Hexamethylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 4th April to 13 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

study performed in GLP certified laboratory, but GLP status not claimed

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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

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

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

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)

Dose / conc.:

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

Dose / conc.:

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

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

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

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.

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

Key result

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

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

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

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

7 day oral (gavage) study with rabbits

GLP compliance:

no

Remarks:

study performed in GLP certified laboratory, but GLP status not claimed

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: crystalline

Details on test material:

Rhodiamine HMD 100% (solid), HMD

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

same species as developmental toxicity study to which this mechanistic study is referring to

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre Lago (Vonnas, France)
- Females (if applicable) nulliparous and non-pregnant: non-pregnant
- Age and weight at study initiation: at the beginning of the treatment period, the females were approximately 19 weeks old and had a mean body weight of 3710 g (range: 3460 g to 3885 g)
- Housing: individually housed in Noryl cages (Tecniplast, 4200 cm²). Individual housing was chosen since rabbits of this age are generally not compatible individuals. The cages were suspended in batteries over trays with absorbent paper and were placed in numerical order on the racks. Each cage contained a platform and a dumbbell, hay (batch No. 8537207) was distributed regularly for the environmental enrichment of the animals. Music was also put on for the same purpose.
- Diet (e.g. ad libitum): ad libitum: pelleted "type 110 C" diet (SAFE, Augy, France),
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 6 days before treatment.

DETAILS OF FOOD AND WATER QUALITY:
pelleted "type 110 C" diet (SAFE, Augy, France), batch No. 17326 and tap water (filtered with a 0.22 µm filter) contained in bottles. The food and water were distributed regularly. Diet controlled for composition and contaminants. Water controlled for bacterial contamination and contaminants.

ENVIRONMENTAL CONDITIONS
temperature : 18 ± 3°C,
relative humidity : 50 ± 20%,
light/dark cycle : 16h/8h,
ventilation : 5 to 15 cycles/hour of filtered, non-recycled air.


IN-LIFE DATES: From: 10 April 2018 To: 17 April 2018

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

same as in developmental toxicity study

Vehicle:

other: water or phosphate buffered Saline (PBS)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared as follows:
On the day of preparation, place the test item aliquot at room temperature for 30 minutes.Then, place it (with cap slightly opened) in a water bath and turn on the water bath to 50°C until complete melting of the test item. No more than two freezing-thawing cycle was done per aliquot. Weigh the amount of test item needed. Mix the test item with around 60-80% of the required quantity of vehicle and adjust the pH to 7.2 ± 0.5 using HCl (batch No.: 17070008 and expiry date: 31 July 2020), pure and diluted to 1N concentration, under magnetic stirring. Complete the formulation with vehicle to the target concentration. Place the formulation under argon atmosphere

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time each day.
The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
Care was taken to correctly wipe the gavage tube before administering the animals in order to remove all dose formulation at the exterior surface of the tube.
The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.


VEHICLE
- Justification for use and choice of vehicle (if other than water): PBS was used in developmental toxixity study
- Concentration in vehicle: 0, 15, 20, 30 mg/mL for both vehicles
- Amount of vehicle (if gavage): A constant dosage-volume of 5 mL/kg/day was used.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

daily for 7 days.

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1; vehicle group 1 (PBS)

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

group 2; vehicle PBS

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

group 3; vehicle PBS

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

group 4; vehicle PBS

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 5; vehicle group 2 (water)

Doses / concentrations 6

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

group 6; vehicle water

Doses / concentrations 7

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

group 7; vehicle water

Doses / concentrations 8

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

group 8; vehicle water

No. of animals per sex per dose:

