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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 11 October 2017and xx month 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
EDA-BADGE-EDA
Cas Number:
854009-15-7
Molecular formula:
C25H40N4O4
IUPAC Name:
EDA-BADGE-EDA
Constituent 2
Chemical structure
Reference substance name:
EDA-BADGE-EDA-BADGE-EDA
Molecular formula:
C48H72N6O8
IUPAC Name:
EDA-BADGE-EDA-BADGE-EDA
Constituent 3
Chemical structure
Reference substance name:
EDA-BADGE α-glycol
Molecular formula:
C23H34N2O5
IUPAC Name:
EDA-BADGE α-glycol
Constituent 4
Reference substance name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Constituent 5
Reference substance name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Test material form:
liquid: viscous
Specific details on test material used for the study:
Identification : 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypr
opane, reaction products with ethylene diamine
Physical State/Appearance: Yellow/brown extremely viscous liquid
Chemical Name: Pheno1,4,4'-(1-methylethylidene)bis-,polymer with N-(2-aminoethyl)-1,2 -ethanedi
amine and (chloromethyl)oxirane
Purity : UVCB substance, purity not applicable
Batch Number : BBF01102V1
Label : 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane,
reaction products with ethylene diamine Batch BBF01102V1 Expiry date 01.01.2021
Date Received : 30 August 2016
Storage Conditions: In the dark at room temperature
Expiry Date : 01 January 2021

