Tris(2,4-ditert-butylphenyl) phosphite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was negative in the Ames test (similar to OECD 471) with Salmonella strains TA 98, TA 100, TA 1535 and TA 1537 (with and without microsomal activation).

No genotoxic findings are reported in vitro and the test substance is considered to be not genotoxic based on studies on mutagenicity in bacteria.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains tested.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomes and co-factors

Test concentrations with justification for top dose:

1, 3, 9, 27, and 81 µg/0.1 mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance is insoluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): growth in absence of histidine

NUMBER OF CELLS EVALUATED:
In the experiments with and without the addition of microsomal activation mixture, three Petri dishes were prepared per strain and per group

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

CONTROLS:
Positive control experiments were carried out simultaneously with the following substances:
1) for Strain TA 1535: N-methyl-N'- nitro-N-nitrosoguanidine, 3 and 5 µg/0.1 ml phosphate buffer
2) for Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate, 25, 50, and 100 µg/0.l ml DMSO
3) for Strain TA 98: daunoblastin, 2.5, 5, and 10 µg/0.l ml phosphate buffer
4) for Strain TA 100: 4-nitroquinoline-N-oxide, 0.0625, 0.125, and 0.25 µg/0.l ml phosphate buffer
The activation mixture was tested with Strain TA 1535 and cyclophosphamide 100 and 250 µg/0.1 ml phosphate buffer

Evaluation criteria:

The test substances was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration

Statistics:

arithmetic mean

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

SUMMARY OF EXPERIMENTAL RESULT

	TA 98		TA 100		TA 1535		TA 1537
Dose (µg/0.1 ml)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	13	26	78	74	8	13	6	9
1	17	17	80	84	11	14	7	10
3	17	32	75	84	11	14	4	8
9	17	26	79	87	10	12	6	9
27	21	29	78	92	10	12	9	9
81	16	28	66	84	10	13	8	10
positive controls:
solvent control	25		74		14	11	5
concentration A	63		281		68	296	43
concentration B	211		398		1067	549	396
concentration C	229		707		-		>1900
solvent control						19
concentration A						256
concentration B						478

Conclusions:

Negative with and without metabolic activation.
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with test material revealed no marked differences.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo, no clastogenic or induced aneugenic potential of the test substance was reported in the following assay:

Negative findings in the micronucleus assay (similar to OECD 474) in chinese hamster bone marrow cells.

Negative findings in the chromosome aberration test (similar to OECD 475) in chinese hamster bone marrow cells.

Negative findings in two chromosome aberration test (similar to OECD 483) in Tif: MAG f [SPF] spermatogonia and spermatocytes.

Further in vivo investigation for a presumably DNA breakage and reunion (sister chromatid exchange) and to determine the potential to produce mutations caused by chromosomal aberrations in germ cells (dominant lethal test) were performed and found to be negative:

Negative findings in the sister chromatid exchange assay in chinese hamster bone marrow cells.

Negative findings in the dominant lethal study (similar to OECD 478) in Tif: MAG f [SPF] mice.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

1000 bone marrow cells (type not specified). No distinction between immature and mature erythrocytes.

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 18 - 26 g (females) and 19 - 27 g (males)
- Diet: ad libitum, standard diet: NAFAG No.924
- Water: ad libitum, tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol 400 (PEG 400)
- Concentration of test material in vehicle: 500, 1000, and 2000 mg/kg in 20 mL/kg PEG 400
- Amount of vehicle (if gavage or dermal): 20 mL/kg

Duration of treatment / exposure:

48 h

Frequency of treatment:

once daily

Post exposure period:

24 h

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide, 128 mg/kg in 20 mL/kg PEG 400
- Route of administration: gavage
- Doses / concentrations: 128 mg/kg

Tissues and cell types examined:

Bone marrow from the shafts of both femurs

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times.