2 (vehicle controls) or 3 (dose groups) animals

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected based on results obtained from a Maximum Tolerated Dose study by oral route (application of neutralized HMD in sodium phosphate buffer by gavage) in non-pregnant rabbits (Citoxlab France/Study No. 41983 TSL) conducted at 50, 125 or 200 mg/kg/d and a prenatal developmental toxicity study by oral route (application of neutralized HMD in PBS buffer by gavage) in rabbits conducted at 12.5, 25 or 50 mg/kg/d.
Severe toxicity was observed in the MTD study at 125 mg/kg/d (NOAEL 50 mg/kg/d!) and in the prenatal devtox study at 50 mg/kg/d (NOAEL 25 mg/kg/d!). This study was designed to investigate a potential impact of using PBS as buffer and to get information on target organs.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Morbidity and mortality
Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends.
Attention was paid to humane endpoints defined in Citoxlab France quality document LI SES 0004. Endpoints specific to the species used in the study were available in the animal facility.
A macroscopic post-mortem examination was performed for animals prematurely euthanized and the required tissues were preserved for a microscopic examination (see § Pathology).
A complete blood biochemistry investigation was performed on moribund animals (see § Blood and urine collection).

Clinical signs
From arrival, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

Body weight
The body weight of each animal was recorded once before the beginning of the treatment period, on the first day and on the last day of treatment.

Food consumption
The quantity of food consumed by each female was recorded for the following intervals (see § study plan adherence):
Days 1-3, 3-5, 5-7.
Food consumption was calculated per animal and per day.

Blood and urine collection
Laboratory investigations were performed on all surviving animals before euthanasia (Day 8) and urine collection was also performed overnight between Days 3-4 and at necropsy for animals prematurely sacrificed on Day 3.

Prior to blood sampling and during urine collection (Day 8 only), the animals were deprived of food for an overnight period of at least 14 hours.
Blood samples were taken from the auricular artery, and collected into tubes containing the appropriate anticoagulant (see following tables).
Thymol was used as a preservative for urine collection.

Complete blood biochemistry investigation was performed on Day 3 on animals prematurely sacrificed after three dosings (in that single case, blood was collected from the most appropriate vein or artery just before or during necropsy).
At urinalysis, pH and semi-quantitative parameters were also performed on Day 3 on animals prematurely sacrificed after three dosings.

Hematology
No hematology investigations were performed.

Blood biochemistry
A large set parameters were determined for all surviving animals and prematurely euthanized animals.
Blood was collected into lithium heparin tubes and analyzed using the ADVIA 1800 blood biochemistry analyzer.

Urinalysis
Urine was collected on the last dosing day (thymol was used as a preservative). Urine collection was also performed overnight between Day 3 to Day 4.
pH and semi-quantitative parameters were also performed on prematurely euthanized animals on Day 3. 
The large set of parameters was determined for all surviving animals on Day 4 and before euthanasia (Day 8).

Sacrifice and pathology:

Euthanasia
On completion of the treatment period and after at least 14 hours fasting, all surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. The same procedure was followed for prematurely euthanized animals.

Organ weights
The body weight of each animal was recorded before euthanasia. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of tissues
For all animals, the tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except, when applicable for the eyes with optic nerves, which were fixed in Modified Davidson's Fixative).

Preparation of histological slides
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin and eosin.

Microscopic examination
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table for all animals euthanized at the end of the treatment period and for animals found dead or euthanized prematurely.

TISSUE PROCEDURE TABLE

Organs Organ Microscopic examination
weights Preservation of tissues

Macroscopic lesions X X
Adrenals X X
Aorta X
Brain (including medulla/pons
cerebellar and cerebral cortex) X X

Cecum X X
Colon X X
Duodenum X X
Esophagus X X
Eyes X
Femoral bone with articulation X
Gall bladder X
Gut-Associated Lymphoid Tissue (GALT) X
Heart X X
Ileum X X
Jejunum X X
Kidneys X X X
Larynx X
Liver X X
Lungs with bronchi X
Lymph nodes (mesenteric and mandibular) X
Mammary gland area X
Optic nerves X
Ovaries X X
Oviducts X
Pancreas X
Pituitary gland X
Rectum X X
Salivary glands (parotid and mandibular, sublingual) X
Sciatic nerve X
Skeletal muscle X
Skin X
Spinal cord (cervical, thoracic and lumbar) X
Spleen X X
Sternum with bone marrow X
Stomach X X
Thymus X X
Thyroids with parathyroids X
Tongue X
Trachea X
Ureters X X
Urinary bladder X X
Uterus (horns and cervix) X X X
Vagina X

