]
-S9 mix
6.25, 12.5, 25.0, 50.0, 100, 200 μg/plate (TA100),
3.13, 6.25, 12.5, 25.0, 50.0, 100 μg/plate (TA98),
0.0977, 0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5 μg/plate (TA1537)
+S9 mix:
6.25, 12.5, 25.0, 50.0, 100, 200 μg/plate (TA1537)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance was not cytotoxic to tester strains TA1535 and WP2uvrA at any concentration with and without metabolic activation.
In the dose-finding studies and in the main study, the minimum cytotoxic concentrations determined for the tester strains TA100, TA98 and TA1537 were as follows:

[Dose-finding study]
-S9 mix: 8.19 (TA1537) and 128 (TA100) μg/plate
+S9 mix: 128 (TA1537) and 320 (TA100) μg/plate
[Dose-finding restudy]
-S9 mix: 8.19 (TA1537), 51.2 (TA98) and 128 (TA100) μg/plate
+S9 mix: 128 (TA1537), 320 (TA100) μg/plate
[Dose-finding additional study]
-S9 mix: 3.13 (TA1537) and 100 (TA100, TA98) μg/plate
+S9 mix: 200 (TA1537) μg/plate
[Main study]
-S9 mix: 4.88 (TA1537), 78.1 (TA98) and 156 (TA100) µg/plate
+S9 mix: 156 (TA100) and 313 (TA1537) µg/plate

Table 1. Results of the bacterial reverse mutation test

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2urvA	TA98	TA1537
–	0	129 ± 7	13 ± 2	25 ± 5	21 ± 2	9 ± 1
–	0.153					9 ± 2
–	0.305					7 ± 1
–	0.610					9 ± 1
–	1.22					12 ± 1
–	2.44					7 ± 2
–	4.88	129 ± 5			21 ± 1	10 ± 2*
–	9.77	139 ± 4			18 ± 3	10 ± 3*
–	19.5	125 ± 5			20 ± 4
–	39.1	134 ± 3			17 ± 2
–	78.1	142 ± 6			18 ± 3*
–	156	131 ± 6 *	12 ± 2	16 ± 3	24 ± 6*
–	313		10 ± 3	22 ± 3
–	625		12 ± 2	21 ± 4
–	1250 (+)		10 ± 2	16 ± 2
–	2500 (+)		10 ± 2	16 ± 2
–	5000 (+)		9 ± 1	13 ± 3
Positive controls, –S9	Name	AF2	SA	AF2	AF2	9AA
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	80
	Mean No. of colonies/plate (average of 3 plates ± SD)	886 ± 5	549 ± 8	148 ± 19	689 ± 53	200 ±7
+	0	121 ± 4	13 ± 1	26 ± 5	29 ± 1	25 ± 3
+	9.77	132 ± 7				20 ± 2
+	19.5	130 ± 6				20 ± 2
+	39.1	125 ± 8				21 ± 2
+	78.1	121 ± 4				13 ± 4
+	156	123 ± 4*	14 ± 0	25 ± 4	24 ± 3	12 ± 2
+	313	118 ± 5*	12 ± 2	19 ± 5	25 ± 3	10 ± 2*
+	625 (+)	106 ± 8*	13 ± 2	23 ± 4	26 ± 6
+	1250 (+)		8 ± 2	16 ± 2	19 ± 3
+	2500 (+)		9 ± 1	16 ± 2	19 ± 1
+	5000 (+)		11 ± 1	16 ± 2	12 ± 2
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 3 plates ± SD)	1018 ± 35	336 ± 19	732 ± 65	370 ± 10	142 ± 5

(+): Visible precipitation was observed at the end of exposure period.