DETAILS OF SLIDE PREPARATION:
Small drops of the above mentioned mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. At the next day the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40 % for 20 min. After being rinsed in methanol 55 % for 5 - 8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of three female and three male animals each of the negative control group and the treatment groups were examinedIn the positive control group the slides of four female and two male animals were analysed. 1000 bone marrow cells each were scored per animal and the following anomalies were registered:a) Single Jolly bodies, b) fragments of nuclei in erythrocytes,c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.

Statistics:

The significance of difference was assessed by χ2- test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

In the control group one female and one male animal died after the second application. In the positive control group one male animal died after the second application.

In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 7.9, whereas the negative control yielded a percentage of 0.1. The difference was highly significant (p<0.05).

			percent of cells with anomalies of nuclei
	number of animal	sex of animal	single jolly bodies	frasgments of nuclei in erythrocytes	micronuclei in erythroblasts	micronuclei in leucopoietic cells	polyploid cells	total
Control (PEG 400)	1	f	0.1					0.1
	2	f	0.1					0.1
	3	f	0.1		0.1			0.2
	4	m						0
	5	m	0.1					0.1
	6	m	0.1					0.1
Cyclophosphamide (128 mg/kg)	1	f	5.2	0.2	0.6	0.2	0.3	6.5
	2	f	4.4	0.9	0.7	0.3		6.3
	3	f	3	1.2	0.7			4.9
	4	m	4.1	1.9	0.1	0.1		6.2
	5	m	10.6	1.5	1.5	0.8	0.3	14.7
	6	m	6.8	0.7	1.1	0.1	0.1	8.8
test article (500 mg/kg)	1	f	0.2					0.2
	2	f	0.3					0.3
	3	f						0
	4	m	0.1					0.1
	5	m						0
	6	m	0.2					0.2
test article (1000 mg/kg)	1	f	0.2			0.1		0.3
	2	f	0.1					0.1
	3	f	0.2					0.2
	4	m	0.1					0.1
	5	m				0.1		0.1
	6	m	0.1					0.1
test article (2000 mg/kg)	1	f	0.1					0.1
	2	f						0
	3	f	0.3					0.3
	4	m	0.1					0.1
	5	m	0.1					0.1
	6	m					0.1	1

Conclusions:

The MNT assay in chinese hamster was found to be negative.
Under the conditions of this experiment, no evidence of mutagenic effects was obtained with the test article.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Limited housing details. Four instead of five animals per group. Two doses administered instead of single application.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

hamster, Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 22-29g
- Fasting period before study: no data
- Housing: no data
- Diet: ad libitum
- Water): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 C + 1 C
- Humidity (%): 55% + 5%
- Photoperiod (hrs dark / hrs light): room was illuminated for 12 hours daily

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol 400 (PEG 400)

Details on exposure:

The substance was administered orally once daily on 2 consecutive days. The animals were injected intraperitoneally with 10 mg colcemide/kg 2 h after the second dose and sacrificed 4 h later.

Duration of treatment / exposure:

once daily on two consecutive days

Frequency of treatment:

once daily on two consecutive days

Post exposure period:

10 mg colcemide/kg 2 h after the second dose and sacrificed 4 h later

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

4 female/male per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (ENDOXAN ): 64 mg/kg

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Bone marrow from the shafts of both femora was suspended in balanced salt solution and diluted to hypotonicity with distilled water, kept in a water bath at 4 to 6°C for 2 3 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanol acetic acid 3:1 for a period of 30 minat room temperature. After resuspension of the pellet and centrifugation for 5 min at 150 x g, the fixative was changed and the specimen stored overnight at 4 C. Finally, the pellets again were resuspended, centrifuged for 5 min at 150 x g and resuspended in some 0.5 ml fixative in order to obtain a more concentrated cell suspension. These specimens were pipetted onto wet slides and stained with acetic-orcein.

