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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Weakly positive results in an Ames Salmonella test for some Ethylendiamine samples have been shown. However, further in vitro and in vivo testing , using different sources of sample, have given negative results.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
Animals and Animal Care
The male rats used in this study were the F0 males from the EDA.2HCl 2-generation rat reproduction study (F0 Fischer 344 rats received from the Charles River Breeding Laboratories, Portage, Michigan). The F0 male rats were 204 days of age at first mating. At this age, the male rats of the test groups had received diet containing EDA.2HCl at 0.50, 0.15, 0.05 or 0 g/kg/day for 23 weeks. The males were housed for the duration of the study in Room 106 of the CHF Building.

Four hundred five Fischer 344 female rats representing three age groups (135 rats/group) were received from the Charles River Breeding Laboratories, Kingston, New York. The female rats were derived from the same stock as those from the Portage, Michigan facility. The Portage Laboratories were closed for renovation when the order for the female rats was placed on September 29, 1980.

The three age groups were 48, 55, and 62 days of age upon receipt and were 98 days of age at mating. All animals were uniquely identified by toe-clipping. The female rats were held in isolation in Room 106 of the CHF Building for 50, 43 and 36 days respectively prior to mating. Mating of the rats took place in Room 106, CHF Building. The female rats were assigned at random to the dosage groups by a computerized randomization procedure. Those female rats judged not suitable because of body weight (weights greater than + 2 standard deviations from the mean), health or other conditions were euthanatized with CO2 and incinerated.

Evaluation of animal health status was made by visual examination of female rats on receipt. Furthermore, quality control included the following tests on five animals: viral serology and aerobic bacterial cultures of nasopharynx and lung. Also, the liver, lungs, heart, kidneys, salivary glands, nasal cavity and cervical lymph nodes were fixed and examined microscopically.

Until mating, the rats were housed 2 or 3 males or 5 females per individual cage in stainless steel wire-mesh cages suspended in stainless steel racks. At mating, the rats were housed 1 male: 1 female per cage. Males were housed 1 per cage 1 week prior to first mating. A layer of Deotized Animal Cage Board® produced by Upjohn Company (Kalamazoo, MI) was kept under each cage and changed at least 3 times a week. Other animal care procedures were performed regularly by the animal care personnel according to BRRC Standard Operating Procedures. Water and food were available ad libitum. Water, supplied by the Municipal Authority of Westmoreland County, was provided by an automatic dispensing system with demand controlled valves in each cage. The basic diet, Ground Purina Certified Rodent Chow 5002, was supplied in 12 oz. opal glass jars. The Fischer strain was chosen on the basis of BRRC's past experience with this strain in chronic toxicology and because the Fischer 344 rat is the strain used in a current long-term EDA.2HCl feeding study at BRRC. The fact that the Fischer 344 rat is the strain of choice by the National Cancer Institute was also taken into consideration.
Route of administration:
oral: feed
Details on exposure:
F0 male rats from the 2-generation EDA.2HCl reproduction study, selected at random, were removed from their dosage regimens and fed control diet 24 hours prior to mating with naive females. At the start of the study, the male rats had received diets containing EDA.2HCl at 0.5, 0.15, 0.05 or 0 g/kg/day for 23 weeks. Concurrently, a positive control group of male rats (randomized from the F0 control male population on July 30, 1980) was given a single intraperitoneal dose of 0.25 mg/kg TEM.

