Cyclohex-1,2-ylenediamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 2021 to April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25.06.2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohex-1,2-ylenediamine

EC Number:

211-776-7

EC Name:

Cyclohex-1,2-ylenediamine

Cas Number:

694-83-7

Molecular formula:

C6H14N2

IUPAC Name:

cyclohexane-1,2-diamine

Test material form:

liquid

Remarks:

varying colors

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST rat.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 11 to 12 weeks old
- Weight at study initiation: 167 - 224 g
- Housing:
Cages Cages comprised of a polycarbonate body with a stainless
steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the
acclimatization and gestation periods.
Cage distribution The cages constituting each group were blocked by group
and mounted in batteries.
Bedding Solid bottom cages contained softwood based bark-free fiber
bedding (sterilized by autoclaving), which was changed at
appropriate intervals each week.
Number of animals per cage Individually housed.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
The concentration of Total Genistein Equivalents in the diet were below the OECD’s suggested upper limits of 325-350 ppm TGE for the rat uterine bioassay. SDS VRF1 can be considered a low Phytoestrogen diet.
- Water: tap water, ad libitum
- Acclimation period: Minimum of 3 days from arrival on Day 2 after mating to
commencement of treatment on Day 6 after mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): filtered not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 August 2021 To: 07 September 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed.
Approximately 60 to 80% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until it appeared homogeneous. The final pH adjusted formulation was stirred for a minimum of 20 minutes using a magnetic stirrer.
The pH was measured and adjusted to 7.5 ± 0.1 using hydrochloric acid (H2O:HCl) or sodium hydroxide (NaOH) as required.
- Frequency of preparation: Weekly, and were prepared in advance of the first day of dosing. Batches were stored in daily aliquots to cover one week of dosing.
- Appearance of the formulation: Clear-amber solution.
The test item was a clear liquid, however, the Sponsor indicated that a colour change to yellow/amber could occur. The CoA detailed the appearance as a ‘liquid of varying colours’.
- Storage of formulation: Refrigerated (2 to 8°C).

Storage and stability of formulations
- 16 Days following refrigerated storage (2 to 8°C).
- 4 Days following ambient storage (15 to 25°C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations were within 9% of the nominal concentration, within the ±10%
acceptance limits, confirming the accuracy of formulation. The difference from mean
remained within 3%, within the 5% acceptance limits, confirming precise analysis.
Procedural recoveries remained within the validated range (94.9% to 104.9%), except from
the recovery at 5 mg/ml which had a value of 86% which was excluded from the mean as
allowed by the SOP, and the mean recovery value was used to correct for the concentrations
of the samples. For further information please refer to table 1 in section 'Any other information on materials and methods incl. tables'.

Details on mating procedure:

Natural mating with Han Wistar of established fertility at the supplier’s facility.
Males and females not related (Mating records are available.)
Day 0 of gestation: When positive evidence of mating was detected.
Delivery to Labcorp: On Day 2 after mating.

Duration of treatment / exposure:

day 6 to 20 after mating (GD6 to 20)

Frequency of treatment:

once daily

Duration of test:

GD 6-21 (necropsy)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for investigation have been selected in conjunction with the Sponsor and
based on the results of a preliminary embryo-fetal development study Labcorp Study No.
8434915.
In that study dose levels of 50, 150 and 500 mg/kg/day were investigated. There were no
treatment-related clinical signs and no post-dose observations. At 500 mg/kg/day overall
(GD 6 to GD 20) mean maternal adjusted body weight stasis was observed compared to a
mean weight gain of 23g in the Controls and this correlated with reduced food intake at this
dose level. This effect on maternal food intake was also associated with reduced male,
female and overall fetal weights at this dose level. At 150 or 50 mg/kg/day, when taking into
account gravid uterine weight, adjusted maternal body weight change was slightly lower than
Control, however these changes were not associated with any effects on embryo-fetal
survival or development. The extent of the effects on maternal body weight performance,
maternal food intake or fetal weights at 500 mg/kg/day were not considered to preclude the
use of this dose level on the main study. There were no findings at macroscopic examination
of the adult females and no treatment related findings at external examination of the fetuses at
any dose level investigated.
Based on these results, the high dose on the current main OECD 414 study is set at
500 mg/kg/day, with intermediate and low dose levels set at 150 and 50 mg/kg/day,
respectively, in order to fulfil the 2-fold to 4-fold dosing interval as specified in the test
guideline.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3x daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 2x daily
Physical examination Day after arrival, GD 5, 12, 18 and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Day after arrival, GD 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined on Days 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 after mating

