Ethylenediamine, ethoxylated and propoxylated

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Developmental toxicity probe study in rats

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: Guideline study (GLP)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Remarks:

not mentioned

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were supplied by "Charles River, Sulzfeld, Germany" and were about 10 (males) and 15 (females) weeks old and had an initial mean wights of 351 and 197 g, respectively.
Housing singly in Makrolon(R) cages Type IIIh on wood granules. All animals or the study were kept in the same room with no other animals from other studies and in an 20 Pa overpressure environment at 23°C, 55 +/-5% relative humidity and a 12 hour light/dark cycle and low-noise music.
Diet was stadard diet "Provimi Kliva 3883.0.15" pellets supplied by Provimi Kliva SA, Kaiseraugst, Switzerland ad libitum. Water was provided ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

vehicle: demineralized water
administration volume: 10 ml/kg bw

Details on mating procedure:

Pairing was performed overnight by placing one F0 female animal together with one F0 male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation. Animals were paired daily during the 2-week mating and one week remating period. Females in which insemination had not been detected by the end of the 2-week mating period, were mated for another week with another male of the respective dose group which had successfully inseminated a female paired with it. F0 females found sperm-positive after the first matings but where body weight gain did not indicate pregnancy by the end of the 2-week mating period were paired again for 7 days during the remating period.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

up to 58 days.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:actual ingested

Dose / conc.:

100 mg/kg bw/day

Remarks:

Basis:actual ingested

Dose / conc.:

300 mg/kg bw/day

Remarks:

Basis: actual ingested

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:actual ingested

No. of animals per sex per dose:

12 male and 12 female Wistar rats/dose and control group

Control animals:

yes, concurrent vehicle

Details on study design:

ADMINISTRATION/EXPOSURE

- Treatment: The male F0 rats were each given ethylenediamine, ethoxylated and propoxylated (NLP#6) or vehicle (demineralized water) once daily for 2 weeks prior to mating, during the following mating and remating period and up to the day before necropsy (necropsy on day 40 of the study; one male which was sacrificed moribund was necropsied on day 36 of the study). NLP#6 or vehicle was administered to each of the female F0 rats once daily for 2 weeks prior to mating, during the subsequent mating and remating period, during gestation and up to the day before necropsy. Necropsy was either performed on day 4 to 7 p.p. in females which delivered, or in inseminated females which did not deliver some days after the expected day of delivery; i.e. the F0 females were treated individually differently for 42 to 58 days.

Positive control:

No positive control

Parental animals: Observations and examinations:

Clinical examination, mortality, body weight and food intake.
Urinalysis: Urine was collected for about 16 hours from 5 rats per dose and sex*. in males this was done on the first 5 males per groups on day 30. In females this was done in those five females, which had been necropsied first (day 45) mostly after pups had been killed.
Animals were fasted during collection period and water was offered for ad libitum consumption. The following parameters were determined:

- Quantitatively: Volume, density, protein per vol., creatine per vol.
- Semiquantitatively: pH, glucose, protein, blood, bilirubin, ketone bodies, urobilinogen
- microscopy of sediment
*These examinations were done as urinalysis in a subacute study (Report No. AT03512) had been erroneously incomplete.

Sperm parameters (parental animals):

- testes
- epididymides
- histopatholocigal evaluation

Litter observations:

- Birth and viability
- L itter siye at birth and the live index
- Sex ratio
- Clinical observation during first days of lactation
- Pup weights
- Gross pathological changes in the F1 pups.

Postmortem examinations (parental animals):

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC) F0:
Organ weights: testes and epididymides (right and left organs separated)
Histopathology: testes, epididymes, and ovaries with oviducts in the control and high dose group.
Implantation sites in the uterus were counted at necropsy while number of corpora lutea in the ovaries was recorded during histopathological investigations (only control and high dose group

Postmortem examinations (offspring):

PARAMETERS ASSESSED ON F1:
- clinical signs
- number of live and dead pups
- sex of pups
- body weights
- external macroscopical investigations (including apparent malformations)

Statistics:

statistical significance was tested using the TASC-System; Analysis of variance (ANOVA) and in case of significant results DUNNETT´s test; 2/N CHI-Square test; in case of sigificant differences Fishers´s exact test with Bonferroni correction.

