1,2-dichlorobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several valid Ames test in S.typhimurium, E.coli and S.cerevesia are available. All of them were negative. The reliable chromosome abberation tests were also negative. The sister chromatid exchange assay and the HPRT assay were negative without metabolic activation and positive with metabolic activation.

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9: Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate (TA100)
-S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate (TA1535, WP2 uvr A, TA98, TA1537)
+S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156, 313µg/plate (all strains)

Vehicle / solvent:

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

Untreated negative controls:

other: not examinated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98, and WP2 uvrA), Sodium azide (TA1535) and 9-aminoacridine hydrochloride (TA1537). + S9 mix: 2-Aminoantracene (all strains).

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inibition

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results: No increase in revertant colonies was observed in the test with either the non-activation method (-S9) or activation (+S9)

Genetic effects: Salmonella typhimurium TA100, TA1535, TA98, TA1537

+ ? -

Without metabolic activation:        [ ] [ ] [*]

With metabolic activation:             [ ] [ ] [*]

Genetic effects: Escherichia coli WP2 uvrA

+       ?     -

Without metabolic activation:          [ ]    [ ]   [*]

With metabolic activation:               [ ] [ ] [*]

Table 1. Results of the bacterial reversion of test of o-dichlorobenzene (1st trial) [direct method:-S9 mix]

		Revertant colonies per plate [Mean± SD]
Compound	Dose [µg/plate]	TA100	TA1535	WP2 uvrA	TA98	TA1537
Test substance	0	99 88 91 [93±6]	13 15 14 [14±1]	24 23 24 [24±1]	17 24 23 [21±4]	9 11 8 [9± 2]
	2.44	82 90 95 [89 ± 7]
	4.88	100 86 81 [89 ± 10]
	9.77	100 83 95 [93 ± 9]	17 11 11[13 ± 3]	28 22  33[28 ± 6]	14 20 14 [16 ± 3]	10 7 6 [8 ± 2]
	19.5	92 85 99[92 ± 7]	13  19 14[15 ± 3]	31 23 24[26 ± 4]	17 16 13[15 ± 2]	10 9 8 [9 ± 1]
	39.1	85 95 85[88 ± 6]	12 14 10[12 ± 2]	25 25 29 [26 ± 2]	13 18 13[15 ± 3]	7 7 7 [7 ±0]
	78.1	83* 72* 72*[76 ± 6]	11 12 13[12 ± 1]	21 22 25 [23 ± 2]	17 13 13 [14 ± 2]	6 6 8 [7  ± 1]
	156		10* 6 * 9*[8 ± 2]	25 23 23[24 ± 1]	11* 11* 7*[10 ± 2]	3 * 3* 3* [3 ± 0]
	313		8* 7* 7*[7 ± 1]	15 * 20* 13*[16 ± 4]	6* 2* 6*[5 ± 2]	1 * 3* 2*[2 ± 1]
Positive contrrol		548 541 559^a[549 ± 9]	455 473 469^b[466 ± 9]	134 161 153 ^a[149 ± 14]	644 640 655^c[646 ± 8]	609 599 601^d[603 ± 5]

a) AF-2: 2 -(2 -Furyl)-3 -(5 -nitro-2 -furyl)acrylamide, 0.01µ/plate

b) NaN3: Sodium azide, 0.5µg/plate

c) AF-2: 0.1µg/plate

d) 9 -AA: 9 -Aminoacridine hydrochloride, 80µg/plate

*: growth inhibition was observed

Table 2. Results of the bacterial reversion of test of o-dichlorobenzene (1st trial) [activation method:+S9 mix]

		Revertant colonies per plate [Mean± SD]
Compound	Dose [µg/plate]	TA100	TA1535	WP2 uvrA	TA98	TA1537
Test substance	0	107 102 103[104 ± 3]	16 14 11[14 ± 3]	26 23 25[25 ± 2]	29 27 28 [28 ± 1]	13 14 16[14 ± 2]
	9.77	111 108 110 [110 ± 2]	10 11 10[10 ± 1]	26 31 27 [28 ± 3]	24 28 30 [27 ± 3]	10 14 12[12 ± 2]
	19.5	112 117 133[121 ± 11]	14 14 11[13 ± 2]	22 26 27 [25 ± 3]	28 27 27 [27 ± 1]	11 16 10[12 ± 3]
	39.1	127 128 128 [128  ± 1]	16 10 16 [14 ± 3]	33 29 33[32 ± 2]	24 25 27 [25 ± 2]	17 14 17 [16 ± 2]
	78.1	124 135 136[132 ± 7]	14 10 12[12 ± 2]	32 23 27[27 ± 5]	29 31 30[30 ± 1]	10 11 15[12 ± 3]
	156	114 * 119* 138*[124 ± 13]	10* 7* 9*[9 ± 2]	28 35 35[33 ± 1]	33* 33* 34*[33 ± 1]	15* 8* 12*[12 ± 4]
	313	74* 90*91*[85 ± 10]	7* 10* 10*[9 ± 2]	30* 28*28*[29 ± 1]	14* 16* 15*[15 ± 1]	17* 11* 13*[14 ± 3]
Positive control		775 806 840^a[807 ± 33]	361 392 364^b[372 ± 17]	813 833 816^c[821 ± 11]	489 467 403^d[453 ± 45]	179 181 173^b[178 ± 4]

a) 2 -AA: 2 -Aminoanthracene, 1 µg/plate

b) 2 -AA, 2 µg/plate

c) 2 -AA , 10 µg/plate

d) 2 -AA, 0.5 µg/plate

*: Growth inhibition was observed

Table 3. Results of the bacterial reversion of test of o-dichlorobenzene (2nd trial) [direct method:-S9 mix]

		Revertant colonies per plate [Mean± SD]
Compound	Dose [µg/plate]	TA100	TA1535	WP2 uvrA	TA98	TA1537
Test substance	0	104 103 108 [105 ± 3]	13 17 10 [13  ± 4]	30 31 25[29  ± 3]	21 17 18 [19  ± 2]	8 9 7 [8 ± 1]
	2.44	114 95 103 [104 ± 10]
	4.88	91 113 117[107 ± 14]
	9.77	123 108 117 [116 ± 8]	8 10 12[10 ± 2]	28 31 32[30 ± 2]	14 16 17[16 ± 2]	11 13 8[11 ± 3]
	19.5	132 88 116[112 ± 22]	11 12 11[11 ± 1]	33 30 32[32 ± 2]	17 18 15[17 ± 2]	11 11 11[11 ± 0]
	39.1	86 123 112[107  ± 19]	8 14 14[12 ± 3]	37 31 32[33 ± 3]	14 14 15 [14  ± 1]	9 10 9[9 ± 1]
	78.1	103* 92* 97*[97 ± 6]	10 9 12 [10 ± 2]	25 26 26[26  ± 1]	11 14 13 [13 ± 2]	10 11 13[11 ± 2]
	156		8 * 8* 9*[8 ± 1]	30* 35* 31*[32 ± 3]	17* 12* 12*[14 ± 3]	6* 5* 6*[6 ± 1]
	313		5* 8* 3*[5 ± 3]	17* 15* 14*[15 ± 2]	1* 5* 5*[4 ± 2]	1* 3* 2*[2 ± 1]
Positive control		600 554 590^a[581 ± 24]	434 488 449^b[457 ± 28]	158 153 154^a[155 ± 3]	540 544 541^c[542 ± 2]	593 592 579^d[588  ± 8]

a) AF-2: 2 -(2 -Furyl)-3 -(5 -nitro-2 -furyl)acrylamide, 0.01µg/plate

b) NaN3: Sodium azide, 0.5µg/plate

c) AF-2: 0.1µg/plate

d) 9 -AA: 9 -Aminoacridine hydrochloride, 80µg/plate

*: growth inhibition was observed

Table 4 Results of the bacterial reversion test of o-dichlorobenzene (2nd trial) [activation method:+S9 mix]