Statistics:

Citox software was used to perform the statistical analyses of body weight, food consumption, blood biochemistry and urinalysis data

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs in animals treated at 75 or 100 mg/kg bw/day. In the high dose groups only one animal (PBS vehicle group) showed clear clinical signs on day 3: thin appearance, Pallor / extremities, cold to the touch, hypoactivity, loss of balance, abdominal breathing, loud breathing.
All animals of the 150 mg/kg groups were sacrificed at the end of day 3 for humane reasons.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths in control groups and in groups treated at 75 or 100 mg/kg bw/day.

In the 150 mg/kg bw/day treated groups on Day 3:
- female M30861 (group 8, vehicle: water) was found dead with large amount of blood in the bedding.
- female M30851 (group 4, vehicle: PBS) was prematurely sacrificed for humane grounds on Day 3 (after two administrations).
- female M30860 (group 8, vehicle: water) had blood in the bedding and was sacrificed for humane grounds.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Vehicle: PBS
In the group treated at 100 mg/kg bw/day, a body weight loss of -5.3% (vs. -1.1% in controls) was recorded over the Days 1-7 interval and was attributed to the test item treatment.
At 75 mg/kg bw/day, the effect was limited to -2.6% (vs. -1.1% in controls) and was mainly due to the contribution of one animal (M30843) which has lost 215 g (vs. -40 g in control group).

Vehicle: water
In group 7 treated at 100 mg/kg bw/day, a minimal body weight loss of -3.0% (vs. +0.1% in control group) was recorded and was attributed to the test item treatment.
In group 6 treated at 75 mg/kg bw/day, the body weight loss remained limited to -1.6% (vs. +0.1% in controls) and was not considered clinically significant.

No statistically significant difference was seen between the two vehicles, whatever the dose level tested.

Overall, body weight loss remained of too limited in magnitude to be considered adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was not determined in groups 4 and 8, due to premature sacrifice.
In groups 3 and 7 treated at 100 mg/kg bw/day, low food consumptions (-31 and -30% vs. control, respectively) were observed during the treatment period and were attributed to the test item treatment.

In group 2 treated at 75 mg/kg bw/day, the low food consumption (-28% vs. control) during the treatment period was mainly due to animal M30843 (-67% vs. control) and was correlated with the higher body weight loss recorded in this animal (-215 g vs. -40 g in control group). An effect of the test item treatment could not be excluded for this animal.

However, for the remaining animals treated at 75 mg/kg bw/day (groups 2 and 6), the effects on food consumption were of too limited magnitude to be considered toxicologically relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

For prematurely sacrificed animals and for animals sacrificed at the end of the treatment period, no relevant changes were reported in test item-treated groups at 75 or 100 mg/kg bw/day whatever the vehicle and the dose level tested.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

For prematurely sacrificed animals dosed thrice at 150 mg/kg bw/day, a moderate to high presence of blood (for PBS and water vehicle groups, respectively) was detected for 1 out of 2 animals in each group (M30849 and M30860). Moreover, urine pH was measured at Day 6 for 1 out of 2 animals in each group (M30849 and M30862)

For animals sacrificed at the end of the treatment period, urine collected on Day 4 did not reveal relevant changes regardless of the vehicle and the dose level tested.

