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REACH

N-(dodecylphenyl)naphthalen-1-amine

EC number: 437-450-6 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro - Ames Assay

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Genetic toxicity in vitro - Chromosome Aberration Test

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in two separate experiments, using a dose range that included a dose level that was the maximum recommended dose level. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Genetic toxicity in vitro - Gene mutation assay

It is concluded that Naugalube APAN showed weak evidence of inducing mutation at the tk locus of L5178Y mouse lymphoma cells when tested for 3 hours in the presence of a rat liver metabolic activation system (S-9). When tested for 3 hours in the absence of S-9 in the same test system, although some evidence of weak induction of mutation was observed, no increases in mutation frequency which exceeded the Global Evaluation Factor were observed and any observed increases were sporadic and not well reproduced, therefore were considered of uncertain biological relevance.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 1999 and 19 August 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No further details specified

Target gene:

histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia col i strain WP2uvrA was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm
the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the range-finding study. The experiment was repeated on a separate day using the same dose range as the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations.

Vehicle / solvent:

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes on the day of each experiment.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The study was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA),1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested.
In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto sterile Vogel-Bonner Minimal agar plates in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of excessive precipitation.

Mutation Study - Experiment 1 (Range-finding Study)
Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of 59-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 μg/plate because of excessive precipitation.

Mutation Study - Experiment 2 (Main Study)
The second experiment was performed using methodology as described for the range-finding study, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding study (50 to 5000 μg/plate).

Rationale for test conditions:

Not specified

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Study
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Study
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments of the main study was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation
Study.
No toxicity was exhibited to any of the strains of bacteria used. A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Preliminary Toxicity Study

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (μg/plate)
With (+) or without (-) S9-mix	Strain	0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	109	109	104	111	104	110	118	131	112	127	91P
+	TA100	107	124	137	126	128	108	116	111	99	85	87P
-	WP2uvrA	21	26	19	26	24	21	25	24	21	23	26P
+	WP2uvrA	34	33	38	30	30	30	25	37	25	30	39P

P = precipitate

SPONTANEOUS MUTATION RATES

(CONCURRENT NEGATIVE CONTROLS)

RANGE-FINDING STUDY

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
91 93 87	(90)	16 14 10	(13)	27 26 22	(25)	21 13 16	(17)	9 3 6	(6)

MAIN STUDY

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
99 72 74	(82)	14 29 24	(22)	12 19 17	(16)	11 11 14	(12)	15 12 8	(12)
								12 12 13	(12)*

* Experimental procedure performed without S9-mix only.

RANGE-FINDING STUDY – WITHOUT METABOLIC ACTIVATION

With or without S9-mix

Test substance concentration

(μg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

(83)

5.5#

(26)

5.0

(29)

7.4

(24)

7.6

(12)

2.9

(78)

5.9

(19)

0.6

(23)

6.1

(17)

1.0

(11)

1.2

150

(91)

7.5

(22)

3.1

(18)

10.6

(17)

3.6

(8)

2.1

1500

(81)

9.6

(28)

2.5

(26)

3.6

(13)

3.2

(13)

4.2

5000

88P

81P

73P

(81)

7.5

25P

19P

22P

(22)

3.0

12P

15P

18P

(15)

3.0

10P

20P

12P

(14)

5.3

11P

10P

11P

(11)

0.6

Positive controls

Name

ENNG

4NQO

9AA

S9-Mix

Concentration (μg/plate)

0.2

No. colonies per plate

342

329

367

(346)

19.3

219

179

139

(179)

40.0

834

635

629

(699)

116.7

153

150

130

(144)

12.5

1042

1000

689

(910)

192.8

ENNG N-ethyl-N’-nitro-N-nitrsoguanidine # Standard deviation

4NQO 4-Nitroquinolinw-1-oxide C Contaminated

9AA 9-Aminoacridine P Precipitate

RANGE-FINDING STUDY – WITH METABOLIC ACTIVATION

With or without S9-mix

Test substance concentration

(μg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

124

121

(113)

16.5#

(15)

1.0

(24)

2.1

(20)

1.0

(11)

6.6

(79)

3.6

(14)

2.6

(26)

9.8

(23)

3.5

(13)

1.0

150

(85)

7.5

(10)

1.7

(22)

9.1

(20)

1.0

(10)

5.1

1500

(85)

5.0

(14)

3.5

(21)

3.2

(22)

10.7

(13)

1.5

5000

91P

95P

87[

(91)

4.0

13P

11P

10P

(11)

1.5

21P

30P

29P

(27)

4.9

17P

20P

24P

(20)

3.5

15P

12P

(11)

4.6

Positive controls

Name

2AA

S9-Mix

Concentration (μg/plate)

No. colonies per plate

1324

1214

1335

(1291)

66.9

155

318

216

(230)

82.4

1091

1124

1150

(1122)

29.6

550

603

532

(562)

36.9

295

328

(312)

23.3

BP Benzo(a)pyrene # Standard deviation

2AA 2-Aminoanthracene C Contaminated

X Poor lawn and colony growth P Precipitate

MAIN STUDY – WITHOUT METABOLIC ACTIVATION

With or without S9-mix

Test substance concentration

(μg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

(80)

11.8#

(22)

6.1

(17)

4.6

(13)

3.2

(11)

2.1

(84)

12.7

(30)

2.3

(21)

2.6

(12)

1.0

(11)

1.5

150

(87)

5.3

(24)

4.4

(20)

3.8

(18)

4.6

(11)

4.4

500

(92)

1.5

(24)

5.0

(16)

3.2

918)

4.0

(11)

2.5

1500

(83)

6.7

(29)

8.3

(11)

0.6

(14)

1.5

(10)

3.5

5000

76P

74P

87P

(79)]7.0

31P

21P

36P

(29)

7.6

22P

21P

11P

(18)

6.1

19P

14P

(17)

2.9

13P

12P

(10)

4.4

Positive controls

Name

ENNG

4NQO

9AA

S9-Mix

Concentration (μg/plate)

0.2

No. colonies per plate

796

799

684

(760)

65.5

317

256

379

(317)

61.5

973

887

1172

(1011)

146.2

210

188

177

(192)

16.8

979

1070

988

(1012)

50.1

ENNG N-ethyl-N’-nitro-N-nitrsoguanidine # Standard deviation

4NQO 4-Nitroquinolinw-1-oxide P Precipitate

9AA 9-Aminoacridine

MAIN STUDY – WITH METABOLIC ACTIVATION

With or without S9-mix

Test substance concentration

(μg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

(86)

8.2#

(14)

4.2

(23)

3.6

(25)

9.2

(12)

1.7

111

(88)

21.3

(15)

1.5

(20)

2.3

(28)

0.6

(13)

1.7

150

105

(93)

10.4

(18)

7.0

(17)

2.9

(29)

4.2

(16)

2.6

500

111

(97)

14.0

(13)

3.6

(22)

7.0

(27)

6.7

(11)

6.0

1500

(82)

10.0

(15)

2.0

(21)

8.5

(22)

2.9

(11)

1.2

5000

84P

79P

86P

(83)

3.6

18P

20P

13P

(17)

3.6

22P

29P

16P

(22)

6.5

14P

21P

(16)

4.0

15P

11P

14P

(13)

2.1

Positive controls

Name

2AA

S9-Mix

Concentration (μg/plate)

No. colonies per plate

778

919

836

(844)

70.9

279

397

352

(342)

59.6

452

459

411

(441)

25.9

415

453

476

(448)

30.8

255

424

210

(296)

112.9

BP Benzo(a)pyrene # Standard deviation

2AA 2-Aminoanthracene P Precipitate

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requiretnents of the OECD Guidelines for Testing of Chemicals No. 471 "Reverse Mutation Study", ethod B 14 of Commission Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the range-finding study. The experiment was repeated on a separate day using the same dose range as the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 October 2000 to 17 January 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test"

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Method B10 of Commission Directive 92/69/EEC.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Genetic Toxicology: Chromosome Aberration Test

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

Detection of structural chromosomal aberrations in cultured mammalian cells.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

Cell Culture
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum, at 37°C with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 μg/ml final concentration.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Experiment 1: The maximum dose level selected for metaphase analysis was based on the lowest precipitating dose level (1935 μg/ml) in both the absence and presence of S9.
Experiment 2: The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was based on the lowest precipitating dose level (1935 μg/ml), in both treatment groups.

