2-methylpentane-1,5-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2021 to mid 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

revised 2018

Deviations:

no

Remarks:

(There were deviations from the study plan, however these study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.)

GLP compliance:

yes (incl. QA statement)

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:

RccHan:WIST

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited.
- Age at study initiation:43 to 49 days.
- Weight at study initiation:Males: 141 to 196 g; Females: 117 to 153 g
- Fasting period before study: no
- Housing: Polycarbonate body with a stainless-steel mesh lid, Wood based bedding, Aspen gnawing material, Plastic shelter, changed at appropriate intervals or replaced when necessary
- Diet: Teklad 2014C, pelleted diet, ad libitum (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
The concentration of isoflavones (daidzein and genistein) were below the upper limit of 350 µg/gram of rodent diet as suggested in the OECD Test Guideline 408. Teklad 2014C rodent diet can be considered a low phytoestrogen diet.
- Water: tap water, ad libitum (except during the period of urine collection).
- Acclimation period: 2021-05-05 to 2021-05-18

DETAILS OF FOOD AND WATER QUALITY:Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Certificates of analysis are routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen gnawing material.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

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): 12/12

IN-LIFE DATES: From: 2021-05-19 To: 2021-08-19

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gavage, using a suitably graduated syringe and a
flexible cannula inserted via the mouth.
The oral exposure route was selected to simulate the conditions of potential human exposure.
Gavage dosing was selected as no marked differences in the observed effects on the basis of
the exposure route were noted in the preliminary study (14-day study with two exposure
routes, diet and gavage; Labcorp Study Number: 8439540).

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed out
separately for each group. Approximately 60 to 80% 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 it
appeared homogeneous. The final pH adjusted formulation
was stirred for a minimum of 20 minutes using a magnetic
stirrer.
The pH was measured and adjusted to 7.5 ± 0.1 using
hydrochloric acid or sodium hydroxide, as required.
Frequency of preparation: Weekly
Storage of formulation: Refrigerated (2 to 8°C).

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 12 of treatment were analyzed for achieved concentration of the test item.
The mean concentrations of DYTEK A Amine in the doses prepared for administration in Week 1 and 12 were generally in the range -7.6 to +5.3% of the nominal concentration and were therefore within the applied limits of ±10%. The only exception of the result for the 50 mg/mL sample (Group 4) in Week 1, which was above this range (+12.2%). Overall, however, as the majority of the results were within the acceptance criteria and only one result was slightly higher, these results were considered to have confirmed accurate formulation with no impact on study integrity.
The difference from mean remained within 5%, confirming the precision of analysis.
For further details please see respective table in 'Additional information on materials and methods incl tables')

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily, 7 days each week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 + 3 satellite females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses selected for this study were based on the results from a 28-day repeated dose
toxicity study on DYTEK A amine (MPMD) in Sprague-Dawley rats designed to meet the
OECD407 guideline (although that study was conducted in 1990 and therefore predated the
current OECD407 test guideline) and the results from a preliminary toxicity study that was
performed at these laboratories (Labcorp Study Number: 8439540) in which DYTEK A
amine was administered orally, by gavage, or continuously in the diet to RccHan™;WIST
(Han Wistar) rats. The results of Labcorp Study Number 8439540 indicated that the
toxicological profile of DYTEK A amine after either of these forms of administration was
very similar. Liver and spleen weights were decreased and there was a range of
hematological and blood biochemical findings that were similar after gavage or dietary
administration. Given the similarity of the findings seen in the animals by either route of
administration, the decision was taken to administer DYTEK A amine by gavage.
In the earlier four-week study, which had been performed in 1990, the dietary levels were 0,
300, 3000 and 10000 ppm, but analysis of achieved concentration revealed that only 65 to
80% of target concentration was achieved. The results revealed that body weight gain was
reduced by 18% in females given 10000 ppm (approximately 750 mg/kg/day), with a small
reduction of food intake (body weight and food intake increased during the recovery period)
but there was otherwise no evidence of any significant toxicity (Sarver, JW (1990), HLR 366-
90).
DYTEK A amine, when administered orally by gavage to Han Wistar rats for 14 days, caused
low weight gains in Week 1 in males receiving 1000 mg/kg/day, which associated with low
food consumption, and there was also an initial slightly low food consumption in females
receiving this dosage. Slightly low hematocrit, hemoglobin concentration and erythrocyte
count was seen in females receiving 1000 mg/kg/day that associated with an increase of
reticulocyte count. Neutrophil counts were low in males receiving 300 or 1000 mg/kg/day
whilst in the females lymphocyte counts were low at 300 or 1000 mg/kg/day, resulting in the
total leucocyte counts of these animals being low. There was a dose-related increase of
platelet count in males receiving 300 or 1000 mg/kg/day and there was also a small
prolongation of prothrombin times in these animals and a reduction of activated partial thromboplastin times at 1000 mg/kg/day. Activated partial thromboplastin time was also
reduced in females given 1000 mg/kg/day, with one female receiving 300 mg/kg/day being
similarly affected. Blood chemistry findings comprised; high alanine amino-transferase
activity in males receiving 1000 mg/kg/day; high urea at all doses (dose-dependent) in males;
high creatinine in both sexes at 1000 mg/kg/day; high triglyceride concentrations in females
given 300 mg/kg/day and in both sexes given 1000 mg/kg/day; low potassium concentration
in males given 1000 mg/kg/day and at all doses in females; low sodium concentration in
males given 1000 mg/kg/day. Macroscopic examination revealed dark areas on the stomach
of three males given 1000 mg/kg/day, one of which also had depressions in the stomach, and
there were also dark areas on the stomach of two females given 1000 mg/kg/day.
The macroscopic findings that occurred in the stomach, together with the knowledge that the
majority of the potentially adverse findings in hematology and blood chemistry were limited
to animals given this high dose, suggested that the dose of 1000 mg/kg/day may be too high
for longer-term administration and that the highest dose should be between 300 and
1000 mg/kg/day. The following dose levels were therefore selected for this 13-week study: 0,
50, 150 and 500 mg/kg/day.

