Hexamethylenediamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2015 - June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylenediamine

EC Number:

204-679-6

EC Name:

Hexamethylenediamine

Cas Number:

124-09-4

Molecular formula:

C6H16N2

IUPAC Name:

hexane-1,6-diamine

Test material form:

solid

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

New Zealand White, INRA, A 1077, Specific Pathogen Free (S.P.F.)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre LAGO (Vonnas, France)
- Age at study initiation: 18-20 weeks
- Weight at study initiation: mean 3761 g (range: 3070 g to 4460 g)
- Fasting period before study: no
- Housing: individually (noryl cages (Tecniplast, 65.3 cm x 65.3 cm x 45 cm))
- Diet: ad libitum; breeding pelleted diet "type 110C", batch Nos. 15114 and 15159 (SAFE, Augy, France)
- Water: ad libitum, tap water (filtered with a 0.22 µm filter)
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 5 to 15
- Photoperiod (hrs dark / hrs light): 8h dark/16h light

IN-LIFE DATES: From: 22 September 2015 To: 6 November 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Phosphate Buffered Saline (PBS 1X pH 7.4)

Remarks:

pH of vehicle adjusted to pH 7.2 +/- 0.5 with hydrochloric acid after addition of test item

Details on exposure:

Route = oral

VEHICLE
- Justification for use and choice of vehicle (if other than water): to neutralise the alkaline test item
- Concentration in vehicle: 0, 2.5, 5, 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no.: 1691215 and 1685701

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- analytical method: Gas Chromatography with FID detection (GC-FID)
- time schedule: once in Weeks 1 and 5 a sample was taken from control and test item dose formulations and analyzed using the validated method
- acceptance criterion: measured concentration = nominal concentration +/- 10%

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: confirmed mating (visual assessment) was designated as Day 0 p.c.

Duration of treatment / exposure:

Day 6 to Day 28 p.c.

Frequency of treatment:

daily

Duration of test:

till Day 29 p.c. (scheduled sacrifice)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

24 females per dose group in main study, 3 females per dose group in satellite study

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of pre-tests

Maximum Tolerated Dose study: non-pregnant animals, 7 repeated exposures towards
- 200 mg/kg bw/d (all 3 animals died or sacrificed after single exposure),
- 50 mg/kg bw/d (no animal died, no obvious signs of in vivo toxicity); these animals were afterwards treated with 125 mg/kg bw/day (see below)
- 125 mg/kg bw/day (one animal sacrificed after 4 days due to blood loss, two other animals survived till end of exposure period)

Preliminary study: exposure of pregnant animals, Day 6-28 p.c.
- 90 mg/kg bw/day: all animals prematurely sacrificed due to blood loss, loud breathing and/or dyspnea, reduced food consumption and body weight loss
- 60 mg/kg bw/day: one animal prematurely sacrificed due to blood loss, loud breathing and dyspnea, food consumption not clearly affected
- 30 mg/kg bw/day: well tolerated

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes (principal and satellite animals)
- Time schedule: once a day checked for clinical observations; twice a day check for mortality and morbidity during treatment period

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes (principal and satellite animals)
- Time schedule for examinations: on Days 2, 4, 5, 6, 9, 12, 15, 18, 21, 24, 27 and 29 p.c. and prior to premature sacrifice

FOOD CONSUMPTION: Yes (principal and satellite animals)
- Time schedule: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 p.c.
- Food consumption for each animal determined a as g food/day: Yes

WATER CONSUMPTION: No (principal and satellite animals)

POST-MORTEM EXAMINATIONS: Yes (principal animals and prematurely sacrificed animals)
- Sacrifice on gestation day 29
- Organs examined: macroscopic post mortem examinations of the principal thoracic and abdominal organs. Particular attention was paid to the stomach and to the urinary tract, pH of urine measured. Determination of pregnancy status and the numbers of corpora lutea and implantation sites (recorded and classified as live or dead concepti, early or late resorptions or uterine scars)

OTHER:
Urinalysis performed in animals of the satellite group (between Day 25 and 28 p.c.), due to some effects observed in the urinary tract of animals found dead or prematurely sacrificed in the MTD or dose-range finding study
Urines were collected at the end of the treatment period (on Day 26 p.c.) at the following four periods (thymol was used as a preservative):
 [-4h; 0h(a)],
 [0h(a); 4h],
 [4h, 12/14h(b)],
 [12/14h(b); 24h]).
(a): 0h corresponds to the time of dosing.
(b): 12/14h means that for organizational purpose, end and start of collection may be between 12 and 14h after dosing.