Evaluation criteria:

a) Chromatid-type aberrations. These changes include "breaks" and "exchanges". All discontinuations exceeding the diameter of a chromatid were designated as breaks. b) Chromosome-type aberrations. This category comprises "acentric fragments", "minutes" (very small chromosome fragments), "ring chromosomes" and "dicentrics". c) Chromatid gaps. All interruptions of the chromatid continuity which were smaller than the diameter were designated as gaps. d) Chromosome pulverizations. Completely pulverized metaphase chromosomes were distinguished from incomplete pulverizations with recognizable chromosome fragments.

Statistics:

no data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

One male animal of the intermediate-dose group died after the first application. Neither the negative control group nor the dosage groups showed any chromatid-type or chromosome-type aberrations. In contrast to the test article, the positive control caused an increase in all types of aberrations. In addition, pulverizations were recorded.

	CONTROL (PEG 400)				Cyclophosphamide (64 mg/kg)				TEST ARTICLE (500 mg/kg)				TEST ARTICLE (1000 mg/kg)				TEST ARTICLE (2000 mg/kg)
Number of animals	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
Sex of animals	f	f	m	m	f	f	m	m	f	f	m	m	f	f	m	m	f	f	m	m
percent of metaphases with specific aberrations					22	15	25	20
chromatid breaks					16	10	18	16
chromatid exchanges					10	8	15	8
chromatid-type aberrations					21	15	25	20
acentric fragments
minutes					2			1
chromosome-type aberrations					2			1
chromatid gaps	2	1	1		13	10	14	10	1		2		4			2	3	2	2
pulverization (partial)					7	2	7	2
pulverization (complete)						3	5
Number pf individual chromosome aberrations
chromatid breaks 1-2					14	8	14	15
chromatid breaks 3-<					2	2	4	1
chromatid exchanges 1-2					10	8	15	5
chromatid exchanges 3-<								3
acentric fragments
minutes						2		1

Conclusions:

Under the condition of this experiment, no evidence of mutagenic effects was obtained in Chinese hamster treated with the test compound.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

yes

Remarks:

Limited housing details. Four doses administred instead of 1-2 doses.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

other: Tif.MAGf(SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 26-30g
- Fasting period before study: no data
- Housing: no details
- Diet: ad libitum, NAFAG No.890
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C +/- 1°C
- Humidity (%): 50-72%
- Photoperiod (hrs dark / hrs light): room was illuminated for 12 hours daily

Route of administration:

intraperitoneal

Vehicle:

polyethylene glycol 400 (PEG 400)

Details on exposure:

The treatment groups each consisted of 15 male animals, the control group of 12 animals. The substance was administered once daily on five consecutive days (Days 0-4).a) Test substance: 1481 and 4444 mg/kg in 20 ml/kg Polyethylene Glycol (PEG 400). b) 20 ml/kg PEG 400 (negative control).The highest applicable dosage was limited to 4444 mg/kg, because the administration volume is prescribed by standard protocol and because a higher concentration of the substance could not be administered properly.

Duration of treatment / exposure:

once daily on five consecutive days

Frequency of treatment:

once daily on five consecutive days

Post exposure period:

24 h after the last application and three hours after receiving an intraperitoneal injection of 10 mg/kg colcemide

Dose / conc.:

1 481 mg/kg bw/day (actual dose received)

Dose / conc.:

4 444 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15 male (dosed), 12 animal (control)

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin (0.5 mg/kg)

Tissues and cell types examined:

germinal epithelium and in particular spermatogonia

Details of tissue and slide preparation:

The testes of ten animals in each group were processed and drop-preparations made by the air-drying technique ' according to a modification of the method described by SCHLEIERMACHER (1966) . The only departures from method were in the centrifugation process, the first centrifugation being performed at 180 X g for 10 minutes and subsequent centrifugations at 110 X 5 minutes.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

100 spermatogonial metaphase plates per animal were examined in eight control mice and eight mice treated with the two doses of the test substance. The chromosome displays from animals of the control group and from animals treated with the high dosage of test material showed no chromatid-type or chromosome-type aberrations. In the group treated with the low dosage one metaphase showing a chromatid-type aberration in the form of a break was detected. The incidence of the findings in the low-dose group is within the frequency observed in control animals of the breed used and this aberration can therefore be considered spontaneous in origin.

