Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2017 - 19 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: AAF1453400
- Expiration date of the lot/batch: 26 November 2021
- Purity: 100% UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15 to 25C).

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan™:WIST rat
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 41 to 47 days
- Weight at study initiation: Males: 134 to 174 g, Females: 116 to 151 g
- Housing: The cage used was a polycarbonate body with a stainless steel mesh lid, and the bedding was wood based bedding which was changed at appropriate intervals each week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- 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
Details on route of administration:
Oral, by gavage, using a suitably graduated syringe and a
rubber catheter inserted via the mouth.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed. Approximately 40% 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 homogeneous. A series of formulations at the required concentrations were prepared in ascending order.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical procedure was successfully validated for Oxirane, mono[(C12-14-alkyloxy)methyl] derivs in corn oil with respect to the specificity of chromatographic analysis, limits of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision. The homogeneity and stability of the test item in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 200 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature (15 to 25°C) for one day and refrigeration (2 to 8°C) for up to 15 days.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study (0, 100, 300 and 750 mg/kg/day) were selected in conjunction with the Sponsor following the completion of the 14-day preliminary study with Oxirane. In that study, there were no signs observed in association with treatment in any animal, and
body weight gain and food intake were satisfactory. At macroscopic examination, depressions and/or thickening of the non-glandular region of the stomach of all animals treated at 1000 mg/kg/day were observed; for males and females treated at 750 mg/kg/day, instances of thickening of the non-glandular region (four out of ten animals) were observed at macroscopic examination but there was no subsequent incidence of stomach depression seen
in any animal treated at this dose level. There were no treatment-related findings in any animal treated at 500 mg/kg/day.

The stomach changes observed at 750 mg/kg/day or 1000 mg/kg/day suggest an adaptive response to irritancy of the test item, rather than a systemic response to treatment, however the extent and incidence of stomach depressions observed at the high dose level (1000 mg/kg/day), after 14 days of treatment, raised concerns that more severe stomach damage may develop with increasing duration of dosing on this Main (13-Week) OECD 408 Study.

Therefore, a dose level of 750 mg/kg/day was selected for the high dose in order to minimize the risk of more severe stomach lesions occurring while at the same time complying with the guideline requirement that some toxicity was induced at the high dose.

Intermediate and low dose levels of 300 mg/kg/day and 100 mg/kg/day respectively, were selected to allow evaluation of a dose response.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly throughout the study and before necrospy

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment, Week 12
- Dose groups that were examined: Groups 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table [No 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength, motor activity, Others: Approach response, Pinna reflex, Auditory startle reflex, Tail pinch response, Grip strength.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (table 3)

HISTOPATHOLOGY: Yes (table 3)
Statistics:
Data-Types
The following data types will be analyzed at each time-point separately:-
body weight, using gains over appropriate study periods.
blood chemistry and hematology.
grip strength and motor activity examinations.
organ weights, either absolute or adjusted for terminal body weight where
appropriate.

Methods
For categorical data, the proportion of animals will be analyzed for each treated group (as
appropriate) versus the control group.
For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance
between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups
will then be compared with the control group, incorporating adjustment for multiple
comparisons where necessary.
Under the advice of the Head of Statistics and Data Management Department, or other
qualified Statistician, alternative or additional methods may be carried out if deemed
appropriate following data review. Details will be included in the study report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with the controls, statistical significance was attained for several parameters
but the differences were generally minor, confined to one sex and/or within the 5 to 95%
historical control data (HCD) ranges. These included a slight shift upwards in hematocrit
values in males receiving 750 mg/kg/day but all values were within the HCD range (0.431 to
0.498 L/L: n=104) and no similar effect was seen in females, minor reductions were seen in mean cell hemoglobin concentrations in males receiving 100, 300 or 750 mg/kg/day and in
females receiving 750 mg/kg/day, but all individual values were within the HCD ranges (32.8
to 36.2 g/dL: n=104 and 32.7 to 36.2 g/dL; n= 103, for males and females, respectively), and
there was a slight reduction in red cell distribution width in males and females receiving
750 mg/kg/day but all values for the females were within the HCD range (10.1 to 11.7%:
n=103) and although the majority of values were below the range in the males (11.4 to
13.3%: n =104) the effect was of insufficient magnitude to be considered adverse.
Lymphocyte counts were reduced, together with concomitant reductions in total white blood
cell counts, in males receiving 100, 300 or 750 mg/kg/day. When the individual values for
the lymphocyte counts were compared with the HCD, two, five and three males receiving
100, 300 or 750 mg/kg/day, respectively, had values that were below the range (3.67 to
7.34 x109/L; n=104). Given that the response to treatment was not dose-related and that no
similar effect was seen in females these changes were considered not to be related to
treatment. Reductions in large unstained cell counts were seen in males receiving 300 or
750 mg/kg/day; no similar effect was seen in females and all values were within the HCD
range (0.01 to 0.09 x109/L: n=104).
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Mesenteric lymph nodes
Dark mesenteric lymph nodes were seen in two males given 750 mg/kg/day.

Stomach
Thickening of the non-glandular mucosa was seen in most males and females given
750 mg/kg/day and in some males and females given 300 mg/kg/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mesenteric Lymph Node
Erythrocytes, intrasinusoidal were present in five males given 750 mg/kg/day.

