Benzyl salicylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Study period:

1989-06-07 to 1989-06-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study according to principles similar to those of OECD TG 474, but only 1000 instead of 2000 erythrocytes evaluated. To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Ethylhexyl Salicylate (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore, read across is justified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

1000 instead of 2000 erythrocytes were evaluated

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl salicylate

EC Number:

204-263-4

EC Name:

2-ethylhexyl salicylate

Cas Number:

118-60-5

Molecular formula:

C15H22O3

IUPAC Name:

2-ethylhexyl salicylate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): HR 89/131494
- Molecular formula (if other than submission substance): C15H22O3
- Molecular weight (if other than submission substance): 255.33 g/mol
- Physical state: colourless, oily liquid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): not reported
- Composition of test material, percentage of components: not reported
- Purity test date: not reported
- Lot/batch No.: 37798
- Expiration date of the lot/batch: not reported

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SAVO GmbH, Kisslegg, Germany
- Age at study initiation: 6 to 10 weeks old
- Weight at study initiation: 25 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: in groups of up to five animals in Makrolon cages of size II with Altromin woodshaving
- Diet (e.g. ad libitum): Altromin 1324 standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no justification provided
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the solutions were prepared freshly directly before application

Duration of treatment / exposure:

Groups were treated with the substance and sampling was performed after 24, 48 and 72 hours

Frequency of treatment:

One treatment at the beginning of the test.

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Five males and five females

Control animals:

yes, concurrent vehicle

Positive control(s):

Five males and five females treated with 50 mg/kg 9,10-dimethyl-1,2-benzanthracene (DMBA) by oral gavage with sampling time after 48 hours

Examinations

Tissues and cell types examined:

Bone marrow smears and polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a limit test was performed showing that a dose of 2000 mg/kg bw was the maximum tolerated dose not leading to cytotoxicity.
DETAILS OF SLIDE PREPARATION: animals were killed by cervical dislocation and bone marrow was removed from both femora by rinsing with foetal calf serum. Bone marrow cells were centrifuged at 150 g for 10 minutes and the supernatant was discarded. From the pellet, smears were made on slides and air dried. Two slides were made per animal. The slides were stained by the May-Gruenwald-Giemsa method according to Schmid (1973) by staining for 3 minutes in undiluted May-Gruenwald solution, then staining for 2 minutes in May-Gruenwald diluted with distilled water to a ratio 1:1, rinsing briefly in distilled water, staining for 10 minutes in Giemsa diluted with distilled water to a ratio 1:6, rinsing thoroughly in distilled water, drying in air, cleaning the back side of the slides with methanol, cleaning in xylene for 5 minutes and mounting in Eukitt.

METHOD OF ANALYSIS: slides were coded and observed blindly under a microscope with 100x oil immersion objective lens at 1250 fold magnification. At least 1000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes was also recorded. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Evaluation criteria:

An increased frequency of micronucleated polychromatic erythrocytes among treated animals compared to control animal values is taken as an indication of treatment-induced genetic damage.

Statistics:

Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

According to an initial toxicity test the maximum tolerated dose was 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Both negative and positive control frequencies of micronucleated polychromatic erythrocytes agreed with the values previously established in the testing laboratory.

Any other information on results incl. tables

To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Ethylhexyl Salicylate (Source Substance).

The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible.

The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific.

Therefore, read across is justified.

Table: Detailed results of analysis for single animals used in the micronucleus test

Dose and sampling time	Sex	Animal identification	PE examined	PE with MN		NE with MN (‰)	Ratio PE/NE
				Total	‰
Vehicle control, 24 hours after oral application by gavage	female	A1	1086	2	1.84	0.00	1.76
		A2	1071	2	1.87	1.00	2.05
		A3	1010	0	0.00	0.98	1.88
		A4	1016	2	1.97	3.00	2.34
		A5	1004	3	2.99	2.98	2.24
	male	B1	1010	1	0.99	1.94	1.52
		B2	1019	1	0.98	0.00	2.08
		B3	1087	1	0.92	1.98	1.85
		B4	1002	2	2.00	1.97	1.56
		B5	1020	2	1.96	0.00	2.07
Test substance at 2000 mg/kg bw, 24 hours after oral application by gavage	female	C1	1032	1	0.97	0.99	2.24
		C2	1018	2	1.96	0.00	2.15
		C3	1002	2	2.00	0.00	1.83
		C4	1014	1	0.99	1.99	1.32
		C5	1006	2	1.99	0.00	1.89
	male	D1	1005	2	1.99	0.98	1.16
		D2	1003	2	1.99	1.98	1.10
		D3	1004	1	1.00	1.99	1.96
		D4	1003	0	0.00	1.90	1.29
		D5	1003	1	1.00	2.00	1.50
Test substance at 2000 mg/kg bw, 48 hours after oral application by gavage	female	E1	1006	1	0.99	1.96	1.15
		E2	1001	1	1.00	0.00	1.45
		E3	1032	2	1.94	0.98	1.62
		E4	1010	3	2.97	0.00	1.20
		E5	1002	0	0.00	0.99	1.69
	male	G1	1012	3	2.96	2.96	1.27
		G2	1003	1	1.00	1.00	1.56
		G3	1030	2	1.94	2.94	1.17
		G4	1013	1	0.99	2.00	2.02
		G5	1006	1	0.99	0.99	1.96
Test substance at 2000 mg/kg bw, 72 hours after oral application by gavage	female	H1	1006	1	1.00	0.00	1.22
		H2	1005	2	1.99	2.96	1.16
		H3	1046	2	1.91	1.00	1.48
		H4	1033	3	2.90	1.99	1.74
		H5	1009	1	0.99	0.99	1.76
	male	I1	1008	2	1.98	1.92	1.85
		I2	1075	1	0.93	0.99	1.67
		I3	1043	1	0.96	2.97	1.20
		I4	1029	2	1.94	1.99	1.80
		I5	1009	1	0.99	0.00	1.77
DMBA at 50 mg/kg bw, 24 hours after oral application by gavage	female	L1	1013	10	9.87	5.81	1.27
		L2	1011	12	11.87	4.80	1.28
		L3	1000	8	8.00	6.56	1.19
		L4	1005	10	9.95	4.96	1.33
		L5	1009	13	12.88	4.69	1.30
	male	M1	1019	12	11.78	9.79	1.00
		M2	1001	9	8.99	11.93	1.32
		M3	1003	11	10.97	4.73	0.95
		M4	1017	13	12.78	5.66	1.06
		M5	1009	10	9.91	5.00	1.37
PE = polychromatic erythrocytes; NE = normochromatic erythrocytes; PE with MN = micronucleated polychromatic erythrocytes; NE with MN = micronucleated normochromatic erythrocytes; DMBA = 9,10-dimethyl-1,2-benzanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The substance did not induce chromosomal damage or damage to the mitotic apparatus in bone marrow cells of mice after a single oral application at a dose of 2000 mg/kg bw.

Executive summary:

As discussed as part of the category of salicylate substances (RAAF Document).

Only 2 members of this category have in vivo mutagenicity micronucleus data. However, all 11 of these substances have in vivo mutagenicity micronucleus alerts for H-acceptor-path3-H-acceptor interaction which indicates a possiblechemical interaction with DNA and/or proteinsvianon-covalent binding, such as DNA intercalation or groove-binding.

However, the results of the 2 main source substances both have negative experimental data for this endpoint and therefore it can be assumed that when taken into account with the in vitro data and the experimental data consistencies (negative data) within the category and the total lack of any DNA interactions (in vitro and in vivo), that the target substance benzyl salicylate would also be negative in such a genetox in vivo micronucleus assay.