*: Growth inhibition was observed

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Oct - 29 Oct 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
Liquid growth medium (broth): Oxoid Nutrient Broth No. 2 (2.5% (w/v) in distilled water
Selective agar plates: Vogel-Bonner medium composed of bacto agar (1.5%), D-glucose (2%), MgSO4 x 7H20 (0.02%), citric acid (0.2%), K2HPO4 (1%), NaNH4HPO4 x 4 H2O (0.35%) in distilled water (percentages correspond to w/v)
The components were autoclaved separately and mixed afterwards.
Top-agar: bacto agar (0.6%), NaCl (0.5%) in distilled water (percentages correspond to w/v), supplemented with 0.05 mM histidine and 0.05 mM biotin before use

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: see remarks

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1. in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

2. preincubation

DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments including 1 plate incorporation and 1 preincubation test


DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of the background lawn of non-revertant bacteria, appearance of micro-colonies and/or reduction in the number of revertant colonies on the test plates comparised to the solvent control plates

Evaluation criteria:

The test was considered to be valid when the following criteria were met:
1. negative and positive control data were consistent and within the range of historical control data (see table 4)
2. positive controls revealed a marked increase over the concurrent solvent controls
3. the evaluation was not restricted by loss of plates (e.g. through contamination)

The test material may be considered mutagenic in this test system if all of the following criteria were met:
1. dose-related increases in the number of revertants at one or more test points
2. reproducible increases in revertants between replicate plates
3. statistically significance in the increases of revertants
4. increases count more than twice the corresponding solvent control values

The test material may be considered non-mutagenic in this test system when no increases in the number of revertants is observed which exceed 1.5 times the solvent control values at any test point. Sporadically ocurring statistically significanct increases in the number of revertants which were not dose-related will usually be considered incidental and not relevant for the evaluation.

Increases between 1.5 and 2 fold compared to the respective solvent controls meeting the other criteria for a positive result were considered to demonstrate weak mutagenicity.

Statistics:

Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA).
In detail, the number of revertant colonies at each treatment test point were compared to the corresponding solvent control values using the Analysis of Variance test. Statistically significant differences were further evaluated via Dunnett´s test to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicates.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA98, TA 100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no but tested up to limit concentrations (a small reduction in the number of revertants was induced in TA 100 (5000 µg: -15% (preincubation assay, +S9) and TA 1535 (160 µg: -28%; 1600 µg: -46%; 5000 µg: -30% (plate incorporation assay, + S9))

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item did not induce toxicity within the conducted preliminary toxicity test as evaluated by large reductions in the number of revertants or poor growth of the background lawn of non-revertant bacteria either in the absence or presence of S9 mix (see table 1).

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent and positive control values were acceptable and compatible with the historical control values (slight increases have been determined in number of revertants for TA 102 in the plate incorporation (-S9: 470 ± 6 vs 409) and preincubation test (+S9: 460 ± 4 vs 432) and for TA 1535 in the plate incorporation assay after treatment with sodium azide (-S9: 1063 ± 83 vs 908), see table 4).

Table 1. Test results of the preliminary toxicity test (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard Deviation)
		Frameshift type
		TA98
–	0	36.0 ± 1.7
–	50	30.3 ± 3.1
–	160	28.7 ± 5.1
–	500	33.0 ± 6.0
–	1600	34.3 ± 2.9
–	5000	34.3 ± 7.0
Positive controls, –S9	Name	2NF
	Concentrations (μg/plate)	1
	Mean No. of colonies/plate (average of 3 ± SD)	499.7 ± 88.3
+	0	48 ± 3
+	50	48.3 ± 5.5
+	160	43.3 ± 2.9
+	500	40.0 ± 6.6
+	1600	40.0 ± 1.0
+	5000	39.7 ± 6.1
Positive controls, +S9	Name	2AA
	Concentrations (μg/plate)	2
	Mean No. of colonies/plate (average of 3 ± SD)	682 ± 26.5
2NF = 2 Nitrofluorene
2AA = 2-Aminoanthracene
No statistical analysis was performed.

