Nonylbenzoate, branched and linear

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471): 50 - 5000 µg/plate Benzoesäure isononylester - negative
CA in vitro (OECD 473): not clastogenic up to 2000 µg/mL
MLA in vitro (OECD TG 490): 0.0195 -5 μL/mL not mutagenic; read-across from C12-15 alkyl benzoates 

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

Study period:

19. Jun. 2002 - 09. Jul. 2002

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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His -

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital induced rat liver S9

Test concentrations with justification for top dose:

50 - 5000 µg/plate for plate incorporation test
50 - 1000 µg/plate for pre-incubation test

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Five dose levels of BENZOESÄURE-ISONONYLESTER in the absence and in the presence 01 metabolic activation were spaced at
half-log intervals. The test #AM-02/09.2 was performed with a concentration range based on the results 01 test #AM-02/09.1. In test #AM-02/09.1 the plate incorporation method, and in test #AM-02/09.2 the preincubation method was used.

Plate incorporation test
In a sterile tube,
- 0.1 ml of the appropriately diluted test material (or 0.1 ml of the solvent)
(or 0.1 ml [0.05 ml for TA 1535] of the strain specific positive control item)
(or 0.05 ml of the positive control items for proving metabolie activation)
- 0.5 ml phosphate buffer
(or 0.5 ml 89 mix in the experiment with metabolie activation)
- 2 ml of molten trace histidine supplemented top agar at approx. 45"C
- 0.1 ml of the bacterial overnight culture
were mixed. Mixing was done in triplicate, for each bacterial strain and for each concentration of the test material. The mixture was then poured onto the surface of minimal agar plates. These plates were incubated at 37 °C for 72 hours and then the number of revertant colonies was counted.

Preincubation test
In a sterile tube, a 0.1 ml aliquot of each one of the bacterial overnight cultures was mixed with a 0.5 ml volume of 89 mix (for tests with metabolie activation) or phosphate-buffer (for tests without metabolie activation). Then either 50 µl of the positive controls 2-Aminofluorene
and 2-Aminoanthracene (+89), 50 µl of the solvent, or 50 µl of the test item solution were added. The tubes were incubated at 30°C for 30 min with gentle agitation. At the end of the incubation period, 2 ml of molten trace histidine supplemented top agar was added to each tube, mixed briefly and poured onto minimal agar plates. These plates were incubated at 37 °C for 72 hours and then the nurnber of revertant colonies was counted.

Evaluation criteria:

For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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:

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

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:

valid

Positive controls validity:

valid

Additional information on results:

With two exceptions all five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolie activation by S9 mix. The strain TA 1537 showed a limited response to the presence of the positive control item 2-Aminoanthracene in case of the plate incorporation test with metabolic activation (#AM-02/09.1). But the parallel tested additional positive control item 2-Aminolluorene showed
enough activity. The strain TA 102 also showed a limited response to the positive control item 2-Aminoanthracene (tests #AM-02l09.1 and #AM-02/09.2 with metabolie aetivation), but 2-Aminofluorene showed enough activity.

Plate incorporation test # AM-02/09.1

	µg/ plate	TA 98		TA 100		TA 102		TA 1535		TA 1537
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Water (untreated)		1.2	1.4	1.0	1.1	1.0	1.0	0.9	1.4	1.0	0.8
DMSO		1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Test item	50	1.0	1.3	1.0	1.1	1.1	1.1	0.8	1.1	0.9	0.8
Test item	160	1.5	1.3	1.0	1.1	1.1	1.0	0.6	1.5	1.1	0.7
Test item	500	0.9	1.3	1.0	1.1	1.1	1.1	0.7	1.2	1.1	0.6
Test item	1600	1.1	1.3	1.0	1.3	1.1	0.9	0.8	0.9	0.9	0.9
Test item	5000	1.4	1.6	1.1	1.0	1.1	1.1	0.6	1.1	1.0	1.0
2-Nitrofluorene	2.5	2.5
Sodium azide	5.0			7.6				15.8
Sodium azide	2.5
Mitomycin C	2.5					3.5
9-Aminoacridine	40.0									2.1
2-Aminofluorene	100		102.2		17.6		2.0		2.0		2.5
2-Aminoanthracene	2.5		7.8		3.3		1.1		3.2		1.3