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: All adult animals were subject to a complete macroscopic examination. Any abnormal
tissue was sampled as appropriate and retained in appropriate fixative

Ovaries and uterine content:

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

Blood sampling:

- Serum: Yes
- Volume collected 1ml blood per animal

Fetal examinations:

- External examinations: Yes: [all per litter]
Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses examined externally, with particular attention to external genital organs of male fetuses and with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.
- Soft tissue examinations: Yes: [half per litter ]
Examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouins solution. Bouin’s fixed fetuses had heads removed post-fixation and processed by Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution. For Bouin's fixed material serial sections were examined for visceral abnormalities.
- Skeletal examinations: Yes: [ half per litter ]
Sex confirmed internally. Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
IMS fixed fetuses were processed and stained with Alizarin Red. These fetuses were assessed for skeletal development and abnormalities.
- Head examinations: Yes: [half per litter] (see above)
- Anogenital distance of all live rodent pups: yes (see above)

Findings observed were classified, according to severity and incidence, as:
- Major abnormalities:
normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
- Minor abnormalities:
minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
- Variants:
alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs incomplete ossification of 5th and 6th sternebrae.

Statistics:

For adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Thyroid hormones
Organ weights, absolute

The following comparisons were performed: Group 1 vs 2, 3 and 4
For more details please see also the attachement '8439547-statistical methods' in section 'overall remarks, attachments'.


For the litter average ano genital distance, analysis of covariance (Angervall and Carlstrom, 1963) was performed using the average fetal weight for each litter as the covariate.

Indices:

litter size and survival indices

Pre-implantation loss: ((Number corpora lutea – Number implantations) x 100)/Number corpora lutea

Post implantation loss: ((Number implantations – Number live fetuses) x 100)/Number implantations

All group values and SD were calculated from the individual litter values.

Historical control data:

Historical control data was provided by LabCorp to allow comparison with concurrent controls for major and minor visceral and skeletal fetal abnormalities (see also attachment '8439547HCD' in sectioin 'overall remarks, attachments'.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

one female that received 500 mg/kg/day was observed with decreased
activity, piloerection and partially closed eyelids prior to necropsy on Day 21 of gestation,
however, no similar clinical signs were observed in the remainder of the animals in this group
and these signs were not considered to be treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

From Day 12 of gestation (i.e. 6 days of treatment) mean absolute body weight was statistically significantly lower than Control for all groups of treated females, however, a dose response was not apparent and the difference was always only minor (about 3 to 7% from the Control group values).

Females treated with DYTEK DCH-99 at 500 or 150 mg/kg/day showed statistically
significantly low overall (GD 6 to GD 21) mean body weight gain when compared with
Controls (23% and 14% low at 500 or 150 mg/kg/day, respectively).

Overall body weight gain for females that received 50 mg/kg/day was slightly lower than Control during Days 6 to 21 of gestation but statistical significance was not attained.

There was no effect of treatment on gravid uterine weight at any dose level investigated.

Mean adjusted maternal body weight on Day 21 of gestation was statistically significantly
lower than Control in all treated groups, however, a dose response was not apparent and the
difference was always only minor (about 3 to 8% from the Control group values).
When adjusted for the contribution of the gravid uterine weight, a slight but statistically
significant mean maternal body weight loss was observed at 500 mg/kg/day (-6%) and reduced
mean maternal body weight gain was observed at 150 or 50 mg/kg/day, compared to Control,
albeit not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day food intake was persistently reduced following the commencement of
treatment, compared to Control, with statistical significance attained for most intervals.
Overall food consumption (Days 6 to 21 of gestation) at 500 mg/kg/day was slightly but
statistically significantly low compared with Controls (p>0.01).
There was no effect of treatment on the food consumption of females treated at 150 or
50 mg/kg/day.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mean serum Triiodothyronine (T3), Thyroxine (T4) or Thyroid Stimulating Hormone (TSH) concentrations at any dose level investigated.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid and parathyroid weights were unaffected by treatment with DYTEK DCH-99.
As there were no target organs identified at the macroscopic observation, no further organ
weights were measured in the current study.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings observed at necropsy for females at scheduled
termination on Day 21 after mating.
Macroscopic examination of the fetuses did not reveal any findings that could be related to
maternal treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic findings in the thyroids.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic findings in the thyroids.

