Sodium p-chloro-m-cresolate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Gene mutation in mammalian cells (equivalent to OECD 476): negative

DNA damage repair (equivalent to OECD 482): negative

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

Study period:

7 - 21 May 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1984

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

only four S. typhimurium strains (additional E. coli WP2 strain or S. typhimurium TA 102 is missing)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

First experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Second experiment: 50, 100, 200, 240, 400 and 800 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (10 µg/plate, -S9, TA 1535), nitrofurantoin (0.2 µg/plate, -S9, TA 100), 4-nitro-1,2-phenylene diamine (10 µg/plate, -S9, TA 1537 and 0.5 µg/plate, -S9, TA 98), 2-aminoanthracene (3 µg/plate, +S9, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 4 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background growth; cloning efficiency; other: titer

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigation should continue, possibly with modifications, until a final evaluation is possible.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 800 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 400 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 800 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 800 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was soluble in water.

HISTORICAL CONTROL DATA
Summaries of historical negative and positive controls of experiments performed within 6 months each from January 1987 to June 1991 are provided. Please refer to Table 1 under "Any other informations on results incl. tables" for the summarized historical control data of January to June 1991.

Table 1: Summary of historical negative and positive controls of experiments performed from January to June 1991

Compound	S9 mix	Strain
		TA 1535		TA 100		TA 1537		TA 98
		median	semi-Q range	median	semi-Q range	median	semi-Q range	median	semi-Q range
water	-	12	3	111	10	9	2	28	5
Na azide	-	623	102
NF	-			398	56
4-NPDA	-					49	10	89	20
30% water	+	16	3	252	15	12	2	38	7
2-AA	+	182	33	800	163	86	24	472	105

Na azide = sodium azide, NF = nitrofurantoin, 4-NPDA = 4-nitro-1,2-phenylenediamine

 

Table 2: First experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 without S9-mix

Concentration [µg/plate]	Revertants/plate [mean]
	TA 1535	TA 100	TA 1537	TA 98
0	10	130	11	27
8	12	140	13	32
40	11	135	11	29
200	11	128	10	31
1000	6	44	2	19
5000	0	0	0	0
Na-azide^1	479
NF^2		8
4-NPDA^3			55	146

Positive controls:

 ¹Sodium-azide, 10 µg/plate (only TA 1535)

²Nitrofurantoin, 0.2 µg/plate (only TA 100)

³4-nitro-1,2-phenylenediamine, 10 µg/plate (only TA 1537), 0.5 µg/plate (only TA 98)

 

Table 3: First experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 with S9-mix

Concentration [µg/plate]	Revertants/plate [mean]
	TA 1535	TA 100	TA 1537	TA 98
0	18	156	13	37
8	15	143	12	38
40	21	161	18	32
200	16	162	12	40
1000	11	102	5	25
5000	0	0	0	0
2-AA^4	306	959	121	713

Positive controls:

⁴2-aminoanthracene, 3 µg/plate

 

Table 4: Second experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 without S9-mix

Concentration [µg/plate]	Revertants/plate [mean]
	TA 1535	TA 100	TA 1537	TA 98
0	16	121	11	27
50	20	114	13	32
100	15	117	11	29
200	17	118	10	31
400	19	111	2	19
800	12	76	0	0
Na-azide^1	809
NF^2		512
4-NPDA^3			55	146

Positive controls:

¹Sodium-azide, 10 µg/plate (only TA 1535)

²Nitrofurantoin, 0.2 µg/plate (only TA 100)

³4-nitro-1,2-phenylenediamine, 10 µg/plate (only TA 1537), 0.5 µg/plate (only TA 98)

 

Table 5: Second experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 with S9-mix

Concentration [µg/plate]	Revertants/plate [mean]
	TA 1535	TA 100	TA 1537	TA 98
0	20	172	16	48
50	26	176	13	41
100	2	185	15	36
200	25	188	15	44
400	1	176	15	40
800	17	121	6	30
2-AA^4	295	1498	100	828

Positive controls:

⁴2-aminoanthracene, 3 µg/plate

Executive summary:

