Benzene, C10-13-alkyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Read-across to EPA OPPTS 870.5265 studies in Salmonella typhimurium (Ames test) with alkylate 215 and 225, no genotoxic activity were observed.

In Read-across to OECD 476 equivalent studies in CHO cells (mammalian gene mutation, HGPRT) with alkylate 215 and 225; no genotoxic activity were observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor induced rat and mouse livers

Test concentrations with justification for top dose:

10, 40, 200, 1000, 3000, and 10000 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

benzo(a)pyrene

other: sodium nitrite

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

Evaluation criteria:

A response was considered positive if three or more treatments were significantly greater than controls, and if there was a significant dose response.

Statistics:

Significant differences analyzed using Bartlett's test. Comparison to controls analyzed using Dunnett's t test. Dose response analyzed with regression analysis for a log-log straight line and t-test.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Negative with and without activation. A significant difference was seen in strain TA 98 with S9 at all concentrations. There was also a significant response in strain TA 1535 without S9 at a single concentration. Retesting with strain TA 98 showed no significant increase in revertants. A retest of strain TA 1535 was also negative. Due to lack of a dose-response relationship and lack of reproducibility of the positive results, the test substance is considered negative for mutagenicity.

Table 1: Mean Revertants/plate - without S9

Concentration (μg or nl/plate)	TA 1535	TA 100	TA 1537	TA 98
First run
10	28.3 ± 3.51	307 ± 51.5	8.0 ± 1.0	25.7 ± 5.13
40	23.0 ± 2.65	344 ± 33.5	9.0 ± 3.6	19.3 ± 5.51
200	29.0 ± 8.00	310 ± 17.6	10.7 ± 0.58	17.3 ± 3.79
1000	23.3 ± 3.21	318 ± 33.3	10.0 ± 1.0	18.7 ± 1.15
3000	34.7 ± 5.03	296 ± 57.4	9.0 ± 1.7	21.7 ± 2.08
10,000	27.3 ± 4.62	297 ± 59.2	10.3 ± 1.5	27.3 ± 6.03
Solvent control	21.7 ± 0.58	395 ± 30.8	7.3 ± 3.5	23.7 ± 0.58
NaNO2	691
9-aminoacridine			1620
2-nitrofluorene		627		354
Second run
10		321 ± 17.7
40		297 ± 25.1
200		292 ± 39.0
1000		307 ± 20.3
1500	16.0 ± 1.0
3000	14.3 ± 0.58	322 ± 58.2
4000	12.0 ± 4.0
10,000		322 ± 33.2
Solvent control	13.0 ± 3.46	258 ± 25.5
NaNO2	564
2-nitrofluorene		814
Third run
10		245 ± 11.5
40		236 ± 30.7
200		166 ± 57.6
1000		203 ± 49.2
3000		230 ± 11.8
10,000		179 ± 22.3
Solvent control		233 ± 27.0
2-nitrofluorene		506

Table 2: Mean Revertants/plate - with S9

Concentration (μg or nl/plate)	TA 1535	TA 100	TA 1537	TA 98
First run
10	25.7 ± 5.8	309 ± 44.9	11.3 ± 1.5	72.7 ± 5.8
40	21.3 ± 5.8	294 ± 31.0	10.3 ± 2.3	79.3 ± 5.5
200	20.3 ± 1.2	283 ± 12.7	7.3 ± 2.5	63.7 ± 13.9
1000	24.3 ± 2.1	339 ± 38.4	6.7 ± 1.5	71.7 ± 16.0
3000	22.7 ± 3.8	303 ± 27.4	13.7 ± 2.5	71.7 ± 4.2
10,000	28.7 ± 4.9	367 ± 62.4	12.3 ± 3.2	70.7 ± 0.53
Solvent control	24.3 ± 1.5	314 ± 20.3	9.7 ± 2.1	29.0 ± 9.5
B(a)P		938		459
2-AA	661		155
Second run
10	15.7 ± 3.2	280 ± 46.6		27.3 ± 2.5
40	11.0 ± 3.6	299 ± 15.0		33.7 ± 4.9
200	15.3 ± 2.1	294 ± 24.4		39.0 ± 1.0
1000	21.3 ± 3.1	297 ± 26.8		30.0 ± 7.2
3000	21.0 ± 11.5	301 ± 39.9		32.0 ± 7.2
10,000	13.7 ± 2.5	296 ± 6.8		36.0 ± 4.6
Solvent control	13.7 ± 5.7	304 ± 29.0		27.0 ± 1.7
B(a)P		1611
2-AA	511			896
Third run
10		191 ± 43.0
40		210 ± 3.06
200		227 ± 15.0		39.0 ± 6.2
250				35.3 ± 9.3
1000		229 ± 12.9		32.0 ± 5.3
3000		238 ± 29.7
10,000		204 ± 12.2
Solvent control		219 ± 39.8		34.7 ± 3.2
B(a)P		1649		810

