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Carcinogenicity

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Description of key information

There is no evidence of carcinogenity by dermal exposure in mice or oral route (dietary) in rats to ethylenediamine for thier lifetime.

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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP data
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
350 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
100
Control animals:
yes, concurrent no treatment
Details on study design:
The study was a 2-yr chronic toxicity/oncogenicity study of EDA dosed as (EDA_2HCl) in F344 rats. The study consisted of two control groups and three dose group of 100 males and 100females each. The study involved feeding the Ethylenediamine in the diet and included interim sacrifices at 6, 12 and 18months with a terminal sacrifice at 24 months . The interim scarifies were of 10, 10 and 20 rats per sex per group.

All rats were housed in suspended wire-bottom and front stainless-steel cages. Three males or five females were placed in a cage, with water and food available ad lib. Water was provided by an automatic dispensing system with demand-control valves in each cage. Alternatively, water was provided in 10-oz. Macrolon1 bottles fitted with stainless-steel sipper tubes during 5-day water consumption measurement periods conducted prior to each scheduled sacrifice. Ground diet NIH-07 Rat and Mouse Ration was supplied in 12 oz. opal glass jars

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.
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.
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effect type: toxicity (migrated information)
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
Remarks on result:
other: Effect type: toxicity (migrated information)
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
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

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).

 

 

 

Conclusions:
The NOAEL for chronic toxicity was 20 mg/kg/day of EDA*2HCl. This is equivalent to 9mg EDA/kg/day.
Executive summary:

Ethylenediamine dihydrochloride (EDA_2HCl) was incorporated into the diet and fed to Fischer 344 rats for 2 years at target doses of 0, 0.02, 0.10 or 0.35 g/kg/day (equivalent to 0.009, 0.045 and 0.158 g free EDA/kg/day). Two separate untreated control groups were used. Interim sacrifices were at 6, 12 and 18 months and the terminal sacrifice was at 24 months. Under the conditions of this study, EDA_2HCl was not carcinogenic in the Fischer 344 rat. Most toxic response s were observed at the 12-month sacrifice and thereafter. Reductions in mean body weight gain were observed in high dose group male rats throughout most of the study and in the high dose group of female rats after approximately 18 months. Conversely, there was a slight increase in the mean body weight gain for the medium level female rats from about day 21 until 21 months that was of equivocal biological significance. Increased mortality was observed in the high dose group of both sexes and the mid dose group of female rats. The cause of the decreased survival was unclear, but may have been related to the enhancement of background degenerative lesions such as chronic nephropathy. Throughout the study, male rats from the high dose group had decreased erythrocyte counts, haemoglobin concentration and haematocrit. The cause and biological significance of the haematological changes were unknown. Increased water consumption was observed in the high dose group of both sexes during the latter half of the study. Increased urine volume with concurrent decreased urine specific gravity was generally observed in the high dose group of both sexes in the last half of the study and suggested a possible alteration in kidney function. Altered urine volume and specific gravity persisted to termination in female rats only.   Slight increases in absolute and relative kidney weights were also observed in the high dose group of female rats during the latter half of the study. Hepatocellular pleomorphism was observed in the high dose group of both sexes, especially the female rats, and may have contributed to increased mean liver weights observed primarily in female rats from the high dose group. Hepatocellular pleomorphism was first observed in female rats at 12 months but was not observed in male rats until the final sacrifice. Rhinitis and tracheitis were observed with greater frequency in the high dose group of male rats at 12, 18 and 24 months and in high dose group female rats at 18 months. At 24 months, rhinitis, but not tracheitis, persisted at a significantly greater frequency in high dose group female rats. The apparent No Observable effect level (NOEL) of this study was at the lowest dose level, 0.02 g/kg/day (equivalent to 9 mg EDA/kg/day). .    The highest dose level of 0.35g/kg/day equivalent to 159mg/kg/day of Ethylenediamine base showed not increase in the incidence of tumours and therefore a NOAEL for induction of cancer. Ethylenediamine has not demonstrated any potential for carcinogenicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
159 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
a Klimisch 2 study from the published lierature carried out in w ell respected industry laboratory

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:
key 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
Species:
mouse
Strain:
C3H
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
Fifty male C3H/HeJ mice were assigned to the EDA and negative control groups, 40 mice being assigned to the positive control and 5/cage water control groups.
- 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
Remarks:
Doses / Concentrations:
25 μl of 1% aqueous solution/application
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.
Statistics:
The mean survival time of the EDA No. 2 group (598 days) was significantly (p < 0.05) shorter than that of the water controls (626 days) by the Mantel-Cox test but not by the Breslow test. The difference in these statistical results is understandable since the Breslow test gives greater weight to earlier observations. In this case, the survival curves did not differ for the first 600 days of the study. The survival comparisons included the mice sacrificed at 18 months.