		Revertant colonies per plate [Mean± SD]
Compound	Dose[µg/plate]	TA100	TA1535	WP2 uvrA	TA98	TA1537
Test substance	0	108 98 100[102 ± 5]	14 17 18[16 ± 2]	28 29 28[28 ± 1]	32 31 33[32 ± 1]	11 10 12[11 ± 1]
	9.77	100 118 91[103 ± 14]	10 14 12 [12 ± 2]	33 25 36 [31 ± 6]	33 30 28 [30 ± 3]	15 13 14[14 ± 1]
	19.5	115 134 118 [122 ± 10]	13 15 18 [15  ± 3]	42 30 40 [37  ± 6]	39 43 42[41 ± 2]	11 14 13[13 ± 2]
	39.1	147 137 112[132 ± 18]	12 13 12[12 ± 1]	23 28 29[27 ± 3]	30 35 29[31 ± 3]	14 14 13[14 ± 1]
	78.1	123 136 138 [132 ± 8]	15 10 12[12 ± 3]	32 38 32 [34 ± 3]	39 36 44[40  ± 4]	13 13 10[12 ± 2]
	156	152 * 155* 132*[146 ± 13]	8* 13* 11*[11 ± 3]	29 34 29 [31 ± 3]	38 * 40* 33*[37 ± 4]	11* 10* 9*[10 ± 1]
	313	115* 74* 79*[89 ± 22]	8* 7* 5*[7 ± 2]	36* 32* 32*[33 ± 2]	17* 19* 18*[18 ± 1]	13* 10 * 9*[11 ± 2]
Positive control		790 779 783^a[784 ± 6]	373 395 382^b[383 ± 11]	756 816 828^c[800 ± 39]	502 499 480^d[494 ± 12]	162 168 164^b[165 ± 3]

a) 2 -AA: 2 -Aminoanthracene, 1 µg/plate

b) 2 -AA, 2 µg/plate

c) 2 -AA , 10 µg/plate

d) 2 -AA, 0.5 µg/plate

*: Growth inhibition was observed

Conclusions:

Interpretation of results: negative

Executive summary:

Masumori et al., 2001

The mutagenicity of 1,2-dichlorobenzene in was examinated in Salmonella typhimurium, and Escherichia coli WP2 uverA. 5 strains TA98, TA 1537, TA 100 and TA 1535 were used for detection of mutagens that cause frameshift mutations.1,2-dichlorobenzene was tested at the following concentrations:

-S9 mix: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate (TA100)

-S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate (TA1535, WP2 uvr A, TA98, TA1537)

+S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156, 313µg/plate (all strains).

In this study 1,2-dichlorobenzene have been reported to be non-mutagenic in the strains tested. The test was conducted according to OECD Guideline 471 and Japanese "Guidelines for Screening Mutagenicity Testing Of Chemicals" without restrictions restrictions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study with restrictions (often 100 instead of 200 cells scored; negative result was not confirmed by testing with continuous treatment, but test was already conducted longer as required and cell cycle was not delayed according to SCE test results; number of gaps not recorded)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

; often 100 instead of 200 cells scored; negative result was not confirmed by testing with continuous treatment, but test was already conducted longer as required and cell cycle was not delayed according to Sister Chromatid Exchange test results; number

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Stocks of CHO cells were mantained at 37°C in McCoy's 5A (modified) medium (KC Biological, Lenexa, KS) buffered with 20 mM HEPES (Aldrich Chemical Co., Milwaukee, WI) and supplemented with 10% fetal bovine serum (KC Biological and Hazleton, Denver, PA), 2mM L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin (Gibco, Grand Island, NY).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal fraction (S9), was prepared from Aroclor 1254-induced Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.

Test concentrations with justification for top dose:

0, 20.2, 60.5, 202.0 µg/mL (without metabolic activation) and 0, 20.2, 60.5, 152.0, 202.0 ug/mL (with metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen after solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC) was used in the experiment without metabolic activation, and cyclophosphamide (CP) was used in the experiments with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 8 hours; In experiments with metabolic activation, cultures were exposed to 1,2-dichlorobenzene in serum-free medium with S9 and cofactors for 2 hours prior to exposure.
- Fixation time (start of exposure up to harvest of cells): 10 hours (without metabolic activation); 12.5 hours (with metabolic activation)

SPINDLE INHIBITOR (cytogenetic assays): colcemid (10-6 M) added 2-2.5 hours prior to harvest
STAIN (for cytogenetic assays): in 5% Giemsa for 5 min.

NUMBER OF CELLS EVALUATED: 100 cells, with exemption of two positive controls (50 cells for 5.0 µg/mL of MMC; 50 µg/mL of CP) and one tested concentration with metabolic activation (60.5 µg/mL of 1,2-dichlorobenzene).

DETERMINATION OF CYTOTOXICITY
- Method: other: confluence of the cell monolayer in treated flasks in comparison with control flask and noting the presence of mitotic cells.

Evaluation criteria:

Cells were analyzed for different categories of chromosomal aberrations:
- simple: defined as a chromatid gap, break, fragment, and deletion or chromosome gap, break, or double minutes;
- complex: defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes;
- other: defined as pulverized chromosomes or cells with greater than 10 aberrations.
Chromatid and chromosome gaps were recorded but were not used in the analysis. The frequency of polyploid or endoreduplicated cells was noted only when it seemed excessive; however, these categories were not included in the totals or in the statistical analyses.

Statistics:

All categories of aberrations (simple, complex, other) were combined for the statistical analysis, which was based on the percent of total cells with aberrations. The percent of cells with aberrations was used for the analysis, rather than the average number of aberrations per cell.

A binominal sampling assumption was used to examine absolute increases in aberrations over solvent levels at each dose. The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. Only the "total" percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the highest dose level was toxic for the cells

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Test concentrations were empirically chosen based on toxicity and cell delay as noted in the SCE experiments.

Table 1: Summary for chromosome aberration (AB).

Chemical name	Solvent	Chromosome Aberration
1,2 -Dichlorobenzene	DMSO	Results	-S9	+S9
		HT	S	S
		HTD	202 T	202 T

-S9 = without metabolic activation; +S9 = with metabolic activation.

HT = harvest time.

S = standard harvest (10 -12 hours).

HTD = highest dose tested.

T = Toxic

Table 2: Data from AB experiments without metabolic activation

Dose	Cells	Percent Cells With Aberrations
[µg/mL]		Total	Simple	Complex
0.0	100	1.00	1.00	0.00
20.2	100	4.00	3.00	1.00
60.5	100	3.00	3.00	0.00
202.0	100	1.00	1.00	0.00
Positive control - MMC
1.0	100	25.00	22.00	6.00
5.0	50	44.00	32.00	16.00

Trend stastistic: -0.15E+00

Trend probability: 0.56E+00

Cells with pulverized chromosomes or >10 aberrations/cell are included in "total".