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from
Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined
for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time
their health status was assessed. Following the day of arrival, vaginal smears were performed for
all females throughout the acclimatization period and females considered not showing appropriate
estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into
the study. At the start of treatment the males weighed 267 to 357g, and were approximately eleven
weeks old. The females weighed 180 to 229g, and were approximately fourteen weeks old.
Animal Care and Husbandry
Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless
steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase,
animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent
paper on a one male: one female basis within each dose group. Following evidence of successfu
l mating, the males were returned to their original cages. Mated females were housed individually
during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and
softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of
the batches of diet used is given in Annex 6. Mains drinking water was supplied from polycarbonate
bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew
blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mate
d females during gestation and lactation. Mated females were also given softwood flakes, as beddin
g, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichm
ent was considered not to contain any contaminant at a level that might have affected the purpose or
integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shard
low, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes
per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous
light and twelve hours darkness. Environmental conditions were continuously monitored by a com
puterized system, and print-outs of hourly temperatures and humidities are included in the study re
cords. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ±
20% respectively; there were short term deviations from these targets that were considered not to h
ave affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also
randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.
Low 10 4 2.5 12 (25-36) 12 (37-48)
Intermediate 30 4 7.5 12 (49-60) 12 (61-72)
High 60 4 15 12 (73-84) 12 (85-96)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment
group.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposa
ble plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene
glycol 400.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Details on exposure
Test Item Preparation and Analysis
For the purpose of this study the test item was prepared at the appropriate concentrations as a solut
ion in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were d
etermined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the
formulations to be stable for at least twenty one days. Formulations were therefore prepared weekly
and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken on two occasions and analyzed for concentration of
4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction
products with ethylene diamine at Envigo Research Limited, Shardlow, UK, Analytical Services. T
he method used for analysis of formulations and the results obtained are detailed below. The results
indicate that the prepared formulations were within ± 16% of the nominal concentration.
Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL)
Animal Numbers
Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Details on mating procedure
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to
fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation
plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal
smear was prepared for each female and the stage of estrus or the presence of sperm was recorded.
The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive ev
idence of mating (Day 0 of gestation) and the males were subsequently returned to their original ho
lding cages. Mated females were housed individually during the period of gestation and lactation.
Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as
possible during the normal working day) around the period of expected parturition. Observations w
ere carried out at approximately 0830 and as late as possible at weekends and public holidays. The
following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid c
hromatography with UV detection (HPLC/UV) using an external standard technique. The test item
gave a chromatographic profile consisting of a single peak.
Preparation of Calibration Standards
Stock solutions of test item in dilution solvent (methanol) were prepared for external standard calibra
tion. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric fla
sk and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL.
Aliquots of this stock standard solution were used to prepare working standard solutions in dilution
solvent with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calcul
ation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample anal
ysis sequence as a minimum, using the conditions detailed in the instrument parameters section belo
w.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution
solvent from a stock solution of 1.075 mg/mL by serial dilution covering the concentration range 0.05
375 mg/mL to 0.2150 mg/mL.
Preparation of Test Samples
The formulaitons recieved were diluted with diluiton solvent (Methanol). An aliquot of test item
formulation was accurately weighed into a volumetric flaska dn brough to volume with dilution solvent,
this was then shaken and ultrasonicated to dissolve. Where necessary sample solutions were further
diluted with dilution solvent to achieve the worming concnetration.
Preparation of Accuracy and Precision Samples
Samples of PEG 400 were accurately fortified with known amounts of test item equivalent to the lo
west and highest anticipated dose concentrations.
The concentration of Test Item in the final solution was quantified by HPLC using UV detection as
detailed in the instrument parameters section below.
Instrumentation Parameters
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Synergi polar RP (150 x 4.6 mm id) at 40ºC
Mobile phase: Eluent A: 100 mM Ammonium formate + 0.1% formic acid in water
Eluent B: 0.1% formic acid in acetonitrile
Time %B
0 85
12 15
12.1 85
15 85
Flow-rate 0.8 mL/min
UV detector wavelength: 230 nm
Injection volume: 25 #L
Retention time: ~ 5.4 mins
Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured.
Calibration curves were constructed by linear regression of calibration standard response versus cali
bration standard concentration. The area response of the peak observed at the characteristic reten
tion time for Test Item in sample and procedural recovery chromatograms was measured.
Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range.
The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal c
oncentrations of 2.5 mg/mL and 50 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during
the study.
RESULTS
Method Validation
The analytical procedure was successfully validated for Test Item in PEG 400 with respect to the
specificity of chromatographic analysis, the linearity of detector response, method accuracy and
precision. Results are summarized below.
The specificity of the analytical method was demonstrated by the absence of a peak at the character
istic retention time for Test Item in the control sample chromatogram.
The data was found to have a linear correlation within the calibration range of 0.05375 mg/mL to 0.
2150 mg/mL. The R² fit of the calibration curve to the data was 0.9947, and was considered to be a
cceptable.
Method accuracy (recovery) and precision were confirmed and results indicated that a mean recove
ry value of 98% (CV=2.26%, n=5) was obtained for 2.5 mg/mL and 105% (CV=2.26%, n=5) was ob
tained for 50 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during
the study.
Concentration of Dose Formulaitons
Samples of test item formulations were taken on two occasions and analyzed for concentration of
4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction
products with ethylene diamine at Envigo Research Limited, Shardlow, UK, Analytical Services. The
results indicate that the prepared formulations were within ± 16% of the nominal concentration.
CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic
analysis, linearity of detector response, method accuracy and precision.
The concentrations of the test item in the prepared formulations were within ± 16% of the nominal
concentration.
Duration of treatment / exposure:
The test item was administered by gavage to three groups, each of twelve male and twelve female
rats, for approximately six weeks (males) and up to eight weeks
(females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females),
at dose levels of 10, 30 and 60 mg/kg bw/day. A control group of twelve males and twelve females
was dosed with vehicle alone.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two
weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females
not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level
throughout the study (except for females during parturition where applicable). The first day of dosing
was designated as Day 1 of the study.
iii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of
functional/behavioral toxicity.
iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximu
m of fourteen days.
v. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their
original cages and females were transferred to individual cages.
vi. On completion of the pairing phase, five selected males per dose group were evaluated for f
unctional/sensory responses to various stimuli during Week 6.
vii. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum.
Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and
clinical signs were also recorded during this period.
viii. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated
offspring from each litter in order to obtain serum samples.
ix. At Day 12 post partum, five selected females per dose group were evaluated for functional/sensory
responses to various stimuli.
x. Blood samples were taken from five males from each dose group for hematological and blood c
hemical assessments on Day 43. The male dose groups were killed and examined macroscopically
on Day 44 or 45.
xi. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessmen
t of thyroid hormones was performed on two randomly selected offspring (one male and one female)
per litter. Where possible, a further two randomly selected offspring (one male and one female) per
litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained
from one male and one female from each litter where litter sizes allowed. All surviving offspring were
killed and examined externally; where external observations were detected an internal necropsy was
performed.
xii. Blood samples were taken from five randomly selected females from each dose group for hemat
ological and blood chemical assessment on Day 13 post partum. All surviving females were sacrifice
d on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all
females in the morning of the day of necropsy. Any female which did not produce a pregnancy was
also sacrificed and examined macroscopically around the same time as littering females. In addition,
blood samples to produce both serum and plasma were taken from all adult animals at termination.
Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results and discussion

