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Carcinogenicity

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Description of key information

No in vivo data are available concerning carcinogenic effects of the test substance.

Based on the data described in chapter 7.6 the substance shows no genotoxic effects and there is no evidence from the repeated dose study that the substance is able to induce pre-neoplastic lesions. According to Annex X (column 2 of section 8.9.1) and according to Annex X (preface) a carcinogenicity study is not required. With structurally related substances, a combined oral chronic toxicity/carcinogenicity and dermal carcinogenicity studies are available. In the combined oral chronic toxicity and carcinogenicity with the structurally related ethylenediammonium dichloride (EDA*2HCl) there was no evidence for a carcinogenic potential of EDA*2HCl in male and female F344 rats. The NOAEL for chronic toxicity was 20 mg/kg bw/day for EDA*2HCl (corresponding to 9 mg/kg bw/day of EDA). In the dermal carcinogenicity study with the structurally related ethylene diamine (EDA) in mice, there was no evidence for a carcinogenic potential of EDA after life-time dermal administration. The NOAEL was 8.3 mg/kg bw/day (25 µL 1% solution/per mouse).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP data
GLP compliance:
not specified
Specific details on test material used for the study:
High purity EDA was used for synthesizing the chloride salt. Various chromatographic and spectrophotometric analytical methods showed that the obtained EDA di-hydrochloride was of high purity.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Microbiological Associates, Inc. (Walkersville, MD, USA).
- Age at study initiation: 43 days of age at start of study.
- Weight at study initiation: males: 81-141 g , females: 60-112 g,

- Housing: suspended wire-bottom and front stainless-steel cages. Three males or five females were placed in a cage
- Diet: Ground diet (Zeigler Brothers NIH-07 Rat and Mouse Ration, Zeigler Bros., Inc., Gardners, PA, USA) was supplied in 12 oz.
opal glass jars. Prior to use, the diet was evaluated for nutritional content and possible contaminants
- Water : ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Photoperiod (hrs dark / hrs light): 12 h light cycle


Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosed diet was prepared and offered to the animals every 2 wk. Based on the predicted mean body weight gain and diet consumption data, the
concentration of EDA*2HCl in the diet was adjusted to maintain a constant dose level in g/kg.
A HPLC method was used to analyse the dietary concentration of EDA*2HCl. The distribution was homogenous and EDA*2HCl was stable in the diet for 6 months. Random samples during the study confirmed the accuracy of concentrations in diet.
Duration of treatment / exposure:
2 Year
Frequency of treatment:
Every second week
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
No. of animals per sex per dose:
100
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Bi-weekly
FOOD CONSUMPTION AND COMPOUND INTAKE : Diet consumption was determined on animals from the first 10 cages/sex/group biweekly.

WATER CONSUMPTION: Two weeks prior to each scheduled sacrifice, water consumption of animals from the first 10 cages/sex/group was measured.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 week prior to sacrifice
- Anaesthetic used for blood collection: Yes methoxyflurane
- How many animals: 10 per sex/group at 6 and 12 month sacrifice, 20 per sex/group at 18 and 24 months sacrifice
- Parameters examined: Red and white blood cell counts, differental white cell counts, measurement of hemoglobin and mean corpuscular volume, and calculations of hematocrit, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 week prior to sacrifice
- How many animals: 10 per sex/group at 6 and 12 month sacrifice, 20 per sex/group at 18 and 24 months sacrifice
- Parameters examined: Serum concentrations of glucose, urea, nitrogen, aspartate aminotransferase, alanine, aminotransferase, alkaline phosphatase, total protein, albumin, creatinine, bilirubin (conjugated and total) and sorbital dehydrogenase.

URINALYSIS: Yes
- Time schedule for collection of urine: 1 week prior to sacrifice
- Metabolism cages used for collection of urine: Yes
- Parameters examined: The measurements and observations included volume, pH, specific gravity, protein, glucose, ketones, occult blood, turbidity, color, microscopic appearance, bilirubin and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
10 rats/sex/dose and control group were scheduled for sacrifice at 6 and 12 month, 20 rats/sex/dose and control group were scheduled for sacrifice at 18 month.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Relevance of carcinogenic effects / potential:
There was no evidence, under the conditions of this study, that chronic feeding of ethylenediamine dihydrochloride exhibited a carcinogenic effect in the Fischer 344 rat.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Key result
Dose descriptor:
NOAEL
Effect level:
9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Recalculated as ethylenediamine base
Key result
Dose descriptor:
NOAEL
Effect level:
159 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Recalculated as Ethylenediamine base

Doses attained were close to the dose goal, for males, 20, 100 and 350 mg/kg/day, females 20, 100 and 360 mg/kg/day.

A slight increase in diet consumption of EDA treated females between 5 - 15 months, otherwise no significant differences from control.

Increases in water consumption were observed for both males and females from the high dose group at 12 and 18 month and for females from the high dose group at 24 month associated with increased urine volume and decreased urine specific gravity. 