Table 3: Data from AB experiments with metabolic activation

Dose	Cells	Percent Cells With Aberrations
[µg/mL]		Total	Simple	Complex
0.0	100	2.00	1.00	0.00
20.2	100	2.00	3.00	0.00
60.5	200	6.00	3.00	0.00
152.0	100	6.00	1.00	2.00
202.0	100	3.00	3.00	0.00
Positive control - CP
50.0	50	44.00	28.00	24.00

Trend statistic: 0.13E+01

Trend probability: 0.10E+00

Cells with pulverized chromosomes or >10 aberrations/cell are included in "total".

Conclusions:

Interpretation of results: negative

Chromosomal aberrations in Chinese hamster ovary cells with and without exogenous metabolic activation leaded to a negative result.

Executive summary:

Loveday et al., 1990

1,2-Dichlorobenzene was tested for its ability to induce chromosome aberrations (AB) in cultured Chinese hamster ovary (CHO) cells with or without exogenous metabolic activation. The test was conducted similar to OECD Guideline 473 and was regarded as reliable with the following restrictions: often 100 instead of 200 cells scored; negative result was not confirmed by testing with continuous treatment, but test was already conducted longer as required and cell cycle was not delayed according to Sister Chromatid Exchange test results; number of gaps not recorded.

CHO cells were exposed to the test or control chemicals for 10 -12 hours at the following concentrations: 0, 20.2, 60.5, 202.0 µg/mL (without metabolic activation) and 0, 20.2, 60.5, 152.0, 202.0 µg/mL (with metabolic activation).

Toxicity was determined by estimating the percent of confluence of the cell monolayer in treated flasks in comparison with control flask and noting the presence of mitotic cells. The highest dose level of 1,2 -dichlorobenzene was toxic for cells.

Cells were analyzed for different categories of chromosomal aberrations:

- simple: defined as a chromatid gap, break, fragment, and deletion or chromosome gap, break, or double minutes;

- complex: defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes;

- other: defined as pulverized chromosomes or cells with greater than 10 aberrations.

Chromatid and chromosome gaps were recorded but were not used in the analysis.

Chromosomal aberrations in Chinese hamster ovary cells with and without exogenous metabolic activation leaded to a negative result.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study with restrictions (it is not mentioned if duplicate cultures have been used; number of cell cultures per experimental point, number of methaphases analysed, mean number of SCE per chromosome and the statistical evaluation were not reported)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

It is not mentioned if duplicate cultures have been used. The number of cell cultures per experimental point, the number of methaphases analysed, the mean number of SCE per chromosome and the statistical evaluation were not reported.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Stocks of CHO cells were mantained at 37°C in McCoy´s 5A (modified) medium (KC Biological, Lenexa, KS) buffered with 20 mM HEPES (Aldrich Chemical Co., Milwaukee, WI) and supplemented with 10% fetal bovine serum (KC Biological and Hazleton, Denver, PA), 2mM L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin (Gibco, Grand Island, NY).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal fraction (S9), was prepared from Aroclor 1254-induced Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.

Test concentrations with justification for top dose:

0, 5.9, 19.7, 59 µg/mL (without metabolic activation) and 0, 300, 400, 500 ug/mL (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen after solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC) was used in the experiment without metabolic activation, and cyclophosphamide (CP) was used in the experiments with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 2 hours in experiments with metabolic activation and 26 hours in experiments without metabolic activation (2 hours incubation with test substance plus 24 hours incubation with BrdU)
- Fixation time (start of exposure up to fixation or harvest of cells): 28-28.5 hours


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Hoechst 33258 (fluorescence).


DETERMINATION OF CYTOTOXICITY
- Method: other: confluence of the cell monolayer in treated flasks in comparison with control flask and noting the presence of mitotic cells.

Evaluation criteria:

See Any other information on materials and methods incl. tables

Statistics:

Not reported. The procedures for evaluation of test data have been modified from those described in detail by Galloway SM et al 1985, Environ Mutagen 7:1-51; Margolin BH et al, 1985, Environ Mutagen 7:48, and described by Galloway SM et al 1987, Environ Mol Mutagen 10:1-175.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

other: a negative response was observed without metabolic activation, a positive response was observed with metabolic activation

Cytotoxicity / choice of top concentrations:

other: the highest dose level was toxic for the cultures without metabolic activation and was not toxic for the cultures with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: For the SCE assays, a series of dilutions were made from the stock solution to achieve 10 test concentrations in a half-log series covering a range of five logs. The highest dose was used was based on solubility or toxicity, with the highest dose scored being that allowing sufficient M2 cells for analysis at the time of harvest. In the absence of limitations on solubility or toxicity, the maximum test chemical concentration was 5 mg/mL.

Table 1. Summary for sister chromatid exchange (SCE).

Chemical name	Solvent	SCE
1,2 -Dichlorobenzene	DMSO	Results	-	+
		HT	S	S
		HTD	59T	59

-S9: without metabolic activation; +S9: with metabolic activation.

HT =harvest time.

S= standard harvest. For SCEs "S" was 25 -29 hours after addition of BrdUrd.

HTD =highest dose tested

T= Toxic

Table 2. Data from SCE experiment without metabolic activation

Dose µg/mL	Total chromosomes	Total SCE	SCE per cell
0.0	1045	332	6.64
5.9	1048	345	6.90
19.7	1048	317	6.34
59.0	1047	330	6.60
Positive control -MMC
0.0015	1052	569	11.38
0.0100	208	334	33.40

Trend stastistic: -0.46E+00

Trend probability: 0.68 +00

Table 3. Data from SCE experiment with metabolic activation 1

Dose µg/mL	Total chromosomes	Total SCE	SCE per cell
0.0	1042	363	7.26
19.7	1042	432	8.64
59.0	1048	449	8.98*
197.0	1042	445	8.90*
Positive control -CP
0.4	1040	681	13.62
2.5	210	353	35.30

Trend statistic:0.28E+01

Trend probability: 0.24E-02

* 20% increase of SCE/chromosome above control levels

Table 4. Data from SCE experiment with metabolic activation 2

Dose µg/mL	Total chromosomes	Total SCE	SCE per cell
0	1044	424	8.48
300	1048	534	10.68*
400	1048	519	10.38*
500	1048	503	10.06
Positive control-CP
0.4	1048	745	14.90
2.5	208	403	40.30

Trend statistic:0.22E+01

Trend probability:0.14E-01

* 20% increase of SCE/chromosome above control levels

Conclusions:

Interpretation of results:
negative without metabolic activation
positive with metabolic activation

Executive summary:

Loveday et al., 1990

1,2-Dichlorobenzene was tested for its ability to induce sister chromatid exchanges (SCEs) in cultured Chinese hamster ovary (CHO) cells with or without exogenous metabolic activation.

CHO cells were exposed to the test or control chemical for 2 hours at the following concentrations: 0, 5.9, 19.7, 59, 197 µg/mL (without metabolic activation), and 0, 300, 400, 500 µg/mL (with metabolic activation).

Toxicity was determined by estimating the percent of confluence of the cell monolayer in treated flasks in comparison with control flask and noting the presence of mitotic cells.

The highest dose level of 1,2 -dichlorobenzene was toxic for the cultures without metabolic activation and was not toxic for the cultures with metabolic activation.

A negative response was observed without metabolic activation, a positive response was observed with  metabolic activation.