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Preparation of Test and Control Materials
The test material was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial two-fold dilutions prepared. The molecular weight of the test material was 387, therefore the maximum dose level was 3870 μg/ml, which was calculated to be equivalent to 10 mM. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOSM. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.
Vehicle and positive controls were used in parallel with the test material. The positive control materials were as follows:
In the absence of S9, mitomycin C (MMC) (Sigma, Batch Nos. 69H2512 and 119H2540) at 0.4 and 0.2 μg/ml for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.
In the presence of S9, Cyclophosphamide (CP) (Sigma, Batch No. 108H0568) 12.5 μg/ml in both experiments. It was dissolved in dimethyl sulphoxide.

Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at 37 °C, 5% CO2 in humidified air, the cultures were transfered to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 ml of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 10% S9-mix (ie 1% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 ml of 20% S9-mix (ie 2% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.
After 4 hours at 37 °C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37 °C in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at 37 °C with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 ml of the appropriate vehicle control, test material solution or positive control solution. The total volume for each culture was a nominal 10 ml.
After 4 hours at 37 °C, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mi wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 ml of vehicle control, test material dose solution or positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at 37 °C for 24 hours.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 15.12 to 3870 μg/ml. Parallel flasks, containing culture medium without whole blood, were established for all three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main study.

Experiment 1
i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.

Experiment 2
i) 24-hour continuous exposure to the test material prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After approximately fifteen minutes most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 °C for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerised random number generator.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Rationale for test conditions:

Not specified

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
It can be seen that the test material showed no evidence of toxicity in the three treatment groups. A precipitate of the test material was observed in the blood cultures at the end of the exposure, at and above 1935 μg/ml. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at all dose levels in all treatment groups.
Dose selection for Experiment 1 was based on the lowest precipitating dose level which was
1935 μg/ml, for both pulse exposure groups.

Chromosome Aberration Test – Experiment 1
The qualitative assessment of the slides determined that no toxicity was observed and that there were scorable metaphases present at the maximum dose level (1935 μg/ml) of test material, both in the presence and absence of metabolic activation (S9). Precipitate observations were as those observed in the Preliminary Toxicity Test.
The mitotic index data confirm the qualitative toxicity observations in that no dose-related inhibition of mitotic index was observed, either in the presence or absence of S9.
The maximum dose level selected for metaphase analysis was based on the lowest precipitating dose level (1935 μg/ml) in both the absence and presence of S9.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation (S9).
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The qualitative assessment of the slides determined that there were scorable metaphases present at the maximum dose level (1935 μg/ml) of test material, both in the absence and presence of S9.
Precipitate observations were as those observed in the Preliminary Toxicity Test.
The mitotic index data confirm the qualitative toxicity observations in that no dose-related inhibition of mitotic index was observed, in either treatment group. All three upper dose levels in the without-S9 group had MI values that were approximately 50% of the vehicle control group, however, this was considered not to be the result of test material-induced toxicity. The relatively high MI value for the B culture of the vehicle control group was considered to be the cause of the apparent depression of the MI values. No toxicity had been seen in the 24-hour exposure group of the preliminary toxicity test.
The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was based on the lowest precipitating dose level (1935 μg/ml), in both treatment groups.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Mitotic Index – Preliminary Toxicity Test

4-HOUR TREATMENT, 20-HOUR RECOVERY –S9

4-HOUR TREATMENT, 20-HOUR RECOVERY +S9

24-HOUR TREATMENT –S9

CONCENTRATION (μg/ml)	4(20)h WITHOUT S9		4(20)h WITH S9		24h WITHOUT S9
CONCENTRATION (μg/ml)	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL
0	3.80	100	3.95	100	2.35	100
15.12	-	-	-	-	-	-
30.24	-	-	-	-	-	-
60.47	-	-	-	-	-	-
120.94	3.60	95	2.95	75	3.30	140
241.88	4.45	117	3.25	82	2.80	119
483.75	3.55	93	3.45	87	2.55	109
967.50	5.00	132	3.55	90	1.65	70
1935	4.30	113P	2.85	72P	2.65	113P
3870	3.10	82P	3.35	85P	2.40	102P

- = Not assessed for mitotic index

P = Oily precipitate observed at end of treatment period in blood cultures

Mitotic Index – Experiment 1

DOSE LEVEL μg/ml	4 HOURS TREATMENT WITHOUT S9				4 HOURS TREATMENT WITH S9
DOSE LEVEL μg/ml	A	B	MEAN	% OF CONTROL	A	B	MEAN	% OF CONTROL
0	3.70	5.90	4.80	100	3.60	4.05	3.83	100
60.47	-	-	-	-	-	-	-	-
120.49	-	-	-	-	-	-	-	-
241.88	-	-	-	-	-	-	-	-
483.75	3.25	4.20	3.73	78	2.50	3.85	3.18	83
967.5	3.30	4.05	3.68	77	3.00	3.30	3.15	82
1935	5.10P	4.35P	4.73	98	3.45P	3.35P	3.40	89
MMC 0.4	1.90	3.70	2.80	58	NA	NA	NA	NA
CP 12.5	NA	NA	NA	NA	0.90	1.30	1.10	29

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

P = Oily precipitate observed at end of treatment period in blood cultures

- = Not assessed for mitotic index

Mitotic Index – Experiment 2

DOSE LEVEL μg/ml	24 HOURS TREATMENT WITHOUT S9				4 HOURS TREATMENT WITH S9
DOSE LEVEL μg/ml	A	B	MEAN	% OF CONTROL	A	B	MEAN	% OF CONTROL
0	2.25	3.70	2.98	100	2.80	3.30	3.05	100
120.49	-	-	-	-	-	-	-	-
241.88	-	-	-	-	-	-	-	-
483.75	1.70	1.45	1.58	53	1.35	3.70	2.53	83
967.5	1.80	0.95	1.38	46	1.95	2.05	2.00	66
1935	1.70	1.55	1.63	55	2.40	2.20	2.30	75
MMC 0.2	0.85	0.50	0.68	23	NA	NA	NA	NA
CP 12.5	NA	NA	NA	NA	0.50	0.65	0.58	19

MMC = Mitomycin C

CP = Cyclophosphamide

- = Not assessed for mitotic index

Results of Chromosome Aberration Test – Experiment 1 Without Metabolic Activation (S9)