The relevant findings on urine collected over the Days 7-8 interval are described hereafter:
Vehicle 1: PBS
In the group treated at 100 mg/kg bw/day, few erythrocytes were detected in urine of 2/3 animals (group 3: M30847, M30848)

Vehicle 2: water
At 100 mg/kg bw/day, few erythrocytes and/or low to moderate presence of blood were detected for 2/3 animals (group 7: M30859, M30858)
In group treated at 75 mg/kg bw/day on Day 7, low to high presence of blood and/or erythrocyte were for 1/3 animal (M30856). Moreover, few erythrocytes along with few renal cells were also detected in urine of animal M30854.

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 test item-related organ weight differences in animals treated with test item in PBS or in water.
The few organ weight differences were seen in female reproductive tract (ovaries, uterus) that reflected the variable estrus cycle and were unrelated to the test item administration.
All other organ weight changes were minimal, not dose-related and were thus also unrelated to the test item administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Unscheduled deaths: (see Mortality)
Terminal sacrifice:
There were no test item-related changes at macroscopic examination.
The few changes were considered to be part of the background commonly seen in untreated rabbits of these strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths:
At histopathological examination of animals found dead or moribund, the main findings were found in the gastro-intestinal and/or urinary tracts and were of variable severity, ranging from minimal to marked, mainly erosion/ulcer and/or degeneration/necrosis of ileum, cecum, renal tubular cells, urothelium of urinary bladder, kidneys, pelvis, ureters and/or urethra. These changes were considered to be related to the death/moribundity and were associated with infiltrates of mixed cells, sloughing of cells, hemorrhage/congestion (correlated with red discoloration), edema (correlated with gelatinous aspect), blood/clot in lumen (correlated with gross clot/red content), dilation and/or atrophy.

Terminal sacrifice:
The following test item-related changes were noted:
- minimal hyperkeratosis in the esophagus from M30846 treated at 100 mg/kg bw/day test item in PBS,
- minimal congestion/hemorrhage in the jejunum from M30855 treated at 75 mg/kg bw/day in water, from M30846 treated at 100 mg/kg bw/day test item in PBS, and from 2/3 females treated at 100 mg/kg bw/day in water,
- minimal congestion/hemorrhage in the ileum from M30855 treated at 75 mg/kg bw/day in water,
- minimal edema in the urinary bladder submucosa from M30846,
- minimal granulocyte infiltrate in the stomach from M30847 treated at 100 mg/kg bw/day test item in PBS.

On an individual basis, high toxicity in the highest dose group seemed to be more likely linked to changes observed in the urinary tract than to GIT changes: all animals found dead or moribund had severe histopathological changes in the urinary tract (kidney, bladder, or ureter), whereas some of these animals had no or only minimal GIT lesions.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

One female (M30861) treated at 150 mg/kg bw/day with water as vehicle was found dead on Day 3 (after two applications). One female (M30851) treated at 150 mg/kg bw/day with PBS as vehicle was found moribund after two administrations and thus sacrificed. One female (M30860) treated at 150 mg/kg bw/day with water as vehicle was sacrificed after three dose adminstrations due its moribund status. All other animals from the high-dose groups (M30849, M30850 and M30862) were sacrificed on Day 3 without clinical signs.

In the three affected females (M30851, M30860 and M30861) test item-related lesions in the gastro-intestinal and/or urinary tract of variable severity were found, i.e. mainly adverse erosion/ulcer and/or degeneration/necrosis of jejunum, ileum, cecum, stomach, renal tubular cells, urothelium of urinary bladder, kidneys, pelvis, ureters and/or urethra, in association with infiltrates of mixed cells, sloughing of cells, hemorrhage/congestion, edema, blood/clot in lumen, dilation and/or atrophy that were considered as secondary findings.
Findings in animals M30849, M30850 and M30862 sacrificed at day 3 without moribund status were restricted mainly to the intestines (stomach, duodenum, jejunum, ileum and/or cecum) and consisted of minimal to slight erosion/ulcer, atrophy, degeneration/necrosis, edema, congestion/hemorrhage, sloughed cells, infiltrate of mixed cells/heterophils and/or atrophy of villi, with no gross correlates except for M30862.

