2-methylpentane-1,5-diamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05 July 2021 - 11 August 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

LabCorp standard Preliminary Study for Effects on Embryo-Fetal Development in the Han Wistar Rat

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice will be made for this study although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpentane-1,5-diamine

EC Number:

239-556-6

EC Name:

2-methylpentane-1,5-diamine

Cas Number:

15520-10-2

Molecular formula:

C6H16N2

IUPAC Name:

2-methylpentane-1,5-diamine

Test material form:

liquid

Details on test material:

clear colorless liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han Wistar (RccHan;WIST)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 180 to 222 g
- Housing: Cages comprised of a polycarbonate body with a solid
(polycarbonate) bottom and a stainless steel mesh lid;
changed at appropriate intervals
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
(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: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. ad libitum
- Acclimation period: Four days from arrival on Day 2 after mating to
commencement of treatment on Day 6 after mating

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): 12/12

IN-LIFE DATES:
From: 2021-07-12 To: 2021-07-28, Females were treated from Day 6 to Day 20 (inclusive) after mating

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed.
Starting with the highest concentration, approximately 60 to
80% of the final volume of vehicle was added to the test
item and magnetically stirred until uniformly mixed. The
pH was checked and adjusted if necessary to 7.5 ± 0.1 using
hydrochloric acid. The remaining vehicle was added to
achieve the required volume. The pH was checked again
and adjusted as before if necessary. The formulation was
stirred for a minimum of 20 minutes using a magnetic
stirrer, the end result being a solution. The remaining
formulations were prepared by serial dilution with each
being stirred for a minimum of 20 minutes.
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.

Storage of formulation
Refrigerated (2 to 8°C).

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Natural mating with Han Wistar of established fertility at the supplier’s facility. Males and females were not related. Time mated animals arrived at the testing facility on GD2.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 20 (inclusive) after mating

Frequency of treatment:

once daily

Duration of test:

Treatment commenced 12 to 13 July 2021
Necropsy 27 to 28 July 2021

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for investigation (0, 250, 500 and 1000 mg/kg/day) were selected in conjunction with the Sponsor and based on the results of a preliminary 14-day toxicity study Labcorp Study No. 8439540 with non pregnant rats. In that study orally administered dose levels of DYTEK A amine at 100, 300 and 1000 mg/kg/day were investigated.

It that study oral administration of DYTEK A amine at 1000 mg/kg/day caused a reduction in
body weight gain and food intake in males but no similar effect was observed in the females.
The erythrocytes, liver and kidneys were identified as potential targets of toxicity. Low
absolute and body weight-relative liver weights were observed in males and females given
300 or 1000 mg/kg/day, and low absolute and body weight-relative spleen weight were
observed in males and females given 1000 mg/kg/day. Findings of dark areas and depressions
in the stomach were observed macroscopically in both sexes at 1000 mg/kg/day.

Furthermore, the results of a preliminary OECD 414 study in the rat with DYTEK DCH-99 (CAS 694-83-7; a similar compound to DYTEK A amine), Labcorp Study No. 8434915, revealed no severe
effects at 500 mg/kg/day.
Based on these findings, a high dose level of 1000 mg/kg/day was considered suitable for this
preliminary embryo-fetal development study, since the dosing period was very similar to that
of the 14-day study, with low and intermediate dose levels of 250 and 500 mg/kg/day
providing suitable dose increments.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 x daily, Pre-dose observation, One to two hours after completion of dosing, As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on Days 3, 5, 12, 18 and 21
after mating

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 3, 6, 9, 12, 15, 18 and 21 after mating

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes, recorded for the periods Days 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 after mating inclusive

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 after mating
- Organs examined: complete macroscopic examination; kidneys, liver, spleen (weigh)

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
- Other: fetuses live and dead
- Placental weight: yes

Fetal examinations:

all per litter:
- External examinations: Yes
- Sex ratio: yes
- individual fetal weight: yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

signs were either transient in nature and/or not observed in the higher dose groups
(At 1000 mg/kg/day, non-adverse transient post-dose signs were observed comprising dry
rales for one female on Day 17 of gestation, piloerection for another female on Days 9 and 12
of gestation with hunched posture also observed post-dose for this female on Day 9 of gestation.)