Treatment Group

Replicate

Number of Cells Scored

Frequency of Cells with Aberrations (%)

Total Gaps

Chromatid

Chromosome

Others

Total Aberrations

Aberrant Cells

Breaks

Exchanges

Breaks

Exchanges

(+ Gaps)

(- Gaps)

(+ Gaps)

(- Gaps)

Vehicle Control

Total

100

200

(100)

(0.5)

(1.0)

(0.0)

(1.5)

(0.0)

(3.0)

(2.5)

(2.0)

483.75

μg/ml

Total

100

200

(100)

(0.5)

(0.0)

(1.0)

(0.5)

(1.0)

(0.5)

967.6

μg/ml

Total

100

200

(100)

(2.5)

(1.0)

(0.0)

(3.5)

(1.0)

(3.0)

(1.0)

1935

μg/ml

Total

100

200

(100)

(1.0)

(0.5)

(0.0)

(1.0)

(0.0)

(2.5)

(1.5)

(2.5)

(1.5)

Positive Control

MMC 0.4

μg/ml

Total

100

150

(100)

(17.3)

(23.3)

(25.3)

(2.0)

(0.0)

102

(68.0)

(50.7)

(44.0)

56^***

(37.3)

X = >10 aberrations per cell (not included in total aberrations)

(Figures in brackets) = aberrations per 100 cell

MMC = Mitomycin C

^***= P <0.001

Results of Chromosome Aberration Test – Experiment 1 With Metabolic Activation (S9)

Treatment Group

Replicate

Number of Cells Scored

Frequency of Cells with Aberrations (%)

Total Gaps

Chromatid

Chromosome

Others

Total Aberrations

Aberrant Cells

Breaks

Exchanges

Breaks

Exchanges

(+ Gaps)

(- Gaps)

(+ Gaps)

(- Gaps)

Vehicle Control

Total

100

200

(100)

(2.0)

(0.5)

(0.0)

(1.0)

(0.0)

(3.5)

(1.5)

(3.0)

(1.0)

483.75

μg/ml

Total

100

200

(100)

(2.5)

(0.0)

(1.5)

(0.0)

(5.0)

(1.5)

(4.5)

(1.0)

967.6

μg/ml

Total

100

200

(100)

(1.0)

(0.0)

(0.5)

(0.0)

(1.5)

(0.5)

(1.5)

(0.5)

1935

μg/ml

Total

100

200

(100)

(2.0)

(0.0)

(0.5)

(0.0)

(4.5)

(2.5)

(4.0)

(2.0)

Positive Control

CP 12.5

μg/ml

Total

100

(100)

(23.0)

(42.0)

(14.0)

(8.0)

(1.0)

(0.0)

(88.0)

(65.0)

(54.0)

40^***

(40.0)

X = >10 aberrations per cell (not included in total aberrations)

(Figures in brackets) = aberrations per 100 cell

CP = Cyclophosphamide

^***= P <0.001

Results of Chromosome Aberration Test – Experiment 2 Without Metabolic Activation (S9)

Treatment Group

Replicate

Number of Cells Scored

Frequency of Cells with Aberrations (%)

Total Gaps

Chromatid

Chromosome

Others

Total Aberrations

Aberrant Cells

Breaks

Exchanges

Breaks

Exchanges

(+ Gaps)

(- Gaps)

(+ Gaps)

(- Gaps)

Vehicle Control

Total

100

200

(100)

(0.5)

(0.0)

(0.5)

(0.0)

(1.5)

(1.0)

(1.5)

(1.0)

483.75

μg/ml

Total

100

200

(100)

(1.0)

(0.0)

(0.5)

(0.0)

(0.5)

(0.0)

(2.0)

(1.0)

(2.0)

(1.0)

967.6

μg/ml

Total

100

200

(100)

(0.0)

(2.0)

(0.0)

(2.0)

(1.5)

1935

μg/ml

Total

100

200

(100)

(0.5)

(1.5)

(0.0)

(2.0)

(1.5)

(2.0)

(1.5)

Positive Control

MMC 0.2

μg/ml

Total

100

200

(100)

(14.5)

(11.5)

(9.0)

(2.0)

(0.0)

(37.0)

(22.5)

(28.5)

36^***

(18.0)

X = >10 aberrations per cell (not included in total aberrations)

(Figures in brackets) = aberrations per 100 cell

MMC = Mitomycin C

^***= P <0.001

Results of Chromosome Aberration Test – Experiment 2 With Metabolic Activation (S9)

Treatment Group

Replicate

Number of Cells Scored

Frequency of Cells with Aberrations (%)

Total Gaps

Chromatid

Chromosome

Others

Total Aberrations

Aberrant Cells

Breaks

Exchanges

Breaks

Exchanges

(+ Gaps)

(- Gaps)

(+ Gaps)

(- Gaps)

Vehicle Control

Total

100

200

(100)

(1.5)

(0.5)

(0.0)

(2.0)

(0.5)

(2.0)

(0.5)

483.75

μg/ml

Total

100

200

(100)

(0.5)

(0.0)

(1.0)

(0.5)

(0.0)

(2.0)

(1.5)

(2.0)

(1.5)

967.6

μg/ml

Total

100

200

(100)

(3.0)

(0.0)

(3.0)

(0.0)

(3.0)

(0.0)

1935

μg/ml

Total

100

200

(100)

(5.0)

(0.5)

(0.0)

(6.5)

(1.5)

(6.0)

(1.5)

Positive Control

CP 12.5

μg/ml

Total

100

167

(100)

(12.6)

(25.7)

(10.2)

(6.0)

(0.0)

(54.5)

(41.9)

(37.1)

50^***

(29.9)

X = >10 aberrations per cell (not included in total aberrations)

(Figures in brackets) = aberrations per 100 cell

CP = Cyclophosphamide

^***= P <0.001

Mean Frequency of Polyploid Cells (%)

EXPERIMENT 1

DOSE LEVEL μg/ml	HARVEST TIME 24 HOURS
DOSE LEVEL μg/ml	4 HOURS WITHOUT S9	4 HOURS WITH S9
0	0.0	0.0
483.75	0.5	0.0
967.5	0.5	0.0
1935	0.0	0.0
MMC 0.4	0.0	NA
CP 12.5	NA	0.0

EXPERIMENT 2

DOSE LEVEL μg/ml	HARVEST TIME 24 HOURS
DOSE LEVEL μg/ml	4 HOURS WITHOUT S9	4 HOURS WITH S9
0	0.0	0.0
483.75	0.5	0.0
967.5	0.0	0.0
1935	0.5	0.5
MMC 0.4	0.0	NA
CP 12.5	NA	0.6

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

HISTORICAL CONTROL DATA AND EVALUATION CRITERIA

Historical Aberration Ranges for Vehicle Control Cultures

Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures. The current in-house aberration ranges are presented below:

	Without Metabolic Activation		With Metabolic Activation
	% Cells with abs (- gaps)	% Cells with abs (+ gaps)	% Cells with abs (- gaps)	% Cells with abs (+ gaps)
Minimum	0	0	0	0
Maximum	1.5	3.5	1.5	3.0
Mean	0.4	1.0	0.4	1.0
Standard Deviation	0.4	0.8	0.5	0.9
Number	59	59	59	59

Historical Aberration Ranges for Positive Control Cultures

	Without Metabolic Activation		With Metabolic Activation
	% Cells with abs (- gaps)	% Cells with abs (+ gaps)	% Cells with abs (- gaps)	% Cells with abs (+ gaps)
Minimum	12.0	16.5	3.5	7.5
Maximum	54	69	17.8	29.0
Mean	32.1	45.0	9.2	14.9
Standard Deviation	11.2	14.0	3.5	5.5
Number	40	40	40	40

Conclusions:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens which produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B.10 of Commission Directive 92/69/EEC. The study design also meets the requirements of the UK Department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls.