- Fasting period before blood sampling for clinical biochemistry:
overnight withdrawel of food
- Rationale for selecting satellite groups:
Satellite animals (3 per dose group) used for Week 13 estradiol blood sampling and terminated thereafter.
- Post-exposure recovery period in satellite groups: no
- Dose range finding studies: yes, see above (Labcorp Study Number: 8439540)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2-3x daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Week 1 - daily.
Weeks 2 to 4 - twice weekly (middle and end of week).
Weeks 5 to 13 - once each week.
- Frequency:
Pre-dose observation.
1 to 2 hours after completion of dosing of all groups.
As late as possible in the working day.

- Detailed physical examination:
Once each week for all animals throughout the pretreatment and treatment periods

BODY WEIGHT: Yes
- Time schedule for examinations:
one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Water consumption was recorded, each week from Week -1, by weight (over a three day period on each occasion) for each cage of animals, using water bottles fitted with sipper tubes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and Dose groups that were examined:
pretreatment: all animals
Week 12: all main study animals of group 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 main study animals
- Parameters checked:
• Hematocrit (Hct)
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)
• Mean cell hemoglobin concentration (MCHC)
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all main study animals
- Parameters checked:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Total bilirubin (Bili)
• Bile acid (Bi Ac)
• Urea
• Blood urea nitrogen (BUN)
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• High density lipoprotein (HDL)
• Low density lipoprotein (LDL)
• Triglycerides (Trig)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)
• Globulin (Glob)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration

PLASMA/SERUM HORMONES/LIPIDS: Yes
Thyroid hormones:
- Time of blood sample collection: at necropsy
- Animals fasted: No
- How many animals: all main study animals
Estradiol:
- Time of blood sample collection: during week 13, between 13:00 and 14:00 hours; females that were in proestrus.
(the exact days of sampling varied as to when the individual animals were in proestrus).
Female No. 148 showed no evidence of proestrus and, as such, was sampled on the last day of smearing.
- Animals fasted: No
- How many animals: all satellite animals (3 females per dose)

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter
• Bile pigments (Bili)
• Blood pigments (UBld)
• Protein - total (T-Prot) and concentration (Prot)
• Creatinine - total (T-Creat) and concentration (U-Creat)
• Glucose - total (T-Gluc) and concentration (U-Gluc)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12 of treatment (before dosing)
- Dose groups that were examined: all main study animals
- Battery of functions tested: sensory activity / grip strength / motor activity:
*Sensory Reactivity Observations and Grip Strength:
Approach response
Pinna reflex
Auditory startle reflex
Tail pinch response
*Grip strength:
Forelimb and hindlimb grip strength
*Motor Activity:
Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

IMMUNOLOGY: No

OTHER:
ESTROUS CYCLES
Wet smears were taken from the vagina of all main study females using pipette lavage for four days before scheduled termination. The last smear was taken on the morning of necropsy.
Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) at termination and were used to assist in the histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 in 'Any additional information on materials and methods incl. tables')

HISTOPATHOLOGY: Yes (see table 1 in 'Any additional information on materials and methods incl. tables')

Other examinations:

SPERM ANALYSIS
Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed, the following tests were performed:
Sperm motility - all groups
Sperm morphology - all groups
Sperm count - all groups
Homogenization-resistant spermatids count- all groups