The mutagenic potential of the test substance was tested in the Salmonella/Microsome test according to OECD Guideline 471 (1983) and in compliance with GLP. Tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA 1535 and TA 1537. Liver microsomal enzymes were prepared from at least 6 male Sprague-Dawley or male Wistar rats which had received a peritoneal injection of Aroclor 1254 at 500 mg/kg bw (S9 mix). The test substance was solved in water. In a first experiment, 6 dose levels from 0 - 5000 µg/plate were plated with overnight cultures of TA 98, TA 100, TA 1535 and TA 1537 in the presence or absence of rat S9 mix. As a result, the appropriate maximum dose to be plated in the second experiment was determined to be 800 µg/plate with and without metabolic activation, respectively. In the second experiment the test substance was tested at 5 dose levels (50, 100, 200, 400, 800 µg/plate) along with vehicle control (water) and appropriate positive controls in the absence or presence of rat microsomal enzymes. DMSO was used as vehicle for positive controls. 0.1 mL of test-substance in water or positive control in DMSO were added to 0.1 mL of bacteria solution (grown for 17 hours at 37 °C at 90 rpm) and 0.5 mL S9 mix or buffer (for non-activating tests) were added to 2.0 mL molten top-agar. After incubation for a maximum of 30 s at 45 °C in a water bath and mixing, this solution was poured onto solid agar plates. Four replicates were plated for all dose levels and controls. The plates were incubated at 37 °C for 48 h, before the colonies were counted. A result was evaluated as positive when it caused a doubling in the mean number of revertants per plate in at least one tester strain. This increase must be accompanied by a positive dose response. There was no indication of bacteriotoxic effects of the test substances at doses of up to and including 200 µg/plate. Total bacteria counts were comparable to or only slightly different from the negative controls. No inhibition of growth was noted. Doses at and above 400 µg/plate caused bacteriotoxic effects. None of the four strains tested showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls, with and without metabolic activation. The respective positive controls caused an increase in the number of revertants which proved the sensitivity of the test. Therefore, under the conditions of this study, no indications of mutagenic effects of the test substance could be found at doses up to 800 µg/plate in any of the S. typhimurium strains tested.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification document provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 480 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 480 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 480 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 240 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification document provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 200 µg/mL without S9 and at ≥ 100 µg/mL with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification document provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

hepatocytes: rat primary

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10 and 20 µg/mL (67% and 43% survival, respectively)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification document provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+/-S9: at =>313 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+/-S9: at =>313 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+/-S9mix: at =>625 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+/-S9mix: at =>625 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9mix: at=>625 μg/plate, +S9mix: at 1250 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenicity / micronucleus test in mice (OECD 474): negative

Link to relevant study records

Reference

Endpoint:

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

Type of information:

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

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification document provided in IUCLID section 13.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: source, CAS 59-50-7, key rel.2, Herbold 1990

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: source, CAS 59-50-7, rel.2, Herbold 1981

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

 A reliable study regarding gene mutation in bacteria is available for the target substance sodium p-chloro-m-cresolate (CAS 15733-22-9), however, the study lacks the 5th strain required in the current OECD guideline (1997), therefore an additional key study (MHLW, 2007) is provided with the analogue substance p-chloro-m-cresol (CAS 59-50-7), in which five strains were tested. No data are available regarding gene mutation in mammalian cells and in vitro cytogenicity for the target substance, therefore the assessment was based on studies conducted with the analogue substance p-chloro-m-cresol (CAS 59-50-7) as part of a read across approach, which is in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

In vitro:

- Gene mutation in bacteria:

CAS 15733-22-9

The mutagenic potential of the target substance sodium p-chloro-m-cresolate in bacteria was tested in the Salmonella / Microsome test according to OECD Guideline 471 (1983) and in compliance with GLP (Gahlmann, 1991). Tester strains used were the Salmonella typhimurium histidine auxotrophs TA 98, TA 100, TA 1535 and TA 1537. Liver microsomal enzymes were prepared from at least 6 male Sprague-Dawley or male Wistar rats which had received a peritoneal injection of Aroclor 1254 at 500 mg/kg bw (S9 mix). The test substance was solved in water. In a first experiment, 6 dose levels from 0 - 5000 µg/plate were plated with overnight cultures of TA 98, TA 100, TA 1535 and TA 1537 in the presence or absence of rat S9 mix. As a result, the appropriate maximum dose to be plated in the second experiment was determined to be 800 µg/plate with and without metabolic activation, respectively. In the second experiment the test substance was tested at 5 dose levels (50, 100, 200, 400, 800 µg/plate) along with vehicle control (water) and appropriate positive controls in the absence or presence of rat microsomal enzymes. DMSO was used as vehicle for positive controls. 0.1 mL of test-substance in water or positive control in DMSO were added to 0.1 mL of bacteria solution (grown for 17 hours at 37 °C at 90 rpm) and 0.5 mL S9 mix or buffer (for non-activating tests) were added to 2.0 mL molten top-agar. After incubation for a maximum of 30 s at 45 °C in a water bath and mixing, this solution was poured onto solid agar plates. Four replicates were plated for all dose levels and controls. The plates were incubated at 37 °C for 48 h, before the colonies were counted. A result was evaluated as positive when it caused a doubling in the mean number of revertants per plate in at least one tester strain. This increase must be accompanied by a positive dose response. There was no indication of bacteriotoxic effects of the test substances at doses of up to and including 200 µg/plate. Total bacteria counts were comparable to or only slightly different from the negative controls. No inhibition of growth was noted. Doses at and above 400 µg/plate caused bacteriotoxic effects. None of the four strains tested showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls, with and without metabolic activation. The respective positive controls caused an increase in the number of revertants which proved the sensitivity of the test. Therefore, under the conditions of this study, no indications of mutagenic effects of the test substance could be found at doses up to 800 µg/plate in any of the S. typhimurium strains tested.

 

CAS 59-50-7

The mutagenic potential of the source substance p-chloro-m-cresol was tested in the Salmonella / Microsome test according to OECD Guideline 471 (1983) and in compliance with GLP (Herbold, 1991). Tester strains used were the Salmonella typhimurium histidine auxotrophs TA 98, TA 100, TA 1535 and TA 1537. Liver microsomal enzymes were prepared from at least 6 male Sprague-Dawley or male Wistar rats which had received a peritoneal injection of Aroclor 1254 at 500 mg/kg bw (S9 mix). The test substance was solved in ethanol. In a first experiment, 6 dose levels from 0 - 5000 µg/plate were plated with overnight cultures of TA 98, TA 100, TA 1535 and TA 1537 in the presence or absence of rat S9 mix. As a result, the appropriate maximum dose to be plated in the second experiment was determined to be 960 µg/plate with and without metabolic activation, respectively. In the second experiment the test substance was tested at 5 dose levels (30, 60, 120, 240, 480, 960 µg/plate) along with negative control and appropriate positive controls in the absence or presence of rat microsomal enzymes. DMSO was used as vehicle for positive controls. 0.1 mL of test-substance in ethanol or positive control in DMSO were added to 0.1 mL of bacteria solution (grown for 17 hours at 37 °C at 90 rpm) and 0.5 mL S9 mix or buffer (for non-activating tests) were added to 2.0 mL molten top-agar. After incubation for a maximum of 30 s at 45 °C in a water bath and mixing, this solution was poured onto solid agar plates. Four replicates were plated for all dose levels and controls. The plates were incubated at 37 °C for 48 h, before the colonies were counted. A result was evaluated as positive when it caused a doubling in the mean number of revertants per plate in at least one tester strain. This increase must be accompanied by a positive dose response. There was no indication of bacteriotoxic effects of the test substances at doses of up to and including 200 µg/plate. Total bacteria counts were comparable to or only slightly different from the negative controls. No inhibition of growth was noted. Doses at and above 240 µg/plate caused bacteriotoxic effects. None of the four strains tested showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls, with and without metabolic activation. The respective positive controls caused an increase in the number of revertants which proved the sensitivity of the test. Therefore, under the conditions of this study, no indications of mutagenic effects of the test substance could be found at doses up to 960 µg/plate in any of the S. typhimurium strains tested.