The test was performed according to OECD guideline 479 with restrictions (it is not mentioned if duplicate cultures have been used; number of cell cultures per experimental point, number of methaphases analysed, mean number of SCE per chromosome and the statistical evaluation were not reported).

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:

other: Comparable to guideline study with restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

No analitical purity reported. Number of cell cultures was not reported

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fisher´s or RPMI 1640 media were used to maintain the laboratory cultures. Treatment medium was always Fischer´s growth medium with the serum content reduced to 5% by volume, even in experiments where RPMI 1640 medium was used for the colture stock nad for the expression and clonig phases of mutation experiment. Growth medium consisted of Fisher´s medium supplemented with heat-treated horse serum (10% V/V) , 110µg/mL sodium pyruvate, 2 mM L-glutamine , 0.05% Pluronic F68 (nonionic surfactant), and pen-strep (95 U/mL, 95µg/mL).
Cloning medium consisted of growth medium with addition of 0.35% to 0.40% Noble agar (to obtain a soft consistency) , for the cloning efficency dishes, plus 3µg/mL of TFT for the mutant selection dihses, -- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix. S9 was prepared from the livers of Aroclor 1254-induced male Fischer 344 rats as previously described (Myhr et al., 1985, Prog Mutat Res 5:555-568). S9 mix was prepared just prior to use by combining volumes of S9, phosphate-buffered saline (PBS).

Test concentrations with justification for top dose:

2.5-100 nL/mL (3.25-130 ug/ml ; without metabolic activation) and 5-60 nL/mL (6.5-78 ug/ml ; with metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Preliminary studies of test chemical solubility and cytotoxicity were conducted prior to performing the first mutation experiment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methylmethanesulfonate (MMS) in experiment without S9 mix. 3-methylcholanthrene (MCA) for experiment with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2-day expression and growth period.
- Selection time (if incubation with a selection agent): 11-12 days




NUMBER OF REPLICATIONS: 2 or 3 replication for each dilutions


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (CE); relative total growth (RTG)





SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

Evaluation criteria:

See Table I in any other informations on materials and methods incl. tables.

Statistics:

See any other informations on materials and methods incl. tables.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

other: Without S9 activation, 1,2-dichlorobenzene was evaluated as non-mutagenic. With S9 activation mutagenic activity was detected. See remarks

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see remarks

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: 1,2-dichlorobenzene is apparently soluble in culture medium for the assayed dose range. This could indicate an association with serum protein/lipid or an unnoticed suspension of tiny droplets. Variability in dosing or interaction with the cells was indicated by the lethality of 40 nl/ml in the first trial experiment and the survival at 80-100 nl/mL in the next two trial experiments.

Without S9 activation, 1,2 -dichlorobenzene was evaluated as nonmutagenic, although a hint of some activity was indicated in two trials in the high dose range of 80 -100 nl/mL. An interaction between S9 mix and 1,2 -dichlorobenzene occurred such that mutagenic activity was detected at the lowest assayed dose of 5 nl/mL intwo trials. Approximately twofold increases in MF were obtained for an average RTG of about 80% (one culture with a low CE excluded from Trial 1). Successive increases in dose resuklted in lager MFs, which reached 6 -7.5 -fold at 40 -60 nl/mL. Both trials yielded very similar results (if the cultures with low CE vlaues at 5 and 20 nl/mLin trial 1 are excluded). It was concluded that induced rat liver S9 mix activates 1,2 -dichlorobenzene to mutagenic substance(s).

Analyses of the induced mutant colony size showed a large preferential increasein the small colony population (Table 2).

Table 2 Experimental data for 1, 2 -dichlorobenzene

-S9 Trial 1				Eval: -		- S9  Trial 2				Eval: ?		-S9 Trial 3				Eval -		+S9 Trial 1				Eval: +		+S9Trial 2				Eval:+
Conc.NL/ML	CE	RTG	MC	MF	AVGMF	ConcNL/ML	CE	RTG	MC	MF	AVGMF	ConcNL/ML	CE	RTG	MC	MF	AVGMF	ConcNL/ML	CE	RTG	MC	MF	AVGMF	ConcNL/ML	CE	RTG	MC	MF	AVGMF
ETOH	73	69	64#	29		ETOH	79	118	112	47		ETOH	78	92	62	27		ETOH	70	138	102	49		ETOH	86	92	200	77
0	97	130	57#	20		0	93	90	142	51		0	99	130	82	28		0	62	112	148	80		0	82	100	160	65
	82	101	60	24	24		107	100	145	45			91	94	91	33			62	87	98	53			87	104	146	56
2.5	77	88	54	23			89	92	107	40	46		91	84	80	29	29		A75	64	156	69	63		87	104	179	68	67
	86	66	35	14	18	20	94	85	120	42		30	79	68	118	50		5	17	25	75	149		5	79	64	326	138
5	67	66	39	19			93	74	125	45			86	58	89	34			44	75	170	129			64	102	364	189
	73	80	40	18	19		102	98	107	35	41		89	62	98	37	40		60	78	186	103	127		65	76	290	149	159
10	89	66	45	17		30	85	90	90	35		40	83	67	59	24		10	46	75	204	147		10	78	72	373	159
	65	59	42	21	19		75	72	88	39			84	78	71	28			33	38	179	182			64	57	378	196
	C	---	38	---			91	70	107	39	38		113	54	70	21	24		39	55	200	170	166		71	62	333	157	171
20	64	78	53	27		40	93	69	159	57		50	91	62	89	33		20	39	29	453	391		20	80	50	565	237
	84	63	36	14			115	174	140	41			61	42	59	32			15	15	288	647			67	45	534	267
	80	73	51#	21	21		113	75	205	61	53		68	45	70	34	33		45	30	302	222	420		78	43	573	244	249
30	87	33	44	17		50	137r	78	179	43r		60	91	44	57	21		30	48	38	146	101		30	53	23	564	354
	61	30	33	18			132r	70	161	41r			67	44	59	29			34	22	229	223			63	24	680	363
	74	31	58#	26	20		107	57	143	45			73	38	93	43	31		38	17	358#	315	213		A50	8	745	497	404
40	lethal			---		60	114	78	114	33		80	88	66	90	34		40	34	10	410	398		40	39	13	616	533
	lethal			---			100	57	148	49			119	36	79	22			53	18	468	293	346		47	15	658	468	501
	lethal			---			110	66	155	47	43		113	118	80	24	27	60	31	13	381	414		60	lethal			---
MMS5 NL/ML	82#	88	489	199		80	76	40	198	87		100	91	6	81	30			A42	14	477	380			lethal			---
	84	97	531	210	205		87	19	209	80	84		94	10	97	34			38	11	391	343	379		lethal			---
							lethal			---			85	6	140	55	40
Notes:F,b						100	lethal			---		MMS5NL/ML	75	50	514	228		MCA2.5 µg/mL	63	46	499	263		MCA 2.5 µg/mL	57	62	404	236
							lethal			---			74	41	376	169			52	45	634	406	335		41#	55	385	314
							lethal			---			66	40	373	188	195								62	48	682	36	305
						MMS5NL/ML	75	82	582	258		NOTES	R					NOTES:F,b
							69	55	393	190								S9:	LOT	RLI130	15µL/mL			S9:	LOT	RLI 130	15µL/ML
							77	63	581	250	233							Significant trend						Significant trend
						NOTES	R

-S9: Experiments performed without the addition of S9 mix

+S9: Experiments performed with the inclusion of S9 mix

ETOH: negative control containing 1% ethanol as solvents for the test chemical.