Preincubation test # AM-02/09.2

	µg/ plate	TA 98		TA 100		TA 102		TA 1535		TA 1537
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Water (untreated)		1.1	1.0	1.0	1.1	1.0	1.1	1.3	0.8	1.0	1.1
DMSO		1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Test item	50	1.1	0.7	1.0	0.9	1.0	1.0	1.1	0.7	1.2	1.5
Test item	160	1.0	0.7	1.0	1.0	1.0	1.0	0.8	0.9	1.1	1.1
Test item	500	0.9	1.0	0.9	0.9	1.0	1.0	1.4	0.9	1.2	1.5
Test item	750	1.2	0.9	1.0	0.9	1.0	1.0	0.9	0.7	1.0	1.1
Test item	1000	1.1	1.1	1.0	1.0	1.2	1.0	0.7	0.9	1.1	1.5
2-Nitrofluorene	2.5	3.9
Sodium azide	5.0			7.2
Sodium azide	2.5							38.1
Mitomycin C	2.5					3.5
9-Aminoacridine	40.0									3.1
2-Aminofluorene	100		60.2		16.9		2.1		2.6		4.2
2-Aminoanthracene	2.5		3.3		2.3		1.1		4.2		2.1

Conclusions:

BENZOESÄURE-ISONONYLESTER did not induce a mutagenic effect in S. typhimurium. It is therefore not considered to be a bacterial mutagen.

Executive summary:

BENZOESÄURE-ISONONYLESTER was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were treated with the test item by the Ames test plate incorporation (test #AM-02/09.1) as well as by the preincubation method (test #AM-02l09.2). Dose levels covering the range of 50 to 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Phenobarbital/-ß-Naphthoflavone co-induced rat liver S9 mix) were employed.

A reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 102, TA 1535 or TA 1537 was not observed with and without metabolic activation. It is therefore concluded, that Benzoesäure-isononylester is not a bacterial mutagen.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substance have similar toxicological properties, due to the following facts:

The target and source substances are all alkyl esters of benzoic acid, with branched or linear alkyl chains. There are no additional functionalities present that would cause the target substance to behave differently than the source substance.

The target and source substances both contain an ester functionality that is known to be susceptible to hydrolysis, resulting in benzoic acid and the corresponding (branched or linear) fatty alcohol.

Both substances are manufactured from a common (benzoic acid) and a similar (fatty alcohol with different C-chain length) compound in a comparable manufacturing process and, consequently, share common by-products and impurities. Likely impurities include unreacted fatty alcohols (also the known breakdown products of the benzoate esters), as well as other branched and linear isomers.

Based on the available experimental data, the read-across strategy is supported by close structural analogy as well as similar results in other relevant genotoxicity studies.

Overall, read-across from the in vitro gene mutation study in mammalian cells conducted with the source substance C12-15 alkyl benzoates is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance Isononyl benzoate, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively and quantitatively the same type of effects - of the read-across assessment framework i.e. genotoxicity properties of the target substance Isononyl benzoate are predicted to be similar to those of the source substance C12-15 alkyl benzoates.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
please refer to attachment

3. ANALOGUE APPROACH JUSTIFICATION
please refer to attachment

4. DATA MATRIX
please refer to attachment

Reason / purpose for cross-reference:

read-across source

Metabolic activation:

with and without

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Conclusions:

negative based on read-across

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29. Jan. 2003 - 07. May 2003

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

other: Chinese hamster lung cell line CHL

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's Minimal Essential Medium (MEM) with HEPES buffer and Earle's Salts, supplemented "in-house" with 10% foetal bovine serum and antibiotics
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone / ß-naphthoflavone co-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1:
- S9: 0, 2, 4, 8, 12, 16, 32 µg/mL
+ S9: 0, 62.5, 125, 250, 500, 1000 µg/mL
Experiment 2:
- S9: 0, 1, 2, 4, 8, 12, 16 µg/mL
+ S9: 0, 125, 250, 500, 1000, 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

MMC as positive control for assay withou S9, cyclophosphamide for assay with S9 supplementation Migrated to IUCLID6: cyclophosphamide

Details on test system and experimental conditions:

Cultures were establisbed 16 to 72 hours prior to treatment using the appropriate number of cells per flask depending on the pre-exposure culture period. The cells were exposed to at least three doses of the test material, vehicle and positive controls both with and without metabolic activation. All exposures were performed in duplicate (A + B) and cultures were maintained at 37 °C in a humidified atmosphere of 5% C02 in air.
Experiment 1:
6 hours exposure to the test material without S9 mix followed by 18 hours culture in treatment-free media prior to cell harvest.
6 hours exposure to tbe test material with S9 mix (at 5% final concentration) followed by 18 hours culture in treatment-free media prior to cell harvest.
Experiment 2:
24 hours continuous exposure to the test material prior to cell harvest.
6 hours exposure to the test material with S9 mix (at 5% final concentration) followed by 18 hours culture in treatment-free media prior to cell harvestMitosis was arrested by addition of demeeolcine (CoIcemid 0.1 µg/ml) two hours before the required harvest time.