Reproductive indices:

- Insemination index (%): # of Sperm positive females/ # of females co-housed with a male
- Fertility index (%): # of pregnant females / # of sperm positive females
- Gestation index (%): # of females wiht live pups / # of pregnant females

Offspring viability indices:

- Live birth index (%): # of live pups at birth / total # of pups born
- Viability index (%): # of live pups on day 4 / # of live pups born

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

During the study no animal died in the study groups up to 1000 mg/kg.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no decreases in body weights or body weight gain up to and including 1000 mg/kg in males and females during the premating period. Lactating 1000 mg/kg females exhibited a mild (6-7%) body weight depression (p<0.05), while body weight gain during lactation was unaffected.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The semiquantitative urinalyses including the sediment showed no toxicologically relevant findings up to and including 1000 mg/kg

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mild body weight depression during lactation at 1000 mg/kg were reported. The registrant considers the mild body weight depression of 6 to 7% reported in the 1000 mg/kg bw/day in female during lactation as not adverse as the body weight gain during lactation was unaffected. An adverse effect is usually considered to be equal to or greater than 10% reduction in body weight.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: NOAEL as reported in the study report due to body weight depression during lactation at 1000 mg/kg. However, see comment from registrant.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: Salivation at 1000 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: limit dose

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: Salivation at 1000 mg/kg bw/day

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproduction/developmental toxicity

Critical effects observed:

not specified

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Survival rate of F0 animals was not reduced by treatment with ethylenediamine, ethoxylated and propoxylated at a dose level up to and including 1000 mg/kg bw/day.

Salivation directly after the administration was observed in both genders at the 1000 mg/kg dose level but an adverse effect is not concluded from this finding.

The lowest-observed-effect-level (LOEL) and the no-observed-effect level (NOEL) for salivation after administration were as follows:

Males: LOEL: 1000 mg/kg bw/day

NOEL: 300 mg/kg bw/day

Females LOEL: 1000 mg/kg bw/day

NOEL: 300 mg/kg bw/day

Body weight development was not affected to a toxicologically relevant extent in males at a dose level up to and including 1000 mg/kg. Females of the 1000 mg/kg group revealed a mild body weight gain depression during lactation.

There was no difference in food intake among groups up to and including 1000 mg/kg bw/day.

Urine parameters were not changed up to and including 1000 mg/kg bw/day.

PARAMETERS OF REPRODUCTION

Treatment related effects on reproductive parameters, i.e. insemination, fertility and gestation index, time to insemination, duration of gestation, course of birth, lactation behavior, number of corpora lutea, implantation sites, prenatal loss, litter size, sex ratio of pups, pup mortality (live birth index, viability index), pup body weight, and clinical findings of pups were not evident at a dose level up to and including 1000 mg/kg.

An indication for a teratogenic potential of ethylendiamine, ethoxylated and propoxylated, was not evident in this study at a dose level up to and including 1000 mg/kg bw/day.

PATHOLOGY

Necropsy, evaluation of male reproductive organ weight (testes, epididymides) and histopathology of reproductive organs (testes, epididymides, ovaries and oviducts) revealed no treatment related findings up to and including 1000 mg/kg.

Conclusions:

The registrant considers the mild body weight depression of 6 to 7% reported in the 1000 mg/kg bw/day in female during lactation as not adverse as the body weight gain during lactation was unaffected. An adverse effect is usually considered to be equal to or greater than 10% reduction in body weight.