CE: Cloning efficiency in soft agar

RTG Relative total growth

MC Total numeber of mutant colonies in three dishes.

MF Mutant frequency expressed as mutants per 10⁶ cells.

MF Underlined average mutant frequency is greater than the average solvent control value at p=0.05.

LETHAL Lethal or excessively toxic treatment

r Rejected value according to the quality control table 1 in any other informations on materials and methods incl. tables

# Loss of one culture from set, usually due to mold contamination

C Loss of sample set due to contamination

A Culture analyzed for the proportion of large and small mutant colonies

R RPMI 1640 medium used for growth, expression, and cloning

F Fischer´s medium used for growth, expression, and cloning

a Horse serum used at 10% v/v during treatment rather than at 5% v/v

b Horse serum used at 20% v/v for soft agar cloning rather than at 10% v/v.

Conclusions:

Interpretation of results:
negative without metabolic activation
positive with metabolic activation

Executive summary:

Myhr BC and Caspary WJ, 1991.

1,2 -dichlorobenzene was tested for mutagenicity at the thymidine kinase locus in L5178Y mouse lymphoma cells according to OECD guideline 476 (In vitro Mammalian Cell Gene Mutation Test) with deviations (analytical purity and number of cell cultures was not reported).

Cultures were exposed to 2.5-100 nL/mL (3.25-130 µg/ml; without metabolic activation) and 5-60 nL/mL (6.5-78 ug/ml; with metabolic activation) of 1,2-dichlorobenzene for 4 hours, then cultured for 2 days before plating in soft agar medium with 3µg/mL trifluorothymidine (TFT).

Without S9 activation, 1,2-dichlorobenzene was evaluated as nonmutagenic, although a hint of some activity was indicated in two trials in the high dose range of 80 -100 nl/mL. An interaction between S9 mix and 1,2-dichlorobenzene occurred such that mutagenic activity was detected at the lowest assayed dose of 5 nl/mL in two trials. Approximately twofold increases in MF were obtained for an average RTG of about 80% (one culture with a low CE excluded from Trial 1). Successive increases in dose resulted in lager MFs, which reached 6- to 7.5-fold at 40-60 nl/mL. Both trials yielded very similar results (if the cultures with low CE values at 5 and 20 nl/mL in trial 1 are excluded). It was concluded that induced rat liver S9 mix activates 1,2-dichlorobenzene to mutagenic substance(s). Analyses of the induced mutant colony size showed a large preferential increase in the small colony population.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In the MNT (Charles River, 2018) 1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). The negative result is consistent with an earlier MNT (Shelby, 1993).

There are 2 in vivo Comet Tests available (Charles River, 2018a and 2018):

Two in vivo Comet tests are performed with 1,2-Dichlorobenzene on systemic organs liver and bone marrow (bone marrow only 1. experiment with negative result) and local tissues stomach and duodenum. Clearly negative results are observed for the systemic organs in all tests. The results for stomach and duodenum gave “equivocal” results in one test and negative results in the other test. Based on the arguments mentioned above we consider 1,2-Dichlorobenzene as not genotoxic and any remaining uncertainty concerning the “equivocal” results as low. In addition, the “equivocal” observations are not relevant for human risk assessment because they were seen after high oral doses of 250 and 500 mg/kg/day in a local gastric tissue directly exposed to high oral gavage doses. When evaluating the results of the Comet Assays and the relevance for human risk assessment it should be also considered that worker exposure is very unlikely.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study has been requested by the European Chemicals Agency (ECHA Decision SEV-D-2114325960-51-01/F.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Principles of method if other than guideline:

The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Purity/Composition: 99.9%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). The animals were provided by Charles River, Sulzfeld, Germany.

Sex:

male

Details on test animals or test system and environmental conditions:

Test System

The study plan of this type of study was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). The animals were provided by Charles River, Sulzfeld, Germany.
Young adult animals were selected (9-11 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 4 and in the main study 25. In the main study 5 male rats were treated per sampling time in each treatment group.
The body weights of the rats at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights were 274.2 ± 9.3 g and the range 256 – 289 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
The acclimatization period was at least 6 days before the start of treatment under laboratory conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.

Husbandry

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19.3 to 20.2°C with an actual daily mean relative humidity of 47 to 70%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Accommodation
Group housing of maximum 5 animals in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material.
Diet
The animals had free access to pelleted rodent diet. Results of analyses for nutrients and contaminants of each batch were examined and archived.
Water
The animals had free access to tap-water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

Preparation of Test Item
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item was dissolved corn oil. The specific gravity of corn oil is 0.9 g/ml. The density of corn oil was taken into account when preparing the formulations.
The test item concentrations were dosed within 3 hours after preparation.

Details on exposure:

The rats were dosed for three consecutive days (once daily) using an oral intubation of a maximum tolerated (high), an intermediate and a low dose of the test item. The rats were dosed twice with the positive control EMS (Ethyl Methane sulfonate) and once with CP (cyclophosphamide).
A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat). The route of administration of the test item was selected taking into account the possible route of human exposure during manufacture, handling and use.
The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=45 h, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 and t=45. The animals were sacrificed at by abdominal aorta bleeding under isoflurane anesthesia at t=45h.
The dosing volume was 10 ml/kg.
1,2-Dichlorobenzene concentrations were used within 3 hours after preparation.
CP is the positive control for the MN assay, and EMS the positive control for the Comet assay.

Duration of treatment / exposure:

The animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia at t=45h.

Frequency of treatment:

The rats were dosed for three consecutive days (once daily).

Post exposure period:

None

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Dose / conc.:

125 mg/kg bw/day

Remarks:

1,2-dichlorobenzene

Dose / conc.:

250 mg/kg bw/day

Remarks:

1,2-dichlorobenzene

Dose / conc.:

500 mg/kg bw/day

Remarks:

1,2-dichlorobenzene

Dose / conc.:

200 mg/kg bw/day

Remarks:

EMS
Comet assay

No. of animals per sex per dose:

5 male animals/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

EMS (Ethyl Methane sulfonate)

Tissues and cell types examined:

The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum tolerated dose in accordance with current regulatory guidelines,by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach.

Details of tissue and slide preparation:

Approximately 3-5 hours after the third treatment with the test item bone marrow was isolated for the micronucleus test. In addition bone marrow, liver, duodenum and stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.
The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom).
One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides it was not possible to score 50 comets per agarose circle, therefore the remaining number of comets were scored on an additional agarose circle.

Evaluation criteria:

Acceptability of the Comet Assay
The in vivo comet is considered acceptable if it meets the following criteria:
a) The percentage tail intensity of the solvent control should reasonably be within the laboratory historical control data range.
b) The positive control EMS should produce at least a statistically significant (one-sided, p < 0.05) increase in the percentage Tail Intensity compared to the negative control treated animals using the Student’s t-test.

Statistics:

A test item is considered as giving a positive result in the Comet assay if the following criteria are met:
a) A statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase (indication of strand breaks) in percentage Tail Intensity is detected.
A test item is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
a) None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity.
All data was normally distributed thus no transformation (y = 1/y) of the data was necessary.
A trend test was performed for liver and stomach.