Evaluation criteria:

A total of 1000 cells were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Where possible the first 100 consecutive weIl-spread metaphases from each culture were counted, except where there were approximately 50% of cells with aberrations, then slide evaluation was terminated at 50 cells. If the cell had 23 to 27 chromosomes, any gaps, breaks or re arrangements
were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. A senior cytogeneticist checked the aberrant cells recorded by each slide scorer. Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately. The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. The number of gap-type aberrations was recorded and reported.

Statistics:

Fisher's Exact test

Species / strain:

other: Chinese hamster lung cells (CHL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Experiment 1:

Treatment period 6 h without S9	Number and % of cells showing structural aberrations								Gaps	Cell growth index		Number and % of cells showing numerical aberrations
	Observed	ctb	Cte	csb	cse	others	total	%	G	Cell count [%]	Mitotic index [%]	Polyploids	Others	Total
Negative Control (DMSO)	200	0	0	0	1	0	1	0.5	1	100	9.05	0	0	0
8 µg/mL	200	0	0	0	0	0	0	0.0	3	80	7.25	0	0	0
12 µg/mL	200	2	0	1	0	0	2	1.0	1	59	5.6	0	0	0
16 µg/mL	200	3	0	1	1	0	5	2.5	3	23	5.45	2	0	2
32 µg/mL	toxic	-	-	-	-	-	-	-	-	-	-	-	-	-
Positive Control (MMC)	150^a	22	35	6	1	0	51	34	14	51	6.85	0	0	0

MMC = Mitomycin C;  ^aslide evaluation terminated at 50 cells, because approx. 50 % cells with aberrations had been observed

Ctb: chromatid breaks; Csb: chromosome break; cte and cse: exchanges; g: gaps

Treatment period 6 h with S9	Number and % of cells showing structural aberrations								Gaps	Cell growth index		Number and % of cells showing numerical aberrations
	Observed	ctb	Cte	csb	cse	others	total	%	G	Cell count [%]	Mitotic index [%]	Polyploids	Others	Total
Negative Control (DMSO)	200	0	0	0	0	0	0	0.0	1	100	12.85	3	0	3
500 µg/mL P	200	0	0	0	0	0	0	0.0	2	102	8.4	0	0	0
1000 µg/mL P	200	0	0	1	0	0	1	0.5	0	94	7.8	0	0	0
2000 µg/mL P	200	0	0	0	3	0	3	1.5	1	130	5.9	0	0	0
Positive Control (CP)	200	28	36	16	3	0	70	35	23	43	10.5	0	0	0

P = Precipitate;   CP = Cyclophosphamide

Ctb: chromatid breaks; Csb: chromosome break; cte and cse: exchanges; g: gaps

Experiment 2:

Treatment period 24 h without S9	Number and % of cells showing structural aberrations								Gaps	Cell growth index		Number and % of cells showing numerical aberrations
	Observed	ctb	Cte	csb	cse	others	total	%	G	Cell count [%]	Mitotic index [%]	Polyploids	Others	Total
Negative Control (DMSO)	200	0	0	0	1	0	1	0.5	3	100	1.8	0	0	0
4 µg/mL	200	0	1	0	1	0	2	1.0	3	100	1.45	0	0	0
8 µg/mL	200	0	0	0	1	0	1	0.5	2	73	2.60	2	0	2
12 µg/mL	200	1	0	0	0	0	1	0.5	2	57	2.30	2	0	2
16 µg/mL	toxic	-	-	-	-	-	-	-	-	-	-	-	-	-
Positive Control (MMC)	200^a	41	23	14	4	0	67	33.5	25	43	4.80	0	0	0