 

 

Table 2. Test results of main test 1 (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	210.3 ± 7.5	14.7 ± 1.5	469.7 ± 6.1	30.7 ± 7.6	14.0 ± 2.0
–	50	210.7 ± 7.8	15.7 ± 2.5	474.0 ± 3.0	33.3 ± 7.2	17.7 ± 2.5
–	160	206.0 ± 5.3	15.0 ± 1.0	480.0 ± 6.6	27.7 ± 0.6	18.0 ± 2.0
–	500	205.7 ± 3.8	16.3 ± 2.1	478.7 ± 5.7	27.0 ± 2.6	15.0 ± 2.0
–	1600	208.7 ± 9.3	15.0 ± 3.6	479.0 ± 6.2	26.0 ± 5.0	16.3 ± 2.3
–	5000	208.3 ± 2.9	12.3 ± 1.5	483.7 ± 5.5	32.7 ± 3.1	15.7 ± 2.5
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	100	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1136.7 ± 60.8	1063 ± 82.5	968.3 ± 78.4	749.7 ± 100.5	202.0 ± 7.9
+	0	213.7 ± 3.8	20.3 ± 2.9	405.3 ± 1.5	43.3 ± 2.1	16.7 ± 1.5
+	50	213.3 ± 3.1	19.0 ± 1.0	399.7 ± 6.8	41.0 ± 2.0	20.3 ± 0.6
+	160	215.7 ± 3.8	14.7* ± 1.5	401.3 ± 10.2	44.0 ± 2.0	19.7 ± 0.6
+	500	202.0 ± 5.3	17.0 ± 2.6	405.0 ± 11.5	40.3 ± 2.5	18.7 ± 0.6
+	1600	208.0 ± 11.1	11.0 ± 1.7	406.7 ± 9.7	42.0 ± 3.6	18.7 ± 2.1
+	5000	216.7 ± 4.2	14.3* ± 3.1	405.7 ± 4.7	43.0 ± 1.7	19.0 ± 1.7
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	838.0 ± 71.4	276.0 ± 80.0	1030.3 ± 102.6	597.0 ± 25.6	366.7 ± 34.0
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

Table 3. Test results of main test 2 (preincubation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	214.7 ± 4.0	18.0 ± 3.0	403.7 ± 3.1	36.0 ± 1.7	15.7 ± 1.2
–	50	213.3 ± 6.4	17.3± 0.6	405.0 ± 5.6	30.3 ± 3.1	18.3 ± 0.6
–	160	213.7 ± 6.0	18.3 ± 3.5	407.7 ± 2.3	28.7 ± 5.1	18.3 ± 1.5
–	500	213.0 ± 1.7	21.3 ± 4.2	403.7 ± 1.5	33.0 ± 6.0	19.0 ± 1.0
–	1600	211.0 ± 11.5	21.3 ± 3.2	406.7 ± 4.9	34.3 ± 2.9	18.3 ± 2.1
–	5000	212.3 ± 6.5	17.0 ± 1.7	411.3 ± 13.7	34.3 ± 7.0	17.0 ± 1.0
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	25	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1103.0 ± 25.5	596.3 ± 25.5	1158.0 ± 33.2	499.7 ± 88.3	338.7± 22.7
+	0	203.7 ± 7.5	11.7 ± 1.2	459.7 ± 4.2	48.0 ± 3.0	18.7 ± 1.2
+	50	206.0 ± 12.2	17.0 ± 1.0	460.0 ± 2.6	48.3 ± 5.5	20.0 ± 1.0
+	160	213.7 ± 4.7	16.3 ± 4.2	456.7 ± 4.5	43.3 ± 2.9	19.0 ± 1.0
+	500	207.7 ± 6.8	15.7 ± 2.1	455.3 ± 3.8	40.0 ± 6.6	18.0 ± 1.7
+	1600	188.7 ± 3.1	15.0 ± 4.0	458.3 ± 2.5	40.0 ± 1.0	19.0 ± 1.0
+	5000	173.3** ± 9.5	17.7 ± 2.3	459.7 ± 4.2	39.7 ± 6.1	19.0 ± 1.0
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	574.7 ± 30.2	231.7 ± 18.9	1140.7 ± 98.8	682.0 ± 26.5	210.3 ± 16.0
SA = sodium azide
CH = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

