Bicyclo[2.2.1]heptanebis(methylamine)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2016 to 15 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bicyclo[2.2.1]heptanebis(methylamine)

EC Number:

260-280-7

EC Name:

Bicyclo[2.2.1]heptanebis(methylamine)

Cas Number:

56602-77-8

Molecular formula:

C9H18N2

IUPAC Name:

1-[3-(aminomethyl)bicyclo[2.2.1]heptan-2-yl]methanamine

Test material form:

liquid

Details on test material:

Identity: NBDA
Chemical name: Bicyclo[2.2.1]heptanebis (methylamine)
CAS number: 56602-77-8
Intended use: Hardener of epoxy resin
Appearance: Clear liquid
Storage conditions: Room temperature in the dark, under nitrogen
Lot number: K14B0001
Purity: 99.4 %
Expiry date: 30 June 2016
Date received: 22 June 2015

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 70 days old; Females approximately 63 days old
- Weight at study initiation: Males 326 to 400 g; Females 215 to 255 g
- Housing: Animals were housed in cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified pelleted diet (ad libitum)
- Water: (ad libitum)
- Acclimation period: Five days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The required amount of test material was weighed into a suitable plastic container. Approximately 80% of the final volume of vehicle was added to the test material and stirred
magnetically until uniformly mixed. The pH was adjusted to between 3.5 to 9.0 using HCl. The formulation was made up to final volume and stirred magnetically until homogenous.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test material.

Frequency of preparation: Weekly

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups
- Length of cohabitation: Up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal
smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test material by UPLC.

Instrumentation parameters:
Ultra performance liquid chromatograph (UPLC): Binary solvent manager, Sample manager and TUV detector
Column: Waters Acquity BEH C18, 1.7 μm, 2.1 × 50 mm
Column temperature: 40°C
Sample temperature: Ambient
Mobile Phase A (MPA): ACN/water/o-H3P04 10/90/0.1 v/v/v
Mobile Phase B (MPB): ACN/water/o-H3P04 90/10/0.1 v/v/v
Isocratic conditions: 99% MP A:1% MP B
Flow rate: 0.8 mL/min
Weak rinse solvent: ACN/Water 50/50 v/v, 600 μL
Strong rinse solvent: ACN 100%, 200 μL
Detector wavelength: UV, 195 nm
Injection volume: 7.5 μL
Run time: 2.0 minutes
Approximate retention time: 1.0 minutes

Calculations
The peak area response for the test material in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for test material in sample and procedural recovery chromatograms was measured.
The concentration of test material was determined using the following equation:

Analysed concentration (mg/mL) = [(Y - I) / S] × (V / W) × (D / 1000)


Procedural recovery values were determined using the following equation:

Procedural recovery = [Analysed concentration (mg/mL) / Fortified concentration (mg/mL)] × 100

Where
Y = Peak area response for test material in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution volume of sample (mL)
W = Weight of sample (g)
D = Density of sample (g/mL)

Homogeneity and Stability in 0.5% CMC-Na Aqueous Solution Formulations
The homogeneity and stability of test material in 0.5% CMC-Na aqueous solution formulations was assessed at nominal concentrations of 1 mg/mL and 50 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided (4 × 100 mL) into 4 brown plastic screw-capped containers by Pharmacy personnel and submitted for analysis.

Duration of treatment / exposure:

Males - 15 days before pairing up to necropsy; a minimum of four weeks.
Females - 15 day before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Frequency - Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

selected based on the results of a preliminary study (Envigo study number LBM0101)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

selected based on the results of a preliminary study (Envigo study number LBM0101)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

selected based on the results of a preliminary study (Envigo study number LBM0101)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

selected based on the results of a preliminary study (Envigo study number LBM0101)

No. of animals per sex per dose:

ten males and ten females per dose group.

Control animals:

yes, concurrent vehicle

Positive control:

None.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupants.
A viability check was performed near the start and end of each working day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0 males: Weekly
F0 females: Days 0, 7, 14, and 20 after mating. Day 1 and 7 of lactation

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: On the day of treatment and weekly thereater
F0 females; Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 3, 7, 10, 14, 17, and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males: Weekly, from the day that treatment commenced until pairing.
F0 females: Weekly, from the day that treatment commenced until pairing.
Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating.
Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or
g/animal/day) was calculated for each phase.

Postmortem examinations (parental animals):

SACRIFICE
F0 males: During Week 5 of treatment.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 7: On or after day the last offspring died.
F0 females: Day 7 of lactation.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each
animal, a full macroscopic examination of the tissues was performed. All external features
and orifices were examined visually. Any abnormality in the appearance or size of any organ
and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

- Organ Weights; For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially in modified Davidson’s fluid.

- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Postmortem examinations (offspring):

SACRIFICE
F1 offspring: Day 7 of age

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.

Offspring at scheduled termination: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

Statistics:

All statistical analyses were carried out separately for males and females. 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.

Reproductive indices:

- Mating Performance and Fertility
Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals pairing) x 100

- Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Number pregnant) x 100

- Sex ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1 and 7 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Offspring viability indices:

The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 7 / Number live offspring on Day 1 after littering) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no changes observed in association with the time of dosing. The clinical signs detected were minor and not attributed to treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Following the start of treatment there was an indication of a small reduction in body weight gain for males receiving 300 mg/kg/day however this difference did not attain statistical significance and was considered not adverse at the degree observed. There was no effect on body weight before pairing on females at any dose level. During gestation body weight gain was lower in females receiving test material at 300 mg/kg/day, this difference attained statistical
significance. A difference in body weight was not apparent on Day 1 of lactation and weight gain thereafter was similar to the control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect on food consumption before pairing in males or females at any dose level. During gestation, food consumption was lower in females receiving 300 mg/kg/day, this difference attained statistical significance. During lactation food consumption at 300 mg/kg/day was slightly lower between Day 4-6 of lactation but this difference did not attain statistical significance and is considered non-adverse at the degree 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:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- Treatment Related Findings
A limited list of tissues was selected for histopathological examination, comprising testes and epididymides for the males, ovaries for the females, and abnormalities for all animals. The histopathological examination performed after 4 weeks of treatment revealed no test material related lesions.

