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Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan.
- Age at study initiation: (P) 43 wks;
- Weight at study initiation: (P) Males: 112-141 g; Females: 85-111 g;
- Housing: Two or three males or three or five females were housed per cage prior to mating;
- Diet : ad libitum, Purina Certified Rodent Chow 5002.
- Water :ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 55
- Photoperiod (hrs dark / hrs light): 12 hr light cycle
Route of administration:
oral: feed
Details on exposure:
Fresh diet was prepared biweekly with the percentage of EDA*2HCl in the diet adjusted to maintain a constant dosage level in grams per kilogram
for each sex according to the average animal body weight gain and diet consumption. At monthly intervals two diet samples were analysed.
Details on mating procedure:
The F0 parents were mated afier having received dietary EDA * 2HCl for 100 days. Twenty-six females and 13 males (randomly selected from 25 males) were mated per treated group while 52 females and 26 males (randomly selected from 50 males) were mated for the control group. During mating, two females were placed in a cage with one male; at this time all rats received EDA * 2HCl at the concentration in the diet for the respective female groups. Fifteen days after cohabitation, mating was discontinued and the females were placed in individual shoebox cages fitted with wire rod metal tops.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At monthly intervals two diet samples were analysed.
Duration of treatment / exposure:
for two generations
Frequency of treatment:
daily
Details on study schedule:
At each dose level 13 male and 26 female rats were mated in both F0 and F1 generation (control group: 26 male and 52 female rats each); continuous treatment starting 100 days prior to cohabitation of F0 rats until weaning of F2 rats.
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:
13 male and 26 female. Control group: 26 male and 52 female rats
Control animals:
yes, concurrent no treatment
Parental animals: Observations and examinations:

BODY WEIGHT: Yes, measured weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption determined on each cage of animals every 2 week. Dosage adjusted to keep a constant dosage level.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Effect level:
23 mg/kg bw/day
Basis for effect level:
other: Body weight; food consumption; organ weights; (Reclaculated to EDA base)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
227 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Body weight; food consumption; organ weights; histopathology: Hepatocellular pleomorphism; (Recalculated to EDA base)
Reproductive effects observed:
not specified

Significant reduction in parental body weight gain of female rats in the intermediate and high dose group of the F0 generation, in the high dose group of the F1 generation and of male rats in the high dose group of both F0 and F1 generations.

No substance-related parental deaths in the F0 or F1 generation.

Significant decrease of absolute liver weight in male rats of the high dose F1 generation.

No macroscopic or histopathologic findings except a significant higher incidence of hepatocellular pleomorphism in both sexes of the high dose group of the F1 generation (6/10 male, 10/10 female; control: 0/20 each) and a significant decreased prevalence of kidney tubular mineralization in female rats of the high dose group of the F1 generation (0/10 female; control: 10/20).

In conclusion there was no evidence of fertility impairment or embryotoxic effect at dose levels that show maternal or paternal toxicity.

REPRODUCTIVE INDICES        
         
  Dosage (g/kg,lday)
  0.50 0.15 0.05 0
  F0 → F1
Fertility index (%)        
Male 92 100 92 92
Female 81 77 73 77
Gestation index (%)  100 100 100 100
Gestation survival index (%)  99.0 ± 3.4  100.0 ± 0.0  97.2 ± 11.8  99.2 ± 5.2
0- to 4-Day survival index (%)  90.0 ± 30.0  100.0 ± 0.0  94.4 ± 23.6  93.2 ± 22.4
4- to 14-day survival indexb (%)  99.4 ± 2.5  100.0 ± 0.0  94.1 ± 24.3  100.0 ± 0.0
4- to 2I-day survival indexb (%)  99.4 ± 2.5  100.0 ± 0.0  94.1 ± 24.3  99.7 ± 2.0
Pups born alive/litter  6.8 ± 3.5  8.6 ± 2.8  6.2 ± 3.9  8.4 ± 3.0
Days from first mating to parturition 28.3 ± 3.8 29.6 ± 2.0  29.8 ± 3.0  28.1 ± 3.0
  F1 → F2
Fertility index (%)        
Male  100 92 92 100
Female  92 88 73 88
Gestation index (%)  100 100 100 100
Gestation survival index (%)  98.6 ± 6.7  99.1 ± 3.1  99.4 ± 2.5  99.5 ± 2.0
0- to 4-Day survival index (%) 98.0 ± 4.1  99.3 ± 3.5 98.6 ± 3.3  98.5 ± 4.1
4- to 14-day survival index (%) 100.0 ± 0.0  98.7 ± 6.3 100.0 ± 0.0  99.6 ± 3.0
4- to 21-day survival index (%) 100.0 ± 0.0  98.3 ± 8.3 100.0 ± 0.0  99.6 ± 3.0
Pups born alive/litter 8.5 ± 3.0  8.8 ± 2.2 9.4 ± 2.8 10.0 ± 2.5
Days from first mating to parturition 28.5 ± 3.2  28.8 ± 3.3 27.3 ± 3.6  28.4 ± 3.8
Conclusions:
There was no indication of reproductive toxicity in Fischer 344 rats, following exposure to dietary EDA for two generations.
Executive summary:

In a two-generation study, male and female Fischer 344 rats were fed diets containing 0, 50, 150 or 500 mg/kg/day of EDA*2HCl. At each dose level 13 male and 26 female rats were mated in both F0 and F1 generation. In the high dose group effects noted were decreased body weight gain in both F0 and F1 generations, a higher incidence of hepatocellular pleomorphism in F1 generation, and decreased liver weights in male rats in F1 generation. In the intermediate dose group female rats of F0 generation showed a decreased body weight gain. No evidence of impaired fertility or embryotoxicity was seen at any dose level. NOAEL parental was 50 mg/kg (23 mg/kg calculated as Ethylenediamine base) and NOAEL F1 offspring was 500 mg/kg (227mg/kg calculated as ethylenediamine base).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A Klimisch 2 literature reported 2 generation reproduction study, with sufficient data to consider the study vaid.
Additional information

In a two-generation reproduction study (Yang et al., 1984a) there was no reproductive effect noted in any dose group as regards fertility, pup survival, number of pups born alive and number of pups weaned per litter

There was no evidence of reproductive toxicity at levels of 500 mg/kg/day in rats.

Short description of key information:
There was no evidence of reproductive toxicity at levels of 500 mg/kg/day in rats in a two generation study.

Effects on developmental toxicity

Description of key information
Growth retardation was observed in fetuses from dams receiving 1000 mg/kg/day, levels which resulted in maternal toxicity. However there was no evidence of a teratogenic effect.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were dosed via diet during gestation day 6-15. Standard endpoints for teratogenicity were evaluated.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY).
- Age at study initiation: 100 days
- Housing: stainless-steel cages
- Diet : Purina Certified Rodent Chow 5002
- Water (e.g. ad libitum): Yes
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis indicated that the test diet preparations were accurate and homogeneous. The stability of the test diets was also confirmed. Substance was added to diet based on established diet consumption data and an established growth curve to produce the dosage goals.
Details on mating procedure:
At approximately 100 days of age, female rats were introduced into the male rats' cages. The rats were mated one male to one female to achieve a goal of approximately 20 pregnant females per treatment group, and 40 in the negative control group. Expelled vaginal plugs were used as evidence that mating had occurred
Duration of treatment / exposure:
Gestation day 6 - 15
Frequency of treatment:
daily
Duration of test:
cesarean section on gestation day 21
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
20 and 40 in control group.
Control animals:
yes, concurrent no treatment
Details on maternal toxic effects:
Maternal toxic effects:yes
Dose descriptor:
NOAEL
Effect level:
23 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
114 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
114 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
LOAEL
Effect level:
454 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Significant reduction in maternal body weight gain in the intermediate dose group during gestation day 6 - 15 and in the high dose group during gestation day 6 - 21; significant decreased diet consumption in the intermediate and high dose group during gestation day 6 - 15; significant increased number of resorptions/litter and significant decreased mean fetal body weight and reduced fetal crown-rump length in the high dose group; significant higher incidence of a shortened mandible, edematous eye bulge, shortened or missing innominate artery, unossified sternebrae in fetuses of the high dose group.

    Number of pups affected
    Dosage level (mg/kg/day)
           Control 50 250 1000
No. of litters   40 23 21 24
No. of pups   379 232 201 242
           
Pup body weight, males (g)        4.5+0.3 4.5+0.2  4.5+0.2  4.1+0.3
 Pup body weight, females (g)   4.2+0.2  4.2+0.2 4.2+0.3  3.8+0.3
           
Fetal crown rump length, males (mm)    40+2    40+2     40+2      39+2
 Fetal crown rump length, females (mm)   38+1    39+2     39+2      37+2
           
Slightly edematous eye bulge          
  F 0 0 0 4
  L 0 0 0 4
Shortened mandible           
  F 0 0 0 18
  L 0 0 0 4
Missing innominate artery          
  F 0 0 1 9
  L 0 0 1 6
Shortened innominate artery          
  F 4 2 0 27
  L 4 1 0 14

F = fetuses L = litters

The first artery to branch off of the aorta is the brachiocephalic which becomes the innominate after the left carotid branches off.  The innominate then branches into the right subclavian and right carotid artery.  When the authors stated it was missing, they really mean that the right and left carotid branch off of the brachiocephalic artery at the same time.  Thus there is no innominate.  However, this would not affect blood supply to areas served by these arteries.                                                                                                                           

Conclusions:
The fetal alterations were either of questionable teratologic significance or indicative of growth retardation, possibly as a result of reduced food consumption.
The most important finding was an increase in the incidence of missing and shortened innominate artery. Further testing was performed to determine if this was an effect of malnutrition from reduced food consumption.
Executive summary:

Fischer 344 rats were fed EDA*2HCl on gestation day 6-15. Groups of 20 rats were fed doses 50, 250, or 1000 mg/kg. Two control groups were used. Standard endpoints for teratogenicity were evaluated.