MMC = Mitomycin C

Ctb: chromatid breaks; Csb: chromosome break; cte and cse: exchanges; g: gaps

Treatment period 6 h with S9	Number and % of cells showing structural aberrations								Gaps	Cell growth index		Number and % of cells showing numerical aberrations
	Observed	ctb	Cte	csb	cse	others	total	%	G	Cell count [%]	Mitotic index [%]	Polyploids	Others	Total
Negative Control (DMSO)	200	0	0	0	0	0	0	0.0	1	100	6.95	0	0	0
500 µg/mL P	200	1	1	0	0	0	2	1.0	1	102	8.10	1	0	1
1000 µg/mL P	200	0	0	1	0	0	1	0.5	0	89	7.00	1	0	1
1500 µg/mL P	200	1	1	3	2	0	6	3.0	0	93	8.35	0	0	0
2000 µg/mL P	200	0	0	1	0	0	1	0.5	0	96	5.8	0	0	0
Positive Control (CP)	150^a	18	39	4	6	0	57	38	19	54	8.00	0	0	0

P = Precipitate;   CP = Cyclophosphamide;  ^aslide evaluation terminated at 50 cells, because approx. 50 % cells with aberrations had been observed

Ctb: chromatid breaks; Csb: chromosome break; cte and cse: exchanges; g: gaps

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce any toxicologically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times. The test material
was therefore considered to be non-dastogenic to CHL cells in vitro.

Executive summary:

The study was conducted to assess the potential chromosomal mutagenicity of Benzoic acid isononylester on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to the requirements of the Japanese New Chemical Substance Law (METI), OECD 473 and updated Annex V B.10 Method.

Duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Four treatment regimens were used: in Experiment 1 a 6(18)-hours exposure, both with and without the addition of an induced rat liver homogenate metabolising system (S9); and Experiment 2 included a 24- hour continuous exposure in the absence of S9 and a repeat of the 6(18)-hours exposure with metabolic activation. The dose levels used were selected on the basis of the results of a preliminary toxicity test. The dose range for the 6(18)-hour exposure with S9 was 62.5 to 2000 µg/ml, 2 to 32 µg/ml for the 6(18)-hour exposure without S9 and 1 to 16 µg/ml for the 24-hour exposure group.

The vehicle (solvent) control had frequencies of cells with aberrations within the range expected for the CHL cell line. All of the positive control materials induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant increases in the freguency of cells with aberrations in any of the exposure groups. The dose levels of the test material were shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all of the exposure conditions.

The test material was considered to be non-clastogenic to CHL cells in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test in vivo (OECD 474): not clastogenic/mutagenic in vivo
The ability of BENZOIC ACID ISONONYLESTER to cause chromosomal damage in vivo was investigated in a micronucleus test in rats. Based on results obtained in a preliminary toxicity test, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. The test item was administered orally.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.Mar. 2005 - 06. Jun. 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 7-10 weeks
- Weight at study initiation: between 185.7 and 255.7 grams
- Assigned to test groups randomly: no data
- Fasting period before study: animals were fasted overnight before dosing
- Housing: The animals were housed 5 animals/cage, by sexes, in clear polycarbonate cages measuring 59 x 20 x 39 cm with a stainless steel mesh lid and floor (Type 4: Techniplast). Each cage held absorbent bedding which was inspected daily and changed as necessary.
- Diet (e.g. ad libitum): 4RF21, Mucedola S.r.l., Settimo Milanese, MI, Italy ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%,
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: standard vehicle for these kind of studies
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): batch no. 103K0107 obtained from Sigma
- Purity:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions were prepared in corn oil on a weight/volume basis with correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of materials as received. No assay of test item stability, nor its concentration and homogeneity in vehicle were undertaken as not requested by the Sponsor. All dose-levels in this report are expressed to three significant figures.

Duration of treatment / exposure:

24h and 48 h

Frequency of treatment:

Animals were treated once

Post exposure period:

none

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

- Positive control: mitomycin C
- Justification for choice of positive control(s): standard positive control for these kind of studies
- Route of administration: intraperitoneal injection
- Doses / concentrations: 2.0 mg/kg

Tissues and cell types examined:

polychromatic cells from bone marrow of femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of dose-levels was based on the results obtained in a preliminary toxicity assay. A group of two male and two female rats was dosed by oral gavage with the test item at 2000 and 1000 mg/kg body weight. The animals were observed for 48 hours and sacrificed. Scoring was performed on slides prepared from the femurs of animals.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The vehicle and the test item solutions were administered by oral gavage at the volume-dosage of 10 ml/kg bodyweight. The positive control treatment with Mitomycin-C was administered via intraperitoneal injection at the volume-dosage of 10 ml/kg bodyweight. Animals from vehicle treatment group and the test item treatment groups were fasted overnight
before dosing. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time.