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no adverse effect of treatment on body weight gain at 500 or 250 mg/kg/day.

There was no effect of treatment on adjusted maternal body weight or body weight gain at 500 or 250 mg/kg/day.

Overall body weight gain (Days 6-21 of gestation) was slightly lower than Control at 1000 mg/kg/day, although statistical significance was not attained.

When adjusted for the contribution of the gravid uterine weight, a mean adjusted maternal body weight loss of 6g was observed between Days 6-21 of gestation at 1000 mg/kg/day. Review of the individual data revealed a loss of 30g in 1 female at 1000 mg/kg/day (4F 28).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on food intake at 250 mg/kg/day.

Overall food intake (Days 6-21 of gestation) at 500 mg/kg/day was only marginally, and not statistically significantly, lower than Control.

Overall food intake (Days 6-21 of gestation) at 1000 mg/kg/day was statistically significantly lower than Control.

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean absolute spleen weights were statistically significantly lower than Control. Mean absolute spleen weights were also slightly lower than Control at 500 and 250 mg/kg/day, however, statistical significance was not attained and no dose response was apparent.
There was no effect of treatment on mean absolute kidney or liver weights at any dose level investigated.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related findings observed at necropsy for females at scheduled termination on Day 21 after mating.
Macroscopic findings were limited to 1 female (4F 28) with a distended cecum with
abnormal cecum contents at 1000 mg/kg/day. This may be related to the reduced food intake and higher adjusted maternal body weight loss in this female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Post-implantation loss was higher than Control at 1000 and 500 mg/kg/day, however, statistical significance was not attained. This effect may be associated with the slightly higher litter size at these dose levels.

There was no clear effect of treatment on the extent of pre-implantation loss at any dose level investigated.

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 was found not to be pregnant at macroscopic examination.

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a decrease in male, female and overall fetal weights observed in all treated groups, compared to Control, with statistical significance attained at 1000 mg/kg/day (16%, 15% and 16% decrease at 1000 mg/kg/day for male, female and overall fetal weights, respectively).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 and 500 mg/kg/day, sex ratio (% males) was decreased compared to Control, however, statistical significance was not attained. Also the difference from 50% (% males) was similar at 500 mg/kg/day as it was at 250 mg/kg/day and in the absence of a decrease in litter size, this effect was considered unlikely to be related to treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mean total litter weights at any dose level investigated.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of the fetuses did not reveal any findings that could be related to maternal treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Applicant's summary and conclusion

Conclusions:

Based on the results of this Preliminary Study for Effects on Embryo-Fetal Development in the Han Wistar Rat by Oral Gavage Administration, a high dose level of 1000 mg/kg/day was considered a suitable high dose level for use on the main OECD 414 study in rats as the effects observed at 1000 mg/kg/day in this preliminary study were not considered to preclude the use of this dose level. A dose sequence of 0, 100, 300 and 1000 mg/kg/day could be considered.

Executive summary:

The purpose of this study was to make a preliminary assessment of the influence of the submission substance (DYTEK A amine, MPMD) on embryo-fetal survival and development in the rat and to establish suitable doses for a main embryo-fetal toxicity study.

Three groups of seven pregnant females received DYTEK A amine at doses of 250, 500 or
1000 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, water at
the same volume dose as the 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
and organ weights were recorded. All fetuses were examined macroscopically at necropsy.

There were only minor findings in the dose groups (0, 250, 500, 1000 mg/kg/day) investigated. There was an adjusted maternal body weight loss at 1000 mg/kg/day which could be attributed to one female (loss of 30 g). Additionally food consumption was decreased at 1000 mg/kg/day. These maternal effects were also likely associated with the reduced placental weights and reduced male, female and overall fetal weights observed at this dose, however, the extent of these effects were not considered to preclude the use of this dose level on the main study.

Therefore, a dose sequence of 0, 100, 300 and 1000 mg/kg/day could be considered for the main study.