Four treatment conditions were used for the study, ie. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 1% final concentration, with cell harvest after a 20-hour expression period, and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 2% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control treatments gave statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Conclusion. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2017 to 18 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD (2016). Guideline for the testing of chemicals, No 490: In vitro mammalian cell gene mutation test using the thymidine kinase gene

Deviations:

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells is capable of detecting both gene mutations and chromosome aberrations. The mutation system works by placing treated cells under selective pressure so that only mutant cells are able to survive. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-), producing the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products but if a toxic base analogue (5-trifluorothymidine, TFT) is present in the medium, the enzyme will incorporate the analogue into the cells. Thus the cells die unless the enzyme is rendered inactive, by mutation for example. Resistance to TFT results from a lack of thymidine kinase (TK) activity. Thus, the mutants (tk-/-) are unable to use the toxic analogue and survive in its presence.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1°C. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

In the Mutation Experiment, from observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and TFT resistance:
Mutation Experiment (μg/mL):
3 hour -S-9: 0, 10, 20, 40, 70, 100, 125, 150, 200
3 hour +S-9: 0, 10, 20, 40, 70, 100, 125, 150, 200

Vehicle / solvent:

Preliminary solubility data indicated that Naugalube APAN was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 241.7 mg/mL. The solubility limit in culture medium was in the range of 37.77 to 75.55 μg/mL, as indicated by precipitation at the higher concentration which persisted for 3 hours after test article addition, with warming at 37°C. A maximum concentration of 300 μg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating concentration (OECD, 2016). Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
Test article stock solutions were prepared by formulating Naugalube APAN under subdued lighting in DMSO, with the aid of vortex mixing, warming at 37°C and sonication as required, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3 hours of initial formulation.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Test System
The test system was suitably labelled (using a colour-coded procedure) to clearly identify the study number, test article (if required), test article concentration (if applicable) positive and vehicle controls, absence or presence of S-9 mix.

Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL. In the absence and presence of S-9, 3 hour treatment incubation periods were used.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 50 mL RPMI 10.
All cultures were incubated at 37±1°C for 1 day, recounted and diluted to 2 x 105 cells/mL. Cultures were incubated for a further day and counted.

Mutation Experiment
Treatment of Cell Cultures
At least 107 cells in a volume of 17.8 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A to give a final concentration of 5% serum) were used. The cell suspensions were placed in a series of labelled sterile disposable 50 mL centrifuge tubes. 0.2 mL vehicle, test article, or positive control solution was added. S-9 mix or 150 mM KCl was added, as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment culture volume was 20 mL.
After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 50 mL RPMI 10 medium.
Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder and Mutation Experiments.

Expression Period
Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of retaining an appropriate number of cells/flask.
In the Range-Finder Experiment, at the end of the expression period, toxicity was assessed by measuring suspension growth and hence relative suspension growth (RSG), compared to the concurrent vehicle control values. Cultures were not plated for viability assessment.

Plating for Viability – Mutation Experiment
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 104 cells/mL in readiness for plating for TFT resistance.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for TFT Resistance - Mutation Experiment
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 104 cells/mL. TFT (300 μg/mL) was diluted approximately 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using an eight channel pipette, 0.2 mL of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x 103 cells/well). Plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (13 days) and wells containing clones were identified as above and counted.
In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for concentrations of test article where a marked increase in mutant frequency (close to or exceeding the GEF, as considered appropriate) was observed.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was statistically significant.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance.

Statistics:

The relevance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and the global evaluation factor (GEF), was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003 (Moore et al., 2006). Linear regression was performed on ranked mutant frequency against ranked concentration to test for a linear trend. The linear trend was performed on the overall MF per concentration level, not individual replicates (i.e. one MF value per concentration). The test for linear trend is one-tailed, therefore negative trend was not considered relevant. The positive control was excluded from the test for linear trend.
For microwell assays, the GEF is defined as 126 mutants per 106 viable cells.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 9.375 to 300 μg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (75 to 300 μg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration tested in the absence and presence of S-9 (300 μg/mL). All concentrations were retained in the absence and presence of S-9. The highest concentration tested, 300 μg/mL, gave 15% and 17% RSG in the absence and presence of S-9, respectively.
No marked changes in pH, compared to the concurrent vehicle controls, were observed in the Range-Finder at the highest concentration tested (300 μg/mL, individual data not reported). Increases in osmolality, compared to the concurrent vehicle controls, were observed in the absence and presence of S-9 in the Range-Finder but the increases were sporadic and were not reproduced between replicate measurements (individual data not reported), therefore further measurements were made in the Mutation Experiment.
In the Mutation Experiment ten concentrations, ranging from 10 to 300 μg/mL, were tested in the absence and presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest seven concentrations tested in the absence and presence of S-9 (70 to 300 μg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (200 to 300 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and the higher concentration was discarded.
Two days after treatment all remaining concentrations in the absence and presence of S-9 were selected to determine viability and TFT resistance. The highest concentration analysed was 200 μg/mL, which gave 20% and 24% RTG in the absence and presence of S-9, respectively.
No marked changes in pH, compared to the concurrent vehicle controls, were observed in the Mutation Experiment at the highest concentration analysed (200 μg/mL, individual data not reported). There were again increases in osmolality, compared to the concurrent vehicle controls, in the absence and presence of S-9 in the Mutation Experiment but these were sporadic and poorly reproduced between replicate measurements and between concentrations (individual data not reported), therefore were considered of questionable biological relevance.

Mutation
The acceptance criteria were met and the study was accepted as valid.
When tested up to toxic and precipitating concentrations for 3 hours in the absence of S-9, no increases in MF which exceeded the GEF of 126 mutants per 106 viable cells (compared to concurrent controls) were observed in any treated cultures. A statistically significant linear trend (p ≤ 0.001) was observed and the maximum increase in mean MF over the concurrent mean MF observed in any treated culture was approximately 124 (at 150 μg/mL), but the individual MF values at this concentration (159.50 and 283.69) differed markedly and were approximately 71 and 195 above the concurrent mean MF of 88.61. Although the increase in mean MF observed at 150 μg/mL was very close to the GEF, indicating weak evidence of mutation induction, the observation was considered of uncertain biological relevance, particularly as the increase in mean MF over the concurrent mean MF at 200 μg/mL was approximately 93, with good agreement between replicate cultures.
When tested up to toxic and precipitating concentrations for 3 hours in the presence of S-9, an increase in mean MF of approximately 129 mutants per 106 viable cells (compared to concurrent controls), which exceeded the GEF of 126, was observed at one intermediate concentration (100 μg/mL), giving 30% RTG and there was a statistically significant linear trend (p≤0.01). Although the mean MF exceeded the GEF only at 100 μg/mL, increases in mean MF over the concurrent mean MF of approximately 124, 102 and 120 (which were very close to the GEF) were observed at 125, 150 and 200 μg/mL, respectively, and increases in MF which exceeded the GEF were observed in single cultures treated at 125 and 200 μg/mL, thus providing further evidence of weak induction of mutation under this treatment condition.
It may be noted that the mean MF value observed at 40 μg/mL for the 3 hour treatment in the presence of S-9 was uncharacteristically low by comparison with the data observed at lower and higher concentrations (i.e. 20 and 70 μg/mL) under this treatment condition. This was due to very high viability plate counts in both replicate cultures at 40 μg/mL (Table 8.6) which were most likely attributable to a dilution error prior to viability plating, resulting in high % viability and, in turn, high % RTG, therefore the discrepancy is for technical, rather than scientific, reasons.
For the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the vehicle controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 33% to 35%. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P. At concentrations of Naugalube APAN which exceeded (or were close to) the GEF in the presence of S-9 (100, 125, 150 and 200 μg/mL), increases in both small and large colony mutants were observed at each concentration analysed but there were no marked, concentration-related increases in the proportions of small colony mutants.