Executive summary:

NLP#6 (Ethylenediamine, Ethoxylated and Propoxylated Polyol, molecular mass 280 g/mol) was administered daily via gavage in demineralized water to 12 male and 12 female Wistar rats per dose group, in doses of 0, 100, 300 or 1000 mg/kg body weight for a period of 2 weeks prior to a 2 week mating period. Males were treated then further up to necropsy (4 weeks at a minimum) and females during gestation and lactation up to their necropsy after day 4 p.p. of their pups. Investigations were performed on general tolerance of the test compound by the parental animals as well as on effects on reproduction and early postnatal development of Fl pups. The animals were regularly observed and weighed, food intake as well as urine and reproduction parameters were determined. Selected organs were weighed and organs were subjected to macroscopical and histopathological investigations. The test substance was stable in the vehicle for the duration of use. Formulations given to the rats were prepared appropriately. Survival rate of FO rats was not reduced up to and including 1000 mg/kg. At 1000 mg/kg FO rats exhibited salivation directly after the administration. An adverse effect is not concluded from this finding. In lactating females a mild body weight depression was seen at 1000 mg/kg. There was no difference in food intake among groups up to and including 1000 mg/kg. Urine parameters were not changed up to and including 1000 mg/kg. No adverse effects were seen in the weights of the testes, epididymides or ovaries. Histopathology revealed no evidence of test substance induced changes in male and female rats with respect to testes, epididymides, ovaries and oviducts at 1000 mg/kg. Up to and including 1000 mg/kg no effects on mating behavior, fertility and live birth indices, gestation parameters, number of implantation sites or prenatal loss, litter size, number of pups born, sex ratio, viability of pups and pup weights were seen. There were also no remarkable clinical or necropsy findings in pups and no adverse effect on the course of birth or lactation behavior of the dams in these groups.

Thus, the following no-observed-(adverse) effect levels were determined:

Females (body weight depression during lactation) NOAELand NOEL 300 mg/kg

Males NOEL: 1000 mg/kg NOAEL 300 mg/kg

Reproduction/Developmental Toxicity: NOAEL 1000 mg/kg

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

A reproduction toxicity screening study (oral gavage) following the OECD guideline 421 is available for the endpoint.

Survival rate of F0 animals was not reduced by treatment with ethylenediamine, ethoxylated and propoxylated at a dose level up to and including 1000 mg/kg bw/day. Salivation directly after the administration was observed in both genders at the 1000 mg/kg dose level but an adverse effect is not concluded from this finding. Body weight development was not affected to a toxicologically relevant extent in males at a dose level up to and including 1000 mg/kg. Females of the 1000 mg/kg group revealed a mild body weight gain depression during lactation.

The lowest-observed-effect-level (LOEL) and the no-observed-effect level (NOEL) for salivation after administration were as follows:

Males: LOEL: 1000 mg/kg bw/day; NOEL: 300 mg/kg bw/day

Females LOEL: 1000 mg/kg bw/day; NOEL: 300 mg/kg bw/day

There were no detectable effects in offspring. The NOAEL (reproduction/development) is >1000 mg/kg bw/day.

Adequate testing has been undertaken on a sufficient number of the core substances and repeating units. None of the tested core substances and none of the repeating units is classifiable as a reproductive toxin. Hence it would be anticipated that the NLP polyols, as a category, would also not be reproductive toxins. Three of the NLP polyols, nitrilotriethanol, propoxylated, diaminotoluene, propoxylated and ethylenediamine, ethoxylated and propoxylated, were tested to fulfil the requirements of Annex VIII (>10 tonnes/y). The results from these screening reproductive tests confirmed the pre-existing information. The NLP polyols are not reproductive or developmental toxins. Furthermore, a range of studies has been conducted on core substances and repeating units and screening tests have been conducted on most substances in the NLP polyol categories. It is possible to ‘read across’ the results from all of these sources to all substances in these categories. Sufficient data exist to permit robust conclusions that the substances are not reproductive or developmental toxins and that no further testing is required.

