7-azatridecane-1,13-diamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 JUN 1983 to 29 JUL 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency. Seection 83–3: Teratogencity Study. Subdivision F. for Hazard Evaluation: Human and Domestic Animals. issued November, 1982.

Deviations:

not applicable

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

substance exposure only on GD 6-15 instead until one day prior to the schedule kill (i.e. GD20 in this study), slightly higher temperature (1 °C) as indicated in guideline on one day due to power outage

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

EC Number:

907-605-7

Molecular formula:

C6H16N2 (HMD) C12H29N3 (BHT)

IUPAC Name:

Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Bis-(Hexamethylene)-triamine (i.e. BHMT)
- Substance type: grey paste

Test animals

Species:

rat

Strain:

other: Charles River COBS CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 12 weeks (at the time of mating)
- Weight at study initiation: females: 213-274 g (on GD 0)
- Housing: individually housed in hanging wire-mesh cages
- Diet: Purina Certified Rodent chow #5002 (Ralston Purina Company, ST. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26
- Humidity (%): 51-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of the test item for each day was weighed into a graduated mixing cylinder. A suffucient quantity of the vehicle, deionized water, was added to yield a total volume of 25 ml of prepared material. The cylinder was shaken by hand and dispensed into a capped container.
The test material was prepared fresh daily at a concentration of 0,2 g/ml to permit the administration of dose levels of 50, 100 and 250 mg test material/kg/day at dosage volumes of 0.25, 0.5 and 1.25 ml/kg, respectively.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: until copulatory plug was seen at the daily inspection. The day the vaginal plug was observed was defined as gestation day (GD 0)

Duration of treatment / exposure:

from day 6 to 15 of gestation

Frequency of treatment:

once daily

Duration of test:

animals were sacrificed on day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of range finding study

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 16, 20 (day of scheduled kill)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:abdominal and thoracic cavities were examined for grossly evident morphological changes. If any changes were visual, the respective maternal tissues were preserved in 10 % neutral buffered formalin for histopathological examinations. Uteri from females which appeared nongravid were opened and placed in 10 % ammonium sulfide solution for detection of implantations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of viable and nonviable fetuses

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No

Statistics:

All statistical analyses compared the treatment groups to the control with the level of significance at p<0.05 and p<0.01. All means were accompanied by standard deviations.
Male to female fetal sex ratios and the proportions of litters with malformations were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.
The proportions of resorbed and dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
Mean numbers of corpora lutea, total implantations, postimplantation loss, live fetuses and mean fetal body weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.

Indices:

Group mean preimplantation loss (%) = (total no. corpora lutea - total no. implantations)/total no. corpora lutea*100
Group mean postimplantation loss (%) = (total no. implantations - total no. viable fetuses)/total no.implantations*100

Historical control data:

Available for maternal and fetal observations at cesarean section and for incidence of malformations, developmental and genetic variations.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Respiratory rales occurred in all treated groups and was considered compound related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Control group: Survival was 100% during this study.
250 mg/kg/day: 4 animals died between GD 7 and 12, 1 rat was sacrificed in extremis on GD 14
100 mg/kg/day : 5 rats died between GD 7 and 20, 1 rat was sacrificed in extremis on GD 12
50 mg/kg/day: 1 rat died at GD 11
The cause of death could not be determined for any of these animals.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The antemortem observations of the animals that failed to survive to study termination included dried black or red matter around one or both eyes, forelimbs, and nose and/or mouth; moist, black material around the vagina; respiratory rales; nasal discharge; labored breathing; vocalization during breathing; emaciated appearance and moribundity (rats that were sacrificed only).

Necropsy findings:
250 mg/kg/day: One female was observed to have dried, red staining around nose and mouth, hydronephrosis and pitting of the right distended right ureter and calculi in the urinary bladder.
100 mg/kg/day animals that died or were sacrificed in extremis had stomach erosions; distended esophagus, stomach, intestines ureter; hydronephrosis; and mucoid material in the intestines.
50 mg/kg/day: No lesions were exhibited in the rat of this dosage group that died prior to study termination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

further details see below

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

further details see below

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Cesarean section was performed on gestation day 20 of all surviving rats
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): Cesarean section was performed on gestation day 20 of all surviving rats

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

50 and 100 mg/kg/day: slight decrease for mean numbers of corpora lutea and total implantations and increases in postimplantation losses resulted in decreased numbers of viable fetuses in these groups. Effect in low dose group is based on findings in only one out of 25 animals which had 11 late resorptions while all the other animals in the group had no late resorptions. In the mid dose group two out of 25 animals showed either 13 or 11 late resorptions while all other animals showed no increase compared to controls.
250 mg/kg/day: No notable differences were seen in the above mentioned parameters in comparison to the controls.