Conclusions:

Under the conditions described in this study Alkylate 215 did not show mutagenic properties.

Executive summary:

This EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test) and GLP compliant study was performed to investigate the mutagenicity of the test substance alkylate 215. Salmonella typhimurium strains TA 1535, TA 100, TA 1537, and TA 98 were exposed to concentrations of 10, 40, 200, 1,000, 3,000, and 10,000 μg/plate of test substance. DMSO was used as a solvent. 9-aminoacridine, sodium nitrite, benzo(a)pyrene, 2 -acetylaminofluorene, and 2 -nitrofluorene were used as positive controls substances. No reproducible, dose-response related positive results were seen in the treatment groups. A significant increase in revertants per plate was seen in the positive controls. The test substance is not mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

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:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from aroclor induced rat and mouse liver

Test concentrations with justification for top dose:

3, 12, 60, 300, 1000, 3000 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

benzo(a)pyrene

other: sodium nitrite

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

Evaluation criteria:

A response was considered positive if three or more treatments were significantly greater than controls, and if there was a significant dose response.

Statistics:

Significant differences analyzed using Bartlett's test. Comparison to controls analyzed using Dunnett's t-test. Dose response analyzed with regression analysis for a log-log straight line and t-test.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Negative with and without activation

Table 1: Mean Revertants/plate - without S9

Concentration (μg or nl/plate)	TA 1535	TA 100	TA 1537	TA 98
3	25.3 ± 2.5	109.0 ± 15.7	8.7 ± 4.0	20.0 ± 2.0
12	24.0 ± 1.0	106.7 ± 7.6	8.3 ± 7.1	22.3 ± 4.5
60	23.3 ± 1.2	97.7 ± 12.4	7.0 ± 2.0	19.3 ± 4.0
300	22.7 ± 4.5	83.0 ± 12.5	10.0 ± 2.8	17.7 ± 3.2
1,000	27.7 ± 1.5	103.0 ± 6.1	4.7 ± 3.1	19.0 ± 4.0
3,000	25.7 ± 1.5	103.0 ± 12.1	9.3 ± 1.5	20.3 ± 4.6
Solvent control	24.0 ± 6.0	101.0 ± 15.1	11.0 ± 1.0	22.0 ± 2.0
4-NQ 0.1		858		172
4-NQ 0.05		475		118
4-NQ 0.01		191		47
NaNO2 5000	587
NaNO2 2500	387
NaNO2 500	85
9-AA 30		164
9-AA 15		20
9-AA 3		10

Table 2: Mean Revertants/plate - with S9

Concentration (μg or nl/plate)	TA 1535	TA 100	TA 1537	TA 98
3	12.7 ± 3.2	98. ± 0 7.0	8.7 ± 4.0	34.7 ± 6.8
12	15.7 ± 2.1	94.0 ± 8.7	9.7 ± 2.1	46.0 ± 12.1
60	10.3 ± 4.2	92.0 ± 12.5	12.0 ± 3.0	39.3 ± 17.0
300	15.0 ± 3.5	95.0 ± 15.9	10.3 ± 3.1	29.0 ± 4.4
1,000	13.7 ± 2.1	100.3 ± 6.4	12.0 ± 3.0	33.3 ± 7.4
3,000	10.7 ± 4.5	94.7 ± 12.6	8.7 ± 4.0	32.7 ± 6.5
Solvent control	14.0 ± 2.6	103.3 ± 12.1	11.3 ± 2.1	33.7 ± 6.7
B(a)P 2		342
B(a)P 1		183
B(a)P 0.2		128
2AA 30	Toxic		Toxic
2AA 15	Toxic		173
2AA 3	392		99
2AAF 30				1038
2AAF 15				877
2AAF 3				366

Conclusions:

In the current study an Ames test using Alkylate 225 did not show mutagenic properties.