Short description of key information:
The NOAEL (reproduction/development, oral gavage - OECD 421) is >= 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

Developmental toxicity study in rats, OECD 414

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and Guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: sexually mature adult
- Weight at study initiation: 200-250 g
- Housing: 1/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (range of 20-26)
- Humidity (%): 50 (range of 30-70)
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared by mixing the test material in ultrapure water at concentrations of 0, 25, 75 or 250 mg/ml and administered at a dose volume of 4 ml/kg body weight. Dose volumes were adjusted daily based on individual body weights. The control rats were dosed with ultrapure water at 4 ml/kg body weight. Dose solutions were prepared periodically throughout the study based on the established stability.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation of dose solutions from the first mix was performed pre-exposure. The homogeneity of the low- and high-dose test solutions was determined concurrent with dose confirmation. The method used for analyzing the test material in ultrapure water was liquid chromatography-mass spectrometry (LC/MS).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

GD 6-20

Frequency of treatment:

once daily, 7 days/week

Duration of test:

up to GD 21

No. of animals per sex per dose:

24/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for this study were selected on the basis of the developmental toxicity probe study. Oral administration of Ethylenediamine, ethoxylated and propoxylated (>1 – <8.5 mol of EO and PO) to time-mated Crl:CD(SD) rats up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity and no indication of embryo/fetal lethality; therefore, no observable toxicity was expected. The high-dose of 1000 mg/kg/day represents a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study). The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a no-observed-effect level (NOEL).
- Rationale for animal assignment: Animals were stratified by GD 0 body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform mean group weights and standard deviations at the start of the study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- All rats were observed in their cages for significant clinical abnormalities clearly visible upon a limited examination and to monitor the general health of the animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily; approximately 1 hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on GD 0 by the supplier and daily from GD 6-21. Statistical analysis of body weights was performed using data collected on GD 0, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

FOOD CONSUMPTION: Yes, recorded every 3 days from GD 3-21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The stomach, liver, and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded.

OTHER: The weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative portions of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Miccoscopic examination of the liver, kidneys, and gross lesions was not conducted.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

OTHER: The uteri of females lacking visible implantations was stained with a 10% aqueous solution of sodium sulfide based on (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Maternal body weights, maternal body weight gains, maternal organ weights, gravid uterine weights, fetal body weights, feed consumption: Bartlett's test, parametric or non-parametric ANOVA, Dunnett's test or Wilcoxon Rank-Sum test with Bonferroni's correction
Frequency of pre- and post-implantation loss and fetal alterations: censored Wilcoxon test with Bonferroni's correction
Number of corpora lutea, implantations, and litter size: non-parametric ANOVA, Wilcoxon Rank-Sum test with Bonferroni's correction
Pregnancy rates: Fisher exact probability test with Bonferroni's correction
Fetal sex ratios: binomial distribution test
Statistical outliers were identified using a sequential method and, if excluded, were excluded for sound scientific reasons.

Indices:

Pre/post-implantation loss

Historical control data:

Yes

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical and cage-side examinations performed on all animals revealed no treatment-related findings. Observations were sporadic and transient and were therefore considered unrelated to treatment. See 'Overall remarks, attachments' below for data.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no deaths. See 'Overall remarks, attachments' below for data.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in the body weights or body weight gains in any of the treated groups when compared to controls. See 'Overall remarks, attachments' below for data.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related differences in the amount of feed consumed in any of the treated groups when compared to controls. See 'Overall remarks, attachments' below for data.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in any of the measured parameters in any of the treated groups when compared to controls. See 'Overall remarks, attachments' below for data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on numbers of implantations, percent pre-implantation loss, percent postimplantation loss, in any of the treated groups compared to controls. Mean percent post-implantation loss was statistically identified as higher in the 1000 mg/kg/day group when compared to the control group; however, this value was well within the range of historical control data and, therefore, was not considered treatment related. See 'any other information on results incl. tables' and 'Overall remarks, attachments' below for data.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on resorption rates. See 'Overall remarks, attachments' below for data.