Pancreas
Vacuolation of the acinar cells was present in the majority of males and females given
750 mg/kg/day and in three males given 300 mg/kg/day.

Hyperplasia of the non-glandular epithelium was present in the majority of males and females
given 750 mg/kg/day. It was also present in two males and three females given
300 mg/kg/day. Hypertrophy of the antral mucosa was present in the majority of males and
females given 750 mg/kg/day. Erosion of the antral mucosa was present in five males given
750 mg/kg/day.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
pancreas
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
It was concluded that the repeated oral administration of Oxirane,
mono[(C12-14-alkyloxy)methyl] derivs to Han Wistar (RccHan™;WIST) rats for 13 weeks
at doses of 100, 300 or 750 mg/kg/day produced lesions in the stomachs of males and females
given 300 or 750 mg/kg/day. Intrasinusoidal erythrocytes were present in the mesenteric
lymph node of males given 750 mg/kg/day and this finding was likely to be secondary to the
lesions seen in the stomach. These changes were considered to result from gastric irritation
occurring at point-of-contact and as such were not attributed to systemic toxicity. A
degenerative change (vacuolation) was seen in the pancreas of animals given 750 mg/kg/day
and males given 300 mg/kg/day and this was considered adverse.
The No-Observed-Effect level (NOEL) for systemic toxicity was 100 mg/kg/day for males
and 300 mg/kg/day for females based on the vacuolation reported in the pancreas.
The No-Observed-Effect level (NOEL) for the irritant effects seen in the stomach and the
associated findings was 100 mg/kg/day for males and females.
Executive summary:

The purpose of this study was to assess the systemic toxic potential of Oxirane,

mono[(C12-14-alkyloxy)methyl] derivs (a substance used in industry) in a 13 week

oral gavage study in Han Wistar (RccHan™;WIST) rats.

Three groups, each comprising ten male and ten female Han Wistar rats, received Oxirane,

mono[(C12-14-alkyloxy)methyl] derivs at doses of 100, 300 or 750 mg/kg/day. A similarly

constituted control group received the vehicle, corn oil, at the same volume dose as treated

groups.

During the study, detailed physical examination and arena observations, sensory reactivity,

grip strength, motor activity, body weight, food consumption, water consumption (by visual

assessment only), ophthalmoscopy, hematology (peripheral blood), blood chemistry, organ

weight, macropathology and histopathology investigations were undertaken.

Results

No animals died prematurely and the general appearance and behavior of the animals were

unaffected by treatment. Similarly, the neurobehavioral investigations (sensory reactivity,

grip strength and motor activity assessments) did not indicate any treatment-related findings.

Body weight gains, food consumption and water consumption were all unaffected by

treatment.

No treatment-related ophthalmoscopic abnormalities were detected.

There were no test article-related effects on hematology parameters.

Changes in blood chemistry that were attributed to treatment comprised decreased plasma

cholesterol concentrations in females receiving 300 mg/kg/day and in males and females

receiving 750 mg/kg/day and marginally decreased total plasma protein concentrations in

males and females receiving 750 mg/kg/day which led to an increase in albumin: globulin

ratios in both sexes.

After 13 weeks of treatment, adrenal weights were high in males given 100, 300 or

750 mg/kg/day, when compared with controls. Liver weights were also higher than controls

at the end of the treatment period for males and females treated at 750 mg/kg/day. Spleen

weights were low in males treated at 750 mg/kg/day.

The macroscopic examination performed after 13 weeks of treatment revealed dark

mesenteric lymph nodes in two males given 750 mg/kg/day and thickening of the

non-glandular mucosa of the stomach in the majority of animals given 750 mg/kg/day and in

some animals given 300 mg/kg/day.

Histopathological changes related to treatment were seen in the mesenteric lymph nodes,

pancreas and stomach and comprised the following: in the mesenteric lymph nodes

intrasinusoidal erythrocytes were present in five males given 750 mg/kg/day; in the pancreas,

vacuolation of the acinar cells was present in the majority of males and females given

750 mg/kg/day and in some males given 300 mg/kg/day. Treatment-related findings of

hyperplasia of the non-glandular epithelium and hypertrophy of the antral mucosa were seen

in the stomach of the majority of animals given 750 mg/kg/day with hyperplasia also reported

in two males and three females given 300 mg/kg/day. Erosion of the antral mucosa was seen

in five males given 750 mg/kg/day.

Conclusion

It was concluded that the repeated oral administration of Oxirane,

mono[(C12-14-alkyloxy)methyl] derivs to Han Wistar (RccHan™;WIST) rats for 13 weeks

at doses of 100, 300 or 750 mg/kg/day produced lesions in the stomachs of males and females

given 300 or 750 mg/kg/day. Intrasinusoidal erythrocytes were present in the mesenteric

lymph node of males given 750 mg/kg/day and this finding was likely to be secondary to the

lesions seen in the stomach. These changes were considered to result from gastric irritation

occurring at point-of-contact and as such were not attributed to systemic toxicity. A minor

degenerative change (vacuolation) was seen in the pancreas of animals given 750 mg/kg/day

and males given 300 mg/kg/day and this was considered adverse.

The No-Observed-Effect level (NOEL) for systemic toxicity was 100 mg/kg/day for males

and 300 mg/kg/day for females based on the vacuolation reported in the pancreas.

The No-Observed-Effect level (NOEL) for the irritant effects seen in the stomach and the

associated findings was 100 mg/kg/day for males and females.