 

Table 4. Historical control values
Strain	Treatment (µg/mL)	S9 mix		Number of revertant colonies/plate				Number of plates
				Mean	Standard Deviation	Minimum	Maximum
TA 102	solvent	-		305	40	249	409	114
	solvent	+		321	52	224	432	117
	CHP (25)	-		1262	346	836	2487	54
	CHP (100)	-		1252	258	903	2000	48
	2AA (4)	+		1076	197	500	1522	105
TA 100	solvent	-		144	33	91	221	162
	solvent	+		143	35	90	234	168
	SA (1)	-		1006	196	578	1460	150
	2AA (2)	+		1007	362	123	2163	156
TA 98	solvent	-		51	16	19	82	159
	solvent	+		61	17	21	97	162
	2NF (1)	-		658	247	216	1189	147
	2AA (2)	+		851	284	411	1622	150
TA 1537	solvent	-		15	5	5	28	111
	solvent	+		19	7	7	43	117
	2NF (1)	-		357	154	121	919	99
	2AA (2)	+		301	202	67	1121	105
TA 1535	solvent	-		20	8	7	38	117
	solvent	+		18	7	7	42	123
	SA (1)	-		541	169	206	908	105
	2AA (2)	+		272	129	110	648	111
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Oct - 18 Apr 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome abberation

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases

Evaluation criteria:

EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:

When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.

Key result

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)	Mitotic index (MI)
	Without S9			With S9
	Individual values		Relative mean MI (%)	Individual values		Relative mean MI (%)
0	4.8	5.3	100	3.8	2.9	100
313	4.8	4.3	90	3.1	3.5	99
625	4.3	4.1	83	2.9	3.5	96
1250	3.4	3.7	70	3.4	2.8	93
2500	3.7	3.3	69	2.1	1.8	58
5000	3.6	2.9	64	0.4	0.3	10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	1 / 0	2 / 1	1 / 0		1 / 0
1250	-	110	1 / 1	0 / 2	2 / 1
2500	-	75	1 / 4	1 / 3	1 / 4
5000	-	70	2 / 3	2 / 1	2 / 4
Daunomycin (0.015 µg/mL)	-	91	17 / 14 **	9 / 5	20 / 14	2 / 1
0	2%	100	0 / 0	1 / 2	0 / 0
625	2%	67	0 / 1	0 / 0	0 / 1
1250	2%	51	0 / 0	2 / 1	0 / 0
2500	2%	42	0 / 1	1 / 1	0 / 1
Cyclophosphamide (6 µg/mL)	2%	20	33a /19b **	16 / 3	36 / 20	15 / 9

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	0 / 3	1 / 5	0 / 3
40	-	84	1 / 4	3 / 6	1 / 4		1 / 0
80	-	59	1 / 2	0 / 2	1 / 2
160	-	49	0 / 2	3 / 1	0 / 2		0 / 1
Daunomycin (0.015 µg/mL)	-	102	13 / 14 **	6 / 9	13 / 13	1 / 1
0	4%	100	2 / 1	3 / 2	1 / 1	1 / 0
625	4%	66	2 / 4	3 / 1	5 / 1		0 / 1
1250	4%	61	1 / 1	3 / 4	1 / 1
2500	4%	33	5 / 1	3 / 0	3 / 4
Cyclophosphamide (6 µg/mL)	4%	77	36 / 33 **	8 / 8	37 / 33	11 / 11

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)	S9 mix	Frequency of metaphases with aberrant chromosomes excluding gaps (%)				Number of cultures
		Mean	SD	Mimimum	Maximum
Negative control	-	0.8	0.8	0	3	32
Daunomycin (0.015 µg/mL)	-	15.1	7.4	7	34	32
Negative control	+	0.7	0.9	0	3	32
Cyclophosphamide (6 µg/mL)	+	43.0	13.6	23	70	32

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL

Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)

- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period

When the above mentioned criteria were not met, the test item was considered as non-mutagenic.