Early or late resorptions:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Dead fetuses:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): See 'Overall remarks, attachments' below for data.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical and cage-side examinations performed on all animals revealed no treatment-related findings. Observations were sporadic and transient and were therefore considered unrelated to treatment.
There were no treatment-related differences in the body weights or body weight gains in any of the treated groups when compared to controls.
There were no treatment-related differences in teh amount of feed consumed in any of the treated groups when compared to controls.
There were no treatment-related differences in terminal body, liver, or kidney weights in any of the treated groups when compared to controls.
There were no treatment-related gross pathological observations.
There were no treatment-related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent pre-implantation loss, percent post-implantation loss, fetal sex ratios, fetal body weights or gravid uterine weights in any of the treated groups compared to controls. Mean percent post-implantation loss was statistically identified as higher in the 1000 mg/kg/day group when compared to the control group; however, this value was well within the range of historical control data and, therefore, was not considered treatment related. See 'any other information on results incl. tables' and 'Overall remarks, attachments' below for data.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related effects on fetal body weights. See 'Overall remarks, attachments' below for data.

                                              
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See 'Overall remarks, attachments' below for data.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

See 'Overall remarks, attachments' below for data.

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related external alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformations of agnathia and astomia in a single control fetus. See 'any other information on results incl. tables' and 'Overall remarks, attachments' below for data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statically significant or treatment-related skeletal alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformations sternoschisis and fused ribs and the variations delayed ossification (DO) interparietal, supernumerary skull bone, DO cervical centra, extra site of ossification sternebrae, irregular pattern of ossification sternebrae, extra 1st lumbar rib, DO thoracic centra, and DO pubis. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment. See 'any other information on results incl. tables' and 'Overall remarks, attachments' below for data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related visceral alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformations situs inversus, retroesophageal aortic arch, and hydronephosis and the variations fused lung, hemorrhage adrenal, pale liver, and supernumerary hepatic liver lobule. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment. See 'any other information on results incl. tables' and 'Overall remarks, attachments' below for data.

Other effects:

no effects observed

Description (incidence and severity):

Craniofacial Examination
There were no treatment-related craniofacial alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformation
microphthalmia in a single control fetus.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no statisticaly signficant differences in the incidence of any fetal alteration in any of the treatment groups compared to controls. The small number of alterations observed in fetuses from dams administered Ethylenediamine, ethoxylated and propoxylated either occurred at low frequences, were within recent historical control values, and/or were not dose related.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: embryofetal development

Abnormalities:

no effects observed

Developmental effects observed:

no

Post-implantation Loss:

Dose Level (mg/kg/day)	0	100	300	1000
Mean % Post-implantation Loss	3.3 +/- 3.7	3.5 +/- 7.7	2.2 +/- 3.9	6.2* +/- 5.1

*Statistically different from control mean by Consored Wilcoxon's test alpha =0.05

Historal Control Post-implantation Loss:

(D=Dietary, G=Gavage)

Study Route Year

1 D 2010

2 D 2010

3 D 2010

4 D 2012

5 D 2012

6 D 2014

7 G 2014

8 D 2014

9 G 2014

10 G 2015

11 G 2015

12 D 2015

13 G 2015

Mean % Post-implantation Loss

1.9 +/- 4.3

1.4 +/-2.8

2.2 +/-3.4

4.1 +/-9.8

2.0 +/-3.5

4.0 +/-6.5

5.1 +/-4.0

4.2 +/-6.0

3.9 +/-11.1

3.4 +/-4.9

8.8 +/-16.8

2.3 +/-5.3

3.7 +/-5.9

D=Dietary, G=Gavage

Bold type indicates the highest observed value for the endpoint.

Incidences of External Malformations

Dose (mg/kg/day)		0	100	300	1000
Agnathia	F L	1/303^a  1/22	0/326 0/24	0/303 0/22	0/303 0/22
Astomia	F L	1/303^a  1/22	0/326 0/24	0/303 0/22	0/303 0/23

F = fetuses; L = litters

^aMalformations denoted with the same superscript were noted in a single fetus.