Executive summary:

This EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test) and GLP compliant study was carried out to assess the mutagenicity of the test substance alkylate 225. Salmonella typhimurium strains TA 1535, TA 100, TA 1537, and TA 98 were exposed to concentrations of 3, 12, 60, 300, 1000, and 3000 μg/plate of test substance. DMSO was used as a solvent. 2-acetylaminofluorene, 9-aminoacridine, 4-nitroquinoline-N-oxide, sodium nitrite, and benzo(a)pyrene were used as positive controls substances. No positive results were seen in the treatment groups. A significant increase in revertants per plate was seen in the positive controls. The test substance is not mutagenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

HGPRT point mutation

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver induced with aroclor 1254

Test concentrations with justification for top dose:

100 - 2000 μL/mL

Vehicle / solvent:

Ethyl alcohol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

benzo(a)pyrene

ethylmethanesulphonate

Evaluation criteria:

A response was considered positive if one of the three highest concentrations with survival of at least 10% had a mean mutation frequency significantly greater than that of controls, and if there was a dose-response relationship.

Statistics:

Student's t-test was used to compare mutation frequency. Dose-response was analyzed using one-way analysis of variance.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1.0 mg/mL with activation; 0.25 mg/mL without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no significant increase in mutation frequencies as compared to controls.

Table 1: Mutagenicity

Concentration	Mutagenicity (mutation frequency x E^-6)
Without S9
100	19.0
150	18.1
250	24.0
500	7.8
750	10.2
1000	-
1100	-
1250	-
1500	-
1750	-
2000	-
Solvent control	21.6
Ethyl methanesulfonate	284.5
1 % S9
100	13.2
250	12.6
500	20.0
750	41.6
1000	24.8
Solvent control	17.6
B(a)P	415.8
5 % S9
100	-
500	-
1000	-
1250	-
1500	-
1750	-
2000	-
Solvent control	-
DMN	-

Conclusions:

Under the conditions described for this CHO/HGPRT assay Alkylate 215 did not show mutagenic properties.

Executive summary:

This GLP compliant study and similar to the OECD 476 TG (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes) was carried out to assess the mutagenicity of the test substance in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 100 - 2000 μL/mL of test substance both in the presence and absence of metabolic activation. Ethanol was used as a vehicle. Ethylmethanesulfonate, benzo(a)pyrene, and dimethylnitrosamine were used as positive controls. Cytotoxicity was seen at concentrations of 1.0 mg/mL and above with metabolic activation and 0.25 mg/mL and above without metabolic activation. No significant increase in mutation frequencies was seen in treatment groups. The test substance is not mutagenic to mammalian cells.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

HGPRT point mutation

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

100 - 2000 μL/mL

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

other: dimethylnitrosamine

Evaluation criteria:

A response was considered positive if one of the three highest concentrations with survival of at least 10% had a mean mutation frequency significantly greater than that of controls, and if there was a dose-response relationship.

Statistics:

Student's t-test was used to compare mutation frequency. Dose-response was analyzed using one-way analysis of variance.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.5 mg/mL both with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No significant increase in mutation frequency was seen in the treatment groups.

Table 1: Mutagenicity

Concentration	Mutagenicity (mutation frequency x E-6)
Without S9
100	2.2
150	-
250	-
500	2.3
750	-
1000	3.0
1100	13.2
1250	1.1
1500	1.6
1750	-
2000	-
Solvent control	3.5
Ethyl methanesulfonate	237.9
5 % S9
100	0.4
500	0.8
1000	8.1
1250	3.7
1500	0.8
1750	-
2000	-
Solvent control	4.3
DMN	202.8

Conclusions:

Under the conditions described for this CHO/HGPRT assay Alkylate 225 did not reveal mutagenic properties.

