Brominated chlorinated copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of bromines is equal to or more than 10 and less than 16, and the number of chlorines is equal to or more than 0 and less than 7

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study report meets basic scientific principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)

Version / remarks:

adopted 23 Oct 1986

Deviations:

yes

Remarks:

Historical control data not included in the report. MDS not scored, Limit dose exceeded by factor of 5

GLP compliance:

yes

Type of assay:

mouse spot test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Impurities: in all likelihood solvents (nitrobenzene or chlorobenzene; concentration not known)
- Several batches were created from different raw material suppliers. These were tested in the Ames test to screen for quality of the raw materials

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK 13 143 (crude copper phthalocyanine)
- Analytical purity: commercial grade
- Lot/batch No.: F 53 / H 91375

Test animals

Species:

mouse

Strain:

other: C57/Bl/6, males: T-stock

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bomholtgard Ltd. Denmark
- Age at study initiation: 3 - 4 months
- Weight at study initiation: females 20 - 23 g in toleerability test and 19 - 30 g in mutagenicity test; male body weight was not determined. (Males were not treated; males were included to mate with females and then only pregnant females were treated.)
- Assigned to test groups randomly: yes
- Diet: NAFAG No. 890 pellets standard diet, ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 48 - 56 %
- Air conditioned room
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Sesame oil was used as vehicle for the test material, Hank´s BSS was used as vehicle control for the positive control.

Details on exposure:

Tolerability test:
A preliminary test was conducted to determine the highest dosage of the test material to be applied in the mutgenicity test. 3 groups of 4 female mice were treated with 3 different single doses.The observation period lased 2 weeks. Depending on the outcome, the highest dose causing no deaths was used as the highest in the mutagenicity test or, if neccessary, the test was repeated with lower doses. Doses tested for tolerability were 200, 1000 and 5000 mg/kg bw.

Mutagenicity test:
One untreated male was placed in a cage with 2 untreated females. The females were inspected daily for successful mating. The day on which a vaginal plug was observed was designated as "day 1/2 of gestation". The females presumed to be pregnant were removed and the procedure was repeated for 4 consecutive days (4 mating nights). Subsequently the presumably pregnant females were uniformely distributed among the respective groups by random.
The test material preparation was administered intraperitoneally to groups of 71 successfully mated females. All presumably pregnant females were treated on the 10th day after conception. Treatment consisted of a single i.p. injection of the respective dose. The animals of the control group received vehicle only.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

once

Post exposure period:

until the birth of the offspring

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Tolerability test: 4 females per group
Mutagenicity test: 48 males and 96 females per group

Control animals:

yes, concurrent vehicle

Positive control(s):

N-Nitroso-N-ethylurea (ENU), 50 mg/kg bw in Hank´s BSS was administered intraperitoneally in parallel.

Examinations

Tissues and cell types examined:

Animals treated as embryos were allowed to come to birth. The number of live and dead offspring was listed. The pups were inspected for external visible morphological changes. The examination upon spots began at the age of 12 - 14 days and was carried out twice per week during 3 weeks. 2 classes fo spots were distinguished and registered: pigmented and white spots, randomly distributed on the coat (recessive spots RS) and white mid-ventral spots (WMVS) within 5 mm of the mid-ventral line presumably arising from cell killing and thus not a result of mutagenic effects. Yellow, agouti-like spots in the vicinity of the mammae, genitalia, throat, axillary and inguinal areas and on the mid-forehead, which are presumed to result from misdifferentiation (MDS) are omitted from scoring. The pelts from the animals with spots were preserved.

Statistics:

The statisitcal analysis was conducted in 2 parts, Firstly the numbers of recessive spots in the control group and in the treatment groups were compared by a chi-squared test. Secondly, a test was carried out to determine whether the frequency of recessive spots increases with increasing doses.
If the effect of the substance increased with the dose, the trend test was preferable. The tests were applied on the condition that the proportions of recessive spots were constant over litters.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tolerability test:
The dose of 5000 mg/kg bw was found to be the highest applicable in the mutagenicity test and was administered, together with 2 further doses, diminishing by a factor of 0.5.

Mutagenicity test:
From 71 presumably pregnant females per dose group, the following numbers actually were pregnant and gave birth to litters: Control: 56; 1250 mg/kg bw: 45, 2; 2500 mg/kg bw: 48; 5000 mg/kg bw: 44.
The average littersizes registered were: Control: 6.45; 1250 mg/kg bw: 5.93; 2500 mg/kg bw: 5.29; 5000 mg/kg bw: 6.07. 343 animals from the control group, 216, 171 and 169 animals from the groups, treated with 1250, 2500 and 5000 mg/kg bw were examined for colour spots.
The following percentages of animals with recessive (RS) and mid-ventral (WMVS) spots were recorded from gross observations:
RS: Control 0.29 %, 1250 mg/kg bw: 0.93 %, 2500 mg/kg bw: 0 %, 5000 mg/kg bw: 0 %
WMVS: Control 1.17 %, 1250 mg/kg bw: 3.24 %, 2500 mg/kg bw: 2.34 %, 5000 mg/kg bw: 1.78 %
In the positive control, the mean percentage of RS spots was 4.75 and of WMVS spots 2.71.
Statistical analysis for RS revealed the following results: Overall test X2 (3) = 3.82,p = 0.2819; Trend test (one-sided): Z = -0.7637, p = 0.7775

Any other information on results incl. tables

Table 1: THE EFFECT ON OFFSPRING FROM C57 Bl/6 x T CROSS TREATED IN UTERO

dose	vehicle	route of application	treated females with vaginal plug	% females with litters	average litter size	number of offspring examined	offspring with recessive spots (total)	%	number of offspring with intraventral spots (total)	%
negative control	Sesame oil	i .p .	71	62.0	6.1	169	0.0	0.0	3	1.8
1250	Sesame oil	i .p .	71	63.4	5.9	216	2.0	0.9	7	3.2
2500	Sesame oil	i .p .	71	67.6	5.3	171	0.0	0.0	4	2.3
5000	Sesame oil	i .p .	71	62.0	6.1	169	0.0	0.0	3	1.8
50 mg/kg positive control	Hank's	i .p .	71	66.2	6.5	295	14.0	4.8	8	2.7

Table 2: Results of positive control experiment

dose (mg/kg bw) vehicle or N-Nitroso-N-ethylurea	vehicle	route of application	treated females with vaginal plug	% females with litters	average litter size	number of offspring examined	offspring with recessive spots (total)	%	number of offspring with intraventral spots (total)	%
negative control	Hank's BSS	i .p .	66	68.2	5.6	277	0.0	0.0	3	1.1
25	Hank's BSS	i .p .	66	74.2	7.4	350	14.0	4.0	10	2.9
50	Hank's BSS	i .p .	66	66.7	7.2	298	20.0	6.7	5	1.7
75	Hank's BSS	i .p .	66	71.2	7.2	270	27.0	10.0	18	6.7

Applicant's summary and conclusion

Conclusions:

Mated female mice received a single intraperitoneal injection of 1250, 2500 or 5000 mg/kg bw on day 10 of pregnancy. An similar number of litters and litter size was observed for all treatment groups indicating absence of embryotoxicity. The number of offspring with recessive spots (indicators of mutagenicity) was increased in the positive control group, but not in the treatment groups. The number of intraventral spots (indicators of toxicity) showed a higher a variability without dose-dependency.