Incidences of External Malformations:

Dose (mg/kg/day)		0	100	300	1000
Microphthalmia	F L	1/153 1/22	0/160 0/24	0/150 0/22	0/151 0/23

F=fetuses; L= litters

Incidences of Visceral Malformations:

Dose (mg/kg/day)		0	100	300	1000
Situs Inversus	F L	0/153 0/22	0/160 0/24	1/150 1/22	0/151 0/23
Retroesophageal Aortic Arch	F L	0/153 0/22	0/160 0/24	0/150 0/22	1/151 1/23
Hydronephrosis	F L	1/153 0/22	1/160 1/24	1/150 0/22	0/151 0/23

F=fetuses; L=litters

Incidences of Accessory Blood Vessel Kidney:

Dose (mg/kg/day)		0	100	300	1000
Accessory Blood Vessel Kidney	F L	1/153 (0.7%) 1/22 (4.5%)	1/160 (0.6%) 1/24(4.2%)	4/150 (2.7%) 2/22 (9.1%)	3/151 (2.0%) 3/23 (13.0%)

F=fetuses; L=litters

Historical Control Data for Accessory Blood Vessel Kidney:

Study    Route Year

1 D 2010

2 D 2010

3 D 2010

4 D 2012

5 D 2012

6 D 2014

7 G 2014

8 D 2014

9 G 2014

10 G 2015

11 G 2015

12 D 2015

13 G 2015

Accessory Blood Vessel Kidney

F L

0/138 (0.0%) 0/22 (0.0%)

0/168 (0.0%) 0/24 (0.0%)

0/179 (0.0%) 0/26 (0.0)

0/123 (0.0%) 0/22 (0.0%)

0/150 (0.0%) 0/24 (0.0%)

0/137 (0.0%)  0/23 (0.0%)

0/136 (0.0%) 0/23 (0.0%)

0/133 (0.0%) 0/24 (0.0%)

0/140 (0.0%) 0/24 (0.0%)

0/148 (0.0%)   0/24 (0.0%)

0/124 (0.0%) 0/21 (0.0%)

0/153 (0.0%) 0/23 (0.0%)

3/146 (2.1%) 3/24 (12.5%)

D=Dietary, G=Gavage

F = fetuses; L = litters

Bold type indicates the highest observed value for the endpoint.

Incidences of Skeletal Malformations

Dose (mg/kg/day)		0	100	300	1000
Sternoschisis	F L	1/150 1/22	0/166 0/24	0/153  0/22	0/152 0/23
Fused Ribs	F L	0/150 0/22	1/166 1/24	0/153 0/22	0/152 0/23

F=fetuses; L=litters

Incidences of Selected Skeletal Variations:

Dose (mg/kg/day)		0	100	300	1000
DO Sternebrae	F L	0/150 (0.0%) 0/22 (0.0%)	1/166 (0.6%) 1/24 (4.2%)	3/153 (2.0%) 1/22 (4.5%)	3/152 (2.0%) 3/23 (13.0%)
Class I Wavy Ribs	F L	0/150 (0.0%) 0/22 (0.0%)	1/166 (0.6%) 1/24 (0.0%)	0/153 (0.0%) 0/22 (0.0%)	2/152 (1.3%) 2/23 (8.7%)
Class II Wavy Ribs	F L	0/150 (0.0%) 0/22 (0.0.0%)	1/166 (0.6%) 1/24 (4.2%)	0/153 (0.0%) 0/22 (0.0%)	1/152 (0.7%) 1/23 (4.3%)
Calloused Ribs	F L	1/150 (0.7%) 1/22 (4.5%)	2/166 (1.2%) 1/24 (4.2%)	1/153 (0.7%) 1/24 (4.5%)	2/152 (1.3%) 2/23 (8.7%)