STAGE-DEPENDENT EVALUATION OF SPERMATOGENESIS
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all animals prepared and stained using the PAS method. A qualitative examination of spermatogenic stages was made for normal progression of the stages of the spermatogenic cycle, cell associations, and proportions expected to be present during normal spermatogenesis.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
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
Urinalysis
Sperm analysis
Thyroid hormones (T3, T4 and TSH)
Organ weights, absolute or adjusted for terminal body weight and relative to body weight
The following comparisons were performed:
Group 1 vs 2, 3 and 4
The following statistical tests were used for grip strength, motor activity, body weight, organ weight, sperm analysis and clinical pathology data where required:
Bartlett's test
analysis of variance
inter group comparisons using t-tests, with the error mean square from the
one-way analysis of variance
F1 approximate test
Williams' test for a monotonic trend
Dunnett's test
logarithmic and square-root transformations.
Kruskal-Wallis’ test
Wilcoxon rank sum tests
H1 approximate test
Shirley's test
Steel's test
The Reviewer considers the analyses used to be appropriate.
For more detailed information please refer to the attached file type 'full statistical method' in section 'overall remarks, attachments'.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the weekly clinical observation, two males and two females receiving 500 mg/kg/day had
salivation but this is a common sign in studies where the test material is administered by
gavage and, as such, is considered of no toxicological significance.
For more details on the results in tabular form, please refer to table 12.1 in the attached file type 'full results tables'.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female No. 127 (Group 4; 500 mg/kg/day) was found dead in Week 5 but there had been no
clinical signs reported ante mortem. The macroscopic findings were limited to an inability to
flush the nasal turbinates with fixative. Histopathological examination revealed severe
autolysis in multiple organs, and agonal congestion in liver and lung, but no significant
microscopic findings. The cause of death was therefore undetermined, but as there had been
no other premature deaths or marked toxicity at this dose, the death of this animal was most
likely incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Between Week 5 and 13 there was a statistically significant reduction of weight gain by
males receiving 150 or 500 mg/kg/day (25 and 21%, respectively) and there was also a small
(approximately 13%) reduction of weight gain during this period by males receiving
50 mg/kg/day. Overall (Week 0-13) weight gain was 12% lower than control in males
receiving 150 or 500 mg/kg/day and 8% lower than control in males receiving 50 mg/kg/day.
All individual gains (Week 0-13) were within the historical control data (98-percentile range:
160-310 g; n=69).
Body weight gain was unaffected in females.
For more details on the results in tabular form, please refer to table 12.4 in the attached file type 'full results tables'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment. For more details on the results in tabular form, please refer to table 12.5 in the attached file type 'full results tables'.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Overall group mean water intake (Week 0-13) was 14% lower than control in females
receiving 500 mg/kg/day, with values generally being low from Week 2 onwards.
Conversely, water intake was 13% higher than control in females receiving 150 mg/kg/day
but there was a high degree of variation in this dose group. Statistical significance was not
attained for any of the differences from control and there was no similar finding in males.
For more details on the results in tabular form, please refer to table 12.6 in the attached file type 'full results tables'.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmoscopic findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematological examination performed during Week 13 revealed, compared to controls,
slightly, but statistically significantly, low hematocrit, hemoglobin concentration and
erythrocyte count and statistically significantly high reticulocyte count, mean cell
hemoglobin and mean cell volume in females receiving 500 mg/kg/day. Reticulocyte count
was also slightly higher than control in females receiving 150 mg/kg/day. All individual
values were, however, within the historical control data (98-percentile range: hematocrit:
0.384 to 0.502 L/L; hemoglobin concentration: 13.6 to 16.1 g/dL; erythrocyte count:
6.90x1012 to 8.73x1012/L; reticulocyte count: 0.096x1012 to 0.237x1012/L; mean cell
hemoglobin: 17.3 to 21.3 pg; mean cell volume: 52.5 to 62.9 fL; n=241) except for the
erythrocyte count, mean cell hemoglobin and mean cell volume for one female receiving
500 mg/kg/day (No. 122). There were no similar findings in males.
All other inter-group differences, including those attaining statistical significance, were minor
or lacked dose-relationship and were therefore attributed to normal biological variation. Such
differences included the high total leucocyte, neutrophil, eosinophil and basophil counts in
males receiving 500 mg/kg/day, which were attributed to one individual (No. 12) with
particularly high leucocyte counts. They also included the statistically significantly low
hematocrit, hemoglobin concentration and erythrocyte count in females receiving
50 mg/kg/day where, in contrast to the findings in the high dose group, the other red cell
indices were unaffected and there were no similar findings at 150 mg/kg/day.
For more details on the results in tabular form, please refer to table 12.7 in the attached file type 'full results tables'.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed during Week 13 revealed,
compared to controls, slightly high alanine amino-transferase activities in males receiving
500 mg/kg/day, with statistical significance being attained. All individual values were,
however, within the background control range (21 to 84 U/L; n=245) and there was no
similar finding in females.
There was a statistically significant and dose-related increase of bile acid concentrations at all
doses in males, with females receiving 500 mg/kg/day being similarly affected. All individual
values were within the historical control data (98-percentile range: 4 to 68 µmol/L in males
(n=170) and 5 to 130 µmol/L in females (n=167)) except for two males receiving
500 mg/kg/day which had values that were slightly above this range.
In males, total cholesterol and high-density lipoprotein concentrations were lower than
control at 500 mg/kg/day but all individual values were within the historical control data (98-
percentile range: total cholesterol concentration: 1.34 to 3.50 mmol/L (n=245); high density
lipoprotein concentration: 1.11 to 3.05 mmol/L (n=120)) except for two males receiving
500 mg/kg/day and one control animal which had high density lipoprotein concentrations that
were slightly below the historical control data. In both sexes receiving 500 mg/kg/day,
triglyceride concentrations were higher than control, with statistical significance being
attained in females, but all individual values were within or, in one individual, slightly below
the historical control data (98-percentile range: 0.32 to 1.54 mmol/L in males (n=115) and
0.20 to 1.28 mmol/L in females (n=114)) with the exception of one male receiving
500 mg/kg/day with a high value (No. 16; 2.07 mmol/L).
There were some variations to plasma electrolyte concentrations in females, compared with
control, where chloride (statistically significant) and potassium concentrations were low at
50, 150 or 500 mg/kg/day and sodium concentration was low at 500 mg/kg/day, but all
individual values were within the historical control data (98-percentile range: chloride: 94.0
to 105.0 mmol/L (n=104); potassium: 3.04 to 5.26 mmol/L; sodium: 134 to 147 mmol/L
(n=243)). Males were unaffected. In males there was an increase of plasma phosphorus
concentration at 500 mg/kg/day, where all individual values were within the historical control
data (98-percentile range 1.31-2.96 mmol/L (n=105)) and there was no similar finding in
females.
Total protein concentration was lower than controls in males receiving 500 mg/kg/day and
this was attributed to a reduction of both the albumin and globulin fractions. Females were
unaffected.
All other inter-group differences, including those attaining statistical significance, were minor
or lacked dose-relationship and were therefore attributed to normal biological variation.
These differences included the statistically significantly high albumin to globulin ratio at all
doses in females, where there was no dose-response and the extent of the difference from
controls was small. They also included the statistically significantly low glucose
concentrations in males receiving 500 mg/kg/day, where the control group mean values was
increased by several individuals with high values, some of which were above the background
control range).
For more details on the results in tabular form, please refer to table 12.8 in the attached file type 'full results tables'.