The mutagenic potential of the source substance p-chloro-m-cresol was tested in a Bacterial Reverse Mutation Assay according to OECD Guideline 471 (1997) and in compliance with GLP (MHLW, 2007). Tester strains used were the S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA (pKM101). Liver microsomal enzymes were prepared from male Sprague-Dawley induced with phenobarbital and 5,6-benzoflavone. The test substance was dissolved in DMSO, which also acted as the vehicle control. The positive control substances 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA 100, TA 98 and WP2 uvrA (pKM101)), 9-aminoacridine hydrochloride (TA 1537) and sodium azide (TA 1535) were tested in the absence of metabolic activation and 2-aminoanthracene (all strains) was tested in the presence of metabolic activation. A preliminary experiment was conducted using concentrations from 1.22 - 5000 μg/plate. Based on the presence of cytotoxicity in the preliminary experiment, the following concentrations were selected for the main experiment:

9.77, 19.5, 39.1, 78.1, 156, 313 and 625 μg/plate (with and without metabolic activation) for strains TA 100 and TA 1535.

19.5, 39.1, 78.1, 156, 313, 625 and 1250 μg/plate (with and without metabolic activation) for strains WP2 uvrA (pKM101), TA 98 and TA 1537.

Two independent experiments were performed using the pre-incubation method and triplicate plates were included per experiment. In strains TA 100 and TA 1535, cytotoxicity was seen at concentrations ≥313 μg/plate with and without metabolic activation. For strains TA 98 and TA 1537, cytotoxicity was seen at concentration ≥625 μg/plate with and without metabolic activation. For strain WP2 uvrA (pKM101), cytotoxicity was seen at concentrations ≥625 μg/plate without metabolic activation and at 1250 μg/plate with metabolic activation. The vehicle and positive controls were valid, with all positive control substances inducing a marked increase in the number of revertant colonies when compared to the vehicle controls. Under the conditions tested, p-chloro-m-cresol was negative for gene mutation in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA (pKM101) with or without metabolic activation.

The endpoint summary for this additional study has been prepared from the available information on the Japan CHEmicals Collaborative Knowledge database (J-CHECK) for 4-Chloro-m-cresol (CAS 59-50-7 ): https://dra4.nihs.go.jp/mhlw_data/jsp/FileListPageENG.jsp?parameter_csno=59-50-7

  

- Gene mutation in mammalian cells:

The potential of the source substance p-chloro-m-cresol to induce gene mutations at the HPRT locus in Chinese hamster ovarian (CHO; subclone CHO-K1-BH4) cells was assessed in a study performed equivalent to OECD Guideline 476 (2016) and in compliance with GLP (forward mutation assay; Lehn, 1989). The test substance was suspended in DMSO. In a dose range-finding test (cytotoxicity test), 9 dose levels from 1.25 - 320 µg/mL were tested in the presence or absence of rat microsomal enzymes. As a result, the appropriate maximum dose to be plated in the main study would be 300 µg/mL with and without metabolic activation, respectively. The main study for evaluation of mutagenic effects was performed in two independent experiments, using two parallel cultures each. In both experiments cells were treated with concentrations of 50, 100, 150, 200, 250 and 300 µg/mL test substance in the presence in the absence of S9 mix for 6 h. Dimethylbenzanthracene (DMBA) and ethylmethanesulfonate (EMS) were used as positive controls in activation and non-activation assays, respectively. Negative controls included both untreated controls and vehicle (DMSO) controls. Cytotoxic effects were noted at doses of 200 µg/mL and above without metabolic activation and at 100 µg/mL and above with metabolic activation. There was no statistically significant increase in mutant frequency noted at any of the dose levels, under both activating and non-activating conditions. The positive controls EMS and DMBA showed a clear mutagenic effect. According to the results of the study and the relating evaluation criteria, the test substance is considered to be non-mutagenic in the CHO-HGPRT Forward Mutation Assay.