Conclusions:

The test compound exerted no mutagenic action in the mouse under these experimental conditions.

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

yes

Remarks:

Limited housing details. Five doses administred instead of 1-2 doses. No positive control

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

other: Tif: MAG f [SPF]

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 25 to 34 g
- Fasting period before study: no data
- Housing: no details
- Diet: ad libitum, NAFAG No.890
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C +/- 1°C
- Humidity (%): 50-72%
- Photoperiod (hrs dark / hrs light): room was illuminated for 12 hours daily

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol 400 (PEG 400)

Details on exposure:

Five doses of the substance were administered intermittently by intubation on Days 0, 2, 3, 5 and 9. Three days after the final dose, the animals were given an intraperitoneal injection of 10 mg/kg colcemide. Three hours thereafter they were sacrificed within a fixed period of two hours in the forenoon.

Duration of treatment / exposure:

once daily on five consecutive days

Frequency of treatment:

once daily on five consecutive days

Post exposure period:

Three days after the final dose, the animals were given an intraperitoneal injection of 10 mg/kg colcemide.

Dose / conc.:

1 481 mg/kg bw/day (actual dose received)

Dose / conc.:

4 444 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15 male (dosed), 12 animal (control)

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

testes, primary and secondary spermatocytes

Details of tissue and slide preparation:

The testes of ten animals in each group were processed and drop-praparations made by the air-drying technique according to a modification of the method described by SCHLEIERMACHER (1966) . The only departures from method were in the centrifugation process, the first centrifugation being performed at 180 X g for 10 minutes and subsequent centrifugations at 110 X g for 5 minutes.

Evaluation criteria:

Among the spermatocyte I metaphases the following aberrant forms were searched for: a) breaks, b) fragments, c) minutes, d) chromosome exchanges, e) atypical aberrations. Among the spermatocyte II metaphases: a) breaks, b) fragments and c) atypical aberrations were looked for.

Statistics:

x2-test

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

not examined

In the low-dose group two animals died after the fifth application.

In the spermatocyte-I metaphases one aberration in the form of a break was found in the control. In the low-dose group five aberrations were detected, four in the form of a fragment and one in the form of a minute. In the high-dose group one aberration in the form of a fragment was found. Examination of the spermatocyte-II metaphases revealed four aberrations in the form of a fragment in the control group, and one fragment each in the lowdose and high-dose groups.

Statistical comparison of the values for the low-dose group with those recorded in the concurrently studied negative-control group showed no significant difference. This was the case in both instances, i.e. upon comparison of the values from examination of the primary spermatocytes alone and, on the other hand, upon comparison of the values from examination of the primary and secondary spermatocytes together.

The incidence of changes observed in the controls and in the lowdose group is within the frequency observed in animals of the breed used and considered spontaneous in origin.

OVERVIEW OF RESULTS

	CONTROL (PEG 400)								TEST ARTICLE (1481 mg/kg)								TEST ARTICLE (4444 mg/kg)
Number of animals	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
METAPHASE I
% metaphases with aberrations	1									1			1	2		1				1
% metaphases with
breaks 1-2	1
breaks 3-<
fragments 1-2										1			1	2						1
fragments 3-<
exchanges 1-2
exchanges 3-<
atypical aberrations
METAPHASE II
% metaphases with aberrations		2		2								1					1
% metaphases with
breaks 1-2
breaks 3-<
fragments 1-2		2		2								1					1
fragments 3-<

Conclusions:

Under the given experimental conditions, there was no evidence of mutagenic effect of the test substance on spermatocytes of the mouse treated.