In addition, esophageal hyperkeratosis was noted in 2/6 prematurely sacrificed females, indicating possible irritation of the test item.
Macroscopically, brown content was noted in the lumen of cecum of 2/3 females treated with test item in water, together with many findings in gastro-intestinal and urinary tracts, i.e. mainly red discoloration, gelatinous aspect and gross clot/red content.
The spleen of 1/6 females was small and had moderate atrophy of red and white pulp. This finding was likely related to stress.
The edema, fibrosis and/or pigment seen in the abdominal adipose tissue were considered to be contiguous changes to urinary tract lesions.
The presence of blood in urine was partially related to the microscopic lesions, i.e. there were no associated lesions in M30856, M30858 or M30859 while there was slight congestion in the renal medulla of female M30849 and minimal or slight erosion/hemorrhage in the urinary bladder of female M30860. The observation of blood in urine during urinanalysis might indicate that – in absence of macroscopically visible lesions - existing localized lesions in the urinary tract were not included in the samples taken for histopathology.

In surviving animals, hyperkeratosis in the esophagus, congestion/hemorrhage in the jejunum/ileum, edema in the urinary bladder and granulocyte infiltrate in the stomach were noted in isolated females treated with test item at 75 or 100 mg/kg bw/day in water or with test item at 100 mg/kg bw/day in PBS, suggesting a better tolerability of the test item in PBS than in water.

The test item-related findings seen in surviving animals at 75 or 100 mg/kg bw/day were of minimal magnitude.

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

kidney

ureter

urethra

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

ileum

intestine

jejunum

oesophagus

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Study not designed to identify NOAEL/LOAEL

Applicant's summary and conclusion

Conclusions:

Severe toxicity was obseved with both vehicles at 150 mg/kg bw/day.
There were no clinical signs in animals treated at 75 or 100 mg/kg bw/day. Microscopic examination revealed only minimal changes in the GIT and urinary tract in these dose groups.
Hematuria was reported at 75 or 100 mg/kg bw/day in the water vehicle groups, and at 100 mg/kg bw/day in the PBS vehicle group.
Adverse macroscopic and microscopic findings were mainly reported in the urinary and gastrointestinal tracts and were comparable with both vehicles. Use of PBS buffer as vehicle cannot explain the high toxicity observed in the prenatal developmental toxicity study.
Severe clinical conditions were observed in animals with histopathological findings in the urinary tract, but were not clearly associated with gastrointestinal lesions.

Executive summary:

Neutralized hexamethylenediamine was administered daily (gavage) to non-pregnant female New Zealand white rabbits at dose levels of 75, 100 and 150 mg/kg bw/day for 3 or 7 days. Phosphate Buffered Saline (PBS) or water were used as vehicles.

At 150 mg/kg bw/day in both vehicles, one out of three animals in each group was prematurely sacrificed or found dead on Day 3 (before the third administration). One animal of the test group with water as vehicle (M30860) showed blood in the bedding at day 3 and was sacrificed after the third administration. The remaining animals were without clinical signs, but were sacrificed for ethical reasons on Day 3 (after three administrations, based on observations on animals found dead or moribund). Adverse macroscopic and microscopic findings were mainly reported in the urinary and gastrointestinal tracts and were comparable with both vehicles.

There were no clinical signs in animals treated at 75 or 100 mg/kg bw/day. Microscopic examination revealed only minimal changes in the GIT and urinary tract in these dose groups.

Hematuria was reported in several animals in groups treated at 75 or 100 mg/kg bw/day in the water vehicle groups, while in the PBS vehicle groups, only animals treated at 100 mg/kg were affected.

The absence of findings in animals treated at 75 mg/kg bw/day in PBS could suggest a better tolerability of the test item in this vehicle than in water.