Endocrine findings:

no effects observed

Description (incidence and severity):

IMPORTANT NOTE: T3/T4 data was QC checked, but not audited at the time point the update was requiered and thus minimal changes might still occur once the final report is available.

The analysis of serum thyroid hormone concentrations after 13 weeks of treatment showed that TSH, T3 and T4 levles were unaffected by treatment.
Plasma estradiol concentration at proestrus was unaffected by treatment.
(Note: Insufficient sample volume was received for estradiol analysis from animal No. 145 (Group 2) and animal No. 147 (Group 3). As a result, only two animals were reported for Group 2 and 3. Furthermore, the result for animal No. 148 (Group 3) was below the lower limit of quantification and, as such, no mean could be calculated for Group 3.)
Please refer to table 2 in 'Any other information on results incl. tables'.

Generally it should be stressed that fluctuation in hormone levels are well known and even statistical significant effects are often not biologically relevant (Beekhuijzen M, Rijk JCW, Meijer M, de Raaf MA and Pelgrom SMG, 2019. A critical evaluation of thyroid hormone measurements in OECD test guideline studies: Is there any added value? Reproductive Toxicology, 88, 56-66.).

Estrous cyclicity was unaffected by treatment.
For more details on the results in tabular form, please refer to table 12.10 in the attached file type 'full results tables'.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The examination of the urine performed during Week 13 revealed, compared with controls,
low volume associated with high specific gravity in males receiving 500 mg/kg/day but all
individual values were within the historical control data (98-percentile range: volume: 1.5 to
9.0 mL; specific gravity: 1024 to 1069 g/L; n=125), although there were two control animals
with urine volume values that were above the historical control data range and one control
male and one high dose male with specific gravity results that were outside of this range.
There was a statistically significant, dose-related decrease of pH in males receiving 50, 150
or 500 mg/kg/day. The individual values at 50 or 150 mg/kg/day were within the historical
control data, but at 500 mg/kg/day, 9/10 values were below the range (98-percentile range:
5.7-8.6 (n=125)).
Total urinary protein output was higher than controls in both sexes receiving 500 mg/kg/day,
with two males and three females having protein output that was above the background
control range (1.793 to 10.500 mg in males (n=115) and 0.197 to 1.053 mg in females
(n=128)).
There was a dose-related increase of chloride output at all doses and in both sexes, though the
increase at 50 mg/kg/day in females was minimal and was not statistically significant. This
associated with low sodium output in females receiving 500 mg/kg/day.
There was a reduction of creatinine output, compared to controls, in both sexes receiving
500 mg/kg/day but all individual creatinine values were within the background control range
(21.998 to 77.939 µmol in males (n=20) and 0.569 to 46.997 µmol in females (n=44)).
All other inter-group differences, including those attaining statistical significance, were minor
or lacked dose-relationship and were therefore attributed to normal biological variation. Such
differences included the low specific gravity in females receiving 50 or 150 mg/kg/day and
the variations to glucose output in both sexes, where there was clearly no dose-response.
For more details on the results in tabular form, please refer to table 12.9 in the attached file type 'full results tables'.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Motor activity:
In males, there was a treatment-related reduction of total low (cage-floor) and high (rearing)
beam-breaks, with reductions at several timepoints attaining statistical significance compared
with control. Statistical significance was also attained for the total high beam-breaks at
500 mg/kg/day and at all dose levels for the total low beam-breaks, although the difference
from control at 50 or 150 mg/kg/day was slight. Several of the individual total beam-breaks
were also below the historical control data (98-percentile range: high beam-breaks: 207.9 to
358.3; low beam-breaks: 514.3 to 947.6; 37 studies), particularly at 150 or 500 mg/kg/day.
However, several control animals had values that were above this range and the mean of
concurrent controls for both high and low beam-breaks (320.5 and 860.9, respectively), were
above the mean of historical controls (288.3 and 704.6, respectively).
In females, there was a reduction of low (cage-floor) and high (rearing) beam-breaks at
500 mg/kg/day, but statistical significance was not attained and the difference from control
was slight (10 and 29% reductions, respectively).

Sensory Reactivity and Grip Strength:
Sensory reactivity was considered to have been unaffected by treatment.
Hind limb grip strength was lower than concurrent control at all doses in males, though the
extent of the reduction was not dose-related, the means for all dose groups were
approximately the same as the mean of the historical control mean of 37 studies and fore limb
grip strength (the more sensitive marker of any effect on grip strength) was lower than
controls in males receiving 500 mg/kg/day. The majority of individual mean values were
within the historical control data (98-percentile range: 0.88 to 1.30 kg; 37 studies; two males
receiving 500 mg/kg/day had values that were slightly below the background control range
but a further two males in this group had mean values above the range). Moreover, the means
of the concurrent controls for both fore and hind limb grip strength were above the historical
control means. There was no similar finding in females.
When taking the historical control data into consideration, grip strength was considered to
have been unaffected by treatment.
For more details on the results in tabular form, please refer to tables 12.2 and 12.3 in the attached file type 'full results tables'.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Please refer to table 3 in 'Any other information on results incl. tables'.
The analysis of organ weights performed after 13 weeks of treatment revealed, compared
with control, statistically significantly high body weight adjusted liver weights in males
receiving 500 mg/kg/day and in females receiving 150 or 500 mg/kg/day, and high body
weight adjusted kidney weights in females receiving 150 or 500 mg/kg/day. All individual
values were within the historical control data (98-percentile range for absolute weights: liver: 9.241 to 19.304 g in males and 6.400 to 11.435 g in females; kidneys: 1.599 to 3.096 g in
males and 1.059 to 1.946 g in females (n=180)), except for the liver weight of one control
female and one female receiving 50 mg/kg/day, which had values that were below the
historical control data.
Heart weights were slightly lower than control in both sexes receiving 500 mg/kg/day and in
females receiving 150 mg/kg/day, with statistical significance being attained for the body
weight adjusted values. All individual values were within the historical control data (98-
percentile range for absolute weight: 0.786 to 1.742 g in males and 0.618 to 1.105 g in
females (n=180)), except for one female receiving 500 mg/kg/day (No. 122; 0.601 g).
No histopathological correlate was identified to account for the variations to liver, kidney and
heart weights and, consequently, they were considered non-adverse.
All differences in organ weights, statistically significant or not, were consistent with normal
variation and considered incidental. These differences were characterized by one or more of
the following: inconsistency between sexes; lack of a dose relationship or correlative
findings; and/or the magnitude was considered small.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Please refer to table 4 in 'Any other information on results incl. tables'.
Changes related to treatment with DYTEK A amine were confined to the stomach of males
administered 500 mg/kg/day.
The macroscopic examination performed after 13 weeks of treatment revealed the presence of
dark areas (correlated histologically with congestion/hemorrhage) and thickening of the
stomach (correlated histologically with edema) in a low number of males given
500 mg/kg/day.
All other macroscopic findings were considered spontaneous or incidental because they
occurred at a low incidence, were randomly distributed across groups (including concurrent
controls), and/or were as expected for Han Wistar Rats of this age. Therefore, they were
considered not test article related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please refer to table 5 in 'Any other information on results incl. tables'.
Changes related to treatment with DYTEK A amine were confined to the stomach of males
only.
In the glandular stomach, focal slight congestion/hemorrhage, edema and mixed
inflammatory cell infiltrate associated with focal minimally increased zymogen granules
(chief cells) were observed in two males administered 500 mg/kg/day. These changes
correlated with the dark areas and thickening seen macroscopically in this group.
Mononuclear inflammatory cell infiltrate was present in one male administered
150 mg/kg/day, but in the absence of any other findings this was considered likely to be
incidental rather than test item related.
All other microscopic findings were considered spontaneous or incidental because they
occurred at a low incidence, were randomly distributed across groups (including concurrent
controls), and/or were as expected for Han Wistar Rats of this age. Therefore, they were
considered not test article related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