Reduction in maternal body weight gain was noted in the high and intermediate dose group. Reduced diet consumption was also noted in those groups. In the high dose group there was an increased number of resorptions/litter, decreased mean fetal weight and length, higher incidence of shortened mandible, edematous eye bulge, shortened or missing sternebrae, delayed ossification and missing, or shortened innominate arteries.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The results in the study were further investigate in two smaller studies which established that the most serious findings were due to compund induced reduced food consumption (not unpalitability of the diet). The three studies taken togther allow a scientifically valid interpretation of the study.
Additional information

There are both rat and rabbit pre-natal developmental toxicity (teratology)studies. The rabbit study consisted of New Zealand White rabbits, which were dosed by gavage with Ethylenediamine dihydrochoride (EDA*2HCL) on gestation days 6-19. The dose levels were 0, 10, 40 and 80mg/kg bw, calculated as Ethylenediamine base. The top dose was selected due to 20% mortality in a dose ranging study at 100mg/kg bw/day. There were no adverse effect on the maternal females and no indication of any developmental toxicity/teratogenicity on the foetuses.

 

In a rat teratology study Ethylenediamine dihydrochoride was administered in the diet at 0, 50, 250 and 1000mg/kg bw/day for days 6-15 of gestation. These doses were 25, 114 and 454 mg/kg bw/day calculated as ethylenediamine base. There was significantly reduced body weight gain in the intermediate and high dose groups together with reduced food consumption. There were several findings in the offspring at the high dose, such as reduced ossification, increased resorptions, decreased foetal weight and length. The most important finding was an increased incidence of missing and shortened innominate artery. By missing the authors meant that the right and left carotid branched off the brachiocephalic artery, rather than it becoming the innominate after the left carotid branches. So there is no innominate but this would not affect blood supply.  As missing innominate artery has been observed in offspring of rats on diets deficient in folic acid, further studies were performed to determine if this effect was due to malnutrition due to decreased diet consumption. A first step was to establish if the reduced food consumption was due to unpalatability of the diet, 10 pregnant rats were dosed by gavage from days 6-15 of gestation at 1000mg/kg Ethylenediamine dihydrochloride (454mg/kg bw/day calculated as Ethylenediamine base).These rats showed reduced bodyweight gain and food consumption, even though there was no ethylenediamine dihydrochoride in the diet. The foetuses were not examine internally, but there was a decrease in live foetuses/litter and increase in resorptions.  .  The NOAEL for developmental toxicity was 250mg/kg EDA*2HCL (114mg/k bw/day calculated as Ethylenediamine base.

 

This demonstrated that the reduced food consumption seen in the dietary study was not due to unpalatability of the diet. This study was followed by a paired feeding study where a group of pregnant females were fed the same amount of control diet aswas consumed by the rats who were receiving 1000mg/kg EDA*2HCL (454mg/kg Ethylenediamine base). The paired fed controls had the same incidence of missing innominate arteries as those fed Ethylenediamine. This indicated that this effect was as suspected due to a folic acid deficiency caused by reduced food consumption  The ethylenediamine dosed rat did however still show shortened innominate arteries, the authors concluded that this shortening was not a teratogenic effect as it would not result in a functional deficit and may not be irreversible but rather a result of the growth retardation seen together with reduced foetal weights which only occurred in the presence of maternal toxicity.


Justification for selection of Effect on developmental toxicity: via oral route:
This study was described adequately ina publication but was not a modern OECD guideline study but was very similar. It was Klimisch 2.

Justification for classification or non-classification

The two generation study in rats showed clearly that there were no adverse effects on reproduction either on fertility or embryotoxicity from Ethylenediamine, administered in the diet up to 500mg/kg bw/day od EDA*2HCL, (227mg/kg bw/day calculated as Ethylenediamine base) the highest dose group.

 

There were no indications of any developmental toxicity / teratogenicity seen in rabbits at a maximal dose of 80mg/kg bw/day of Ethylenediamine by gavage.

 

In the rat teratogenicity study there were some findings interpreted as being due to malnutrition (low folic acid) which was the case for the missing innominate arteries, due to reduced maternal food consumption, caused by the ethylene diamine but not due to unpalatability of the diet. Other findings of shortened innominate arteries, delayed ossification etc. were considered to be due to delayed development linked to reduced foetal weight at a maternally toxic dose level. The NOAEL for effects in the foetuses was 250mg/kg EDA*2HCL (114mg/k bw/day calculated as

Ethylenediamine base.

 

Based on these findings it is concluded that ethylene diamine does not require any classification under the EU CLP(GHS) criteria for adverse effects on reproduction or foetal development.

Additional information