Executive summary:

This GLP compliant study and similar to the OECD 476 TG (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes) examined the potential of the test substance to cause mutations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 100 - 2000 μL/mL of test substance both in the presence and absence of metabolic activation. Ethanol was used as a vehicle. Ethylmethanesulfonate, benzo(a)pyrene, and dimethylnitrosamine were used as positive controls. Cytotoxicity was seen at concentrations of 0.5 mg/mL and above both with and without metabolic activation. No significant increase in mutation frequencies was seen in treatment groups. The test substance is not mutagenic to mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In read-across to OECD TG 475 studies in rats (bone marrow chromosome aberrations) no genotoxic activity were observed

In an OECD TG 474 study (Erythrocyte micronucleus formation;) in mice no genotoxic activity were observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Jun 1989 to 8 Jun 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: BRO:NMRI (SPF Han.)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: FA. Winkelmann
- Age at study initiation: young adult
- Weight at study initiation: about 26 g
- Fasting period before study: 18 hours
- Housing: 5 animals per cage in Makrolon Type III cages, identified by cage signs
- Diet: Ssniff R 10 - Alleindiat fur Ratten
- Water: Tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 1
- Humidity (%): 60 ± 10
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: gavage

Duration of treatment / exposure:

Single oral gavage exposure

Frequency of treatment:

Once

Post exposure period:

Animals were sacrificed at 24, 48, and 72 hours after exposure.

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15 including negative controls and 5 in positive control group

Control animals:

yes

Positive control(s):

Positive control animals were given a single dose of 100 mg/kg cyclophosphamide. Negative control animals were given water instead of test substance.

Tissues and cell types examined:

After sacrifice, the marrow from the thighs of each animal were removed and 2 mL fetal bovine serum was added. This suspension was centrifuged for 5 minutes. Marrow from each animals was divided into 3 slides, dried, and stained.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
Animals were given a single oral dose, and 5 animals of each sex per dose were sacrificed at 24, 48, and 72 hours after exposure.

DETAILS OF SLIDE PREPARATION:
Marrow from each animals was divided into 3 slides, dried, and stained.

METHOD OF ANALYSIS:
Cells were analysed by microscope ,1000 cells were analyzed per sample (3000/animal).

Evaluation criteria:

The nucleus of each cell was examined for size (micronucleus, which was considered 1/20th of the largest nucleus), and the number of polychromatid erythrocytes (PCE) to normochromatid erythrocytes (NCE).

Statistics:

F-test, t-test, and U-test were used to analyze the data.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

diarrhea and diuresis was seen in test group animals

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Animals in the treatment group exhibited diarrhea and diuresis during the treatment period. There was a significant increase in the PCE/NCE ratio and PCE cells with micronucleus in the positive control group. There was no significant increase in either of these parameters in the treatment group.

Table 1: Results of Mouse Micronucleus Assay

	Dose (mg/kg)	Hours after application	% PCE with micronucleus	PCE/NCE
Positive control	100	24	3.34 ± 0.97	0.97 ± 0.07
Negative control	-	24	0.18 ± 0.06	1.15 ± 0.10
	-	48	0.25 ± 0.12	1.31 ± 0.09
	-	72	0.28 ± 0.10	1.20 ± 0.33
Treatment group	5000	24	0.20 ± 0.07	1.07 ± 0.10
	5000	48	0.18 ± 0.08	1.26 ± 0.16
	5000	72	0.18 ± 0.11	1.38 ± 0.20

Conclusions:

Under the conditions described for this Mammalian Erythrocyte Micronucleus assay the tested substance did not reveal mutagenic properties.

Executive summary:

This study in accordance with OECD 474 and in compliance with GLP was performed to examine the mutagenic potential of the test substance in vivo. A groups of 15 female and 15 male mice were given a single dose of test substance to determine the effect on bone marrow. 5 animals of each sex were sacrificed at 24, 48, and 72 hours after exposure. Similar groups were exposed to cyclophosphamide (positive control), and water (negative control). All animals in the positive control group were sacrificed at 24 hours. Analysis of the bone marrow showed no significant increase in either micronucleus or polychromatid erythrocytes in the treatment group as compared to negative controls. The test substance is not mutagenic.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

EPA/TSCA

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

Corn oil

Frequency of treatment:

Single treatment

Post exposure period:

Animals were sacrificed at 6, 12, 24, and 48 hours after exposure (6 animals at each sacrifice)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Dose / conc.:

6 000 mg/kg bw/day (actual dose received)

Dose / conc.:

12 700 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

18 - 24, negative control 24

Control animals:

yes, concurrent vehicle

Positive control(s):

6 males and 6 females were given 40 mg/kg of cyclophosphamide and sacrificed at 24 hours.