Statistics:

Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid.

Table 1: First Experiment - 4 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.36	1
625	91	116	57	2.04	0.86
1250	108	94	65	2.44	1.03
2500	106	83	36	2.66	1.12
3600	89	76	11	0.30*	0.12
5000	74	83	8	0.04*	0.017
ENU (50)	53	62	45	#
Table 2: First Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.39	1
625	75	81	65	2.59	1.08
1250	92	44	46	3.04	1.27
2500	93	30	9	0.9	0.38
3600	90	5	1	0.2	0.08
5000	84	4	1	0.47	0.2
DMBA (3.3)	24	50	29	#
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	not reported	not reported
313	82	9	2	not reported	not reported
625	73	4	1	not reported	not reported
1250	68	4	1	not reported	not reported
2500	38	4	1	not reported	not reported
3600	31	1	0	not reported	not reported
ENU (50)	53	35	9	not reported	not reported
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2	1
156	83	95	62	1.95	0.98
313	84	101	70	1.84	0.92
625	82	105	65	1.79	0.9
1250	46	98.5	65	1.84	0.92
2500	38	88	37	1.93	0.97
3600	36	4	0	0	0
DMBA (3.3)	28	52	27	37.3	18.65
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.17	1
`2.5	102	100	62	2.16	1
5	100	114	151	2.02	0.93
10	92	95	145	1.97	0.91
20	105	100	60	2.11	0.97
40	82	95	103	2.17	1
80	69	111	97	1.82	0.84
160	25	44	16	2.32	1.07
320	11	31	6	1.94	0.89
ENU (50)	16	31	9	51.8	23.87

 

ENU: N-ethyl-N-nitrosourea

DMBA: 7,12-dimethyl-1,2-benzanthracene

*: statistically significant with p < 0.01

#: could not be calculated as all wells contained mutant colonies

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

lack of cytotoxicity data

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome abberation test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

no data

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

1600, 3000, 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 10 h without S9, 2 h with S9

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

Cells were arrested in first metaphase by addition of colcemid and harvested by mitotic shake off, fixed, and stained in 6% Giemsa.

In the absence of S9, cells were incubated with test compound or solvent for 10 h at 37°C. Cells were then washed and fresh medium containing colcemid was added for an additional 3 h followed by harvest.

In the presence of S9, cells were incubated with test compound or solvent for 2 h at 37°C. Cells were then washed, medium without test compound was added, and incubation was continued for 10 h. Colcemid was added for the last 3 h of incubation before harvest. S9 was from the livers of Aroclor 1254-induced male Sprague Dawley rats.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

.

Table 1:Summary of results of the chromosome aberration study with Castor oil*

 

	S9	Harvest time (h)	Conc. in μg/mL	Cells scored	No of aberrations	Aberrations /Cell	Percent Cells with aberrations
DMSO	-	12		200	2	0.01	1.0
Test item	-	12	1600	200	1	0.01	0.5
Test item	-	12	3000	200	2	0.01	1.0
Test item	-	12	5000	200	1	0.01	0.5
Pos. control (MMC)	-	12	0.0625	200	48	0.24	15.5
DMSO	+	13		200	3	0.02	1.5
Test item	+	13	1600	200	4	0.02	2.0
Test item	+	13	3000	200	4	0.02	2.0
Test item	+	13	5000	200	3	0.02	1.5
Pos. control (CP)	+	13	2.5	200	44	0.22	16.0

MMC = Mitomycin C

CP = Cyclophosphamide

*In the absence of S9, cells were incubated with study compound or solvent for 10 h at 37°C. Cells were then washed and fresh medium containing colcemid was added for an additional 3 h followed by harvest. In the presence of S9, cells were incubated with study compound or solvent for 2 h at 37°C. Cells were then washed, medium without test compound was added, and incubation was continued for 10 h. Colcemid was added for the last 3 h of incubation before harvest.