Reference 5

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Hamsters (4 female/male) were treated orally by a single dose of the test material 2h after subcutaneous implantation of a 45 mg-tablet of 5-bromodeoxyuridine (BUdR) in the neck. 24 h after administration of the test material and 2 h after intraperitoneal injection of 10 mg colcemide/kg the animals were sacrificed by dislocation of the cervical vertebrae.The slides of two or three female/male animals (treatment and control groups) were examined. Twenty-five differently stained metaphases of the second cell cycle with BUdR-substitution were analysed per animal for the number of SCE's following specific criteria.

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 21-32g
- Fasting period before study: no data
- Housing: no details
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 24-72%
- Photoperiod (hrs dark / hrs light): room was illuminated for 12 hours daily

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol 400 (PEG 400)

Details on exposure:

On the first occasion the treated groups and the control groups consisted of four female and four male animals each; on the second occasion all groups consisted of six female and six male animals. The animals were treated orally by a single dose of the test material two hours after subcutaneousimplantation of a 45 mg-tablet of 5-bromodeoxyuridine (BUdR) in the neck. 24 hours after the administration of the test material and 2 hours after, intraperitoneal injection of 10 mg colcemide/ kg the animals were sacrificed by dislocation of the cervical vertebrae.

Duration of treatment / exposure:

single exposure

Frequency of treatment:

single exposure

Dose / conc.:

1 111 mg/kg bw/day (actual dose received)

Remarks:

original study

Dose / conc.:

2 222 mg/kg bw/day (actual dose received)

Remarks:

original study

Dose / conc.:

4 444 mg/kg bw/day (actual dose received)

Remarks:

original study

Dose / conc.:

1 777 mg/kg bw/day (actual dose received)

Remarks:

Additonal study

Dose / conc.:

2 666 mg/kg bw/day (actual dose received)

Remarks:

Additonal study

Dose / conc.:

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

Remarks:

Additonal study

Dose / conc.:

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

Remarks:

Additonal study

No. of animals per sex per dose:

first occasion: four female and four male
second occasion: all groups consisted of six female and six male

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-Dimethylbenz(a)anthracene (DMBA)

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Bone marrow from the shafts of both femurs was suspended in 1% sodium citrate solution for hypotonic treatment, kept in a waterbath at 4 to 6 C for 45 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanolacetic acid 3:1 for a period of 30 min, resuspended, centrifuged for 5 min at 150 x g, and stored in fresh fixative overnight at 4 C. Finally the pellets again were centrifuged for 5 min at 150 x g, and resuspended in some 0.5 ml fixative in order to obtain a more concentrated cell suspension.These specimens were pipetted onto wet slides and air-dried. The air-dried slides then were treated with a solution of bisbenzimide (H 33258) for 15 min, rinsed in Mcllvaine-buffer pH 8.0 and irradiated in this buffer at 50°C with UV-light of 350 nm. Following the development of the fluorochrome-UVlight reaction in 60 C 2 x SSC (standard sodium citrate) for 90 min, the slides were stained in 40% Giemsa for 20-40 min, well rinsed, cleared in Xylol and mounted in Eukitt.

Evaluation criteria:

On the first occasion the slides of two female and two male animals each of the treatment groups and of the control groups were examined. In the additional study the slides of three female and three male animals in each group were examined. Twenty-five differently stained metaphases of the second cell cycle with BUdR-substitution were analysed per animal for the number of SCE's following specific criteria.

Statistics:

The significance of differences of the treatment groups compared to the negative control group was assessed by t-test on the level of one percent.As a non-parametric test for a dose-dependent trend on the numbers of SCE's per cell the JONCKHEERE test was used.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

In the first experiment no statistically significant difference in the number of SCE's was found in the low-dose group (1111 mg/kg) or in the intermediate-dose group (2222 mg/kg) by comparison with the negative control. In the high-dose group (4444 mg/kg), one of the four animals examined showed an increase in the number of SCE's. Statistical analysis by the t-test revealed that the difference between the mean number of SCE's in this group (6.26) and in the concurrent negative control group (4.21) was significant. A dose-dependent trend of the group medians (JONCKHEERE-test) was statistically significant.