- Incidental Findings
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.
The nature, incidence and distribution of all the findings were consistent with the common background of changes seen in these laboratories.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Pre-Coital Interval, Mating Performance, Fertility and Gestation Length
There was no effect of the test material on pre-coital interval, mating performance, and conception rate or fertility index. Gestation length was slightly longer in females receiving 300 mg/kg/day attaining statistical significance, actual percentages of animal in each category were within the historical control ranges with one exception. One female (Number 75) at this dose had a gestation length above the expected range (22-23.5 days) observed at this laboratory, this was attributed to an atypically low litter size.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Results: P1 (second parental generation)

General toxicity (P1)

Dermal irritation (if dermal study):

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Offspring did not display any clinical signs indicative of a reaction to parental exposure to the test material.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

All control and females receiving 15 mg/kg/day were pregnant and reared a live litter to Day 7 of lactation. At 70 mg/kg/day one female was found to be not pregnant at necropsy on Day 25 after mating, this was not attributed to treatment with the test material. At 300 mg/kg/day one female failed to litter and at necropsy on Day 25 after mating was identified to have one early resorption within the uterus, a second female gave birth to two pups which were cannibalised before Day 1 this female was identified to have three uterine implantations. These two atypical pregnancies are of uncertain relationship to treatment.
There were a lower number of implantation in females receiving 300 mg/kg/day as a result total and live litter size was slightly low up to Day 7. There was no effect of test material on sex ratio.
Post implantation survival was low at 300 mg/kg/day this was due to the total litter resorption and total litter loss detailed above. There was no effect on live birth index or viability index survival indices.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of offspring derived from animals receiving 300 mg/kg/day was slightly superior to Control on Day 1 of age, possibly related to the slight extension in gestation length. There was no effect of test material on weight gain from Day 1 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring did not display any necropsy signs indicative of a reaction to parental exposure to the test material.

Histopathological findings:

not examined

Other effects:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

In conclusion within the limitation of this OECD 421 screening study there was considered to be no adverse effect of the test material administered orally to adult rats for two weeks before pairing and to females throughout gestation and lactation until Day 6 on reproductive capability, maternal toxicity or offspring growth and development until Day 7 of age at doses up to 300 mg/kg/day.

Executive summary:

A screening study on reproductive/developmental toxicity was conducted under GLP conditions and in accordance with the standardised guideline OECD 421.

During the study, three groups of ten male and ten female rats received test material at doses of 15, 70 or 300 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before

pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 7 of lactation. The F1 generation received no direct administration of the test material; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, 0.5% sodium carboxymethyl cellulose, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed.

There were no unscheduled deaths, no changes observed in association with the time of dosing and clinical signs detected were minor and not attributed to treatment with the test material.

There was no adverse effect on body weight performance or food consumption of males during the study or females before pairing or during lactation. Body weight gain and food consumption during gestation were low in females receiving 300 mg/kg/day.

Litter size was lower in these females and the reduced weight of fetuses being carried and lower physiological demands may be the cause of these differences rather than direct maternal toxicity.

There was no adverse effect of treatment on male reproductive organ weights. Ovary weights were marginally but statistically lower than Control for females receiving 300 mg/kg/day but there was no correlating histopathology finding for this tissue, therefore this difference is considered not to be adverse.

The macroscopic and microscopic examination performed after 4 weeks of treatment in males and for females dosed from 15 days before pairing, throughout gestation, until Day 6 after the

birth of the F1 generation, revealed no test material related lesions.

Investigation of reproductive endpoints indicated there was no effect of NBDA on pre-coital interval, mating performance, and conception rate or fertility index. There was no effect on the live birth index or the viability index survival indices. Offspring did not display any clinical or necropsy signs indicative of a reaction to parental exposure to the test material. Offspring bodyweight gain was not affected by parental treatment.

At 300 mg/kg/day one female failed to litter and at necropsy on Day 25 after mating was identified to have one early resorption within the uterus, a second female gave birth to two pups which were cannibalized before Day 1 this female was identified to have three uterine implantations. These two atypical pregnancies are of uncertain relationship to treatment but in the absences of any changes in mating performance and fertility or the male or female reproductive organs detected at histopathology, and as the death of the conceptuses in the two litters occurred at different stages of the life cycle, these findings were considered unlikely to be related to treatment.

There were a lower number of implantations in females receiving 300 mg/kg/day and as a result total and live litter size was slightly low up to Day 7 of age. Post implantation survival was lower than control at 300 mg/kg/day. This was due to the total litter resorption and total litter loss detailed above.

Gestation length was slightly longer in females receiving 300 mg/kg/day attaining statistical significance; this was considered not adverse at the degree observed, was generally within historical control ranges and attributed to the lower litter size for these females.

Body weight of offspring derived from animal receiving 300 mg/kg/day was slightly superior to Control on Day 1 of age. This difference was attributed to the slightly longer gestation length for these females. There was no effect of test material on weight gain from Day 1 of age at any dose level.

Therefore, under the conditions of the study, the No Adverse Effect Level was considered to be 300 mg/kg bw/day.