Starting January 7, 1981 a mating regimen was followed sequentially for three consecutive weeks with each of the 3 female age groups mated for one week, thereby assuring that each male had the opportunity to be mated with three females. Each male was mated with only one female during each weekly mating period. The females were introduced into the male cages. The naive, virgin females for the three weekly mating regimens were assigned to dosage groups by a computerized randomization procedure. The female rats were not dosed at any time during the study. Each female was euthanatized by severing the cervical cord 13 days after the appearance of a vaginal plug. If no vaginal plug was seen, the female was euthanatized on day 17 from the first day of cohabitation with the male. With the exception of the counting of corpora lutea, all observations were made without using optical aids. Corpora lutea were counted after the mesovarium was totally removed. The uteri, with urinary bladders attached, were fixed in 10% neutral buffered formalin and saved.
Duration of treatment / exposure:
23 weeks pre treament of the males only, no treament during this study
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Table 1
Exposure Levels
(g/kg/day for F0 Males for 23 weeks a No of Rats / Group
Male (F0) Female (Naive)
0.50 20 20
0.15 20 20
0.05 20 20
0 (control) 40 20
Positive Control (TEM 0.25mg/kg) 20 20
a These exposure levels were those used in the EDA.2HCL 2-generation reproduction study; no animals were exposed to EDA.2HCL in the present study.
Control animals:
yes, plain diet
Positive control(s):
- Route of administration: intraperitoneal
- Doses / concentrations: a single dose of 0.25 mg/kg triethylenemelamine
Evaluation criteria:
The terminology, criteria and procedures used for determining dominant lethality in this study were derived basically from those of Bateman and Epstein (1971) and Bateman (1977). In certain instances where detailed information was not available in the literature, our own criteria based on past experience were incorporated. The criteria for determining the different types of implantation were as follows:
1) Normal implantation (live embryo): any implantation of pink colour, largest ones in size for that female rat.
2) Early death (mole); any implantation, generally lucid and pale, substantially smaller than the normal implantation for that female rat.
3) Late death: any implantation with size intermediate between the normal implantation and early death, or of the normal size for that female rat but appearing pale and sometimes lucid.

In the literature, dominant lethality was often associated with early fetal deaths (Bateman, 1977; Bateman and Epstein, 1971; Epstein and Legator, 1971; Green et al., 1976). However, in certain studies no differentiation between early and late foetal deaths was made and dominant lethality was therefore evaluated on the basis of early and late foetal deaths (Beall, 1974; Green et al., 1977; Moreland et al., 1979). From the point of view of safety evaluation, it is unwise to ignore the late fetal deaths. Accordingly, in the present study, the differences between the dosed groups and the control group were evaluated on the basis of early fetal deaths, late fetal deaths and the total fetal deaths (i.e., pooled early and late deaths).
Treatment effects were determined by statistical evaluation of fertility (male and female), number of total implants, number of viable implants, litters with all fetuses viable, preimplantation loss, number of fetal deaths (early, late, early and/or late), number of females with early fetal deaths, number of females with late fetal deaths and number of females with early and/or late fetal deaths.
Statistics:
For every experimental parameter measured, the results of the three test levels (high, medium, low) were compared with those of the control group. The positive control group data were kept separately, and they were compared only with the negative control group data. The female was used as the sampling unit (Weil, 1969). Enumerative data were evaluated statistically by the R X C Chi Square Test (Snedecor and Cochran, 1967); differences between groups were delineated by Fischer's Exact Test (Sokal and Rohlf, 1969). Nonparametric data were compared by the Kruskal-Wallis Test (Zar, 1974); differences between groups were delineated by a distribution—free multiple comparison method (Zar, 1974). The fiducial limit of 0.05 was employed as the critical level of difference not attributed to chance.
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Decrease of body weight gain at highest dose level
Positive controls validity:
valid
Additional information on results:
Summaries of the number of pregnant females, fertility index, the median numbers of implants per pregnant female, the median number of viable implants per pregnant female, the number of litters with all fetuses viable, the median preimplantation loss, the median number of foetal deaths per pregnant female and the median percentage of foetal deaths are presented in Table 2. The fertility index is presented in Table 3. Ratios of the total number of females with early foetal deaths to the number of pregnant females are shown in Table 4 while ratios of the total number of females with late foetal deaths to the number of pregnant females are shown in Table 5. Table 6 is a presentation of the ratios of total number of females with early and/or late foetal deaths to the number of pregnant females.

With one exception, all parameters for the EDA.2HCl-treated rats were statistically similar to those of the negative control group (Tables 2-6). This exception was a marginal increase in the number of females with early foetal deaths/number of pregnant females for the intermediate dosage group during week 1 (Table 4). This change was considered to be an artifact attributable to chance because of the lack of a dose-response relationship. This conclusion is corroborated by the negative results for weeks 2, 3, and the total frequencies for early foetal deaths for this group (Table 4). Conversely, most if not all, parameters in the TEM positive controls were severely affected (Tables 2, 4, 5, 6). These data indicate that, under the experimental conditions, while a known mutagen would be able to elicit dominant lethal effect in these Fischer 344 rats, there was no indication of any mutagenic response in the EDA.2HCl treated rats.

Table 2 Summary of Experimental Parameters Measured

Week

Dose level g/kg/day

No of pregnant females

Fertility Index

Median No of implants/ pregnant female

Median viable implants/ pregnant female

Litters with all Foetuses viable

Median pre implantation loss!