DETAILS OF SLIDE PREPARATION: Animals were sacrificed at appropriate sampling times as indicated in the experimental schemes. The femurs of animals were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. Three slides from each animal were air-dried, fixed in methanol for 10 minutes and then stained with haematoxylin and eosin, and mounted with Eukitt.

METHOD OF ANALYSIS: The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. Two thousand polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.

Evaluation criteria:

Provided that the slides are of an adequate quality and a sufficient number of cells can be scored, it may only be necessary to score one of series. Scoring is effected using a microscope and high-power objective. Immature polychromatic erythrocytes (PCE's) stain a blue-grey colour (since they retain basic ribosomal material for approximately 24 h after enucleation), and can be distinguished from the orange-red normochromatic erythrocytes (NCE's). Erythrocytes lack nuclei, making micronuclei obvious when present; tbe criteria of Schmid (1976) will be used to score micronuclei. At least two thousand polychromatic erythrocytes per animal are scored for the presence of
micronuclei (unlcss thcrc is a marked depression in PCE numbers). At the same time the number of normochromatic erythrocytes is recorded, as we as the number of micronucleated NCE's. The proportion of immature erythrocytes among total erythrocytes gives an indication of the
toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. The incidence of micronucleated NCE's gives an indication of the pre-treatment status of the animals. Finally, tbe incidence of micronucleated PCE's provides an index of induced genetic damage.

Statistics:

Only counts from polychromatic cells are subjected to statistical analysis. Using the original observations (and not the micronueleus frequencies per 1000 cells) a modified chi-squared calculation is employed to compare treated and control groups. The degree of heterogeneity within each group is first calculated, and where it is significant it is taken into account in the comparison between groups. If there is no significant within-group heterogeneity, the chi-squared test is used to compare treated groups with the controls. If there is significant within-groups heterogeneity, then that group is compared with the controls using a variance ratio (F) value calculated from the between-group and within-group Chi-squared values.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Soft faeces were observed for all treated animals at 1000 and 2000 mg/kg bodyweight. No depression of bone marrow erythropoietic cell division was observed. Body weight loss was observed for one male animal only dosed at 2000 mg/kg. Based on the above results, dose-levels of 500, 1000 and 2000 mg/kg bodyweight were selected for the Main assay.

 

Treatment	Dose level (mg/kg)	Incidence of micronucleated PCE´s			PCE (PCE +NCE)  % over the mean negative control value
	M/F	Mean	SE	Range
24 hr sampling time
Vehicle	10 mL/kg	0.8	0.2	2.5	100
Test item	500	0.6	0.1	1.5	100
Test item	1000	0.6	0.1	1.0	95
Test item	2000	0.9	0.2	2.5	99
Mitomycin-C	2.0	6.2 ***	0.7	9.5	88
48 hr sampling time
Vehicle	10 mL/kg	0.6	0.2	1.5	100
Test item	2000	0.8	0.2	2.5	92

PCE = polychromatic erythrocytes, NCE = normochromatic erythrocytes

*** Significantly different at p < 0.001

Mucus in the cage litter was the only sign observed in some animals from the high dose group. No relevant increase in the incidence of micronucleated PCE’s over the control values was observed in any test item treatment group at any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system. The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, a slight inhibitory effect on erythropoietic cell division was observed at the high and intermediate dose-levels of treatment for female animals from the 24 hour sampling time. A slight depression of bone marrow erythropoietic cell division was also observed at the 48 hour sampling time for both male and female animals from the high dose-group.

Conclusions:

Interpretation of results (migrated information): negative
On the basis of the results obtained, it is concluded that BENZOIC ACID ISONONYLESTER, administered orally, does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Executive summary:

The ability of BENZOIC ACID ISONONYLESTER to cause chromosomal damage in vivo was investigated in a micronucleus test in rats. Based on results obtained in a preliminary toxicity test, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. The test item was administered orally as indicated in the Study Protocol.

Sprague-Dawley SD rats were dosed once only with the vehicle corn oil, Benzoic Acid Isononylester at the selected dose-levels or with the positive control substance Mitomycin-C. Each group consisted of five male and five female animals with the exception of the control and high-dose group, which included an additional five animals of each sex per group. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Bone-marrow smear slides were made and stained with haematoxylin and eosin stains. The slides were coded prior to scoring and two thousand polychromatic cells per animal were examined for the presence of micronuclei. The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test.