There was a significant reduction in body weight gain in male rats of the high dose group throughout the whole study course and in female rats of the high dose group from the 18th month until termination. A significant increase of body weight gain in female rats of the intermediate dose group from day 21 until the 21st month was noted. 

 

As shown in Tables 1 and 2, mortality for groups ingesting EDA was comparable to control values during the first 18 months of the study. After 22 months, mortality in males and females ingesting EDA were elevated from both control groups. In addition, the mortality rate in female rats ingesting 100 mg/kg/day was increased after 24 months.

Significant reduction in the absolute weights of liver, kidney, spleen (male) and increase of the relative weights of liver, kidney, heart, brain (females) in rats of the high dose group. No substance-related changes in hematologic data, clinical chemistry values and urinalysis except a decrease in erythrocyte count, hemoglobin concentration, hematocrit (male) and serum albumin concentration (female) in rats of the high dose group. Significantly higher incidence of hepatocellular pleomorphism in female rats of the intermediate and the high dose group; rhinitis and tracheitis were seen with greater frequency in high dose males at 12, 18 and 24 months and in high dose females at 18 months; at 24 months, rhinitis persisted at a significantly greater frequency in high dose females while tracheitis did not; lower incidence of pituitary adenomas and testicular interstitial cell adenoma in the high dose group (incidence ratio: 2/4 in comparison to 25/26 and 12/15 in the control groups); all other tumor incidences did not differ significantly from control. 

Table 1: Cumulative % Mortality of Male Rats after 18 Months

 

   Dose Level, mg/kg/day

Month

Control-A

Control-B

20

100

350

18

5.6

5.9

2.5

6.2

12.0

19

9.7

7.3

2.5

7.7

13.4

20

16.4

14.1

5.8

12.9

20.0

21

21.4

17.6

10.9

21.6

29.8

22

26.4

24.4

15.9

33.8

44.5a,a

23

34.8

41.6

27.7

40.8

65.7a,a

24

46.5

67.4-a

54.6

61.7

88.6a,a

25

63.4

78.7

77.3

68.1

95.4a,a

a,a First letter denotes significantly different from control group A and second letter denotes same from control group B (p0.05).

Table 2:Cumulative % Mortality of Female Rats after 18 Months

 

   Dose Level, mg/kg/day

Month

Control-A

Control-B

20

100

350

18

2.1

2.4

6.2

4.6

3.2

19

6.2

3.7

7.7

6.0

7.4

20

7.9

7.1

9.4

12.6

10.6

21

9.6

10.3

17.8

15.9

20.3

22

11.3

12.0

21.1

19.2

25.2a,a

23

14.6

16.8

21.1

27.5

33.3a,a

24

18.0

21.7

26.1

39.0a,a

41.5a,a

25

30.4

24.9

29.4

56.0a,a

55.7a,a

a,a First letter denotes significantly different from control group A and second letter denotes same from control group B (p0.05).

able 3  Selected tumor incidences in rats fed EDA-2HCl

 

Dose

Pituitary

Testes/interstitial

mg/kg/day

adenomas

cell adenomas

0 (A)

15/61

49/60

0 (B)

21/60

44/59

20

14/58

48/58

100

14/59

47/58

350

5/59*

7/60*


* Statistically significant as compared to both control groups (P0.01).

 

 

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
9 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Please refer to section 13 for read across justification.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-GLP
Qualifier:
no guideline followed
Principles of method if other than guideline:
The maximum tolerated dose (for local effects) was applied to the back of 50 male mice three times weekly throughout their lifespan. Positive (3-methylcholanthrene) and negative (water) control groups (50 mice/group) were included.
GLP compliance:
not specified
Specific details on test material used for the study:
Substance no 1:
Impurity: 0,07% ammonia
Substance no 2:
Impurities:
0.54 % pyrazine
0.08 % ammonia
0.03 % water
0.02 % monomethylamine
0.02 % ethylamine
0.02 % N-methyl-piperazine
0.02 % methylpyrazine
trace dimethylamine
trace ethanol
trace N-ethylpiperazine
trace ethylpyrazine
Species:
mouse
Strain:
C3H
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: C3H/HeJ mice from Jackson Laboratories, Bar Harbor, Maine
- Age at study initiation: 74 to 79 days of age
- Housing: Housed individually in stainless steel cages with wire mesh floors. All mice were housed in the same room with
controlled lighting
- Diet (e.g. ad libitum): Ziegler Bros. NIH 07 pellets (Gardners, Pa.)
- Water (e.g. ad libitum): water from an automatic watering system
Route of administration:
dermal
Vehicle:
water
Details on exposure:
Mice were treated three times weekly for their complete life span with 25 μl per application of each substance. Substances were applied with an Eppendorf pipet to the back of each mouse, from which the fur was clipped once weekly.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations verified monthly during the study
Duration of treatment / exposure:
complete life span
Frequency of treatment:
3x/wk
Dose / conc.:
1 other: % Solution/application
Remarks:
Basis:analytical conc.
No. of animals per sex per dose:
Treatment group singly housed: 50 mice
Control animals:
yes, concurrent vehicle
Details on study design:
Control group singly housed: 50 mice received distilled water. Control group housed 5/cage: 40 mice received water
Positive control:
Positive control group housed 5/cage: 40 mice received 0.1% 3-methylcholanthrene in acetone.
Observations and examinations performed and frequency:
All mice were examined daily, and the onset and progress of tumor growth were recorded monthly. 
Sacrifice and pathology:
Ten mice from the EDA and individually housed water control groups were scheduled for sacrifice at 18 months to evaluate their tissues for possible pathologic changes.  Complete necropsies were performed on all mice.  The dorsal skin from all animals plus all gross lesions were examined histologically after sectioning and staining with hematoxylin and eosin.  In addition, all livers, kidneys and lungs from the 18 month sacrifice were fixed for histopathologic examination.
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % solution 25μL
Based on:
test mat.
Sex:
male
Key result
Dose descriptor:
NOAEL
Effect level:
8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Highest dose tested
Critical effects observed:
no