SPERM ANALYSIS
The morphological examination of the sperm revealed, compared with control, a slight but
statistically significant increase in total abnormal sperm, associated with a statistically
significant decrease in normal sperm, in males receiving 500 mg/kg/day. The control group
mean value was, however, lower than the historical control data range. There was also a
minimal increase in the incidence of abnormalities, including decapitates, head, neck and tail
abnormalities at all dose levels but this was unlikely to be of any toxicological significance.
All values were within or below the background control range (mean total abnormal sperm
3.0-5.5% (n=97 animals from 4 studies)).
At 150 mg/kg/day testis spermatid count (millions /g) and total (million) was lower than
control and outside the historical control range (spermatid count 84-101 millions/g and 167-199 million total). In the absence of any effect on sperm motility in the high dose group, this
was considered unrelated to treatment.
For more details on the results in tabular form, please refer to tables 12.11, 12.12 and 12.13 in the attached file type 'full results tables'.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity

1

Critical effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

2

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

other: unspecific toxicity

Organ:

other: adverse weight gain reduction

Treatment related:

yes

Dose response relationship:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Statistically significant compared to Group 1 (* p≤0.05; ** p≤0.01)

 

Table 2: Summary of Thyroid Hormone Levels after 13 Weeks of Treatment.

T3/T4 and TSH: Dosed groups compared to the Control group using Williams’ test.

Estradiol: Analysis of sattelite animals: as a deviation from the study plan - as there were insufficient data points for analysis, statistical
analysis was therefore, not performed. These study deviations neither affected the overall interpretation of study findings nor
compromised the integrity of the study. Full tabular results are provided in the attached file type 'estradiol full results' in section 'overall remarks, attachments'.

For detailed information on statistical evaluation please refer to the attached file type 'full statistical method' in section 'overall remarks, attachments'.

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose level (mg/kg/day)	0	50	150	500	0	50	150	500
Thyroxine (pg/mL) (T4)	30610	46250	42020	35570	42360	31443	35690	39389
Triiodothyronine (pg/mL)(T3)	507	606	572	443	747	933	832	831
Thyroid stimulating hormone (pg/mL)(TSH)	921	1020	944	793	503	563	537	433
Estradiol concentration (pg/mL)					29.7	42.8	-	36.3

Table 3: Test Article-Related Effects in Organ Weights – Terminal Sacrifice After 13 Weeks of Treatment. Dosed groups compared to the Control group using Williams’ test. For detailed information on statistical evaluation please refer to the attached file type 'full statistical method' in section 'overall remarks, attachments'. Statistically significant compared with control (* = p≤0.05; ** = p≤0.01)

Body weight adjusted organ weights are presented here and in the attachment file type 'organ weights full tables' in table 5.1. Body weight relative organ weights are presented and can be assessed in the attachment file type 'organ weights full tables' in table 5.2. The emphasis in this section and for the assessment is laid on body weight adjusted organ weights, since there is considerable experimental and observational evidence, that the assumption of organ weight as a simple proportion of the terminal body weight (= body weight relative organ weight) does not generally hold. According to Angervall and Carlstorm (ANGERVALL, L. and CARLSTROM, E. (1963) Theoretical criteria for the use of relative organ weights and similar ratios in biology.  Journal of Theoretical Biology, 4, 254-259), analysis of covariance (ANCOVA) is the method of choice to overcome the inherent inaccuracy in interpreting body weight relative organ weights and was chosen here, to adjust the organ weight group means according to how far the terminal body weight deviates from the average body weight over all groups.