Since there was no effect at the high dose and no dose-related trend as the 50 mg/kg/day appeared to be marginally more affected than the 100 mg/kg/day group with respect to the decrease in mean corpura lutea number, total implantations and numbers of viable fetuses, the findings were not considered to be related to treatment.

Details on maternal toxic effects:

mortality (but no particular cause of early death could be identified at necropsy), respiratory difficulties

Other than the above described findings (e.g. hair loss, dry matter around eyes, nose, slightly reduced body weight gains during first treatment interval (GD6 to9) etc.) were not considered meaningful, as they were either not dose-dependent, the findings exhibited a high degree of variability or were observed also in the negative control group.

Effect levels (maternal animals)

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): fetal body weights were recorded on the day of cesarean section only

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

50 mg/kg/day: significantly decreased number of viable fetuses/dam compared to control
100 mg/kg/day: slight, non-significant decrease for number of viable fetuses/dam
250 mg/kg/day: no difference to control

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

litter size and weights were recorded on the day of cesarean section only

Changes in postnatal survival:

not examined

Description (incidence and severity):

postnatal survival is not examoned in a teratogenicity study

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related external malformations were found.
Further details on embryotoxic/teratogenic effects in the respective field below.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related effects on fetal ossification variations, external,or skeletal malformations were found.
Further details on embryotoxic/teratogenic effects in the respective field below.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related effects on soft tissue were found.
Further details on embryotoxic/teratogenic effects in the respective field below.

Details on embryotoxic / teratogenic effects:

The malformations observed among control and treated groups included:
anasarca, localized edema, omphalocele, bent tail, microphthalmia, interventricular septal defect, vestigial uterine horn, bent ribs, interrupted ossification of ribs, bent scapula and bent limb bones. The malformations were considered incidental and occurred in no more than two fetuses per group. Although the mid-dose group was observed to have more litters with malformations than the control group, this was not considered meaningful as similar increases did not occur in the high-dose group.
Moreover all the above mentioned anomalies found could also be seen in the data of historical controls.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test item to the rat at doses of 0, 50, 100 or 250 mg/kg body weight from gestation day 6 - 15 caused early deaths as well respiratory rales in the maternal animals. In the foetuses no treatment related effects on foetal ossification variations, external, soft tissue or skeletal malformations could be found. With regard to the present study a No Observed Adverse Effect Level (NOAEL) for maternal toxicity could not be established. The respective maternal LOAEL (including effects on reproduction and fertility) in this study is 50 mg/kg/day. Based on the lack of any effects concerning the foetal development the developmental NOAEL of this study is 250 mg/kg/day.

Executive summary:

Groups of 25 mated female CD rats received the test material by oral gavage once daily at the dose levels of 0, 50, 100 or 250 mg/kg body weight from gestation day 6 to 15 (day 0 = day the vaginal plug was observed) and were sacrificed an day 20 of pregnancy (equivalent to OECD 414, GLP).

Four rats in the 250 mg/kg/day dosage group, five rats in the 100 mg/kg bw/day dosage group and one rat in the 50 mg/kg bw/day dosage group died on study during gestation days 7 to 12, 7 to 20, and day 11, respectively. The cause of death could not be determined for any of these animals. One rat each in the 100 and 250 mg/kg/day dosage groups were sacrificed in extremis. Survival was 100% in the control group. Breathing difficulties occurred in all treated groups and were considered a result of treatment. Necropsy observations were comparable between the control group and all treated groups. Body weight gains in the all treated groups were reduced when compared to the control group values during the early treatment period (GD 6 to 9). However, a high degree of variability precluded the assessment of any meaningful relation to the test article at any specific interval.

Slightly increased postimplantation losses coupled with a decrease in corpora lutea were observed in the 50 and 100 mg/kg bw/day dosage groups when compared to the control group values. Consequently, the mean viable foetuses values for these groups were lower than the control group value. As these parameter values for the 250 mg/kg bw/day dosage group were comparable to the control group values, the findings in the 50 and 100 mg/kg bw/day dosage groups were not considered treatment-related. The mean foetal body weight and foetal sex distribution values for all treated groups were comparable to the group values.

There were no biologically meaningful differences in the incidence of foetal malformations and developmental variations between the control and treated groups.

Under the tested conditions the submission substance, did not produce a developmental/teratogenic effect (NOEAL (developmental) = 250 mg/kg bw/day). Nevertheless there was maternal toxicity seen throughout the study therefore resulting in a LOAEL (maternal) of 50 mg/kg bw/day.