Formulations Analysis
Results of formulation analyses demonstrated achieved concentrations within 100±10% of the nominal test article concentrations and were considered acceptable (Formulations Analysis Contributory Report). No test article was detected in the vehicle sample.

RSG Values – 3 Hour Range-Finder Experiment

Concentration

µg/mL

%RSG

-S-9

%RSG

+S-9

9.375

18.75

37.5

75 P

150 P

300 P,PP

100

Mutation Experiment: 3 Hour Treatment in the Absence of S-9

Concentration

µg/mL

%RTG

MF §

(mean)

MF §

(individual values)

70 P

100 P

125 P

150 P

200 P PP

MMS 15.00

MMS 20.00

100

88.61

111.92

91.69

116.24

123.74

138.70

132.07

212.07

181.34

614.67

1622.33

74.08, 103.15

112.13, 110.82

77.86, 105.83

118.25, 113.39

107.54, 144.45

133.85, 139.97

128.74, 135.33

159.50, 283.69 #

180.51, 182.27

Linear trend test on mutant frequency: p-value = 0 (*** P<0.001)

Mutation Experiment: 3 Hour Treatment in the Presence of S-9

Concentration

µg/mL

%RTG

MF §

(mean)

MF §

(individual values)

70 P

100 P

125 P

150 P

200 P PP

B[a]P 2.000

B[a]P 3.000

100

94.58

137.72

167.52

98.91

178.48

223.10 #

218.37

197.01

214.92

1458.69

1372.55

103.36, 87.15

153.95, 122.39

176.17, 158.92

101.25, 94.92

163.05, 194.25

214.70, 231.27 #

202.84, 234.58 #

218.94, 176.60

221.15 #, 208.02

Linear trend test on mutant frequency: p-value = 0.0048 (*** P<0.01)

Symbols and Abbreviations Used in the Tables

β	Positive wells per tray. Total number of wells scored is 96 unless otherwise stated e.g. 52/95 due to contamination
b²/S_b	Test for linear trend, slope b and its variance S_b
B[a]P	Benzo[a]pyrene
MF	Mutant frequency (TFT resistant mutants / 10⁶viable cells 2 days after treatment)
MMS	Methyl methane sulphonate
Nm	Total wells (mutant)
P	Indicates precipitation observed at the beginning of treatment
PP	Indicates precipitation observed following treatment incubation period
RSG	Relative Suspension Growth = (Individual SG / Mean control SG)
RTG	Relative Total Growth = (Individual SG / Mean control SG) x (Individual %V / Mean control %V)
RVc	Relative Viability compared to the control = (Individual %V / Mean control %V)
SG	Suspension growth; calculated as increase in cell numbers from end of treatment period to end of expression period (i.e. over Day 1 and Day 2)
%V	% Day 2 viability
Ym	Wells without colonies (mutant)
#	The MF of the test concentration exceeds the sum of the mean control MF plus GEF (Global Evaluation Factor)
*	Comparison of each treatment with control: Dunnett’s test (one-sided), significant at 5% level
, , **	Test for linear trend: χ²(one-sided), significant at 5%, 1% and 0.1% level respectively

Raw Plate Counts and Data Analysis, Mutation Experiment in the Absence of S-9 (3 Hour Treatment)

Naugalube APAN: Mutation Experiment in the Absence of S-9 (3 Hour Treatment) – Cell Counts and Wells with Clones/Plate

Concentration

µg/mL

Day 0 $

Day 1 $

Day 2 $

Viability β

(Day 2)

Resistant Mutants β

(Day 2)

70 P

100 P

125 P

150 P

200 P,PP

MMS15

MMS20

5.00

6.27

5.73

5.75

5.26

5.15

5.38

3.74

4.11

3.16

3.39

3.13

3.12

2.81

2.98

2.79

2.49

2.11

2.18

4.95

4.11

14.67

12.32

12.25

12.99

11.33

11.22

10.25

11.10

10.82

10.87

10.07

10.15

10.22

9.38

9.17

10.91(2.49)

9.12(2.11)

9.06(2.18)

13.33

11.78

77/94

74/92

76/90

75/92

69/93

58/90

69/91

49/93

74/91

83/92

67/91

65/85

81/94

71/91

75/87

46/90

22/94

7/24

25/82

18/82

7/24

16/95

20/95

26/94

19/93

26/93

$ Cell counts (x10⁵). Setup on previous day to 2x10⁵unless otherwise stated in parentheses

A, B Replicate cultures

Note Concentrations of 250 and 300 µg/mL were tested but discarded following treatment incubation period to precipitation. No data are presented.

Naugalube APAN: Mutation Experiment in the Absence of S-9 (3 Hour Treatment) – Summary of Individual Replicates

Concentration

µg/mL

RSG

Day 0 Factor

%RVc

%RTG

MF §

70 P

100 P

125 P

150 P

200 P,PP

MMS 15

MMS 20

23.00

17.65

17.61

17.08

14.59

15.09

9.58

11.41

8.55

9.21

7.88

7.92

7.18

6.99

6.40

6.45

4.56

4.53

16.50

15.05

113.16

86.84

86.65

84.06

71.78

74.26

47.16

56.12

42.06

45.33

38.77

38.96

35.33

34.39

31.47

26.84

22.44

22.29

81.17

74.05

1.00

106.38

110.06

101.24

118.14

135.40

99.62

124.96

111.98

105.87

116.02

126.10

91.99

102.40

99.93

101.26

73.17

99.62

96.62

77.01

45.76

98.33

101.72

93.57

109.19

125.15

92.07

115.50

103.50

97.85

107.23

116.55

85.02

94.64

92.36

93.59

67.62

92.07

89.30

71.18

42.29

111.26

88.34

81.09

91.78

89.83

68.37

54.47

58.09

41.16

48.61

45.19

33.12

33.44

31.76

29.46

18.15

20.66

19.91

57.78

31.32

74.08

103.15

112.13

110.82

77.86

105.83

118.25

113.39

107.54

144.45

133.85

139.97

128.74

135.33

159.50

283.69

180.51

182.27

614.67

1622.33

A, B Replicate cultures

Naugalube APAN: Mutation Experiment in the Absence of S-9 (3 Hour Treatment) – Summary of Results