Key result

Sex:

male

Genotoxicity:

other: No genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a dose-range finding study 4 animals (group A: 3 males and group B: 1 male) were dosed via oral gavage with 500 and 750 mg/kg body weight of the test item (group A and B, respectively). The animal dosed with 750 mg/ kg body weight showed generally treatment related clinical signs of severe intensity after dosing and didn’t recover before the next dosing. The clinical signs comprised of lethargy, ataxia, rough coat, hunched posture, ventral recumbancy, tremors and not all feed provided overnight was eaten. A dose of 750 mg/kg/day was therefore considered as too high as top dose for the main study.
Three males were dosed with 500 mg/kg body weight. All animals showed clear clinical signs. The effects were generally of slight intensity and comprised lethargy, rough coat, ataxia, hunched posture, ventral recumbency and not all feed provided overnight was eaten. A dose of 500 mg/kg/day was therefore considered as the maximum tolerated dose as defined in the genotoxicity OECD 489 and 474 guideline and selected as top dose for the main study.

RESULTS OF DEFINITIVE STUDY
None of the animals in the vehicle control group, positive control groups and group treated with 125 mg/kg 1,2-dichlorobenzene showed abnormal clinical signs with exception of two animals in the positive control group of the comet assay treated with 200 mg/kg EMS which had a rough coat after the second dosing.
Animals treated with 250 and 500 mg/kg 1,2-dichlorobenzene showed the following signs: lethargy, ataxia (500 mg/kg only), rough coat, hunched posture and not all feed provided overnight was eaten.

Comet Test:
The mean Tail Intensity in bone marrow, liver, duodenum and stomach was all well within the historical control data range. Moreover, the positive control EMS induced a statistically significant increase in the Tail Intensity in bone marrow, liver, duodenum and stomach. Overall it was concluded that the comet assay in bone marrow, liver, duodenum and stomach was valid.
No statistically significant increase in the mean Tail Intensity (%) was observed in bone marrow and duodenum of test item treated male animals compared to the vehicle treated animals. In addition, no statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of 1,2-dichlorobenzene-treated male animals at a dose of 125 and 250 mg/kg/day compared to the vehicle treated animals. Only at 500 mg/kg a statistically significant (slight) increase in the Tail Intensity was observed (2.1-fold increase, p<0.05 Dunnett’s t test) from 2.35% in the vehicle control to 4.87% at 500 mg/kg. A trend test was performed and a statistically significant trend was observed (p<0.05, non-parametric trend analysis by contrast). In stomach cells the 125 and 250 mg/kg treatment group showed a significant increase in mean Tail Intensity (%) from 23.85% in the vehicle control to 57.99% at 125 mg/kg (2.0-fold increase) and to 56.72% (2.4-fold increase) at 250 mg/kg (p<0.05 Dunnett’s t test). At 500 mg/kg no statistically increase was observed. A trend test was performed and a statistically significant trend was observed (p<0.05, non-parametric trend analysis by contrast).

Histopathology
All rats were necropsied and the part of the liver, stomach and duodenum was fixed. Microscopic examination was performed on the liver and stomach of all animals since an increase in DNA damage was observed in these two organs after test item treatment.
Test item-related microscopic findings were present in the

Liver:
• Centrilobular necrosis was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to marked degree.
Centrilobular mononuclear inflammatory cell infiltrate was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to moderate degree.

Glandular stomach:
• Hypertrophy parietal cells was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to slight degree.
Of these findings, the centrilobular necrosis in the liver is the only finding representing cell death and causing increased DNA damage.

•  No increase in DNA damage was observed in the comet assay in bone marrow, and duodenum of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In liver cells a slight increase in DNA damage in the comet assay was observed at 500 mg/kg from 2.35% in the vehicle control to 4.87% at 500 mg/kg. Histopathology showed that at this dose centrilobular necrosis was present up to a marked     degree.  Therefore it can be concluded that the DNA damage observed at 500 mg/kg is caused by necrosis.  Moreover, the Tail Intensity of 4.87% is clearly with in the historical data control range of the comet assay in liver. Thus overall it can be concluded for liver that necrosis is present at a dose of 500 mg/kg but that no genotoxic effect in liver is observed up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In stomach a statistical significant increase in DNA damage was observed at 125 and 250 mg/kg. But since no increase was observed at 500 mg/kg, no dose response was observed.  Histopathological examination showed no necrosis in stomach at any of the tested concentrations.  Thus DNA damage was observed after treatment but since this effect showed no dose response, the overall conclusion is equivocal and additional investigation in a comet assay in stomach is required.

•  Thus overall it can be concluded that no genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.

Conclusions:

Overall it can be concluded that no genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.

Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach.

As a result

•  No increase in DNA damage was observed in the comet assay in bone marrow, and duodenum of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In liver cells a slight increase in DNA damage in the comet assay was observed at 500 mg/kg from 2.35% in the vehicle control to 4.87% at 500 mg/kg. Histopathology showed that at this dose centrilobular necrosis was present up to a marked degree. Therefore it can be concluded that the DNA damage observed at 500 mg/kg is caused by necrosis. Moreover, the Tail Intensity of 4.87% is clearly with in the historical data control range of the comet assay in liver. Thus overall it can be concluded for liver that necrosis is present at a dose of 500 mg/kg but that no genotoxic effect in liver is observed up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In stomach a statistical significant increase in DNA damage was observed at 125 and 250 mg/kg. But since no increase was observed at 500 mg/kg, no dose response was observed.  Histopathological examination showed no necrosis in stomach at any of the tested concentrations.  Thus DNA damage was observed after treatment but since this effect showed no dose response, the overall conclusion is equivocal.

Thus overall it can be concluded that no genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.

Detailed discussion on glandular stomach:

The mean Tail Intensities in stomach cells are as follows:

	Mean Tail Intensity (%) (1)	S.D.
Vehicle Control	23.85	6.93
1,2-Dichlorobenzene 125 mg/kg	47.99 (2)	14.37
1,2-Dichlorobenzene 250 mg/kg	56.72 (2)	7.10
1,2-Dichlorobenzene 500 mg/kg	34.61	19.60
EMS 200 mg/kg	97.32 (3)	0.68

(1)Five animals per treatment group.

(2) Statistically significant from the control group (Dunnett’s t test, p<0.05)

(3) Significantly different from corresponding control group (Students t test, p < 0.001).

The observations in the stomach are considered to be equivocal because:

1) No clear dose response is observed.

in stomach cells the 125 and 250 mg/kg treatment group showed a significant increase in mean Tail Intensity (%) from 23.85% in the vehicle control to 47.99% at 125 mg/kg (2.0-fold increase) and to 56.72% (2.4-fold increase) at 250 mg/kg (p<0.05 Dunnett’s t test). At 500 mg/kg a statistically non-significant 1.5-fold increase was observed.

2) the Tial Intensity reported is within historical control values.

The mean Tail Intensities of the low, mid and high dose group are within the historical control values and far below the historical positive control values observed with EMS.

Historical data Comet assay Negative control data:

	Stomach Tail Intensity (%) Males and Females
Range	2.45 – 72.13
Mean	35.94
SD	19.88
n	44

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed in 2012 – 2017

Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days):

	Stomach Tail Intensity (%) Males and Females
Range	85.69 – 99.19
Mean	93.53
SD	3.67
n	42

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed in 2012 – 2017

3) Histopathology is observed in the Glandular stomach.

Hypertrophy parietal cells was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to slight degree.

Overall it can be concluded: in the MNT (Charles River, 2017) 1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

In the comet assay no genotoxic effect was observed in bone marrow, duodenum and liver. In stomach, the result of the Comet assay was equivocal and additional investigation is required (Charles River, 2017).

