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Administrative data

Description of key information

Two Repeat Dose Studies are available.

In a GLP 28-day oral repeated dose study conducted in accordance with OECD 407 on the structural analogue. The systemic NOAEL was determined to be 50 mg/kg bw/day based on GI, stomach effects and atrophy of the female reproductive organs (uterus, cervix and vagina) observed at all dose levels.

In a GLP, 90 day, repeat dose oral study conducted in accordance with OECD TG 408, administration of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl) aniline to Han Wistar rats for 13 weeks did not cause any toxicologically significant finding. Mucosal hyperplasia was present in the duodenum of males and females which received 5 or 15 mg/kg/day but was considered to be an adaptive response associated with local irritation and non-adverse. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 15 mg/kg/day.

 In both the OECD 407 and 408 there were indications of target organ / portal of entry effects within the duodenum and stomach at 50 mg/kg bw d and 5 mg/kg bw d respectively. This is an indication of a maximum threshold dose of ca. 5 mg/kg bw d. Dosing above this level is likely to result in damage to the GI tract epithelial layer and lead to physiological changes resulting from toxic overload rather than systemic toxicity.


Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st May 2019 - 18th December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: AAH1020800
- Expiration date of the lot/batch: 8 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8°C) and protected from light.
- Solubility and stability of the test substance in the solvent/vehicle: Fully soluable in Propylene glycol.


Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan™:WIST rat.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Supplier: Envigo RMS Limited.
Number of animals: 45 males and 45 females.
Spare animals were removed from the study room after
treatment commenced.
Duration of acclimatization: Approximately 2 weeks before commencement of treatment.
Age at start of treatment: 43 to 49 days.
Weight range at the start of treatment:
Males:137 to 188 g
Females:111 to 153 g

Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Housing specification: Cage floor area: 2065 cm2. Cage height: 21.5 cm.
Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage: Five of the same sex.
Bedding: Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material Provided to each cage throughout the study and replaced when necessary.
Plastic shelter provided to each cage throughout the study and replaced when necessary.
Route of administration:
oral: gavage
Details on route of administration:
Oral, by gavage using a rubber dosing catheter attached to a disposable plastic syringe.
Vehicle:
propylene glycol
Details on oral exposure:
Method of preparation
The required amount of test item was weighed.
Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. The formulation was transferred to the final containers, via syringe whilst magnetically stirring.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item in ascending order of concentration.

Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C) and protected from light.

Formulations prepared at 0.1 and 10 mg/mL were found to be homogeneous and stable at ambient temperature (15 to 25C) for up to three days and refrigerated (2 to 8°C) for up to 17 days. In both cases formulations were protected from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentration Samples of each formulation prepared for administration in Weeks 1, 2, 7 and 13 of treatment were analyzed for achieved concentration of the test item.

Content check issues in Week 1
Before dosing commenced, the Week 1 results for Groups 3 and 4 were within specification (99 and 96% of intended respectively), however Group 2 was at 43%. The pharmacy data were checked and no discrepancies found. Reanalysis
of the samples confirmed the low results. Consequently, the Group 2 dose was discarded and re-formulated. It was not used to dose the animals.
The reformulated dose for Group 2 was found to be at 161% of intended. The pharmacy data were checked and no discrepancies found. Reanalysis of the samples confirmed the high results. This formulation was used to dose Group 2 animals in Week 1.
Due to the analytical problems in Week 1, additional samples were taken and analysed from all groups in Weeks 2 and 7, as well as those planned for Week 13. The potentially higher than intended dose given to Group 2 animals in Week 1 only was not considered to have affected the integrity of the study.

The stability and homogeneity of the test item in the vehicle were determined in Study No.: XT87YV
Duration of treatment / exposure:
Ninety consecutive days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rational:
The dose levels for this study (0, 1.5, 5 and 15 mg/kg/day at 5 mL/kg) were selected following the outcome of a two generation reproductive toxicity study in Han Wistar rats performed at the same laboratories, with particular reference to the responses observed in the F0 generation animals (the animals were of a similar age at the start of treatment to that of the F0 generation and were treated for a similar, although slightly shorter duration).

