2-hydroxybenzoic acid 2-butyloctyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The subject material was negative when tested in a bacterial reverse mutation assay following OECD guidelines 471 and 472. It was negative in vitro in a chromosomal aberration test with human lymphocytes following OECD guideline 473. A surrogate material was negative in an in vitro mammalian cell gene mutation assay with Chinese hamster lung fibroblast (V79) cells.

Link to relevant study records

Reference

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:

The study was conducted from 3 March 1998 to 16 March 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Dated 1983

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

Dated 1983

Qualifier:

according to guideline

Guideline:

other: Proposal for replacement of Guidelines 471 and 472, dated 1997

Qualifier:

according to guideline

Guideline:

other: EEC Annex to Directive 92/69/EEC (1992) Part B: Methods for Determination of Toxicity, B.13 and B.14

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR Part 799 (1997) Toxic Substances Control Act Test Guidelines, Sub-section 799.9510

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium:
TA 1535, TA 100: hisG46
TA 1537: hisC3076
TA 98: hisD3052
Escherichia coli CM891 (WP2uvrA/pKM101):
contains ochre mutation; deficient in DNA repair system 9uvrA); contains the pKM101 plasmid

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat S9

Test concentrations with justification for top dose:

Test 1
5, 15, 50, 150, 500, 1500 and 5000 microg/plate

Test 2
50, 150, 500, 1500 and 5000 microg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO).

The solubility of the test substance was assessed at 50 mg/ml in DMSO, in which it dissolved. Therefore, DMSO (Aldrich, ACS spectrophotometric grade, lot. No. 09347HN, >/= 99.9% pure) was used as solvent in this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Pos. Control for TA98 (+S9), TA100 (+S9), TA1537 (+S9) Migrated to IUCLID6: 5 microg/plate

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Pos Control for TA98 (-S9), Migrated to IUCLID6: 1 microg/plate

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Pos Control for TA100 (-S9), TA1535 (-S9) Migrated to IUCLID6: 3 microg/plate

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2 microg/plate)

Remarks:

Pos Control for TA1535 (+S9)

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Pos Control for TA 1537 (-S9) Migrated to IUCLID6: 80 microg/plate

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (10 microg/plate)

Remarks:

Pos Control for E. coli (+S9)

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Pos Control for E. coli (-S9) Migrated to IUCLID6: 2 microg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Test 1:
Tests were conducted in the presence or absence of S9. Eight dose levels and controls were tested up to and including 5,000 microg/plate at approximately half-log intervals. All tests were performed in triplicate.
- To a series of tubes were added 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH4), 0.1 ml of a 10-hour bacterial culture, and 0.1 ml of the solvent or test chemical mixture followed immediately by the addition of 2.0 ml of molten agar supplemented with 0.5 mM L-histidine / d-biotin / trryptophan. The contents of the resulting tubes were mixed and poured onto previously prepared petri dishes containing 25 ml minimal agar. After solidification, plates were inverted and incubated at 37 deg C for 72 hours. The negative control was the chosen solvent, DMSO. The application of positive controls was also included.

Test 2:
As a clear negative response was obtained in Test 1, a variation to the test procedure was used for the second test. A variation of the pre-incubation assay was used in which the tubes were incubated at 27 deg C for 30 minutes with shaking before the addition of the agar overlay. Only 5 concentrations were used in this second test.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Evaluation criteria:

For a test to be considered valid, the mean of the solvent control and revertant colony numbers for each bacterial strain should lie in the range as stated in the appropriate Standard Operating Procedure or quoted by Gatehouse et al. (1990). Also, the positive control substance must cause at least a doubling of mean revertant colony numbers over the negative control.

The mean number of revertant colonies for all treatment groups were compared with those obtained for the solvent control groups. The mutagenic activity of the test substance was assessed by applying the following criteria:
a) If treatment produced an increase in revertant colony numbers of at least twice the concurrent controls, with some evidence of a positive dose-response, in two separate experiments, with any bacterial strain in the presence or absence of S9 mix, it was considered evidence of a mutagenic response. No statistical analysis was performed.
b) If treatment did not cause a reproducible increase of at least 1.5 times the concurrent solvent controls in either mutation test, it was considered to show no evidence of mutagenic activity. No statistical analysis was performed.
c) If results failed to satisfy the criteria for clear positive or clear negative response, additional testing would be performed to resolve the issue of the mutagenic activity.

If no clear positive or negative response could be obtained, the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures will be those of Mahon et al. (1989) and will usually be analysis of variance followed by Dunnett's test.

Statistics:

See above.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Refer to Tables 1 to 4 for complete study results.