Median No of Foetal Deaths/ Pregnant Female

 

 

 

 

 

 

 

 

 

Early

Late

Early and /or late

**Median % of fetal deaths

1

0.50

16

80

10

10

11

0

0

0

0

0

 

0.15

19

95

11

10

12

8

0

0

0

0

 

0.05

17

85

11

11

8

0

0

1

1

8

 

0

29

72

11

11

22

8

0

0

0

0

 

0.25 mg/kg *TEM

17

85

10

4c

2c

23

1b

2c

5c

58c

2

0.50

17

85

10

10

12

0

0

0

0

0

 

0.15

20

100

11

10

8

0

0

0

1

9

 

0.05

17

85

11

11

12

0

0

0

0

0

 

0

32

80

11

10

17

4

0

0

0

0

 

0.25 mg/kg *TEM

17

85

6c

0c

0c

38c

4c

0

6c

100c

3

0.50

17

85

11

10

10

8

0

0

0

0

 

0.15

17

85

12

11

11

0

0

0

0

0

 

0.05

19

95

12

11

11

8

0

0

0

0

 

0

36

90

11

10

21

8

0

0

0

0

 

0.25 mg/kg *TEM

17

85

8c

1c

0c

30b

6c

0

6c

80c

b : 0.01 > P > 0.001     c : P < 0.001

!Each median was obtained from individual derived from the following calculations:

Pre-implantation loss + corpora lutea – implantations/corpora lutea x 100

*TEM – Triethyenemelamine ** Percentage fetal death=No of fetal deaths/pregnant female    x 100

                                                                                      No of implants/pregnant female

Table 3 Fertility Index

Exposure Levels

(g/kg/day for F0Males for 23 weeksa

Fertility Index*During Week

1

2

3

0.50

80

85

85

0.15

95

100

85

0.05

85

85

95

0 (control)

72

80

90

Positive Control (TEM 0.25mg/kg)

85

85

85

Fertility index = No of pairs mated that produced pregnancies / Total pairs mated x 100

 

Table 4 Ratios of Total No of females with early deaths to no of females pregnant

Exposure Levels

(g/kg/day for F0Males for 23 weeksa

No of females with early foetal deaths to no pregnant females*During Week

1

2

3

Total

0.50

0/16

0/17

2/17

2/50

0.15

6/19a

4/20

2/17

12/56

0.05

0/17

3/17

4/19

7/53

0 (control)

1/29

8/32

11/36

20/97

Positive Control (TEM 0.25mg/kg)

10/17c

14/17

16/17

40/51c

                                                     Percentage of pregnant females with one or more early foetal death during weeks

 

1

2

3

 

0.50

0

0

12

 

0.15

32a

20

12

 

0.05

0

18

21

 

0 (control)

3

25

31

 

Positive Control (TEM 0.25mg/kg)

59c

82c

94c

 

a : 0.05 >P >0.01

c : P <0.001

 

 

 

Table 5 Ratios of Total No of females with late deaths to no of females pregnant

Exposure Levels

(g/kg/day for F0Males for 23 weeksa

No of females with late foetal deaths to no pregnant females*During Week

1

2

3

Total

0.50

5/16

5/17

7/17

17/50

0.15

3/19c

9/20

4/17

16/56

0.05

9/17

2/17

6/19

17/53

0 (control)

6/29

10/32

7/36

23/97

Positive Control (TEM 0.25mg/kg)

14/17c

5/17

1/17

20/51c

                                                     Percentage of pregnant females with one or more late foetal death during weeks

 

1

3

3

 

0.50

31

29

41

 

0.15

16

45

24

 

0.05

53

12

32

 

0 (control)

21

31

19

 

Positive Control (TEM 0.25mg/kg)

82c

29

6

 

 

c : P <0.001

 

 

 

 

Table 6 Ratios of Total No of females with late deaths to no of females pregnant

Exposure Levels

(g/kg/day for F0Males for 23 weeksa

No of females with early and/or late foetal deaths to no pregnant females*During Week

1

2

3

Total

0.50

5/16

5/17

7/17

17/50

0.15

7/19

12/20

6/17

25/56

0.05

9/17

5/17

8/19

22/53

0 (control)

7/29

15/32

15/36

37/97

Positive Control (TEM 0.25mg/kg)