F=fetuses; L=litters

Historical Control Data for Selected Skeletal Variations:

Study Route Year

1 D 2010

2 D 2010

3 D 2010

4 D 2012

5 D 2012

6 D 2014

7 G 2014

8 D 2014

9 G 2014

10 G 2015

11 G 2015

12 D 2015

13 G 2015

DO Sternebrae

F L

1/138 (0.7%)  1/22 (4.5%)

2/152 (1.3%) 2/24 (8.3%)

3/180 (1.7%) 2/26 (7.7%)

2/131 (1.5%) 2/22 (9.1%)

2/151 (1.3%) 2/24 (8.3%)

1/139 (0.7%) 1/23 (4.3%)

0/135 (0.0%) 0/23 (0.0%)

1/134 (0.7%) 1/24 (4.2%)

0/145 (0.0%) 0/24 (0.0%)

2/151 (1.3%) 2/24 (8.3%)

0/125 (0.0%) 0/21 (0.0%)

0/152 (0.0%) 0/23 (0.0%)

2/147 (1.4%) 2/24 (8.3%)

Class I Wavy Ribs

F L

0/138 (0.0%) 0/22 (0.0%)

0/152 (0.0%) 0/24 (0.0%)

2/180 (1.1%) 2/26 (7.7%)

1/131 (0.8%) 1/22 (4.5%)

2/151 (1.3%) 2/24 (8.3%)

0/139 (0.0%) 0/23 (0.0%)

1/135 (0.7%) 1/23 (4.3%)

4/134 (30%) 1/24 (4.2%)

0/145 (0.0%) 0/24 (0.0%)

0/151 (0.0%) 0/24 (0.0%)

0/125 (0.0%) 0/21 (0.0%)

1/152 (0.7%) 1/23 (4.3%)

2/147 (1.4%) 2/24 (8.3%)

Class II Wavy Ribs

F L

0/138 (0.0%) 0/22 (0.0%)

0/152 (0.0%) 0/24 (0.0%)

0/180 (0.0%)  0/26 (0.0%)

0/131 (0.8%) 0/22 (0.0%)

0/151 (0.0%) 0/24 (0.0%)

0/139 (0.0%) 0/23 (0.0%)

0/135 (0.0%) 0/23 (0.0%)

1/134 (0.7%) 1/24 (4.2%)

0/145 (0.0%) 0/24 (0.0%)

0/151 (0.0%) 0/24 (0.0%)

0/125 (0.0%) 0/21 (0.0%)

0/152 (0.0%) 0/23 (0.0%)

2/147 (1.4%) 2/24 (8.3%)

Calloused Ribs

F L

0/138 (0.0%) 0/22 (0.0%)

0/152 (0.0%) 0/24 (0.0%)

1/180 (0.6%) 1/26 (3.8%)

0/131 (0.8%) 0/22 (0.0%)

1/151 (0.7%) 1/24 (4.2%)

0/139 (0.0%) 0/23 (0.0%)

3/135 (2.2%) 3/23 (13.0%)

4/134 (30%) 1/24 (4.2%)

0/145 (0.0%) 0/24 (0.0%)

0/151 (0.0%) 0/24 (0.0%)

0/125 (0.0%) 0/21 (0.0%)

0/152 (0.0%) 0/23 (0.0%)

2/147 (1.4%) 2/24 (8.3%)

D=Dietary, G=Gavage

F = fetuses; L = litters

Bold type indicates the highest observed value for the endpoint.

Conclusions:

Gavage administration of Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) up to, and including the limit dose of 1000 mg/kg/day, resulted in no treatment-related maternal toxicity and no indication of embryo/fetal toxicity or teratogenicity. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity and developmental toxicity was the limit dose of 1000 mg/kg/day.

Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) in Crl:CD(SD) rats following repeated gavage administration.

Groups of 24 time-mated female rats were administered Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) in ultrapure water by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day on gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral and craniofacial alterations while skeletal examinations were conducted on the remaining fetuses.