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

exposure duration 13 weeks

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: 22.6 - 23.0 g (males), 17.2 - 17.7 g (females)
- Housing: rats: 5 per cage, mice individually in Polycarbonate cages lined with heat-treated hardwood chips, covered with polyester filter sheets.
- Diet (Control feed (NIH 07) or diet formulations of castor oil): ad libitum; feeders were changed twice per week throughout the study.
- Water (automatic watering system): ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 42 - 72
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

Vehicle(s)/solvent(s) used: none

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Post exposure period:

none

Dose / conc.:

917 mg/kg bw/day (actual dose received)

Remarks:

0.62% (w/w); Basis: male animals

Dose / conc.:

2 022 mg/kg bw/day (actual dose received)

Remarks:

1.25% (w/w); Basis: male animals

Dose / conc.:

3 800 mg/kg bw/day (actual dose received)

Remarks:

2.50% (w/w); Basis: male animals

Dose / conc.:

7 823 mg/kg bw/day (actual dose received)

Remarks:

5.00% (w/w); Basis: male animals

Dose / conc.:

15 017 mg/kg bw/day (actual dose received)

Remarks:

10.0% (w/w); Basis: male animals

Dose / conc.:

1 153 mg/kg bw/day (actual dose received)

Remarks:

0.62% (w/w); Basis: female animals

Dose / conc.:

2 282 mg/kg bw/day (actual dose received)

Remarks:

1.25% (w/w); Basis: female animals

Dose / conc.:

5 009 mg/kg bw/day (actual dose received)

Remarks:

2.50% (w/w); Basis: female animals

Dose / conc.:

9 627 mg/kg bw/day (actual dose received)

Remarks:

5.00% (w/w); Basis: female animals

Dose / conc.:

16 786 mg/kg bw/day (actual dose received)

Remarks:

10.0% (w/w); Basis: female animals

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control(s):

- Route of administration: Male mice treated for 4 weeks with urethane in the drinking water (0.2%).
- Doses / concentrations: 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:

Tissue: Peripheral erythrocytes
Cells: Peripheral erythrocytes

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No significant elevation in the frequency of micronucleated erythrocytes was observed in either male or female mice administered castor oil in dosed feed.

Table 1: Frequency of Micronuclei in Peripheral Blood Erythrocytes of B6C3F1 Mice Exposed to Castor Oil in Dosed Feed for 13 Weeks

 

sex	% in feed	% Normochromatic erythrocytes with micronuclei (mean values)	% Polychromatic erythrocytes with micronuclei (mean values)	Number of mice
f	0	0.11	1.20	10
f	0.6	0.13	1.18	10
f	1.3	0.11	1.16	10
f	2.5	0.13	1.24	10
f	5.0	0.09	1.40	9
f	10.0	0.09	1.21	10
m	0	0.10	1.18	10
m	0.6	0.09	1.21	10
m	1.3	0.07	1.11	9
m	2.5	0.09	1.11	10
m	5.0	0.09	1.49	10
m	10.0	0.06	1.00	10
m	Urethane 0.2 %	1.68	1.71	3

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Analogue justification

No data on the in vitro genetic toxicity in bacterial and mammalian cells are available for Castor oil, ester with glycerol. The genetic toxicity assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read across approach is provided in the technical dossier (see IUCLID Section 13).