Based on the equivocal results in the Comet assay in the stomach we do propose the following follow-up activities:

Re-conduct a Comet assay in glandular stomach and duodenum in the same strain of rats (Wistar) in another laboratory with more experience and tighter historical control data not as broad as the initial laboratory. In addition, histopathology will be included right from the beginning in the study protocol to set observations into perspective for this irritating compound. The study is scheduled for February 2018 and the audited draft will be available end of March. Based on these results potential tier 2 follow up activities will be considered.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study has been requested by the European Chemicals Agency (ECHA Decision SEV-D-2114325960-51-01/F.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach (see - In Vivo Mammalian Alkaline Comet Assay).

GLP compliance:

yes

Type of assay:

other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Specific details on test material used for the study:

Purity/Composition: 99.9%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). The animals were provided by Charles River, Sulzfeld, Germany.

Sex:

male

Details on test animals or test system and environmental conditions:

Test System

The study plan of this type of study was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). The animals were provided by Charles River, Sulzfeld, Germany.
Young adult animals were selected (9-11 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 4 and in the main study 25. In the main study 5 male rats were treated per sampling time in each treatment group.
The body weights of the rats at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights were 274.2 ± 9.3 g and the range 256 – 289 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
The acclimatization period was at least 6 days before the start of treatment under laboratory conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.

Husbandry

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19.3 to 20.2°C with an actual daily mean relative humidity of 47 to 70%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Accommodation
Group housing of maximum 5 animals in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material.
Diet
The animals had free access to pelleted rodent diet. Results of analyses for nutrients and contaminants of each batch were examined and archived.
Water
The animals had free access to tap-water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

Preparation of Test Item
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item was dissolved corn oil. The specific gravity of corn oil is 0.9 g/ml. The density of corn oil was taken into account when preparing the formulations.
The test item concentrations were dosed within 3 hours after preparation.

Details on exposure:

The rats were dosed for three consecutive days (once daily) using an oral intubation of a maximum tolerated (high), an intermediate and a low dose of the test item. The rats were dosed twice with the positive control EMS (Ethyl Methane sulfonate) and once with CP (cyclophosphamide).
A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat). The route of administration of the test item was selected taking into account the possible route of human exposure during manufacture, handling and use.
The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=45 h, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 and t=45. The animals were sacrificed at by abdominal aorta bleeding under isoflurane anesthesia at t=45h.
The dosing volume was 10 ml/kg.
1,2-Dichlorobenzene concentrations were used within 3 hours after preparation.
CP is the positive control for the MN assay, and EMS the positive control fpor the Comet assay.

Duration of treatment / exposure:

The animals were sacrificed at by abdominal aorta bleeding under isoflurane anesthesia at t=45h.

Frequency of treatment:

The rats were dosed for three consecutive days (once daily).

Post exposure period:

None

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Dose / conc.:

125 mg/kg bw/day

Remarks:

1,2-dichlorobenzene

Dose / conc.:

250 mg/kg bw/day

Remarks:

1,2-dichlorobenzene

Dose / conc.:

500 mg/kg bw/day

Remarks:

1,2-dichlorobenzene

Dose / conc.:

200 mg/kg bw/day

Remarks:

EMS
Comet assay

Dose / conc.:

20 mg/kg bw/day

Remarks:

CP
MN assay

No. of animals per sex per dose:

5 male animals/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

EMS (Ethyl Methane sulfonate)
CP (cyclophosphamide).

Tissues and cell types examined:

The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Details of tissue and slide preparation:

Approximately 3-5 hours after the third treatment with the test item bone marrow was isolated for the micronucleus test. In addition bone marrow, liver, duodenum and stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.
Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.

Evaluation criteria:

Acceptable if:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).



































































































































































































































A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) All results are within the 95% control limits of the negative historical control data range.
Data for number of micronuclei was normally distributed thus no transformation (y = 1/y) of the data was necessary. But was normally distributed thus not transformation of the data was necessary. In addition no trend tests were performed.

Statistics:

Positive:
a) At least one of the treatment groups exhibits a statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Results are outside the 95% control limits of the historical control data range.
Negative:
a) None of the treatment groups exhibits a statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Results are within the 95% control limits of the negative historical control data range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with test item compared to the vehicle treated animals.

Additional information on results:

In a dose-range finding study 4 animals (group A: 3 males and group B: 1 male) were dosed via oral gavage with 500 and 750 mg/kg body weight of the test item (group A and B, respectively). The animal dosed with 750 mg/ kg body weight showed generally treatment related clinical signs of severe intensity after dosing and didn’t recover before the next dosing. The clinical signs comprised of lethargy, ataxia, rough coat, hunched posture, ventral recumbancy, tremors and not all feed provided overnight was eaten. A dose of 750 mg/kg/day was therefore considered as too high as top dose for the main study.
Three males were dosed with 500 mg/kg body weight. All animals showed clear clinical signs. The effects were generally of slight intensity and comprised lethargy, rough coat, ataxia, hunched posture, ventral recumbency and not all feed provided overnight was eaten. A dose of 500 mg/kg/day was therefore considered as the maximum tolerated dose as defined in the genotoxicity OECD 489 and 474 guideline and selected as top dose for the main study.

Bone marrow smears were analysed.  No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with test item compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes.

In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database.  Hence, all criteria for an acceptable assay were met.

The groups that were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis.

The highest dose in the study was the maximum tolerated dose showing clear treatment related clinical signs indicating (indirect) proof of exposure. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

 

Conclusions:

Result: negative
1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.  

The in live phase of the study is terminated and histopathology on liver and glandular stomach is initiated based on initial data observed in the COMET test. All data of this study will be available until end of October and an IUCLID update with all data on this Comet test will be provided to ECHA in a Dossier update on November 17.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

1,2-dichlorobenzene was investigated by different expert committees taking into account the whole data base, positive and negative studies, in weight-of-evidence evaluations (e.g. SCOEL (1995), IARC MONOGRAPHS (1999), NICNAS (2000), OECD SIDS (2001), ATSDR (2007), MAK (2012). Based on the negative carcinogenicity studies and the negative genotoxicity data in the majority of tests none of the mentioned committees considered 1,2-dichlorobenzene to be carcinogenic/mutagenic.

ECHA requested further studies on genotoxicity (ECHA Decision SEV-D-2114325960-51-01/F) as follows:

“Pursuant to Article 46(1) of the REACH Regulation the Registrant(s) shall submit the following information using the indicated test methods/instructions (in accordance with Article 13 (3) and (4) of REACH Regulation) and the registered substance subject to the present decision. The requested tests shall be conducted with the registered substance, 1,2-dichlorobenzene, with the highest possible concentration of the identified impurities as specified in the registration dossiers (EC No 202-425-9). The tests shall be implemented in a tiered approach, as follows:

Tier 1. In vivo Mammalian Alkaline COMET Assay (test method: OECD 489) combined with In vivo Mammalian Erythrocyte Micronucleus Test (test method: OECD 474) (further referred as COMET Assay and Micronucleus Test, respectively)

The tests shall be conducted on rat and by oral route (gavage). For the COMET Assay, the following tissues shall be analysed: liver, glandular stomach, duodenum/jejunum and bone marrow. For the Micronucleus Test, the bone marrow shall be analysed. Animals shall be dosed with 1,2-dichlorobenzene 48, 24 and 3 hours prior to sacrifice.