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine to rats by gavage, at dose levels of 10, 30 and 60 mg/kg bw/day, resulted in histiocyte aggregates present in the mesenteric lymph nodes in both male and females treated with 60 mg/kg bw/day and males treated with 30 mg/kg bw/day at a minimal to mild severity. Without any additional pathological changes this finding was considered to be non adverse. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 60 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 10, 30 and 60 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same period.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all treated females. Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14post partum,respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Results…….

Adult Responses

Mortality

On Day 3 of treatment, one control female was found dead. The clinical and necropsy observations along with the histopathology findings suggested the death may be associated with dosing trauma.

There were no further unscheduled deaths.

Clinical Observations

There were no clinical signs observed that were considered to be indicative of systemic toxicity. 

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There were no treatment-related changes detected in the functional performance parameters.

Sensory Reactivity Assessments

There were no treatment-related changes detected in sensory reactivity.

Body Weight

There were no adverse effects on body weight development detected for males or for females during pre-pairing, gestation or lactation phases.

Food Consumption

There were no adverse effects on food consumption or food conversion efficiency for males or for females during pre-pairing, gestation or lactation phases.


Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Reproductive Performance

Estrous Cycle

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study.

There were also no intergroup differences in the stage of estrous cycle on the day of necropsy.

Mating

There were no treatment-related effects detected on mating performance.

Fertility

There were no treatment-related effects detected in conception rates.

Gestation Lengths

Gestation lengths were essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at any dose level.

Offspring Growth and Development

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13post partumat any dose level.

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 10, 30 or 60 mg/kg bw/day.


Laboratory Investigations

Hematology

There were no toxicologically significant changes identified in the hematological parameters measured.

Blood Chemistry

There were no toxicologically significant changes in the blood chemical parameters measured.

Pathology

Necropsy

Adults

There were no treatment-related abnormalities detected at necropsy.

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Organ Weights

There were no treatment-related changes detected in the organ weights measured.

Histopathology

Treatment-related findings were as follows:

Mesenteric Lymph node

Histiocyte aggregates were present in three males each treated with 30 or 60 mg/kg bw/day at minimal severity. In females, minimal histiocyte aggregates were present in one female each from the control and 10 mg/kg bw/day dose group at minimal severity and in four females treated with 60 mg/kg bw/day at minimal or mild severity.

Conclusion

The oral administration of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine to rats by gavage, at dose levels of 10, 30 and 60 mg/kg bw/day, resulted in histiocyte aggregates present in the mesenteric lymph nodes in both male and females treated with 60 mg/kg bw/day and males treated with 30 mg/kg bw/day at a minimal to mild severity. Without any additional pathological changes this finding was considered to be non adverse. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 60 mg/kg bw/day.

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