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 111-03-5 (source substance)

In a GLP-study performed according to OECD guideline 471, the 2,3-dihydroxypropyl oleate was investigated in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E. coli WP2 uvrA (WoE, 2005). In three dose finding tests with test concentrations up to 5000 µg/plate, suitable concentrations for treatment of the bacterial strains in the main study were selected. Based on the results obtained in the pre-test regarding solubility and/or cytotoxicity of the test substance, concentrations up to 9.77 (TA 1537), 156 (TA 98 and TA 100) and 5000 µg/plate (TA 1535, WP2 uvrA) were chosen in the absence of metabolic activation, whereas concentrations up to 313 (TA 1537), 625 (TA 100) and 5000 µg/plate (TA 1535, WP2 uvrA and TA 98) were chosen in the presence of metabolic activation. Cytotoxic effects were present at 4.88 (TA 1537), 78.1 (TA 98) and 156 (TA 100) µg/plate without metabolic activation as well as at 156 (TA 100) and 313 (TA 1537) µg/plate with metabolic activation. Precipitation was noted at the end of the exposure period at ≥ 625 and ≥ 1250 µg/plate in the presence and absence of metabolic activation, respectively. No increase in the mean number of revertants per plate was observed in any strain compared to controls. The positive and negative controls included in the assay demonstrated the validity of the assay. Under the conditions of this assay, the test substance was not mutagenic in the presence or absence of metabolic activation.

CAS 736150-63-3 (source substance)

The potential mutagenicity of Glycerides, castor-oil, mono, hydrogenated, acetates was assessed in a bacterial mutation assay according to OECD guideline 471 and in compliance with GLP (WoE, 2004). S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to concentrations of 50 - 5000 µg/plate with or without S9-mix in two independent experiments. No dose-dependent increase in the mean number of revertants per plate was observed in any tester strain up to the maximum exposure concentration. Slight cytotoxicity, as indicated by a small reduction in the number of revertants compared to controls, was induced in TA 100 at 5000 µg/plate (with metabolic activation) in the preincubation assay (experiment I) and in TA 1535 at 160 µg, 1600 µg and 5000 µg/plate (with metabolic activation) in the plate incorporation assay (experiment II). The solvent and positive control values were within the historical control values. Based on the results of this study, the test substance did not induce mutagenicity in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

Conclusion:

Based on the available data (weight of evidence) and following the analogue approach,Castor oil, ester with glycerolis not expected to be mutagenic in bacteria.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 736150-63-3 (source substance)

An in vitro mammalian chromosome aberration test was performed with Glycerides, castor-oil, mono, hydrogenated, acetates in human lymphocytes according to OECD guideline 473 and in compliance with GLP (WoE, 2004). An initial cytotoxicity test was performed with concentrations of 313 - 5000 µg/mL in the presence or absence of metabolic activation (S9-mix) for 3 h. A dose-related toxicity was observed. At the highest concentration (5000 µg/mL), the mitotic index was reduced to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation, respectively. Based on these results, concentrations of 625, 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250, 2500, 3600 and 5000 µg/mL (with 2% S9-mix) were chosen for treatment of cells in the main assay for an exposure period of 3 h. Metaphase analysis was performed at concentrations of 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250 and 2500 µg/mL (with 2% S9-mix). In the second main assay, cells were exposed to concentrations of 313-5000 µg/mL (without S9 mix) or 625-5000 µg/mL (with 4% S9 mix) for either 3 or 20 h, respectively. As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups, this part of the experiment was repeated with lower concentrations ranging from 2.5 to 320 µg/mL. Based on the cytotoxicity data obtained, concentrations of 40, 80 and 160 µg/mL (without S9) and 625, 1250 and 2500 µg/mL (with 4% S9-mix) were used for metaphase analysis. The chromosome analysis of the experiments showed no treatment-related increase in the number of cells with chromosomal aberrations compared with controls. The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values and the positive controls were shown to be valid. Based on the results of this chromosome aberration test, the test substance was not clastogenic in human lymphocytes in the presence or absence of metabolic activation under the experimental conditions chosen.