Concentration

µg/mL

RSG

%RVc

%RTG

MF §

70 P

100 P

125 P

150 P

200 P, PP

MMS 15

MMS 20

20.32

17.35

14.84

10.49

8.88

7.90

7.08

5.93

4.55

16.50

15.05

100.00

85.35

73.02

51.64

43.70

38.87

34.86

29.16

22.37

81.17

74.05

108.20

109.12

114.99

118.14

110.84

106.97

101.11

85.65

98.14

77.01

45.76

100.00

100.86

106.28

109.19

102.44

98.87

93.45

79.16

90.70

71.18

42.29

100.00

86.09

77.60

56.39

44.76

38.43

32.57

23.08

20.29

57.78

31.32

88.61

111.92

91.69

116.24

123.74

138.70

132.07

212.07

181.34

614.67

1622.33

Linear trend test on mutant frequency: p-value = 0 (***P<0.001)

Naugalube APAN: Mutation Experiment in the Absence of S-9 (3 Hour Treatment) – Small and Large Colony Plate Counts

Concentration µg/mL		Small Colonies (Day 2) β				Large Colonies (Day 2) β
Concentration µg/mL		A	B	C	D	A	B	C	D
0 MMS 15 MMS 20	A B A A	6 5 40 53	4 7 48 49	4 8 36 50	8 6 35 58	7 8 16 23	7 17 14 24	8 13 23 18	12 14 25 22

A, B Replicate cultures

Naugalube APAN: Mutation Experiment in the Absence of S-9 (3 Hour Treatment) – Small and Large Colony Mutant Frequencies

Concentration µg/mL	Small Colonies			Large Colonies			Proportion of small colony mutants
	Mutants		MF §	Mutants		MF §
	Ym	Nm		Ym	Nm
0 MMS 15 NNS 20	720 225 174	768 384 384	29.82 347.06 864.95	682 306 2.97	768 384 384	54.88 147.42 280.72	0.35 0.70 0.75

Raw Plate Counts and Data Analysis, Mutation Experiment in the Presence of S-9 (3 Hour Treatment)

Naugalube APAN: Mutation Experiment in the Presence of S-9 (3 Hour Treatment) – Cell Counts and Wells with Clones/Plate

Concentration

µg/mL

Day 0 $

Day 1 $

Day 2 $

Viability β

(Day 2)

Resistant Mutants β

(Day 2)

70 P

100 P

125 P

150 P

200 P,PP

B[a]P 2

B[a]P 3

5.00

6.79

6.53

4.61

4.36

3.19

3.25

2.85

2.89

2.92

2.84

2.89

2.70

1.49

2.76

2.19

2.65

2.26

2.47

3.38

3.01

10.72

11.31

9.81

9.73

9.33

9.01

8.74

8.90

8.92

9.04

8.73

7.88

6.07(1.49)

7.50

9.14(2.19)

7.01

8.33(2.26)

8.78(2.47)

8.00

7.88

69/92

78/95

68/95

80/95

79/95

27/94

14/92

27/94

30/95

17/95

24/90

38/91

$ Cell counts (x10⁵). Setup on previous day to 2x10⁵unless otherwise stated in parentheses

A, B Replicate cultures

Note Concentrations of 250 and 300 µg/mL were tested but discarded following treatment incubation period to precipitation. No data are presented.

Naugalube APAN: Mutation Experiment in the Presence of S-9 (3 Hour Treatment) – Summary of Individual Replicates

Concentration

µg/mL

RSG

Day 0 Factor

%RVc

%RTG

MF §

70 P

100 P

125 P

150 P

200 P,PP

B[a]P 2

B[a]P 3

18.20

18.46

11.31

10.61

7.44

7.32

6.23

6.43

6.51

6.42

6.31

5.32

3.04

5.18

4.57

4.64

4.17

4.39

6.76

5.93

99.27

100.73

61.68

57.86

40.59

39.94

33.97

35.08

35.52

35.02

34.41

29.02

16.56

28.23

24.93

25.34

22.72

23.95

36.88

32.35

1.00

98.31

104.30

102.58

122.60

113.97

111.98

228.00

198.63

104.62

106.38

93.52

98.04

102.58

93.19

106.38

118.14

110.06

101.24

63.06

97.09

103.01

101.32

121.09

112.56

110.60

225.18

196.17

103.33

105.07

92.36

96.83

101.32

92.04

105.07

116.68

108.70

99.99

62.28

96.38

103.75

62.49

70.06

45.69

44.17

76.50

68.82

36.71

36.79

31.78

28.10

16.77

25.98

26.19

29.56

24.70

23.95

22.97

20.15

102.36

87.15

153.95

122.39

176.17

158.92

101.25

94.92

163.05

194.25

214.70

231.27

202.84

234.58

218.94

176.60

221.15

208.02

1458.69

1372.55

A, B Replicate cultures

Naugalube APAN: Mutation Experiment in the Presence of S-9 (3 Hour Treatment) – Summary of Results

Concentration

µg/mL

RSG

%RVc

%RTG

MF §

70 P

100 P

125 P

150 P

200 P, PP

B[a]P 2

B[a]P 3

18.33

10.96

7.38

6.33

6.47

5.81

4.11

4.61

4.28

6.76

5.93

100.00

59.77

40.26

34.53

35.27

31.71

22.39

25.13

23.34

26.88

32.35

101.25

111.82

112.97

211.61

105.50

95.74

97.71

111.98

105.50

63.06

100.00

110.44

111.57

208.99

104.19

94.55

96.50

110.60

104.19

62.28

100.00

66.01

44.92

72.15

36.75

29.99

21.61

27.80

24.31

22.97

20.15

94.58

137.72

167.52

98.91

178.48

223.1 #

218.37

197.01

214.92

1458.69

1372.55

Linear trend test on mutant frequency: p-value = 0.0048 (**P<0.01)

Naugalube APAN: Mutation Experiment in the Presence of S-9 (3 Hour Treatment) – Small and Large Colony Plate Counts

Concentration µg/mL		Small Colonies (Day 2) β				Large Colonies (Day 2) β
Concentration µg/mL		A	B	C	D	A	B	C	D
0 100 P 125 P 150 P 200 P,PP B[a]P 2 B[a]P 3	A B A B A B A B A B A A	8 5 12 12 13/94 18 18 16 14 13 55 51	6 5/92 10 19 14 17 9 10 18 25 56 54	4 5 17 14 12/95 18 20 14 21 18 61 45	8 5/95 18 14 22/91 15 14 21 17 11 52 42	9 14 15 17 14/94 16 21 16 18 13 27 33	14 10/92 15 20 19 19 19 12 17 15 27 28	15 8 17 25 18/95 14 25 24 23 15 22 30	7 12/95 23 19 16/91 19 17 18 20 22 31 37

A, B Replicate cultures

Naugalube APAN: Mutation Experiment in the Presence of S-9 (3 Hour Treatment) – Small and Large Colony Mutant Frequencies

Concentration µg/mL	Small Colonies			Large Colonies			Proportion of small colony mutants
	Mutants		MF §	Mutants		MF §
	Ym	Nm		Ym	Nm
0 100 P 125 P 150 P 200 P,PP B[a]P 2 B[a]P 3	717 652 631 646 631 160 192	763 768 760 768 768 384 384	30.71 85.52 95.18 77.24 93.12 694.13 549.57	674 617 625 616 625 277 256	763. 768 760 768 768 384 384	61.25 114.33 100.07 98.47 97.65 258.97 321.48	0.33 0.43 0.49 0.44 0.49 0.73 0.63

Conclusions:

It is concluded that Naugalube APAN showed weak evidence of inducing mutation at the tk locus of L5178Y mouse lymphoma cells when tested for 3 hours in the presence of a rat liver metabolic activation system (S-9. An increase in mutant frequency in excess of the Global Evaluation Factor of 126 mutants per 10e6 viable cells was observed at one intermediate concentration (100 μg/mL), with a statistically significant linear trend.
When tested for 3 hours in the absence of S-9 in the same test system, although some evidence of weak induction of mutation was observed, no increases in mutation frequency which exceeded the Global Evaluation Factor were observed and any observed increases were sporadic and not well reproduced, therefore were considered of uncertain biological relevance.