In the reproductive study, dose levels of 5, 15 and 25 mg/kg/day at 4 mL/kg were administered to the F0 generation animals from 6-7 weeks of age for a minimum of 16 weeks. There were no test item-related premature deaths, changes in general clinical
condition or signs in relation to dose administration. Males given 15 or 25 mg/kg/day showed low mean body weight gain throughout the study, although mean food consumption was not clearly affected; body weight gain and food intake in females was unaffected by treatment during the 10-week pre-pairing treatment period at all dose levels investigated. At scheduled termination, test item-related macroscopic abnormalities were limited to the stomach, where ulceration were observed in males at 25 mg/kg/day. Histopathological evaluation revealed stomach ulceration at 25 mg/kg/day (primarily in males), and a dose-dependent incidence of mucosal degeneration/atrophy of the stomach, duodenum and jejunum (with associated regenerative hyperplasia in the duodenum and jejunum) in both sexes at 15 or 25 mg/kg/day. In addition, erythrocytosis/erthyrophagocytosis, sinusoidal ectasia and/or cortical atrophy were apparent in the lymph nodes in animals given 25 mg/kg/day and minimal splenic white pulp depletion was evident at 25 mg/kg/day. The No Observed Effect Level was 5 mL/kg/day.

The effects on the gastroinestinal tract seen at 15 or 25 mg/kg/day were considered to be associated with local irritancy. At 25 mg/kg/day the changes were considered of sufficient magnitude to exclude this as a high dose in this 13-week study. Consequently 15 mg/kg/day was selected as the high dose and was expected to be well tolerated over 13 weeks with minimal local irritant effects. The low and intermediate doses of 1.5 and 5 mg/kg/day were selected to allow analysis of any dose response relationship and to aid in the determination of a No Observed Adverse Effect Level. As the local irritancy was possibly linked to the dose concentration, the dose volume was increased from 4 mL/kg (used in the two-generation reproductive toxicity) to 5 mL/kg to slightly reduce the concentrations at the selected doses.

Allocation
Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals
Each animal was assigned a number and identified uniquely within the study by a microch/>ip inserted shortly after arrival.

Identification of cages
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.
Positive control:
no (not required)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
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. Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
During the acclimatization period, observations of the animals and their cages were recorded
at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations:
The weight of each animal was recorded one week before treatment commenced, on the day
that treatment commenced (Week 0), weekly throughout the study and before necropsy.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
Pretreatment, Week 12
- Dose groups that were examined:
Pretreatment- all animals
Week 12- all group 1-4 animals.

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Not specified
- Time schedule for examinations: week 13
- Animals fasted: Overnight
- How many animals:
All animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Week 13
- Animals fasted: Overnight
- How many animals:
All animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
Statistics:
The following data types were analyzed at each timepoint separately:
Grip strength and motor activity
Body weight, using gains over appropriate study periods
Hematology
Blood chemistry
Organ weights, absolute or adjusted for terminal body weight
The following comparisons were performed:
Group 1 vs 2, 3 and 4

For Further Details see Additional information
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no deaths and the general appearance and behaviour of the animals were unaffected by treatment.
Chin rubbing and salivation at the time of dose administration were reported on transient occasions in a low number of animals but since these are commonly observed signs in studies where the test item is administered by gavage they are considered of no toxicological
significance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall bodyweight gain (Weeks 0 to 13) was significantly slightly low, when compared to the controls, for males receiving 15 mg/kg/day.
The bodyweight gain of males receiving 1.5 or 5 mg/kg/day and of all groups of treated females was not clearly affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption (Weeks 1 to 13) was slightly low, when compared to the controls,
for males receiving 15 mg/kg/day.
The intake of males receiving 1.5 or 5 mg/kg/day and of all groups of treated females was not
clearly affected by treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The haematological examination during Week 13 did not reveal any toxicologically significant differences from controls.
Lymphocyte counts were slightly low (resulting in a low total white cell count), when compared to the controls, for males receiving 15 mg/kg/day and for all groups of treated females. The difference from controls was small, there was no dose relationship in females
and none of the individual values was outside the background range (98-percentile range; 2.44x109/L to 8.58x109/L; n=104 for males and 1.27x109/L to 7.20x109/L; n=103 for females). Basophil, monocyte and large unstained cell counts were also slightly low, when
compared to the controls, for females receiving 5 or 15 mg/kg/day but none of the individual values was outside the background range (98-percentile range; 0.00x109/L to 0.03x109/L for basophils, 0.04x109/L to 0.22x109/L for monocytes and 0.00x109/L to 0.07x109/L for large
unstained cells ; n=104). Consequently, these inter-group differences from controls were
considered to represent normal biological variation.
All other inter-group differences from controls, including those that attained statistical
significance, were minor, occurred in one sex only or were without dose-relationship and
were therefore considered to represent normal biological variation.
Description (incidence and severity):
The biochemical examination of the blood plasma after 13 weeks of treatment did not reveal any toxicologically significant differences from controls.
All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation. Such differences included the slightly
high alanine amino transferase activity, bile acid concentration and potassium concentration in males receiving 15 mg/kg/day, and the slightly low sodium concentration in females
receiving 15 mg/kg/day.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weights of animals killed after 13 weeks were unaffected by treatment.
All inter-group differences from controls were minor, occurred in one sex (where appropriate) or were associated with the low terminal bodyweights and were therefore considered to represent normal biological variation. These included the slightly low adrenal
and thyroid gland weight in males at 15 mg/kg/day which associated with the low terminal bodyweights, and the slightly high brain weight in females at 15 mg/kg/day as the difference from controls was small and there was no similar finding in males.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The histopathological examination performed on animals killed after 13 weeks of treatment revealed mucosal hyperplasia in the duodenum of males and females which received 5 or 15 mg/kg/day; a dose-relationship in incidence and severity was apparent.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Conclusions:
It is concluded that the oral (by gavage) administration of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl) aniline to Han Wistar rats for 13 did not cause any toxicologically significant finding. Mucosal hyperplasia was present in the duodenum of males and females which received 5 or 15 mg/kg/day but was considered to be an adaptive response associated with local irritation and non-adverse. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 15 mg/kg/day.
Executive summary:

The purpose of this study was to assess the systemic toxic potential of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl) aniline (an industrial chemical), when administered orally (by gavage) to Han Wistar rats (RccHan™;WIST) for 13 weeks.

Three groups, each comprising ten male and ten female rats, received doses of 1.5, 5 or 15 mg/kg/day. A similarly constituted control group received the vehicle (propylene glycol), at the same volume dose as the treated groups. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption,

water consumption (visual assessment only), ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, organ weight, macropathology and histopathology investigations were undertaken.

Results

The general appearance and behaviour of the animals and sensory activity, grip strength and motor activity were unaffected by treatment.

The bodyweight gain and food intake was significantly slightly low for males receiving 15 mg/kg/day.

There were no treatment-related ophthalmoscopic findings.

The haematological and blood chemistry investigation did not indicate any toxicologically significant findings.

Estrous cycles at the end of the treatment period were unaffected.

Serum triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) concentrations were unaffected.

Organ weights were unaffected and there were no treatment-related macroscopic findings.

Histopathology revealed mucosal hyperplasia in the duodenum of males and females which received 5 or 15 mg/kg/day.

Conclusion

It is concluded that the oral (by gavage) administration of p-(2,3-epoxypropoxy)-N,Nbis(2,3-epoxypropyl) aniline to Han Wistar rats for 13 did not cause any toxicologically significant finding. Mucosal hyperplasia was present in the duodenum of males and females which received 5 or 15 mg/kg/day but was considered to be an adaptive response associated with local irritation and non-adverse. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 15 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study Klimisch 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The key repeated dose data is derived from a GLP 28-day oral repeated dose study conducted on the structural analogue in accordance with OECD 407. The oral route of exposure was considered the most appropriate for assessment and the systemic NOAEL was determined to be 50 mg/kg bw/day based on GI, Stomach effects and atrophy of the female reproductive organs (uterus, cervix and vagina) observed at 150 mg/kg/day.

 In both the OECD 407 and 408 there were indications of target organ / portal of entry effects within the duodenum and stomach at 150 mg/kg bw d and 5 mg/kg bw d respectively. This is an indication of a maximum threshold dose of ca. 5 mg/kg bw d. Dosing above this level is likely to result in damage to the GI tract epithelial layer and lead to physiological changes resulting from toxic overload rather than systemic toxicity.

Repeated dose toxicity testing via the inhalation and dermal routes where considered unjustified as oral is the most relevant route.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only this repeated dose study is available. A GLP and OECD test guideline 28-day oral repeated dose toxicity study with rats has been conducted, the systemic NOAEL was determined to be 50 mg/kg bw/day based on atrophy of the female reproductive organs (uterus, cervix and vagina) observed at 150 mg/kg/day. Based on the local effects in the gastrointestinal tract (duodenum, Peyer’s patches and lymph nodes), likely to be secondary effects due to the irritation potential of the test substance, starting at the lowest dose level of 50 mg/kg/day, a local No Observed Adverse Effect Level (NOAEL) could not be established in this study.

Justification for classification or non-classification

Specific Target Organ Toxicity – Repeated Exposure: The structural analogue used as read-aross did exhibit significant effects arising from a repeated exposure at a dose level of 150 mg/kg/day in male and female animals. As a result, the substance does meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.9. The substance is classified as STOT RE Cat 2, H373 May cause damage to organs (gastro-intestinal tract, stomach, female reproductive organs) through prolonged or repeated exposure.