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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 6, 1990 to January 9, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Conclusion based on scoring 1000 PCEs/animal for MNPCEs versus 2000 PCEs/animal as per OECD 474.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
scored fewer PCEs/animal than recommended.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
m-phenylenediamine
EC Number:
203-584-7
EC Name:
m-phenylenediamine
Cas Number:
108-45-2
Molecular formula:
C6H8N2
IUPAC Name:
m-phenylenediamine
Details on test material:
Analytical purity: > 98%
- Name of test material (as cited in study report): meta-phenylenediamine

Test animals

Species:
mouse
Strain:
other: Crl:CD-1(ICR)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc., Breeding Area 62, Montreal, Quebec, Canada.
- Age at study initiation: 50 days old
- Weight at study initiation: Males were 28.6 to 33.5 g, females were 21.3 to 26.1 g
- Assigned to test groups randomly: By computer-generated random numbers
- Housing: Individually housed in standard wire mesh cages
- Diet: Purina certified Rodent Chow #5002, Lots # Aug 07 901A and Aug 10 901B) ad libitum
- Water: Ad libitum
- Acclimation period: Quarantined and acclimated for 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Deionized water
Frequency of treatment:
Twice, with doses administered approximately 24 hours apart.
Doses / concentrations
Remarks:
Doses / Concentrations:
16, 33, 65 mg/kg/day
Basis: actual ingested
No. of animals per sex per dose:
5 males/females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide

Examinations

Details of tissue and slide preparation:
Immediately following sacrifice at 24 or 48 hours after the administration of the second dose, the marrow from both femurs of each animals was collected. A Miniprep automatic blood smearing instrument was used to make the bone marrow smears. At least 2 slides/animal were prepared.
Evaluation criteria:
Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy. Only cells showing good morphology and staining were selected for scoring. Polychromatic erythrocytes (PCEs) were identified by their characteristic reddish staining. Normochromatic erythrocyte (NCEs) appeared dark green. One thousand PCEs/animal were scored for the presence of micronuclei, which are typically round, bright yellow-green fluorescent bodies. Cellular inclusions that were irregularly shaped or stained, or were not in the focal plane of the cell were considered artifacts. The unit of scoring was the micronucleated cell. PCEs containing more than one micronucleus were counted as a single micronucleated cell. The number of micronucleated NCEs seen in the optic fields scored to obtain 1000 PCEs was also recorded. The proportion of PCEs among 1000 erythrocytes was determined for each animal.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 25, 50, 65, 75 mg/kg/day, 2X ~24 hours apart.

- Clinical signs of toxicity in test animals: 75 mg/kg/day group, 3/3 males and 1/3 females died within 30 hours of second dosing.
65 mg/kg/day-lethargic and had labored breathing; these signs were observed in all animals and occurred within 2 hrs of dosing on both days. Mild clinical signs (ruffled fur and partially closed eyes) persisted for 2 days following the final exposure, however, all animals recovered within 4 days post-treatment. 50 and 25 mg/kg/day-face pawing, labored breathing, gasping, lethargy and half closed eyes.

- Dose range: 85, 100, 200 or 300 mg/kg-a second treatment at these levels was not conducted due to excessive mortality or the severity of the clinical signs observed 24 hrs after the initial treatment


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: 65 mg/kg/day-within 1 hour of initial dose, mice exhibited lethargy, gasping, face pawing, spasms, tremors, or half-closed eyes. Lethargy, half-closed eyes and labored and/or rapid breathing were still evident in the majority of the animals, 3-4 hrs after initial observations. Clinical signs following the second dose were similar to those observed the preceding day. One male was found dead 24 hrs after the final treatment. 16 and 33 mg/kg/day-lethargy, half closed eyes and rapid breathing within 24 hrs of final dose. The majority of the 16 mg/kg/day treated group appeared to recover, and only slight clinical sign were still evident in the 33 mg/kg/day treated group.

Weight loss-there was a statistically significant weight loss in 33 and 65 mg/kg/day treated groups at the 24 hour sacrifice, and in the 65 mg/kg/day treated group at the 48 hr sacrifice.

There were no statistically significant increases in MN-PCEs among test substance treated mice at any dose level or sampling interval.

Any other information on results incl. tables

Table 1. Mouse Micronucleus Assay of m-PDA

 

m-PDA Treatment

(mg/kg/day)

Sampling Time

(hrs)

Sex

Animals Per Group

Change in Body Weight (g)

x ± S.E.

0

24

M

5

0.0 ± 0.2

 

 

F

5

-0.4 ± 0.5

16

24

M

4a

-0.9 ± 0.6

 

 

F

5

-0.7 ± 0.4

33

24

M

5

-3.3 ± 0.7**

 

 

F

5

-2.8 ± 0.2**

65

24

M

6

-5.3 ± 0.4**

 

 

F

6

-4.5 ± 0.3**

0

48

M

5

0.4 ± 0.4

 

 

F

5

0.8 ± 0.9

65

24

M

4a,b

-3.4 ± 0.9**

 

 

F

6

-3.3 ± 0.5**

CP, 20

24

M

5

0.2 ± 0.3

 

 

F

5

-0.6 ± 0.2

 

 

 

 

 

 

CP = cyclophosphamide

a animal misdosed and removed from study.

b animal died before scheduled sacrifice time.

** p < 0.01

 

 

Table 2. Data Summary. Mouse Micronucleus Assay of m-PDA.

 

m-PDA Treatement

(mg/kg/day)

Sampling Time

(hrs)

Mean % Micronucleated PCEs ± S.E.a

 

Mean PCE:NCE Ratio ± S.E.

N

Males

N

Females

 

Males

 

Females

0

24

5

0.16 ± 0.05

5

0.02 ± 0.02

 

1.16 ± 0.11

 

1.12 ± 0.03

16

24

4b

0.15 ± 0.09

5

0.16 ± 0.05

 

0.96 ± 0.05

 

1.13 ± 0.08

33

24

5

0.24 ± 0.07

5

0.10 ± 0.04

 

0.91 ± 0.07

 

1.14 ± 0.07

65

24

6

0.28 ± 0.07

6

0.13 ± 0.05

 

0.77 ± 0.10

 

1.13 ± 0.12

 

 

 

 

 

 

 

 

 

 

0

48

5

0.40 ± 0.13

5

0.28 ± 0.05

 

0.98 ± 0.12

 

1.34 ± 0.09

65

48

4b,c

0.50 ± 0.27

6

0.18 ± 0.03

 

0.58 ± 0.08*

 

0.87 ± 0.26

 

 

 

 

 

 

 

 

 

 

CP, 20

24

5

0.96 ± 0.09**

5

0.82 ± 0.09**

 

0.71 ± 0.03**

 

1.00 ± 0.10

 

 

 

 

 

 

 

 

 

 

 

CP = cyclophosphamide

a 1000 PCEs scored per animal.

b animal misdosed and removed from study.

c animal died before scheduled sacrifice time.

*   p < 0.05

** p < 0.01

 

Applicant's summary and conclusion

Conclusions:
Mice exposed to the test substance did not exhibit statistically significant increases in micronucleated polychromatic erythrocytes at any dose level or sampling interval.
Executive summary:

No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance-treated animals at any sampling time. A significant depression in the ratio of young, polychromatic erythrocytes to mature, normochromatic erythrocytes was observed in the high dose males at the 48 -hr sampling interval. Under the conditions of this assay, the test substance did not induce micronuclei; the test substance is negative.

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