The mean survival time of the group-housed water controls (488 days) was significantly reduced compared to the singly housed controls by the Breslow but not the Mantel-Cox test. Since these curves differed primarily in the first 600 days of the study, the difference was significant by the test that gave greater weight to early deaths.
Dose descriptor:
NOAEL
Effect level:
1 other: % solution 25μl
Based on:
test mat.
Sex:
male
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
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
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

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.

Conclusions:
The high incidence of skin tumours seen in the positive controls confirmed the ability of the mice used in this study to detect skin carcinogens. The results indicate no evidence for cutaneous oncogenicity when Ethylenediamine was applied to the skin of mice for their lifetime.
Executive summary:

The dermal oncogenic potential of ethylenediamine (EDA) was assessed by applying 25 µ1 of a I% solution in deionized water to the skin of 50 male C3H/HeJ mice. This was the highest concentration not producing irritation or weight changes in a preliminary 2-week study. Two EDA samples (Nos. 1 and 2) from different production sources were tested. Applications were made thrice weekly until the death of the animals. A negative control group received deionized water only. This group and the EDA-treated groups were individually housed. A fourth group of 40 mice, housed 5 per cage, received 0.1% 3-methylcholanthrene (MC) in acetone as a positive control substance. A fifth group of 40 mice, housed 5 per cage, also received deionized water to determine the effect of group housing on survival. No skin tumors were observed in the EDA-treated groups. In the positive control group, however, 39 animals (98%) had skin tumors including 37 (92%) with confirmed squamous cell carcinomas. Eleven mice (22%) which received EDA No. 1 had dermal fibrosis indicative of probable skin irritation in this group; there was no such lesion in the controls.

No skin tumors were observed in the EDA treated animals. In the positive control group 98% of the animals had skin tumors.

Neither Ethylenediamine sample was oncogenic (carcinogenic) to the skin or systemically under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
8 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
a Klimisch 2 study from the published lierature carried out in w ell respected industry laboratory

Additional information

Ethylenediamine dihydrochloride was fed to Fischer 344 rats in the diet for 2 years with a top dose of ca. 350mg/kg equivalent to 159mg/kg of the Ethylenediamine base. There were increased mortalities at the top dose but no increase in tumour incidence seen at any dose level.  There was a decreased incidence of pituitary adenomas and interstitial cell adenomas of the testis in males, this may have been influenced by the increased mortalities resulting in fewer rats surviving sufficiently long for these tumours to develop spontaneously.

 

In the dermal carcinogenicity study in C3H/HeJ mice, the maximum level of ethylene diamine that could be applied to the skin of the mice without causing significant skin irritation was 25µl of a 1% solution equivalent to 8mg/kg bodyweight. Mice were dosed three times a week for their lifetime with separate groups of 50 mice for each of two samples of Ethylenediamine from two different suppliers both were high purity but had different impurities. The study also included a positive control group dose with a skin carcinogen 3-methylcholanthrene at 0.1% in acetone. Negative controls were dosed with water only.

 

No skin tumours were observed in the EDA treated animals. The positive control group confirmed the sensitivity of the mice to skin carcinogens with 98% of the animal developing skin tumours.


Justification for selection of carcinogenicity via oral route endpoint:
This is valid 2 year carcinogenicity study in rats equivalent to the OECDguideline.

Justification for classification or non-classification

Ethylenediamine (as the dihydrochloride salt) was administered in the diet to Fischer 344 rats up to a dose level that can be seen as a maximum tolerated dose of 159mg/kg/day and there were no indications of any carcinogenicity. Also in a lifetime skin dosing study in C3H/HeJ mice, in which the mice were shown to be sensitive to the skin carcinogen 3-methylcholanthrene, there were no skin tumours observed in the ethylenediamine treated mice at a maximum none irritant dose equivalent to 8mg/kg/day, confirming the lack of carcinogenicity seen in the rats. This lack of carcinogenicity in rats and mice is consistent with the lack of genotoxicty seen in the in-vitro and in-vivo testing. The weight of evidence is that Ethylenediamine is not carcinogenic.