qualitative parameters:
- appearance, colour

Quantitative parameters determined in urine:
- pH, volume, specific gravity

Semi-quantitative parameters determined in urine:
- proteins, glucose, ketones, bilirubin, nitrites, blood (hemoglobin), urobilinogen, cytology of sediment

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter (One half of the fetuses was decapitated and the head was fixed in Harrison’s fluid with decalcification. Serial sectioning was performed for evaluation of brain, nasal passages and tongue (and other structures, where appropriate). In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison’s fluid. Serial section was made for examination of the organ.)

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).

Indices:

none

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

emaciated appearance, absence of feces/urine, nearly no food consumption and/or abortion in prematurely sacrificed animals; no remarkable clinical observations in the surviving animals

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two of the principal animals and one of the satellite animals of the high dose group were prematurely sacrificed due to bad health conditions. Additionally, one principal animal of the high dose group died on Day 10 p.c. during dose formulation administration, possibly due to technical issues during the gavage procedure.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reddish colored area were observed on the stomach in two females treated at 12.5 mg/kg/day, five females treated at 25 mg/kg/day and four females treated at 50 mg/kg/day but not in controls. In one principal high dose female sacrificed on Day 19, an ulcerated focus was seen on the stomach. In control animals, no reddish colored areas of the stomach were observed. Colored foci/deposit(s) and/or area(s) in the stomach were not ulcerated.
Vagina with a liquid brownish colored content, gall bladder with a blackish colored content, dilated ureter, stomach with ulcerated foci and /or gall bladder with dilatation observed in prematurely sacrificed animals

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

There were no remarkable clinical observations in the surviving animals. Single effects observed in treated and control animals were absence of urine, absence of faeces, lought breathing or blood in the bedding.

There were no unscheduled deaths or abortions in control, 12.5 and 25 mg/kg/day groups.

In the 50 mg/kg/day group (principal and satellite animals), there were three prematurely sacrificed females (E32296, E32301 and E32318) and one found dead female (E32300):
female E32296 was sacrificed for human grounds on Day 23 p.c. (emaciated appearance and absence of feces/urine from Day 15 p.c. and blood in the bedding on Day 23 p.c.). This animal lost 15% of body weight from Days 6 to 21 p.c. and had nearly no food consumption on Days 9 to 21 p.c. At necropsy, this female (with 11 corpora lutea and 11 late resorptions) had a vagina with a liquid brownish colored content and a gall bladder with a blackish colored content,

Female E32301 was sacrificed for abortion on Day 11 p.c. (blood in the bedding from Day 10 p.c. and embryos in the bedding on Day 11 p.c.). At necropsy this female (with 11 corpora lutea and 10 uterine scars) had a dilated ureter and 10 placentas in the uterine horns,

Female E32318 (satellite group) was sacrificed for human grounds on Day 19 p.c. (absence of feces from Day 7 p.c., absence of urine on Days 7 to 8 p.c., blood in the bedding on Days 14 to 15 p.c.). This animal lost 13% of body weight from Day 6 to 18 p.c. and had nearly no food consumption on Days 6 to 18 p.c. At necropsy this female was not pregnant and had a stomach with ulcerated foci and a gall bladder with dilatation,

Female E32300 died on Day 10 p.c. during dose formulation administration. At necropsy this female (with 13 corpora lutea and 13 dead implants) had lungs with a reddish diffuse abnormal color, and a stomach with several whitish colored deposit and brownish colored areas in the mucosa.

For females E32296, E32301 and E32318, the deaths were considered to be test item treatment-related while for female E32300 a technical issue during the gavage procedure cannot be excluded.

Macroscopic changes except stomach findings at terminal necropsy of the surviving dams on Day 29 p.c. are commonly observed in this species and strain.

Reddish colored area were observed on the stomach in two females treated at 12.5 mg/kg/day, five females treated at 25 mg/kg/day and four females treated at 50 mg/kg/day but not in controls. These correlated histologically with haemorrhage.

In female E32300 treated at 50 mg/kg/day and found dead on Day 10 p.c. brownish colored foci (without histopathological correlates) and whitish deposits (correlating with increased mucus on surface) were seen on the stomach. Any relationship of these observations with the test item was excluded.

In female E32318 sacrificed on Day 19, an ulcerated focus was seen on the stomach correlating histologically with slight erosion.