Note: Values for absolute weight and ratio body weight-adjusted weights dosed groups expressed as percentage control mean value

Sex	DYTEK A amine
	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Liver
Absolute Weight (g)	12.354	97	96	100	7.368	104	109	110
Body Weight Adjusted (g)	11.682	104	104	107*	7.368	106	109*	109*
Kidney
Absolute Weight (g)	2.249	95	91	94	1.286	105	108	112
Body Weight Adjusted (g)	2.141	101	98	100	1.286	105	108*	111**
Heart
Absolute Weight (g)	1.064	95	94	88	0.779	99	94	92
Body Weight Adjusted (g)	1.020	100	100	93*	0.779	99	94*	92**
Adrenals
Absolute Weight (g)	0.062	95	97	92	0.069	97	99	97
Body Weight Adjusted (g)	0.060	98	102	97	0.069	97	99	97
Brain
Absolute Weight (g)	2.120	100	94	96	1.915	100	100	96
Body Weight Adjusted (g)	2.082	103	97	98	1.915	100	100	96*
Epididymides
Absolute Weight (g)	1.393	98	98	96	NA	NA	NA	NA
Body Weight Adjusted (g)	1.377	99	99	97	NA	NA	NA	NA
Ovaries
Absolute Weight (g)	NA	NA	NA	NA	0.092	95	99	98
Body Weight Adjusted (g)	NA	NA	NA	NA	0.092	95	99	98
Pituitary
Absolute Weight (g)	0.010	90	90	80	0.014	93	100	100
Body Weight Adjusted (g)	0.009	100	100	89	0.014	93	100	93
Prostate, Seminal Vesicles and Coagulating Gland
Absolute Weight (g)	2.370	92	92	99	NA	NA	NA	NA
Body Weight Adjusted (g)	2.270	97	98	105	NA	NA	NA	NA
Spleen
Absolute Weight (g)	0.679	99	93	101	0.486	100	107	101
Body Weight Adjusted (g)	-	-	-	-	0.486	100	107	101
Testes
Absolute Weight (g)	3.819	96	95	101	NA	NA	NA	NA
Body Weight Adjusted (g)	3.732	99	98	104	NA	NA	NA	NA
Thymus
Absolute Weight (g)	0.322	96	101	69	0.258	105	108	83
Body Weight Adjusted (g)	0.308	101	108	73**	0.258	106	107	82
Thyroids and Parathyroids
Absolute Weight (g)	0.019	95	95	100	0.016	94	94	100
Body Weight Adjusted (g)	0.020	90	90	95	0.016	94	94	100
Uterus and Cervix
Absolute Weight (g)	NA	NA	NA	NA	0.634	91	136	104
Body Weight Adjusted (g)	NA	NA	NA	NA	0.598	95	132	108

 

 

 

 

Table 4: Incidence and Severity of Test Article-Related Macroscopic Findings – Terminal Sacrifice After 13 Weeks of Treatment For detailed information on any other organ not reported here because of lacking any adverse effects please refer to the attached file type ‘macropathology data (table)’ in section 'overall remarks, attachments'.

Sex	DYTEK A amine
	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Stomach
Number Examined	10	10	10	10	10	10	10	9
Dark Area	0	0	0	2	0	0	0	0
Thickened	0	0	0	1	0	0	0	0

 

Table 5: Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice After 13 Weeks of Treatment For detailed information on any other organ not reported here because of lacking any adverse effects please refer to the attached file type ‘histopathology data (table)’ in section 'overall remarks, attachments'.

Sex	DYTEK A amine
	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Stomach
Number Examined	10	10	10	10	10	0	0	9
Congestion/Hemorrhage
Slight	0	0	0	2	0	0	0	0
Edema
Slight	0	0	0	1	0	0	0	0
Infiltrate, inflammatory Cells
Slight	0	0	1	1	0	0	0	0
Zymogen Granules, Increased, Chief Cells
Minimal	0	0	0	2	0	0	0	0

 

Applicant's summary and conclusion

Conclusions:

It is concluded that the oral administration of DYTEK A amine to Han Wistar rats at doses of
0, 50, 150 or 500 mg/kg/day were generally tolerated but caused an adverse reduction of
weight gain in males receiving 150 or 500 mg/kg/day and adverse histopathological findings
in the stomach of a few males receiving 500 mg/kg/day that were likely responses to an
irritant formulation. Non-adverse findings included evidence of adaptive responses by the
liver and kidney in both sexes receiving 150 or 500 mg/kg/day. In view of the non-specific
toxicity that occurred in males, the no-observed-adverse-effect level (NOAEL) in this study
was 50 mg/kg/day in males and 500 mg/kg/day in females.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of the submission substance (i.e. MPMD, DYTEK A amine, an industrial chemical), when administered orally (by gavage) to Han Wistar rats for 13 weeks.
Three groups, each comprising ten males and ten females, received DYTEK A amine at doses of 50, 150 or 500 mg/kg/day. A similarly constituted control group received the vehicle (purified water) at the same volume dose as the treated groups. A further three females were allocated to each group of females and were used to provide blood samples for plasma estradiol evaluation.
During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food and water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry,
urinalysis, estradiol analysis, thyroid hormone, estrous cycle, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken.