To ascertain whether this was a fortuitous event, or due to an effect of the test substance, a second experiment with doses of 1777, 2666, 4000 and 6000 mg/kg was performed. In the various dose-groups no statistically significant increase in the numbers of SCE's was found in comparison with the negative control. A statistical analysis of the trends, including all dosage groups, yielded a negative result in this second experiment.

The respective positive control groups in both studies showed highly significant increases in the rates of SCE's per cell (9.07 and 10.5) in comparison with the concurrent negative controls (4.21 SCE's per cell and 4.39 SCE's/cell, respectively) (p<0.01).

EXPERIMENT 1

			Statistics
			SCEs per cell				observed t value
Group	No of animal	Sex	Animal mean	SD	group mean	SD
control	1	f	4.6	1.92	4.21	2.2	---
	2	f	5.16	2.9
	3	m	3.84	1.52
	4	m	3.24	1.83
DMBA (100 mg/kg)	1	f	8.96	2.92	9.07	3.93	10.79*
	2	f	11.52	4.3
	3	m	6.28	2.98
	4	m	9.52	3.65
Test article  (1111 mg/kg)	1	f	4.52	1.5	4.69	2.22	1.54
	2	f	5.2	2.93
	3	m	4.36	1.85
	4	m	4.68	2.37
Test article (2222 mg/kg)	1	f	4.64	1.93	4.62	2.05	1.36
	2	f	4.36	1.85
	3	m	4.24	1.71
	4	m	5.24	2.57
Test article (4444 mg/kg)	1	f	5.52	2.57	6.26	3.61	4.85*
	2	f	4.88	2.68
	3	m	10	4
	4	m	4.64	2

* = statistically significant at 1% (critical t-value = 2.34, d.f. JONCKHEERE trend test significant at 5%.

EXPERIMENT 2

			Statistics
			SCEs per cell				observed t value
Group	No of animal	Sex	Animal mean	SD	group mean	SD
control	1	f	4.04	1.7	4.39	2.35	---
	2	f	3.96	1.95
	3	f	4.88	2.52
	4	m	4.6	2.55
	5	m	4.2	2.75
	6	m	4.64	2.55
DMBA (100 mg/kg)	1	f	10.44	4.91	10.49	6.09	11.45*
	2	f	11.8	9.28
	3	f	9.28	4.8
	4	m	10.64	6.98
	5	m	11.12	4.91
	6	m	9.68	4.41
test article (1777 mg/kg)	1	f	4.56	2.2	4.63	2.14	0.92
	2	f	4.52	2.12
	3	f	4.88	2.45
	4	m	4.4	2.1
	5	m	4.76	1.54
	6	m	4.68	2.5
test article (2666 mg/kg)	1	f	3.68	2.39	4.57	2.57	0.63
	2	f	4.36	1.96
	3	f	3.84	1.91
	4	m	4.28	3.06
	5	m	4.88	1.86
	6	m	6.4	3.16
test article (4000 mg/kg)	1	f	4.8	2.77	4.85	2.81	1.54
	2	f	4.88	2.44
	3	f	4.72	2.82
	4	m	5.6	4.15
	5	m	4.48	1.87
	6	m	4.6	2.43
test article (6000 mg/kg)	1	f	4.64	2.64	4.9	2.62	1.77
	2	f	5.08	1.85
	3	f	4.6	1.87
	4	m	5.2	3.32
	5	m	5.44	3.23
	6	m	4.44	2.52

* = statistically significant at 1% (critical t-value = approx. 2.326, d.f. = 298. JONCKHEERE trend test significant at 5%.

Conclusions:

The SCE assay in chinese hamster was found to be negative.
From the results of both investigations together, it can be concluded that the higher rate of SCE's in one animal in the highdose group in the first experiment was purely fortuitous and not due to the test substance.
Thus, the results obtained indicate that under the given experimental conditions the test substance does not provoke any effect interpretable as being suggestive of a mutagenic property.