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

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One Control female, one female that received 150 mg/kg/day and four females that received 500 mg/kg/day were found to be not pregnant at macroscopic examination, therefore there were 21, 22, 21 and 18 litters available for examination at 0, 50, 150 or 500 mg/kg/day, respectively.
At 500 mg/kg/day, in the absence of any increase in pre- and post-implantation losses in the 18 other pregnant females in this group, this was considered likely due to chance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Mean placental weight was statistically low in females that received 500 mg/kg/day when compared with Controls (-8%).

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male, female and overall fetal weights were slightly but statistically significantly low in
females that received 500 mg/kg/day when compared to Controls (-8%). There was no effect of
treatment on fetal weights at 150 or 50 mg/kg/day.

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

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

When compared with Control, there was an increased incidence of the minor skeletal
abnormalities of medially thickened/kinked ribs (5 fetuses in 3 litters), misaligned
ossification sites of sternebrae (5 fetuses in 4 litters) and short 13th ribs (5 fetuses in 3 litters)
at 500 mg/kg/day that exceeded the Historical Control Data (HCD) range.
When compared with Control, there was an increase in incidence in the minor skeletal
abnormality of short supernumerary cervical ribs across the treated groups that exceeded the
HCD range, however a dose response was not apparent. In addition, one fetus at
500 mg/kg/day had a full length cervical rib.
When compared with Control, there was an increase in incidence of the minor skeletal
variants of delayed/ incomplete ossification/ unossified 5th and/or 6th sternebrae across all
the treated groups that also exceeded the HCD range, however, no clear dose response was
apparent.

Other findings observed (e.g. misaligned hemicentres of sternebrae, misaligned costal cartilage) were not considered treatment-related as they were minor in incidence with no dose response apparent.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 500 mg/kg/day there was one fetus with the major abnormality; microphthalmia (4F 72),
and also in one other litter with an incidence of four fetuses the minor visceral abnormality of
haemorrhage in the vitreous humour of the eye (4F 76) was observed.
At 50 mg/kg/day, there was one fetus (2F 26) with several major abnormalities; misshapen upper lip and basisphenoid; absent nasolabial sulcus, upper incisor socket, and palatine processes; and fused bilateral premaxilla. As these abnormalities only occurred in one litter respectively, these findings were considered incidental and not related to treatment.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: skeletal: cervical rib and 13th ribs

Description (incidence and severity):

At 500 mg/kg/day, the combination of increased incidences of short cervical ribs, one full cervical rib and short 13th ribs without costal cartilages (short ribs in a second area of the skeleton) was considered to represent an adverse effect on fetal development.

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 150 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of DYTEK DCH-99 on embryo-fetal
survival and development when administered during the organogenesis and fetal growth
phases of pregnancy in the Han Wistar rat.
Three groups of 22 females received DYTEK DCH-99 at doses of 50, 150 or 500 mg/kg/day
by oral gavage administration, from Day 6 to 20 after mating at a dose volume of
10 mL/kg/day. A similarly constituted Control group received the vehicle, purified water at
the same volume dose as treated groups. Animals were killed on Day 21 after mating for
reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were
examined macroscopically at necropsy on Day 21 after mating and the gravid uterus weight
was recorded along with pregnancy status (for each ovary/uterine horn; corpora lutea,
implantation sites, resorption sites, fetuses), blood samples were taken for thyroid hormone
analysis and thyroid weight was recorded. Ano-genital distance was measured for fetuses and
all fetuses were examined macroscopically at necropsy and subsequently by detailed internal
visceral examination or skeletal examination.

RESULTS:

There were no unscheduled deaths. No treatment-related signs were observed at routine examination or following dose administration.  There was no effect of treatment on gravid uterine weight.  There were no treatment-related macroscopic findings in any adult female and no effect of treatment on thyroid weight or microscopic appearance of the thyroid.  There were no changes in serum concentration of T3, T4 or TSH.  Implantation count, number of resorptions and live young, the ratio of male to female offspring and total litter weights were unaffected by treatment and fetal ano-genital distance was similar to Control.