 

- DNA repair study:

The source substance p-chloro-m-cresol was evaluated for unscheduled DNA synthesis (UDS) in cultured primary rat hepatocytes equivalent to OECD Guideline 482 (1986) and in compliance with GLP (Cifone, 1988). Freshly prepared hepatocytes from male Fischer 344 rats were exposed to 6 concentrations of the test substance ranging from 0.25 to 20 µg/mL in two trials (trial 2: 0.25, 0.5, 2.5, 7.5, 10, 20 µg/mL and trial 3: 2.53, 5.06, 7.58, 10.1, 15.2, 20.2 µg/mL; the first trial was discarded due to extensive cytotoxicity). The test substance as well as the positive control (2-acetylaminofluorene) was solved in DMSO as vehicle. DMSO was used as vehicle control. In addition to the test substance 5 µCi/mL ³H-thymidine was present in the cell culture medium and cells were exposed for 18-19 h in medium with reduced serum content (1% FCS). Three replications were conducted for the UDS assay and duplicate cell cultures were treated for determination of cytotoxicity via trypan blue exclusion. The cells were fixed, washed and air-dried prior to coating in the dark with photographic emulsion. The coated slides were stored at 4°C for 4-10 days and then developed. The slides were then rinsed and stained with haematoxylin/eosin. Grain counting was done manually using a microscope. Selected concentrations represented a good range of cytotoxicity (100.6 - 43% relative survival). High toxicities were observed at dose levels from 20.0 μg/mL to 10.0 μg/mL in the second trial. In the third trial moderate toxicity was observed at 20.2 µg/mL which was the highest concentration chosen for nuclear grain counting. None of the treatments with the test substance in both trial 2 and trial 3 caused nuclear labeling significantly different from the solvent control. Furthermore, no dose-related trend was evident. Thus, under the conditions of the study, the test substance evaluated as inactive in the Rat Primary Hepatocyte UDS Assay.

  

In vivo:

CAS 59-50-7

- Cytogenicity / micronucleus test in mice:  

The potential of the source substance p-chloro-m-cresol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was assessed in a study performed according to OECD Guideline 474 (1983) and in compliance with GLP (Herbold, 1990). The test substance was suspended in polyethylene glycol 400, which was also used as vehicle control. Five male and five female Bor: NMRI mice per test group received a single intraperitoneal injection of 125 mg/kg bw. The mice were sacrificed at 24, 48 and 72 h after treatment and the bone marrow cells were collected for micronuclei analysis. Animals administered the positive control (20 mg/kg bw cyclophosphamide) or the vehicle were sacrificed at 24 h after treatment. Per animal, 1000 polychromatic erythrocytes (PCEs) were scored for micronuclei. The ratio between PCEs and normochromatic erythrocytes (NCEs) was determined in the same sample and expressed in NCEs per 1000 PCEs. Animals treated with 125 mg/kg bw test substance showed symptoms of toxicity which comprise apathy, roughened fur, staggering gait, prone position, spasm, twitching and diarrhoea. The symptoms lasted until sacrifice. 4/40 mice treated with the test substance died during the study period. The ratio of PCEs to NCEs was not altered in the groups which received the test substance. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any gender used. The positive control caused a clear clastogenic effect which is shown by the significant increase of PCEs with micronuclei. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.

 

With respect to in vivo genotoxicity, an additional micronucleus test conducted with the source substance is considered for support (Herbold, 1981). In fact, this study was performed to assess the potential of the source substance p-chloro-m-cresol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test substance was suspended in Lutrol, which was also used as vehicle control. Five male and five female Bor NMRI mice per test group received the test substance at 200 and 400 mg/kg bw orally via gavage two times 24 h apart. Two groups (5 animals per sex) received the vehicle or the positive control (100 mg/kg bw cyclophosphamide) accordingly. The mice were sacrificed at 6 h after the second treatment and the bone marrow cells were collected for micronuclei analysis. Per animal, 1000 polychromatic erythrocytes (PCEs) were scored for micronuclei. The ratio between PCEs and normochromatic erythrocytes (NCEs) was determined in the same sample and expressed in NCEs per 1000 PCEs. The ratio of PCEs to NCEs was not altered in the groups which received the test substance. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any test substance concentration and with any gender used. The positive control caused a clear clastogenic effect which is shown by the significant increase of PCEs with micronuclei.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No 1907/2006, information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to sodium p-chloro-m-cresolate, data will be generated from data of the reference source substance to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

                                                                   

The available data on genetic toxicity for both the source and the target substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.

This is in line with the existing harmonised classification of the source substance according to Annex VI of Regulation (EC) 1272/2008 as well as with the Opinion of the Committee for Risk Assessment (RAC), adopted 10 March 2016.