In control animals, no reddish colored areas of the stomach were observed.

Colored foci/deposit(s) and/or area(s) in the stomach were not ulcerated. They were observed with dose related increased incidences in surviving females from the test item-treated groups, but not in control animals.

At microscopic examination of the stomachs, minimal to slight mucosal hemorrhages (mainly superficial) were observed in all groups including controls. Although the highest grades were seen in test item-treated animals, the absence of dose-related incidence and severity and the lack of associated changes (e.g. inflammation or pigmented macrophages) suggested these hemorrhages were likely agonal and not induced by the test item.

There were no effects on mean gravid uterus weight, mean carcass weight and net body weight change on Day 29 p.c. from Day 6 p.c..

Urinalysis in satellite animals did not point to any test item related effects.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

There were no effects on hysterectomy data in principal surviving dams on Day 29 p.c.

Hysterectomy data in principal females were the following:

Hysterectomy data in principal surviving dams on Day 29 p.c.

Dose-level (mg/kg/day) 0 (vehicle) 12.5 25 50
Number of females with live fetuses
at termination 24 23 23 20
Mean number of corpora lutea per animal 11.7 12.3 12.0 12.1
Mean number of implantations per animal 10.0 10.6 9.7 10.7
Mean pre-implantation loss (%) 14.6 14.6 18.4 11.3
Mean number of fetuses per animal 9.3 9.8 9.2 10.1
Dead fetuses (%) 0.0 0.0 0.4 0.0
Mean number of implantation scars 0.0 0.0 0.0 0.0
Mean number of early resorptions 0.5 0.4 0.4 0.3
Mean number of late resorptions 0.2 0.3 0.1 0.3
Mean post-implantation loss (%) 6.5 8.5 5.9 5.8

Effect levels (maternal animals)

1

Key result

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

2

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: mortality

Description (incidence and severity):

three of 24 animal of the high dose group were prematurely sacrificed due to poor health conditions

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): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

- fetal body weight and sex-ratio: there was a lower mean fetal body weight in the 50 mg/kg/day group which was considered to be not toxicologically significant (less than 10%),
- external examination: at 50 mg/kg/day, there was a higher percentage of litters with fetuses with malrotated paws,
- soft tissues examination: from 25 mg/kg/day there were increases in litter and fetal incidences of fetuses with colored focus on the gall bladder,
- cartilage and skeletal examination: there were higher litter and fetal incidences of fetuses with unossified 1st metacarpals (metacarpal bone cartilages were present; statistically significant only at 50 mg/kg/day;).

All these external, soft tissue and cartilage/skeletal variations were considered to be test item treatment related but not adverse. There were no tendencies towards a specific trend or syndrome in the distribution of external, soft tissues or skeletal malformations.

See attachment for results of fetal examination

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed in fetuses

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

The test item concentrations in the administered dose formulations analyzed in Weeks 1 and 5 remained within an acceptable range of variations (-6.8% to +8.6%) when compared with the nominal values (± 10% of the nominal concentrations).

 

In Weeks 1 and 5, test item was observed in the control dose formulations with concentrations below the LOQ (< 2.5 µg/mL). As control dose formulations were diluted 10-fold before injection, the quantity of test item observed was estimated below 25 µg/mL, which was considered to be negligible.

 

The interfering peak detected in the control group chromatogram was also detected in the corresponding analytical diluent. Therefore, this interfering peak could be related to analytical process.

Applicant's summary and conclusion

Conclusions:

The test item did not induce effects on embryo-fetal development of rabbits up to maternal toxic doses under the conditions of the test.

Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item, hexamethylenediamine, on the pregnant female and on embryonic and fetal developmentand on urinalysis parameters following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum(p.c.) inclusive).

Methods

Three groups of 24 principal mated female New Zealand White rabbits were administered the test item, hexamethylenediamine(batch No. 14 266 13), once daily from Day 6 to Day 28 p.c., by gavage, at dosages of 12.5, 25 or 50 mg/kg/day (groups 2 to 4). An additional group of 24 mated females received the vehicle, Phosphate Buffered Saline (PBS 1X pH 7.4), under the same experimental conditions and acted as the control group (group 1). A constant dose volume of 5 mL/kg/day was used.

 

In order to ascertain whether or not the test item treatment was associated with urinalysis parameters changes, three satellite animals per group were included in the study and urines were collected at the end of the treatment period (between Days 25 and 28p.c.). These satellite animals were administered the test item or the vehicle within the same experimental conditions.