Tissues and cell types examined:

Bone marrow cells (60 cells per animal)

Details of tissue and slide preparation:

2 mg/kg colchicine was administered 2 hrs before termination. Immediately after termination, cells were collected and processed for slide preparation.

Statistics:

Kruskal-Wallis non-parametric analysis of variance

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lowest dose producing toxicity = 6000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No effect on mitotic index or P/N ratio. Significant mean body weight loss in animals treated with 12700 mg/kg bw. Significant weight loss was also seen in males at the 6000 mg/kg dose level.

Table 1: Results of in vivo bone marrow assay

Concentration	Chromatid gaps	Chromosome gaps	Chromatid breaks	Chromosome breaks	Exchanges	% Aberrant cells
6-hrs
Corn oil	0	0	0	0	0	0
2000 mg/kg	0	0	0	0	0	0
6000 mg/kg	0	0	0	0	0	0
12700 mg/kg	0	0	0	0	0	0
12 hrs
Corn oil	2	1	0	0	0	0.17
2000 mg/kg	2	0	2	0	0	0.35
6000 mg/kg	0	0	0	0	0	0
12700 mg/kg	2	0	5	0	0	1.00
24 hrs
Corn oil	1	0	0	0	0	0
2000 mg/kg	0	0	1	0	0	0.16
6000 mg/kg	2	0	1	0	0	0.18
12700 mg/kg	0	0	2	0	1	0.49
CP 40 mg/kg	6	1	68	3	9	14.80

Conclusions:

Under the conditions described for this rat bone marrow chromosome aberration assay Alkylate 215 did not reveal genotoxic properties.

Executive summary:

This GLP compliant study and similar to the OECD 475 TG was conducted to examine the potential of the test substance to cause chromosome aberrations in vivo. Groups of 18 - 24 male and female rats were given doses of 2000, 6000, or 12700 mg/kg of test substance. Negative control animals were given corn oil (vehicle) only. Positive control animals were given 40 mg/kg cyclophosphamide. Animals were sacrificed at 6, 12, 24, and 48 hours after exposure. The bone marrow cells were then removed and examined for chromosome aberrations. The test substance was toxic (reduced body weight) at the 12700 mg/kg dose in both males and females. Males in the 6000 mg/kg dose level also showed weight loss. No significant increase in chromosomal aberrations was seen in any treatment group. The test substance is not clastogenic.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

EPA/TSCA

GLP compliance:

not specified

Type of assay:

mammalian germ cell cytogenetic assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

Corn oil

Frequency of treatment:

Single treatment

Post exposure period:

Animals were sacrificed at 6, 12, 24, and 48 hours after exposure (6 animals at each sacrifice)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

Dose / conc.:

4 000 mg/kg bw/day (actual dose received)

Dose / conc.:

12 700 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

18 - 24; negative control 24

Control animals:

yes, concurrent vehicle

Positive control(s):

6 males and 6 females were given 40 mg/kg of cyclophosphamide and sacrificed at 24 hours.

Tissues and cell types examined:

Bone marrow cells (60 cells per animal)

Details of tissue and slide preparation:

2 mg/kg colchicine was administered 2 hrs before termination. Immediately after termination, cells were collected and processed for slide preparation.

Statistics:

Kruskal-Wallis non-parametric analysis of variance

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lowest dose producing toxicity = 12,700 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No effect on mitotic index or P/N ratio. Significant mean body weight loss in all treatments groups at the 12700 mg/kg dose level.