From Day 12 of gestation (i.e. 6 days of treatment) mean absolute body weight was statistically significantly lower than Control for all groups of treated females, however, a dose response was not apparent and the difference was always only minor (about 3 to 7% from the Control group values).  Females treated with DYTEK DCH-99 at 500 or 150 mg/kg/day showed statistically significantly low overall (GD 6 to GD 21) mean body weight gain when compared with Controls (23% and 14% low at 500 or 150 mg/kg/day, respectively), with an adjusted mean maternal body weight loss of 1g observed at 500 mg/kg/day compared to a gain of 17g in the Controls and this correlated with the reduced food intake observed at 500 mg/kg/day.  Overall mean body weight gain was slightly low at 50 mg/kg/day and mean maternal adjusted body weight gain was also slightly low at 150 or 50 mg/kg/day.  At 500 mg/kg/day, the 23% reduction in absolute mean body weight gain, the adjusted mean weight loss of 1g, together with the persistent reduction in mean food intake with no consistent improvement as pregnancy progressed, were judged in combination to represent an adverse effect of treatment (maternal animals).  At 150 mg/kg/day the extent of the reduction in mean adjusted maternal body weight gain was not associated with any effect on food intake, maternal clinical condition or reproductive performance and was therefore not judged to represent an adverse effect of treatment.  Therefore, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity will be set at 150 mg/kg/day.  The effect on maternal food intake at 500 mg/kg/day was also likely associated with the slightly reduced placental weights and slightly reduced male, female and overall fetal weights at this dose level.

At 150 mg/kg/day the extent of the reduction in mean adjusted maternal body weight gain was not associated with any effect on food intake, maternal clinical condition or reproductive performance and was therefore not judged to represent an
adverse effect of treatment.

One Control female, one female that received 150 mg/kg/day and four females that received 500 mg/kg/day were found to be not pregnant at macroscopic examination. In the absence of any increase in pre- and post-implantation losses in the 18 other pregnant females in this 500 mg/kg/day group, this was considered likely due to chance.

There were no major fetal abnormalities (visceral or skeletal) or minor visceral fetal abnormalities related to treatment. 

At 500 mg/kg/day there was an increased incidence of the minor skeletal abnormalities of medially thickened/kinked ribs (5 fetuses in 3 litters), misaligned ossification sites of sternebrae (5 fetuses in 4 litters) and short 13th ribs (5 fetuses in 3 litters).  There was also an increased incidence of the minor skeletal abnormality of short supernumerary cervical ribs across the treated groups, however, since a dose response was not apparent, the presence of this individual finding was not considered adverse at 150 or 50 mg/kg/day.  At 500 mg/kg/day, one fetus had a full cervical rib.  The combination of increased incidences of short cervical ribs, one full cervical rib and short 13^th ribs without costal cartilages (short ribs in a second area of the skeleton) was considered to represent an adverse effect on fetal development at 500 mg/kg/day (in the presence of maternal toxic effects).  Cervical ribs can cause severe health effects including thoracic outlet disease (Tyl, Chernoff and Rogers 2007), and a full cervical rib is judged more likely to cause this disease compared with a short rib.  In addition, there was an increased incidence of the minor skeletal variants of delayed/ incomplete ossification/ unossified 5th and/or 6th sternebrae across the treated groups that exceeded the HCD range.  Although a relationship to treatment cannot be completely ruled out, there was no clear dose response apparent (contrary to the increased incidence of the other minor skeletal abnormalities in the high dose; medially thickened/kinked ribs, misaligned ossification sites of sternebrae and short 13th ribs and the dose response observed in the number of litters short cervical ribs).  In addition, these minor variants were only observed in one part of the skeleton and occurred in litters with and without other minor skeletal abnormalities.  Furthermore, given that incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity, this finding was not considered to represent an adverse effect of treatment and was unlikely to be related to treatment.

Conclusion:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 150 mg/kg/day (with developmental effects only being present at maternal toxic doses).

 

References used above:

Tyl, RW. Chernoff, N. Rogers, JM. Altered Axial Skeletal Development. Birth Defects Research (Part B) 80: 451-472 (2007)