15/17c

17/17c

17/17c

49/51c

                                                     Percentage of pregnant females with one or more early and/or late foetal death during weeks

 

1

2

3

 

0.50

31

29

41

 

0.15

37

60

35

 

0.05

53

29

42

 

0 (control)

24

47

42

 

Positive Control (TEM 0.25mg/kg)

88c

100c

100c

 

 

c : P <0.001

 

 

 

Conclusions:
Interpretation of results (migrated information): negative
There were no genotoxic effects observed in this Dominant Lethal Assay.
Executive summary:

In conjunction with a two-generation reproduction study, the possible dominant lethal effects of ethylenediamine (EDA) were evaluated in Fischer 344 rats. Ethylenediamine dihydrochloride (EDA-2HCl) was incorporated in the diet of male and female rats at dosage goals of 0.50, 0.15 or 0.05 g/kg/day during two generations of reproduction. One parallel control group without EDA.2HCl in the diet was observed concurrently. After successful completion of the F0–F1amating, the F0 male rats which had received diets containing EDA.2HCl at 0.5, 0.15, 0.05 or 0 g/kg/day for 23 weeks were transferred to the dominant lethal study. These male rats were removed from their dosage regimens and fed control diet 24 hours prior to mating with naive females. Concurrently, a group of naive male rats was given a single intraperitoneal dose of 0.25 mg/kg triethylenemelamine (TEM) to serve as a positive control. A mating regimen was followed sequentially for three consecutive weeks. Approximately 13 days after conception, the female rats were sacrificed and the uteri examined. The criteria examined included fertility, corpora lutea count, number of implantations/female, late fetal deaths/female and early foetal deaths/female.

No dose-related responses were found in any of the criteria examined. However, marked mutagenic responses were noted in the positive control animals (TEM treated) indicating that the rats used in this study were susceptible to a known mutagen.

There is no evidence in this study implicating EDA as a mutagen.

There were no genotoxic effects observed in a Dominant Lethal Assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Weakly positive results in an Ames test with Salmonella typhimurium strains TA 100, TA 1535 were reported by Haworth (1983). Ambiguous results have been presented in additional test with Salmonella typhimurium (Guzzie, 1987).

However Ames tests with Salmonella typhimurium strains TA 98, 100, 1535 and 1537 reported by Willems (1979) were negative. The cause of the weakly positive response has been hypothesized to be due to an impurity. (Hedenstedt, 1978, as cited in the SIDS Risk Assessment dossier for Ethylenediamine, 2001).

Genotoxic potential of EDA was tested by Slesinski (1983) in different in vivo and in vitro tests. The in vitro tests included a gene mutation assay on Chinese Hamster Ovary cells, a sister chromatid exchange assay on Chinese Hamster Ovary cells and an Unscheduled DNA synthesis on primary rat hepatocytes. All the tests gave negative results.

Negative results were also obtained in "in vivo" tests, a Dominant lethal assay on Fischer 344 rats (Slesinski 1983c) and two Drosophila SLRL tests by oral and injection (Zimmering 1985).

Negative results were also obtained in an "In vitro Mammalian Chromosome Aberration Test" according to OECD Guideline 473 (Morris 2015)

In a chromosomal aberration test in human lymphocytes, there was no statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

There were some weak positive results seen in TA100 and to a lesser extent TA1535 in the Ames test, however in a tests with a higher purity sample no such effects were seen. A comprehensive selection of other in-vitro genotoxicity tests showed no evidence in-vitro of mutagenicity in mammalian cells and no evidence of clastogenicity in human lymphocytes. This was confirmed by a lack of any genotoxicity in-vivo in a dominant lethal study in rats and in drosophila both by administration by feeding and injection. The weight of evidence is clearly that ethylene diamine is not genotoxic (mutagenic or clastogenic) .


Justification for selection of genetic toxicity endpoint
This studyconfirmed that lack of mutagenicity in-vivo that was demonstrated in-vitro in mammalian cells.

Justification for classification or non-classification

The weight of evidence from in-vitro and in-vivo genotoxicity testing is that Ethylenediamine is not genotoxic. Ames results were inconsistent, with some weak positive results reported in TA100 and TA1535 but negative results with a high purity sample, all other in-vitro and in-vivo tests were consistently negative showing no evidence of mutagenicity in mammalian cells and no clastogenicity in human lymphocytes. In-vivo data from a dominant lethal study in rats and SLRL tests in Drosophila were consistently negative.