Table 1: Results Test 1 With S9 Activation

	Revertant colony counts (mean 3 replicates)
Addition (micrograms/plate)	TA98	TA100	TA1535	TA1537	E. coli
Sterility Check	0	0	0	0	0
5000	32	88	23	9	90
1500	43	97	22	14	91
500	37	100	19	11	91
150	45	90	27	9	99
50	38	109	23	13	92
15	30	94	25	11	96
5	32	104	24	14	96
DMSO (0.1 ml)	37	92	21	16	96
2-Benzo[a]pyrene (5)	311	431	-	216
2-Aminoanthracene (2)			200
2-Aminoanthracene (10)					231

Table 2: Results Test 1 Without S9 Activation

	Revertant colony counts (mean 3 replicates)
Addition (micrograms/plate)	TA98	TA100	TA1535	TA1537	E. coli
Sterility Check	0	0	0	0	0
5000	34	86	17	15	92
1500	38	83	14	15	88
500	40	103	15	17	97
150	33	97	16	14	102
50	43	95	19	13	98
15	37	92	21	14	88
5	27	103	18	12	85
DMSO (0.1 ml)	39	100	21	15	103
2-Nitrofluorene (1)	314
ENNG (3)		344
ENNG (5)			109
9-Aminoacridine (80)				3812
ENNG (2)					963

Table 3: Results Test 2 (Pre-incubation) With S9 Activation

	Revertant colony counts (mean 3 replicates)
Addition (micrograms/plate)	TA98	TA100	TA1535	TA1537
Sterility Check	0	0	0	0
5000	34	89	17	8
1500	32	93	19	10
500	35	98	16	9
150	39	91	14	8
50	32	92	17	12
DMSO (0.1 ml)	35	97	19	11
2-Benzo[a]pyrene (5)	291	372		164
2-Aminoanthracene (2)			206

Table 4: Results Test 2 (Pre-incubatioin) Without S9 Activation

	Revertant colony counts (mean 3 replicates)
Addition (micrograms/plate)	TA98	TA100	TA1535	TA1537
Sterility Check	0	0	0	0
5000	29	90	22	8
1500	28	94	25	9
500	36	91	18	10
150	31	98	23	11
50	36	79	14	10
DMSO (0.1 ml)	30	99	18	10
2-Nitrofluorene (1)	308
ENNG (3)		382
ENNG (5)			236
9-Aminoacridine (80)				2532

Conclusions:

Interpretation of results (migrated information):
negative with or without S9 activation

It was concluded that, when tested in DMSO, HallBrite BHB showed no evidence of mutagenic activity in any of the bacterial strains tested.

Executive summary:

In a first test, no substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to HallBrite BHB at any concentration in either the presence or absence of S9 mix. There was no measured cytotoxicity, as judged by thinning of the background lawn of non-revertant cells, following exposures. A top exposure concentration of 5000 microg/plate was then selected for use in a second test.

A second test gave results consistent with the first.

It was concluded that HallBrite showed no evidence of mutagenic activity in the bacterial systems tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In an guideline study (OECD 471/472) in bacterial S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA pkM 101, the subject material was negative for mutagenicity either in the presence or absence of rat liver S9 activation up to a maximum plate incorporation level of 5000 ug/plate.

The subject material was tested for clastogenicity in a guideline study (OECD 473) with cultured human lymphocytes either in the presence or absence of rat liver S9 activation. In a first test, cultures were exposed for 3 hours and harvested 17 hours later. In a second test, cultures receiving S9 were treated in the same way but cultures without S9 were tested continuously for 20 hours before harvest. There were no biologically or statistically significant increases in the frequency of chromosomal aberrations in the first study. In the second study and when gaps were excluded, there were statistically significant increases in aberration frequency at the highest exposure concentration of 2500 ug/mL in the presence of S9. However, the aberration frequency exceeded the historical control range in only one of the two replicates indicating a lack of reproducibility. Under the conditions of this test, the subject material was not considered a clastogen.

A surrogate material, 2 -ethylhexyl salicylate (CAS 118 -60 -5), was tested in a guideline OECD 476 study with Chinese hamster lung fibroblast (V79) cells. In a first experiment, the treatment period was 4 hours with and without metabolic activation. In a second experiment, treatment times of 4 hours with and 24 hours without metabolic activation were employed. No substantial and reproducible dose dependent increase in mutation frequencies (HPRT locus) was observed in either experiment. Under the conditions of the study, the surrogate material was judged to be negative for mutagenicity.

Justification for selection of genetic toxicity endpoint

Well conducted studies on the subject material or a surrogate following OECD guidelines 471, 472, 473 and 476 indicated a lack of genetic toxicity.

Justification for classification or non-classification

Based on the results of in vitro studies with either the subject material or a surrogate, the subject material would not be classified for Germ Cell Mutagenicity according to EU CLP (Regulation (EC) No. 1272/2008).