CAS 8001-79-4 (source substance)

The clastogenic activity of Castor oil was investigated in an in vitro mammalian chromosome aberration test performed similar to OECD guideline 473 and under GLP conditions (WoE, 1992). Chinese hamster Ovary (CHO) cells were treated with the test substance at 1600, 3000 and 5000 µg/mL for 10 h (without metabolic activation) or 2 h (with metabolic activation), respectively. No increase in the number of cells with chromosomal aberrations was observed at any concentration compared to controls. The positive controls showed the expected increase in the number of cells with chromosomal aberrations. Under the conditions of this assay, the test substance was not clastogenic in Chinese hamster Ovary (CHO) cells in the presence and absence of metabolic activation.

Conclusion:

Based on the available data (weight of evidence) and following the analogue approach,Castor oil, ester with glycerolis not expected to be clastogenic in mammalian cells.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 736150-63-3 (source substance)

An in vitro mammalian cell gene mutation assay was performed with glycerides, castor-oil-mono, hydrogenated, acetates according to OECD guideline 476 and under GLP conditions (Key2002). In the first experiment, mutations at the TK locus of mouse lymphoma L5178Y cells were investigated at concentrations of 625, 1250, 2500, 3600 and 5000 µg/mL. The L5178Y cells were exposed to the test material for a period of 3 h in the presence and for 4 h in the absence of metabolic activation (S9-mix), respectively. At 3600 µg/mL, the relative total growth was 1-11% compared with the negative controls. In the second experiment, cells were exposed to a concentration range of 313 to 3600 µg/ for a period of 24 h, without metabolic activation, and to a concentration range of 156-3600 µg/mL for a period of 4 h, with metabolic activation. Since the relative growth without metabolic activation was very low (0-2%) at all test concentrations, the 24-h treatment of cells in the absence of metabolic activation was repeated with concentrations ranging from 2.5-320 µg/mL, which resulted in appropriate levels of cytotoxicity (10-20% relative growth) at 160 µg/mL. In the presence of metabolic activation, the relative total growth was 37 and 0% at 2500 and 3600 µg/mL in the second experiment, respectively. After a 3-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose concentration. The positive controls significantly increased mutant frequency. In conclusion, the test substance did not induce mutations in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

Conclusion:

Based on the available data (weight of evidence) and following the analogue approach,Castor oil, ester with glycerolis not expected to be mutagenic in mammalian cells.

 

Genetic toxicity (cytogenicity) in vivo

CAS 8001-79-4 (source substance)

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with Castor oil in B6C3F1 mice similar to OECD Guideline 474 (Key[GA1] , 1992). Ten animals per group were treated with test substance concentrations of 0, 0.62, 1.25, 2.50, 5.00, 10.0% (w/w) in the diet by oral feeding for 13 weeks (approx. 0, 917, 2022, 3800, 7823, 15017 mg/kg bw/day). Blood smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. At least 2000 PCE and 10000 NCE from each animal were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in Peripheral Blood Erythrocytes of B6C3F1 mice exposed to Castor Oil in doses up to approx. 15000 mg/kg bw/day fed for 13 weeks occurred, whereas the positive control substance (0.2% urethane) significantly increased the number of normochromatic and polychromatic erythrocytes with micronuclei in three control animals. Based on these results, the test substance was not considered to be clastogenic in vivo.

 

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of Castor oil, ester with glycerol in bacterial and mammalian cells. Analogue read-across from 3 source substances was applied from in vitro studies in bacterial cells, from in vitro studies on cytogenicity and from gene mutation in mammalian cells. The results of the available in vitro studies were consistently negative. Based on the available data and following the analogue approach, Castor oil, ester with glycerol is not expected to be mutagenic and/or clastogenic.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Castor oil, ester with glycerol, data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.