Following treatment with Benzoic Acid Isononylester, no statistically significant increase in the incidence of micronucleated PCE's over the control value, was observed in any treatment group. Statistically significant increases in the incidence of micronucleated PCE's over the control values were observed following treatment with the positive control Mitomycin-C, indicating the correct functioning of the test system.

Following treatment with the test item, no remarkable adverse reaction was observed after treatment. A slight depression of bone marrow erythropoietic cell division, was observed at the high and intermediate dose-levels of treatment for female animals from the 24 hour sampling time. A slight depression of bone marrow erythropoietic cell division was also observed at the 48 hour sampling time for both male and female animals from the high dose-group.

It is concluded that, under the reported experimental conditions, BENZOIC ACID ISONONYLESTER administered orally at the selected dose-levels to male and female rats, does not induce micronuclei in the polychromatic erythrocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The target substance Isononyl benzoate was tested for mutagenicity in bacteria, and for the potential to induce chromosome aberrations in mammalian cells. Further, the substance was tested in vivo to determine the ability to cause micronuclei in bone marrow. No gene mutation study in mammalian cells is available for the target substance.

The source substance C12-15 alkyl benzoates was tested in the full battery of in vitro genotoxicity tests, which were all negative. The justification for read-across is attached in the target record.

 

Ames Test (OECD 471): With the bacterial mutagenicity test (Ames Test) according to OECD 471, a reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 102, TA 1535 or TA 1537 was not observed with and without metabolic activation neither with the plate incorporation nor with pre-incubation method. It is therefore concluded, that Benzoesäure-isononylester is not a bacterial mutagen.

For additional information, the source substance C12-15 alkyl benzoates did also not induce reverse mutation in S. typhimurium stains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 in the absence or presence of S9 metabolic activation in a study conducted according to OECD guideline 471.

 

CA in vitro (OECD 473): In a chromosomal aberrations test in vitro with CHL cells according to OECD 473, the test item did not induce any significant increases in the freguency of cells with aberrations in any of the exposure groups. The vehicle (solvent) control had frequencies of cells with aberrations within the range expected for the CHL cell line. All of the positive control materials induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The dose levels of the test material were shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all of the exposure conditions. The test material was considered to be non-clastogenic to CHL cells in vitro.

The source substance C12-15 alkyl benzoates did not induce micronuclei in human lymphocytes in the absence or presence of S9 metabolic activation in a study according to OECD TG 487.

 

MLA in vitro (OECD TG 490): In a mouse lymphoma assay according to OECD 490 the soruce substance C12-15 alkyl benzoates was examined for mutagenic activity mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

A preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 5 μL/mL and a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 μg/mL. Two independent assays for mutation at the TK locus were performed. Based on the results obtained in the preliminary trial, the dose levels described in the following table were selected in order to achieve the appropriate toxicity of 10-20% relative total growth (RTG).

In the presence of S9 metabolism and using the long treatment in the absence of S9 metabolic activation, adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed. Using the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at higher concentrations, however opacity of treatment medium was observed, indicating the adequate selection of dose levels. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that C12-15 alkyl benzoates does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

 

Micronucleus test in vivo (OECD 474): The ability of BENZOIC ACID ISONONYLESTER to cause chromosomal damage in vivo was investigated in a micronucleus test in rats. Based on results obtained in a preliminary toxicity test, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. The test item was administered orally to Sprague-Dawley SD rats once in the vehicle corn oil. Following treatment with Benzoic Acid Isononylester, no statistically significant increase in the incidence of micronucleated PCE's over the control value, was observed in any treatment group. Statistically significant increases in the incidence of micronucleated PCE's over the control values were observed following treatment with the positive control Mitomycin-C, indicating the correct functioning of the test system. Furthermore, a slight depression of bone marrow erythropoietic cell division, was observed at the high and intermediate dose-levels of treatment for female animals from the 24 hour sampling time. A slight depression of bone marrow erythropoietic cell division was also observed at the 48 hour sampling time for both male and female animals from the high dose-group. It is concluded that, under the reported experimental conditions, BENZOIC ACID ISONONYLESTER administered orally at the selected dose-levels to male and female rats, does not induce micronuclei in the polychromatic erythrocytes.

 

Justification for classification or non-classification

GHS classification (GHS UN rev.2, 2007): no classification required