Table 1: Results of in vivo bone marrow assay

Concentration	Chromatid gaps	Chromosome gaps	Chromatid breaks	Chromosome breaks	Exchanges	% Aberrant cells
6-hrs
Corn oil	0	0	0	0	0	0
1200 mg/kg	1	0	1	0	0	0.16
4000 mg/kg	0	0	1	0	0	0.17
12700 mg/kg	0	0	0	0	0	0
12 hrs
Corn oil	1	0	1	0	0	0.16
1200 mg/kg	1	1	1	0	0	0.16
4000 mg/kg	4	0	1	1	0	0.16
12700 mg/kg	2	0	1	0	0	0.16
24 hrs
Corn oil	2	0	1	0	0	0.17
1200 mg/kg	4	0	1	0	0	0.16
4000 mg/kg	3	0	4	0	1	0.54
12700 mg/kg	1	0	2	0	0	0.32
CP 40 mg/kg	8	0	93	4	31	16.25

Conclusions:

Under the conditions described for this rat bone marrow chromosome aberration assay Alkylate 225 did not reveal genotoxic properties.

Executive summary:

This study similar to the OECD 475 was performed to examine the potential of the test substance to cause chromosome aberrations in vivo. Groups of 18 - 24 male and female rats were given doses of 1200, 4000, or 12700 mg/kg of test substance. Negative control animals were given corn oil (vehicle) only. Positive control animals were given 40 mg/kg cyclophosphamide. Animals were sacrificed at 6, 12, 24, and 48 hours after exposure. The bone marrow cells were then removed and examined for chromosome aberrations. The test substance was toxic (reduced body weight) at the 12700 mg/kg dose in both males and females. No significant increase in chromosomal aberrations was seen in any treatment group. The test substance is not clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro mutagenicity in bacteria:

The read-across to the EPA OPPTS 870.5265 guideline study determining the mutagenicity of the test substance alkylate 215 and 225. Salmonella typhimurium strains TA 1535, TA 100, TA 1537, and TA 98 were exposed to concentrations of 10, 40, 200, 1,000, 3,000, and 10,000 ug/plate of test substance. DMSO was used as a solvent. 9-aminoacridine, sodium nitrite, benzo(a)pyrene, 2 -acetylaminofluorene, and 2 -nitrofluorene were used as positive controls substances. No reproducible, dose-response related positive results were seen in the treatment groups. A significant increase in revertants per plate was seen in the positive controls. The test substance is not mutagenic.

In vitro mutagenicity in mammalian cells:

The read-across to the OECD 476 guideline study. This study examined the potential of the alkylates 215 and 225 to cause mutations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.1 to 2 mg/mL of test substance both in the presence and absence of metabolic activation. Ethanol was used as a vehicle. Ethylmethanesulfonate, benzo(a)pyrene, and dimethylnitrosamine were used as positive controls. Cytotoxicity was seen at concentrations of 1.0 mg/mL and above with metabolic activation and 0.25 mg/mL and above without metabolic activation. No significant increase in mutation frequencies was seen in treatment groups. The test substance is not mutagenic to mammalian cells.

In vivo mutagenicity:

In the OECD 474 guideline study, groups of 15 female and 15 male mice were given a single dose of test substance to determine the effect on bone marrow. Five animals of each sex were sacrificed at 24, 48, and 72 hours after exposure. Similar groups were exposed to cyclophosphamide (positive control), and water (negative control). All animals in the positive control group were sacrificed at 24 hours. Analysis of the bone marrow showed no significant increase in either micronucleus or polychromatid erythrocytes in the treatment group as compared to negative controls. The test substance is not mutagenic.

The read-across to the OECD 475 guideline study performed with alkylates 215 and 225 which examined the potential of the test substance to cause chromosome aberrations in vivo. Groups of 18 -24 male and female rats were given doses of 1200, 4000, or 12700 mg/kg of test substance. Negative control animals were given corn oil (vehicle) only. Positive control animals were given 40 mg/kg cyclophosphamide. Animals were sacrificed at 6, 12, 24, and 48 hours after exposure. The bone marrow cells were then removed and examined for chromosome aberrations. The test substance was toxic (reduced body weight) at the 12700 mg/kg dose in both males and females. No significant increase in chromosomal aberrations was seen in any treatment group. The test substance is not clastogenic.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.