Gavage administration of Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) up to, and including the limit dose of 1000 mg/kg/day, resulted in no treatment-related maternal toxicity and no indication of embryo/fetal toxicity or teratogenicity.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity and developmental toxicity was the limit dose of 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

The purpose of this study was to evaluate the maternal and developmental toxicity of Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) in Crl:CD(SD) rats following repeated gavage administration.

Groups of 24 time-mated female rats were administered Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) in ultrapure water by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day on gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral and craniofacial alterations while skeletal examinations were conducted on the remaining fetuses. Gavage administration of Ethylenediamine, ethoxylated and propoxylated (>1-<8.5 mol of EO and PO) up to, and including the limit dose of 1000 mg/kg/day, resulted in no treatment-related maternal toxicity and no indication of embryo/fetal toxicity or teratogenicity.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity and developmental toxicity was the limit dose of 1000 mg/kg/day.

Ethylenediamine, ethoxylated and propoxylated is not classifiable as hazardous in respect to its developmental toxicity based on the OECD 414 study in rats. There is sufficient information from a qualitative and quantitative understanding of the toxicological properties of the core substance, the repeating unit, and OECD 414 study with the substance. There is also negligible human exposure based on the uses with the registered substance.

In addition, there were no observations of adverse effects on reproductive organs such as testes, epididymides, ovaries, oviducts, uterus in the 28- nor 90-day repeated dose studies and reproductive/developmental toxicity screen (OECD TG 421) resulting from EDA/EO/PO MW 280 g/mole treatment. No effects on mating behavior, fertility, live birth incidences, gestation parameters, number of implantation sites or prenatal loss, litter size, number of pups born, sex ratio, viability of pups or pup weights were seen. Clinical or necropsy findings in pups were not shown for the substance nor were adverse effects during birth or lactation behaviors of dams. The reproductive/developmental toxicity NOAEL with EDA/EO/PO substance was 1000 mkd.

Note that none of the registrants has sales or imports above 1000 t/yr. Annex IX requires a pre-natal developmental toxicity study in one species, with the decision on the need to perform a study at this tonnage level or the next on a second species based on the outcome of the first species developmental toxicity study and all other relevant available data. Based on all available data described above, there is no requirement to conduct a pre-natal developmental toxicity study in a second species.

Toxicity to reproduction: other studies

Additional information

The purpose of this study was to make a preliminary evaluation of the maternal toxicity and embryo/fetal lethality potential of Ethylenediamine, ethoxylated and propoxylated (>1 – <8.5 mol of EO and PO) in Crl:CD(SD) rats following repeated gavage administration. Results from this study were used to set dose levels for a subsequent developmental toxicity study in Crl:CD(SD) rats.

Groups of five time-mated female Cr1:CD(SD) rats were administered 0 or 1000 mg/kg/day Ethylenediamine, ethoxylated and propoxylated (>1 – <8.5 mol of EO and PO) in ultrapure water by oral gavage at a dose volume of 4 ml/kg on gestation day (GD) 6 through 20. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed consumption. On GD 21 all surviving animals were euthanized and examined for gross pathologic alterations. Liver and kidney weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead fetuses.

Oral administration of Ethylenediamine, ethoxylated and propoxylated (>1 – <8.5 mol of EO and PO) to time-mated Crl:CD(SD) rats up to and including the limit dose of 1000 mg/kg/day resulted in no maternal toxicity and no indication of embryonal/fetal lethality.

Justification for classification or non-classification

A reproduction toxicity screening study (oral gavage) following the OECD guideline 421 is available for the endpoint. Possible minor effects on body weight and salivation do not constitute 'serious damage', the regulatory requirement relevant to classification.

No effects were observed at any dose level in the developmental toxicity OECD 414 study or in the developmental toxicity probe study. Based on the results of these studies, ethylenediamine, ethoxylated and propoxylated, will not be classified for reproduction toxicity.