Reference 6

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978-07-10 - 1978-09-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

yes

Remarks:

Two doses tested. No positive control.

Principles of method if other than guideline:

The test material was administered orally in single doses to male albino mice (Tif: MAG f [SPF]) which were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones. Doses of 1000 and 3000 mg/kg were given. The experiment was done to evaluate any cytotoxic or mutagenic effects on the male germinal cells as expressed by the loss of pre-implantation zygotes as well as by the rate of deaths of post-implantation stages of embryonic development.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: Tif: MAG f [SPF]

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: closed SPF breeding colony
- Age at study initiation: about 2 ½ - 6 months
- Diet: Standard diet: NAFAG No. 890
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 60 ± 5
- Photoperiod (hrs dark / hrs light): 14 / 10

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose) and water
- Concentration of test material in vehicle: 1000 and 3000 mg/kg in 0.2 mL vehicle /10 g bw
- Amount of vehicle (if gavage or dermal): 0.2 mL/g bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The compound was administered orally by intubation in single doses of 1000 and 3000 mg/kg to each 20 males. An aqueous solution of carboxymethylcellulose served as the vehicle (0.2 mL/10 g of body weight). The control group was treated with the vehicle only. The females remained untreated.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

6 w

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 males each of which was placed in a cage with 2 untreated females immediately after treatment. At the end of 1 week, the females were removed and replaced by another group of 2 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

The first week after administration of the drug general condition and symptomatology were checked on the males.

Statistics:

To compare the totals of the number of implantations - indicating possible pre-implantation losses - Student's t test or Mann-Whitney' U-test was used. The totals of the numbers of mated and pregnant dams or embryonic deaths were compared with the aid of the x^2-test or Fisher's exact test. Whenever necessary, a test on the heterogeneity of the material was performed. Experimental data, particularly on the numbers of implantations and early embryonic deaths were compared with "spontaneous data" of a cumulative of untreated controls observed over a longer period of time (autopsy on day 18 of pregnancy).

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

The data on mating ratio, on the numbers of implantations, and embryonic deaths are comparable for all groups.

SUMMARY OF RESULTS

			Number of females mated		number of females with deciduomata only		number of females pregnant		number of implantations			Live embryos		Embryonic death
	Dose group (mg/kg)	Number of females	total	%	total	%	total	%	total	average	SD	total	%	total	%
Mating 1	0	40	31	77.5	0	0	27	87.1	253	9.37	2.31	228	90.1	25	9.9
	1000	40	38	95	1	2.6	34	89.5	322	9.47	3.13	298	92.5	24	7.5
	3000	40	34	85	0	0	29	85.3	295	10.17	2.61	264	89.5	31	10.5
Mating 2	0	38	34	89.5	0	0	27	79.4	288	10.67	2.67	258	89.6	30	10.4
	1000	40	37	92.5	0	0	34	91.9	326	9.59	3.08	297	91.1	29	8.9
	3000	40	31	77.5	0	0	28	90.3	269	9.61	2.33	238	88.5	31	11.5
Mating 3	0	38	35	92.1	1	2.9	32	31.4	343	10.72	1.69	304	88.6	39	11.4
	1000	40	39	97.5	0	0	34	87.2	341	10.03	1.96	315	92.4	26	7.6
	3000	40	32	80	0	0	28	87.5	268	9.57	3.07	248	92.5	20	7.5
Mating 4	0	38	33	68.8	0	0	27	81.8	280	10.37	3.01	267	95.4	13	4.6
	1000	40	37	92.5	0	0	35	94.6	354	10.11	2.26	325	91.8	29	8.2
	3000	40	31	77.5	0	0	29	93.5	294	10.14	1.83	263	89.5	31	10.5
Mating 5	0	38	31	81.6	0	0	28	90.3	288	10.29	1.88	264	91.7	24	8.3
	1000	40	36	90	0	0	28	77.8	292	10.43	2.56	275	94.2	17	5.8
	3000	40	32	80	0	0	28	87.5	305	10.89	1.69	285	93.4	20	6.6
Mating 6	0	38	31	81.6	1	3.2	26	83.9	282	10.85	2.74	258	91.5	24	8.5
	1000	40	35	87.5	0	0	31	88.6	346	11.16	2.34	315	91	31	9
	3000	40	31	77.5	0	0	23	74.2	252	10.96	1.99	231	91.7	21	8.3
Cumulative control			450		4	0.9	378	84	4062	10.77	2.29	3717	91.5	345	8.5