 

The animals were checked daily for mortality and/or clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29p.c.animals were sacrificed and submitted to macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. Kidneys, ureters and urinary bladder were sampled and kept preserved in a fixative. The fetuses from principal animals were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

 

Results

 

Chemical analysis of dose formulations: the test item concentrations in the administered dose formulations analyzed in Weeks 1 and 5 remained within an acceptable range of variations (-6.8% to +8.6%) when compared with the nominal values (± 10% of the nominal concentrations). In Weeks 1 and 5, test item was observed in the control dose formulations with concentrations below the LOQ (< 2.5 µg/mL). As control dose formulations were diluted 10-fold before injection, the quantity of test item observed was estimated below 25 µg/mL which was considered to be negligible.

 

Pregnancy status: in the control, 12.5, 25 and 50 mg/kg/day groups, there were 24, 23, 23 and 20 principal females and 3, 2, 3 and 2 satellite females with live fetuses at hysterectomy, respectively.

 

Mortality/abortion: there were no unscheduled deaths or abortions in control, 12.5 and 25 mg/kg/day groups.

In the 50 mg/kg/day group (principal and satellite animals), three prematurely sacrifices for poor health condition (e.g. emaciated appearance, absence of feces/urine, nearly no food consumption and/or abortion) were considered to be test item treatment-related. At necropsy of prematurely sacrificied animals a range of findings was recorded (e.g. vagina with a liquid brownish colored content, gall bladder with a blackish colored content, dilated ureter, stomach with ulcerated foci and /or gall bladder with dilatation) for which a test item-relationship was not excluded.

 

Clinical signs: there were no remarkable clinical observations in the surviving animals.

 

Body weights and body weight changes: there were no test item treatment-related effects on mean body weights and mean body weight changes.

Food consumption: there were no toxicologically significant effects on mean food consumption.

 

Urinalysis: there were no statistically significant differences in mean urinalysis parameters recorded in satellite surviving dams or on urinary pH recorded at necropsy.

 

Maternal terminal examination:

.            at necropsy, there were non-ulcerated colored foci/deposit(s) and/or area(s) in the stomach of treated animals. These findings were observed with increased incidences in surviving females from the test item-treated groups (without dose response), but not in controls. Histological findings indicate that they are not test item related and therefore not adverse, which is supported by the absence of clinical signs and significant effects on body weight or food consumption,

.            there were no effects on mean gravid uterus weight, mean carcass weight and net body weight change on Day 29p.c.from Day 6p.c.

 

Hysterectomy data: there were no effect on hysterectomy data in principal surviving dams on Day 29p.c.

 

Fetal examination:

.            fetal body weight and sex-ratio: there was a lower mean fetal body weight in the 50 mg/kg/day group which was considered to be not toxicologically significant (less than 10%),

.            external examination: at 50 mg/kg/day, there was a higher percentage of litters with fetuses with malrotated paws,

.            soft tissues examination: from 25 mg/kg/day there were increases in litter and fetal incidences of fetuses with colored focus on the gall bladder,

.            cartilage and skeletal examination: there were higher litter and fetal incidences of fetuses with unossified 1^stmetacarpals (metacarpal bone cartilages were present; statistically significant only at 50 mg/kg/day;).

 

All these external, soft tissue and cartilage/skeletal variations were considered to be test item treatment‑related but not adverse. There were no tendencies towards a specific trend or syndrome in the distribution of external, soft tissues or skeletal malformations.

 

Pathology:

No test item-related changes were observed in the stomach at any of the dose-levels.

Minimal to slight hemorrhages correlating with reddish colored area were observed at necropsy in several animals treated at 12.5, 25 or 50 mg/kg/day. In the absence of dose-related incidence or severity and of associated changes (e.g inflammation or pigmented macrophages) any relationship to the test item was considered to be unlikely.

 

Conclusion

The test item, hexamethylenediamine (batch No. 14 266 13), was administered by gavage, once daily, from Days 6 to 28p.c., inclusive, to mated female New Zealand White rabbits at dosages of 12.5, 25 or 50 mg/kg/day.

 

On the basis of the results obtained in this study:

.            the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 25 mg/kg/day (based on mortality at 50 mg/kg/day),

.            the NOAEL for embryo-fetal development was considered to be 50 mg/kg/day based on absence of adverse effects at this dose-level.

Hexamethylenediamine did not elicit a teratogenic potential.