Mean survival time of the exposure group for substance 2 (598 days) was shorter than that of the singly housed control group (626 days); no treatment-related macroscopic or histopathologic findings; one mouse of the exposure group had a dermal fibrosis at application site and another one had a mammary adenocarcinoma.  One sebaceous adenoma of the skin of the thorax was noted in the control group individually housed. In the exposure group for substance 1, 1 mouse had a myosarcoma at the base of the tail, and 11 animals had mild to moderate dermal fibrosis, suggesting skin irritation. Survival time did not differ from negative control groups.

In the 3-methylcholanthrene group, 39 of 40 mice had skin tumors including 37 with confirmed squamous cell carcinomas.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
mouse
Quality of whole database:
Please refer to section 13 for read across justification.

Justification for classification or non-classification

Additional information

No in vivo carcinogenicity data are available for the test substance. An in vitro cell transformation test (Huntsman-1982, validity 2) was

conducted with balb/3T3 mouse cells, similarly to the EU Method B.21 (In Vitro Mammalian Cell Transformation Test). The test

concentrations were 4.38, 17.5, 35, 70, 105 nL/mL. No indication of cell transformation could be evidenced at

all tested concentrations. Thus, the test substance was negative in the cell transformation test with balb/3T3 mouse cells.

oral

With the structurally related ethylenediammonium dichloride (EDA*2HCL) there is a combined chronic toxicity and carcinogenicity study available (Hermansky et al., 1999, reliability score: 2, see section 13 for read across justification). Groups of each 100 F344 rats per sex received the test substance orally via the feed at adjusted dose levels of 0, 20, 100, 350 mg/kg bw/day (males) or 0, 20, 100 and 360 mg/kg bw/day (females). Mortality was comparable to controls during the first 18 months of the study. After 22 months, mortality in males and females increased at the high dose and in females at 100 mg/kg bw/day after 24 months. There was a significant reduction in body weight gain in male rats and female rats of the high dose group. Significant increases of the relative weights of predominantly the liver and kidney were noted in rats of the high dose group. Significantly higher incidence of hepatocellular pleomorphism in female rats of the intermediate and the high dose groups; rhinitis and tracheitis were also observed in high dose males and females. There was no evidence for a carcinogenic potential of EDA*2HCLin male and female F344 rats, when administered via the diet over the course of 2 years. The NOAEL for chronic toxicity was 20 mg/kg bw/day for EDA*2HCl (corresponding to 9 mg/kg bw/day of EDA).

dermal

With the structurally related ethylene diamine (EDA) there is a dermal carcinogenicity study available

(DePass et al., 1984, reliability score: 2, see section 13 for read across justification). Male CH3 mice received 25μl of an 1% aqueous solution of EDA, the maximum tolerated dose for local effects,to the clipped skin on the backthree times weekly for their complete life span. Two EDA samples from different producers were tested on groups of 50 mice. A negative control group was treated with water and a positive control group received 0.1% of 3 -methylcholanthrene in acetone.Ten mice were intermittently sacrificed at 18 months. Complete necropsies were performed on all mice at intermittent and terminal sacrifice.  The dorsal skin from all animals plus all gross lesions of animals at terminal sacrifice as well as all livers, kidneys and lungs from the 18 month sacrifice were examined histopathologically.There were no treatment-related macroscopic or histopathological findings. No skin tumors were observed in the EDA treated animals. In the positive control group, 98% of the animals revealed skin tumors. Thus, there wasno evidence for a carcinogenic potential of EDA after life-time dermal administration in male C3H mice. The NOAEL was 8.3 mg/kg bw/day (25 µL 1% solution/per mouse).