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Genetic toxicity: in vivo

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in vivo mammalian germ cell study: gene mutation
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)
Version / remarks:
adopted 23 Oct 1986
Historical control data not included in the report. MDS not scored, Limit dose exceeded by factor of 5
GLP compliance:
Type of assay:
mouse spot test

Test material

Constituent 1
Chemical structure
Reference substance name:
29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper
EC Number:
EC Name:
29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper
Cas Number:
Molecular formula:
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):
- Analytical purity: commercial grade
- Lot/batch No.: F 53 / H 91375

Test animals

other: C57/Bl/6, males: T-stock
Details on test animals or test system and environmental conditions:
- 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

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

Administration / exposure

Route of administration:
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:
Post exposure period:
until the birth of the offspring
Doses / concentrationsopen allclose all
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
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.


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.
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
no effects
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
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


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

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.