In this study, doses of 50, 150 or 500 mg/kg/day were tolerated though there was the development of a non-specific toxic response in males. The stomach was the main target organ and there were findings in this study that were indicative of an effect on liver and kidney function. There was no evidence that DYTEK A amine affected the hormonal control of the thyroid gland, since serum thyroid hormone levels were unaffected (TSH, T3, T4 and estradiol) and there was no effect on thyroid gland weight and pathology. In addition, there was no effect on the female reproductive system since estrus cyclicity and estradiol levels at proestrus were unaffected and there were no histopathological findings in the reproductive system of females.

There was an adverse, progressive reduction of weight gain from Week 5 in males receiving 150 or 500 mg/kg/day that was indicative of a non-specific response to the toxicity of DYTEK A amine. Between Week 5 and 13 the gains of these animals were reduced by <20%
compared to controls during the same period, although the overall body weight gain of all animals during the 13-week treatment period was within the historical control data range.
There was no associated reduction of food intake and no clinical signs (treatment-related clinical signs in this study were limited to salivation in a few animals, which is a common finding in studies where the test material is administered by gavage and, as such, is of no
toxicological significance). In addition, there were no deaths that were attributable to treatment. It is possible that the slight reductions of motor activity in males given 150 or 500 mg/kg/day and the increase of abnormal sperm at the highest dose may be associated
with this non-specific toxic response.
Histopathological findings that were due to treatment were confined to the stomach, where there was slight congestion/hemorrhage, edema and mixed inflammatory cell infiltrate associated with minimal increase of zymogen granules (chief cells) reported in males
receiving 500 mg/kg/day. This affected only a few high dose males and the findings may have been a response to a slightly irritant test formulation. However, over a longer treatment period these findings can be adverse.
There were findings that were indicative of an effect upon liver and kidney function.
Liver weights were high after 13 weeks in males given 500 mg/kg/day and in females given 150 or 500 mg/kg/day. There were some findings in the blood plasma (high alanine aminotransferase
activities in males receiving 500 mg/kg/day, high bile acid concentrations at all doses in males and at 500 mg/kg/day in females, low total cholesterol and high-density lipoprotein concentrations at 500 mg/kg/day in males, high triglyceride concentrations in both
sexes receiving 500 mg/kg/day and low protein concentration in males receiving 500 mg/kg/day) that were indicative of an effect upon liver function. The majority of individual values were within the historical control data range, with the exception of a few animals with bile acid, high density lipoprotein and triglyceride concentrations that were slightly outside of the range. There was no associated histopathological finding, despite the increased plasma alanine amino-transferase activities in the high dose males. There was no evidence of any increased hepatocellular vacuolation associated with the effect on cholesterol and triglyceride. Consequently, these findings were considered non-adverse and were likely secondary to a change in liver function as a result of metabolism of high doses of a xenobiotic.

Kidney weights were high after 13 weeks in females given 150 or 500 mg/kg/day. There was a reduction of water intake in the high dose females and some findings in the blood plasma (low sodium, chloride and potassium concentrations) and changes in urinary composition (high urinary protein output and increased chloride and decreased sodium output at 500 mg/kg/day) that were consequences of an effect on the kidneys. All individual values for the blood plasma parameters were within the historical control data range, but several values for the urinalysis parameters were outside of the range. In males there were findings at 500 mg/kg/day, which comprised low volume associated with high specific gravity and high urinary protein output with several values being outside of the historical control data range.
For both sexes, there was, however, no associated histopathological finding. Consequently, these findings were considered non-adverse and were likely secondary to a change in kidney function as a result of renal excretion of DYTEK A amine and/or its metabolites.

There was no histopathological correlate for the alterations to the red cell indices in females receiving 500 mg/kg/day (low hematocrit, hemoglobin concentration and erythrocyte count and high reticulocyte count, mean cell hemoglobin and mean cell volume), or the high reticulocyte count in females receiving 150 mg/kg/day, i.e. there was no evidence of compensatory extramedullary hemopoiesis and there were no findings in the bone marrow.
There was no increase of plasma bilirubin concentration that would indicate increased erythrocyte turnover. The majority of individual values were within the historical control data range. Similarly, there was no histopathological finding that accounted for the low heart
weights in both sexes given 500 mg/kg/day and in females given 150 mg/kg/day. Consequently, these variations were considered non-adverse.

In summary due to the non-specific toxicity that occurred in males, the no-observed-adverse-effect level (NOAEL) in this study was 50 mg/kg/day in males and due to no adverse findings in females the NOAEL was 500 mg/kg/day, i.e. the highest dose tested,  in females.