Depending on the results of these tests, the following tests shall also be conducted:

Tier 2a. In case the Micronucleus Test is positive the Mammalian Spermatogonial Chromosome Aberration Test (OECD 483), oral route, in rat is requested (further referred as the Chromosome Aberration Test).

Tier 2b. In case the COMET Assay is positive, Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay (OECD 488) also oral route, in rat with 70 days exposure is requested (further referred as the TGR).

In this test the liver, glandular stomach, duodenum/jejunum, bone marrow and male germ cells shall be analysed.”

The Tier 1 study is finalized with the following results:

Micronucleus assay:

1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

Comet assay:

There are 2 in vivo Comet test available (Charles River, 2018a and 2018b).

In the first comet assay no genotoxic effect was observed in bone marrow, duodenum and liver. In stomach, the result was equivocal (Charles River, 2018a).

A repeat study was conducted to investigate the equivocal results in the first test in detail (Charles River, 2018b). The repeat study investigated the same rat strain as the earlier study and the dosing regime and dose levels are identical with the doses investigated in the first study (125, 250 and 500 mg/kg/day). Systemic effects observed included, reduction in body weight gain and clinical observations, which confirm that this study, as the earlier study, was conducted at the MTD; the maximum tolerated dose in accordance with current regulatory guideline.

The following organs were investigated in both studies:

Liver:

Initial study; “In liver cells a slight increase in DNA damage in the comet assay was observed at 500 mg/kg from 2.35% in the vehicle control to 4.87% at 500 mg/kg. Moreover, the Tail Intensity of 4.87% is clearly with in the historical data control range of the comet assay in liver. Histopathology showed that at this dose centrilobular necrosis was present up to a marked degree. Therefore it can be concluded that the DNA damage observed at 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) is caused by necrosis.”

Repeat study: 1,2-dichlorobenzene did not induce any DNA damage in the liver. Histopathology showed centrilobular necrosis, consistent with the initial study.

Overall conclusion on the liver is that 1,2-dichlorobenzene does not induce any DNA damage in liver.

Stomach:

The repeat study was conducted because the initial in vivo Comet test was “equivocal” for this tissue.

Initial study: “In stomach a statistical significant increase in DNA damage was observed at 125 and 250 mg/kg. But since no significant increase was observed at 500 mg/kg, no dose response was observed. Histopathological examination showed hypertrophy but no necrosis in stomach at any of the tested concentrations. Thus DNA damage was observed after treatment but since this effect showed no dose response and the negative control values were high, the overall conclusion is equivocal and additional investigation in a comet assay in stomach is initiated.”

Repeat study: 1,2-dichlorobenzene, did not induce any DNA damage in the stomach. The animals treated with 1,2-Dichlorobenzene showed statistically significant decrease in % Tail DNA at the high dose (500 mg/kg/day). An increase in % Tail DNA would indicate potential genotoxicity but a decrease in %Tail DNA is not biologically relevant.

Overall, conclusion on the stomach is that 1,2-dichlorobenzene does not induce DNA damage. The uncertainty on potential genotoxicity based on the initial experiment could be experimentally clarified in a follow up study. The follow up study was conducted under more stringent conditions compared to the initial study. The negative control % Tail DNA data are more tight in the repeat study (2.9% compared to 23.8% in the initial study) and within the range defined by ECHA as acceptable (<20% % Tail DNA).

Duodenum:

Initial study: “No increase in DNA damage was observed in the comet assay in bone marrow, and duodenum of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).”

Repeat study: The data obtained for 1,2-dichlorobenzene for increase in mean of % Tail DNA in the duodenum are considered to be “equivocal” by the laboratory because the criteria defined for a negative result include that “All results are inside the distribution of the historical negative control data (e.g. 95% control limits),“. The individual mean values for control, 125, 250, and 500 mg/kg/day Test Item groups were 1.43, 0.43, 4.45, and 3.73%, respectively, and the historical control values were between 0.43 and 2.63 %. The individual mean values for the 250 and 500 mg/kg/day groups were outside the historical control values and consequently the data are interpreted as “equivocal” although the observation is not statistically significant.

For interpreting the data the following aspects have to be considered:

- The increase in mean of % Tail DNA in the mid and high dose groups is not dose dependent.

- The increase in mean of % Tail DNA did not show any statistical significance.

- Historical control data for this tissue is available for a limited number of studies only (9 studies are available for duodenum in contrast to 21 studies for stomach and 23 studies for liver).

- Standard deviations of increase in mean of % Tail DNA for the mid and high dose groups are high compared to the control group (control group 1.43 ±0.56%, low dose group 0.43 ±0.11%, mid dose group 4.45 ±2.11%, high dose 3.73 ±2.60%, positive control group 21.85 ±5.59%).

- Values observed for mid and high dose groups are substantially lower than the positive control value (21.8%).

- In the histopathology examination similar findings were noted as reported for the positive control EMS in animals receiving 250 and 500 mg/kg/day 1,2-dichlorobenzene; mild decreased mitoses in crypts (250 and 500 mg/kg/day) and mild decreased staining in crypts at 500 mg/kg/day.

Overall, the data obtained for 1,2-dichlorobenzene for increase in % Tail DNA in the duodenum are considered to be “equivocal” for the repeat study because the individual mean values for the 250 and 500 mg/kg/day groups were outside the historical control values. Since the values are not statistically significant, not dose dependent, only a limited number of historical control data are available for duodenum, the variability of the mid and high dose groups values are high, and the values are substantially lower than the values observed for the positive control group, this equivocal finding might be considered as biologically questionable. Together with the negative data in the initial study the remaining uncertainty on the “equivocal” result observation in the repeat experiment as low.

In particular it should be considered that these “equivocal” observations are not relevant for human risk assessment in the REACH context because they were seen after high oral doses of 250 and 500 mg/kg/day in a local gastric tissue directly exposed to high oral gavage doses. Local effects after oral high dose exposure are not relevant in our mind for human risk assessment of 1,2-dichlorobenzene taking into account that the compound is irritating (skin irrit. Cat 2 and eye irrit. Cat 2) and sensitising (Skin sens 1B) and consequently allocated to the medium hazard band for local effects (systemic oral short term DNEL for the general population is 3 mg/kg) with limited human exposure.

Justification for classification or non-classification

1,2-dichlorobenzene was investigated by different expert committees taking into account the whole data base, positive and negative studies, in weight-of-evidence evaluations (e.g. SCOEL (1995), IARC MONOGRAPHS (1999), NICNAS (2000), OECD SIDS (2001), ATSDR (2007), MAK (2012). Based on the negative carcinogenicity studies and the negative genotoxicity data in the majority of tests none of the mentioned committees considered 1,2-dichlorobenzene to be carcinogenic/mutagenic.

ECHA requested further studies on genotoxicity (ECHA Decision SEV-D-2114325960-51-01/F). Overall it can be concluded on these new studies:

In the MNT 1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

Two in vivo Comet tests are performed with 1,2-Dichlorobenzene on systemic organs liver and bone marrow (bone marrow only 1. experiment with negative result) and local tissues stomach and duodenum. Clearly negative results are observed for the systemic organs in all tests. The results for stomach and duodenum gave “equivocal” results in one test and negative results in the other test (Charles River, 2017, 2018). Overall, 1,2-Dichlorobenzene is considered as not genotoxic and any remaining uncertainty concerning the “equivocal” results as low.