Executive summary:

Naugalube APAN was assayed for the ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used in the absence and presence of S-9.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 9.375 to 300 μg/mL (limited by solubility of the formulated test article in culture medium). The highest concentration tested, 300 μg/mL, gave 15% and 17% relative suspension growth (RSG) in the absence and presence of S-9, respectively.

In the Mutation Experiment ten concentrations, ranging from 10 to 300 μg/mL, were tested in the absence and presence of S-9. The highest concentration analysed, 200 μg/mL (limited by the appearance of post treatment precipitate), gave 20% and 24% relative total growth (RTG) in the absence and presence of S-9, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.

When tested up to toxic and precipitating concentrations for 3 hours in the absence of S-9, no increases in MF which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 106 viable cells (compared to concurrent controls) were observed in any treated cultures. A statistically significant linear trend (p≤0.001) was observed and the maximum increase in mean MF over the concurrent mean MF observed in any treated culture was very close to the GEF (approximately 124 at 150 μg/mL), but the individual MF values at this concentration differed markedly and only one exceeded the GEF by comparison with the concurrent mean MF. This observation was considered of uncertain biological relevance, particularly as the increase in mean MF over the concurrent mean MF at 200 μg/mL was approximately 93, with good agreement between replicate cultures.

When tested up to toxic and precipitating concentrations for 3 hours in the presence of S-9, an increase in mean MF of approximately 129 mutants per 10⁶viable cells (compared to concurrent controls), which exceeded the GEF of 126, was observed at one intermediate concentration (100 μg/mL), giving 30% RTG. Although the mean MF exceeded the GEF only at 100 μg/mL, increases in mean MF over the concurrent mean MF of approximately 124, 102 and 120 (which were very close to the GEF) were observed at 125, 150 and 200 μg/mL, respectively and there was a statistically significant linear trend (p≤0.01), thus providing further evidence of weak induction of mutation under this treatment condition. Increases in the proportions of both small and large colony mutants were observed at 100, 125, 150 and 200 μg/mL but there were no marked, concentration-related increases in the proportion of small colony mutants.

Results of formulation analyses demonstrated achieved concentrations within 100 ± 10% of the nominal test article concentrations and were considered acceptable.

No test article was detected in the vehicle sample.

When tested for 3 hours in the absence of S-9 in the same test system, although some evidence of weak induction of mutation was observed, no increases in mutation frequency which exceeded the Global Evaluation Factor were observed and any observed increases were sporadic and not well reproduced, therefore were considered of uncertain biological relevance.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo - Micronuclues Test

The test material was considered to be non-genotoxic under the conditions of the test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2001 to 12 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test",

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Method B12 of the EEC Commission Directive 92/69/EEC

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The results of the test are believed to be of value in predicting the mutagenic potential of the test material to man. The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sufficient albino Crl:CD-1 TM(ICR)BRs train mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main study the mice weighed 24 to 30g and were approximately five to eight weeks old. After a minimum acclimatisation period of seven days the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.
The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding. Free access to mains drinking water and food (Certified Rat and Mouse Diet 5LF2, PMI Nutrition International, Nottingham, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

intraperitoneal

Vehicle:

For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

Details on exposure:

Groups, each of seven mice, were dosed once only via the intraperitoneal route with the test material.

Duration of treatment / exposure:

Single treatment, 24 or 48 hours exposure.

Frequency of treatment:

All animals were dosed once only.

Post exposure period:

48 hours

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Groups, each of seven mice, were dosed with the test material.
Three further groups of mice were included in the study; two groups (seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide.

Control animals:

yes

Positive control(s):

Supplier's identification: Cyclophosphamide
Supplier's lot number: 108H0568
Safepharm serial number: R-2 1 89
Date received: 05 June 2001
Storage conditions: 4°C in the dark
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Parkfield Pharmaceuticals batch no. A1 147).

Tissues and cell types examined:

In mitotic cells in which chromosome damage has been caused by the test material or its metabolites, fragments (centric or acentric) or whole chromosomes tend to lag behind in the anaphase stage of cell division. After telophase, a large proportion of the fragments are not included in the nuclei of the daughter cells and hence form a single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. These micronuclei are seen in a wide variety of cell types, but erythrocytes are chosen since micronuclei are easily detected in these cells.

Details of tissue and slide preparation:

Slide Preparation
Immediately following termination (ie. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

Slide Evaluation
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part 111 (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Study
In animals dosed with test material via the oral and intraperitoneal routes no premature deaths or clinical signs were observed.
The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main study. Adequate evidence of test material toxicity was not demonstrated using either route of administration. Therefore, the intraperitoneal route was selected for use in the main study in an attempt to maximise exposure of the animals to the test material. The maximum recommended (MRD) of the test material, 2000 mg/kg, was selected for use in the main study, with 500 and 1000 mg/kg as the lower dose levels.

Micronucleus Study
Mortality Data and Clinical Observations
There were no premature deaths or clinical signs seen in any of the dose groups.

Evaluation of Bone Marrow Slides
Statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 2000 mg/kg test material groups when compared to their concurrent vehicle control groups. This was taken to indicate that a cytotoxic response had been achieved in the target issue (bone marrow).
There were statistically significant increases in the frequency of micronucleated PCEs at all three of the 24-hour test material dose groups when compared to their concurrent vehicle control group. However, all three values were well within the normal historical range for vehicle controls, and were only significant because of the unusually low vehicle control value.
Therefore, the increases were considered to have no toxicological significance.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophospharnide under the conditions of the test.
The test material was found not to produce any toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Micronucleus Study – Summary of Group Mean Data

TREATMENT GROUP	NUMBER OF PCE WITH MICRONUCLEI PER 200 PCE		PCE/NCE RATIO
TREATMENT GROUP	GROUP MEAN	SD	GROUP MEAN	SD
1. Vehicle Control 48-hour sampling time	0.9	1.2	0.91	0.21
2. Vehicle Control 24-hour sampling time	0.1	0.4	1.09	0.46
3. Positive Control 24-hour sampling	58.6***	36.2	1.23	0.39
4. APAN 2000 mg/kg 48-hour sampling time	0.3	0.5	0.58**	0.16
5. APAN 2000 mg/kg 24-hour sampling time	1.7***	0.8	0.62*	0.23
6. APAN 1000 mg/kg 24-hour sampling time	1.6**	0.8	0.90	0.22
7. APAN 500 mg/kg 24-hour sampling time	2.3*	2.4	0.77	0.18