Conclusions:

No evidence of dominant lethal effect was observed in the progeny of male mice treated with the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance was evaluated for its mutagenic and genotoxic potential in-vitro and in-vivo. Overall the data do not indicate any mutagenic or genotoxic potential of the substance.

 

in-vitro-Tests:

No evidence for a mutagenic effect was obtained in Salmonella strains TA 98, TA 100, TA 1535 and TA 1537 treated with up to 81 µg per plate in experiments with and without microsomal activation.

No evidence for a mutagenic effect was obtained in Saccharomyces cerevisiae MP-1 treated with up to 10000 µg per plate in experiments with and without microsomal activation.

 

in-vivo-Tests:

No evidence for a clastogenic effect in Chinese Hamster bone marrow cells was obtained after treatment for two consecutive days by gavage with up to 2000 mg/kg bw/day.

No induction of SCE's in Chinese Hamster bone marrow cells occurred after treatment by gavage with up to 6000 mg/kg bw. In the first experiment one of four animals of the high dose group (4444 mg/kg) showed a statistically significantly increased frequency of SCE's. A statistical analysis of the trends, including all dosage groups, also yielded a positive result. In the repeat experiment performed with doses of up to 6000 mg/kg, the number of SCE's in all treatment groups showed no significant increase in comparison with the concurrent negative control. A statistical analysis of the trends, likewise, yielded a negative result in this second experiment. From the results of both investigations together, it can be concluded that the higher rate of SCE's in one animal in the high-dose group in the first experiment was purely fortuitous and not due to the test substance.

No evidence for a clastogenic effect or induced aneuploidy in Chinese Hamster bone marrow cells was obtained after treatment by gavage with 500, 1000 or 2000 mg/kg test substance.

No evidence for a clastogenic effect or induced aneuploidy in male mice spermatogonia and spermatocytes was obtained after treatment by gavage with 1481 and 4444 mg/kg.

No evidence for Dominant Lethal effects or reduced male fertility was obtained in male mice treated by gavage with either 1000 or 3000 mg/kg bw.

The studies were found to be adequate to fulfill the purposes of this endpoint.

 

Assessment of the need of performing studies on mutagenicity in mammalian cells in vitro with test substance

The test on genotoxicity in mammalian cells in vitro will not give any further useful information on genotoxicity. The main argument is that the substance was found to be non carcinogenic in a carcinogenicity study in rats. The study is adequate in design and quality to assess the endpoint of carcinogenicity.

Mutagenicity is one mode of action for the development of cancer and so a carcinogenicity study may give an indication on in-vivo mutagenicity.

In addition, the following arguments are given:

a) The water solubility of the substance is <0.005mg/l (at 20°C) and therefore, due to the sensitivity of cultivated cells to precipitates, the mutagenicity test with mammalian cells in vitro could only be performed with very low concentrations.

b) The substance does not carry structural alerts as identified by the rules of Zeiger (Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans, K., (1992). Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ Mol Mutagen, 19 Suppl 21, 2-141.)

c) The substance does not carry structural alerts as identified in the knowledge base of DEREK (Lhasa Inc.).

d) The substance is not mutagenic in the Ames test.

e) No adverse effects were observed in the dominant lethal study in rats.

f) The substance neither induces sister-chromatide-exchanges nor chromosome-aberrations in-vivo.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480.

As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.