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

* = P <0.05

** = P <0.01

*** = P <0.001

Micronucleus Study – Individual and Group Means and Standard Deviations: Vehicle Control (10 ml/kg) 48-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
1. VEHICLE CONTROL 10 ml/kg 48-hour sampling time	1	28	2000	0	0.00	626	0	0.60
	2	27	2000	0	0.00	493	0	1.03
	3	27	2000	0	0.00	468	0	1.14
	4	28	2000	3	0.15	529	1	0.89
	5	29	2000	1	0.05	522	0	0.92
	6	29	2000	2	0.10	606	0	0.65
	7	29	2000	0	0.00	471	1	1.12
	Group Mean	28.1	2000	0.9	0.04	531	0.3	0.91
	SD	0.9	0	1.2	0.06	63	0.5	0.21

Micronucleus Study – Individual and Group Means and Standard Deviations: Vehicle Control (10 ml/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
2. VEHICLE CONTROL 10 ml/kg 24-hour sampling time	8	27	2000	0	0.00	590	0	0.69
	9	25	2000	0	0.00	378	0	1.65
	10	29	2000	0	0.00	375	0	1.67
	11	29	2000	0	0.00	541	0	0.85
	12	27	2000	0	0.00	630	0	0.59
	13	29	2000	1	0.05	423	0	1.36
	14	29	2000	0	0.00	547	0	0.83
	Group Mean	27.9	2000	0.1	0.01	498	0.0	1.09
	SD	1.6	0	0.4	0.02	104	0.0	0.46

Micronucleus Study – Individual and Group Means and Standard Deviations: Cyclophosphamide (50 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
3. CYCLOPHOSPHAMIDE 50 mg/kg 24-hour sampling time	15	28	2000	22	1.10	413	0	1.42
	16	30	2000	32	1.60	622	1	0.61
	17	30	2000	49	2.45	442	0	1.26
	18	28	2000	80	4.00	376	0	1.66
	19	29	2000	110	5.50	459	1	1.18
	Group Mean	29.0	2000	58.6	2.92	462	0.4	1.23
	SD	1.0	0	36.2	1.81	95	0.5	0.39

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (2000 mg/kg) 48-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
4. APAN 2000 mg/kg 48-hour sampling time	20	29	2000	1	0.05	698	2	0.43
	21	29	2000	0	0.00	661	1	0.51
	22	26	2000	0	0.00	565	0	0.77
	23	27	2000	0	0.00	556	1	0.80
	24	24	2000	0	0.00	615	0	0.63
	25	27	2000	1	0.05	657	1	0.52
	26	29	2000	0	0.00	728	0	0.37
	Group Mean	27.3	2000	0.3	0.01	640	0.7	0.58
	SD	1.9	0	0.5	0.02	65	0.8	0.16

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (2000 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
5. APAN 2000 mg/kg 24-hour sampling time	27	29	2000	2	0.10	590	1	0.69
	28	28	2000	1	0.05	514	1	0.95
	29	24	2000	3	0.15	714	0	0.40
	30	29	2000	2	0.10	743	0	0.35
	31	27	2000	2	0.10	550	2	0.82
	32	29	2000	1	0.05	583	0	0.72
	33	25	2000	1	0.05	699	1	0.43
	Group Mean	27.3	2000	1.7	0.09	628	0.7	0.62
	SD	2.1	0	0.8	0.04	90	0.8	0.23

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (1000 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
6. APAN 1000 mg/kg 24-hour sampling time	34	28	2000	2	0.10	481	0	1.08
	35	29	2000	0	0.00	535	1	0.87
	36	27	2000	1	0.05	593	1	0.69
	37	30	2000	2	0.10	456	0	1.19
	38	26	2000	2	0.10	487	0	1.05
	39	27	2000	2	0.10	623	0	0.61
	40	30	2000	2	0.10	557	0	0.80
	Group Mean	28.1	2000	1.6	0.08	533	0.3	0.90
	SD	1.6	0	0.8	0.04	62	0.5	0.22

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (500 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM	PCE/NCE RATIO
7. APAN 500 mg/kg 24-hour sampling time	41	28	2000	6	0.30	620	1	0.61
	42	28	2000	0	0.00	574	0	0.74
	43	27	2000	0	0.00	477	0	1.10
	44	26	2000	4	0.20	518	0	0.93
	45	28	2000	3	0.15	633	0	0.58
	46	30	2000	0	0.00	590	2	0.69
	47	27	2000	3	0.15	579	0	0.73
	Group Mean	27.7	2000	2.3	0.11	570	0.4	0.77
	SD	1.3	0	2.4	0.12	55	0.8	0.18

Conclusions:

The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the revised OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of the EEC Commission Directive 92/69/EEC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METIMHLW guidelines for testing of new chemical substances.

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum recommended dose (2000 mg/kg) with 1000 and 500 mg/kg as the two lower dose levels.

Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.

Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 2000 mg/kg test material dose groups when compared to their concurrent control groups.

This was taken to indicate that a cytotoxic response had been achieved in the target tissue (bone marrow).

There were statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material at all three dose levels used when compared to the concurrent vehicle control group. However, all three values were well within the normal historical range for vehicle controls (Appendix I), and were only significant because of the unusually low vehicle control value. Therefore, the increases were considered to have no toxicological significance.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

Conclusion. The test material was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro - Ames Assay

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Genetic toxicity in vitro - Chromosome Aberration Test

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls.

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

Genetic toxicity in vitro - Gene Mutation Assay

When tested up to toxic and precipitating concentrations for 3 hours in the absence of S-9, no increases in MF which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 10⁶viable cells (compared to concurrent controls) were observed in any treated cultures. A statistically significant linear trend (p≤0.001) was observed and the maximum increase in mean MF over the concurrent mean MF observed in any treated culture was very close to the GEF (approximately 124 at 150 μg/mL), but the individual MF values at this concentration differed markedly and only one exceeded the GEF by comparison with the concurrent mean MF. This observation was considered of uncertain biological relevance, particularly as the increase in mean MF over the concurrent mean MF at 200 μg/mL was approximately 93, with good agreement between replicate cultures.

Genetic toxicity in vivo - Micronucleus Test

The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum recommended dose (2000 mg/kg) with 1000 and 500 mg/kg as the two lower dose levels.

Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.

Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 2000 mg/kg test material dose groups when compared to their concurrent control groups.

This was taken to indicate that a cytotoxic response had been achieved in the target tissue (bone marrow).

There were statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material at all three dose levels used when compared to the concurrent vehicle control group. However, all three values were well within the normal historical range for vehicle controls, and were only significant because of the unusually low vehicle control value. Therefore, the increases were considered to have no toxicological significance.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

Conclusion. The test material was considered to be non-genotoxic under the conditions of the test.

Justification for classification or non-classification

Genetic toxicity in vitro - Ames Assay

The test material was considered to be non-mutagenic under the conditions of this test, and thus does not fill the criteria required for classification.

Genetic toxicity in vitro - Chromosome Aberration Test

The test material was shown to be non-clastogenic to human lymphocytes in vitro, and thus does not fill the criteria required for classification.

Genetic toxicity in vivo - Micronucleus Test

The test material was considered to be non-genotoxic under the conditions of the test and thus does not fill the criteria required for classification.

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N-(dodecylphenyl)naphthalen-1-amine

Endpoint summary

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Endpoint conclusion

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