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REACH

Cyclohex-1,2-ylenediamine

EC number: 211-776-7 | CAS number: 694-83-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

DCH (DYTEK DCH-99) was applied to male and female Han Wistar rats via gavage (vehicle water, pH adjusted formulation) at doses of 0, 50, 150 or 500 mg/kg bw/day according to OECD TG 408 (daily exposure for 13 weeks). In view of the various effects seen at the high and mid dose groups tested, the no-observed-adverse-effect level (NOAEL) in this oral gavage study was 50 mg/kg bw/day for males and 150 mg/kg bw/day for females.

In a subacute inhalation toxicity study male rats were exposed for 6 hours per day and overall 10 times within two weeks to an aerosol/vapour mixture of DCH. Concentrations used were 0, 10, 49 and 240 mg/m³ (analytical). Local effects on the upper respiratory tract were observed in each dose group. Therefore, a LOAEC of 10 mg/m³ was established and classification with respect to specific target organ toxicity after short-term exposure is proposed: (STOT SE Cat 3, H335, together with Corr. Cat 1A).

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:

From 4th May 2021 to mid 2022

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)

Version / remarks:

revised 2018

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Remarks:

RccHan:WIST rat

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: 41 to 47 days
- Weight at study initiation: Males: 134 to 176 g; Females: 112 to 144 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 and blood chemistry and during the period of urine collection).
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
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 2021-05-16

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: 2021-05-17 To: 2021-08-17

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 results from a preliminary toxicity study that was performed at these laboratories (Labcorp Study Number: 8439542), in which DYTEK DCH-99 was administered orally, by gavage, or continuously in the diet to RccHan™;WIST (Han Wistar) rats, indicated that the toxicological profile of DYTEK DCH-99 after either of these forms of administration was very similar, with liver and kidney weights being increased and the range of hematological and blood biochemical findings also being similar. However, a problem was encountered when diet samples that had been taken from the diet formulations prepared for the study, were analyzed. The samples were analyzed for achieved content of the test item and the results obtained were not consistent and substantially below the nominal concentrations. Given the similarity of the findings seen in the animals by either route of administration, the decision was taken to conduct the 13-week study by the oral gavage route, for which there was no significant variation from the intended formulation concentrations.

The preliminary study concluded that oral gavage administration of the test item caused low
weight gain in males and findings in males and females that indicated that the liver and
kidneys were potential target organs. The range of findings seen at 1000 mg/kg/day in males
suggested that this dose would be too high for longer-term administration and that the highest
dose should be between 300 and 1000 mg/kg/day; the dose of 1000 mg/kg/day would be
expected to be tolerated for 13-weeks by females.

In combination with the knowledge from a Combined 28-day Repeated Dose Toxicity Study
with the Reproduction/Developmental Toxicity Screening Test of DYTEK® DCH-99 in Rats
by Oral Gavage 28 (oral gavage; NOTOX B. V. Project No. 479003, 2007), the following
dose levels were selected: 0, 50, 150 and 500 mg/kg/day.

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, and was prepared in advance of the first day of dosing.
Storage of formulation: Refrigerated temperature (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 DCH-99 in the doses prepared for administration in Week 1 and 12 were within the range -5.2 to -0.2% of the nominal concentration and were therefore within the applied limits of ±10%, confirming accurate formulation. The difference from mean remained within 5%, confirming the precision of analysis.
For further information please refer to table 'Analyzed Concentrations for DYTEK DCH-99 in Water' in section 'Additional information on materials and methods incl tables'.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily, 7 days each week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The preliminary study (Labcorp Study Number: 8439542) concluded that oral gavage administration of the test item caused low weight gain in males and findings in males and females that indicated that the liver and kidneys were potential target organs. The range of findings seen at 1000 mg/kg/day in males suggested that this dose would be too high for longer-term administration and that the highest dose should be between 300 and 1000 mg/kg/day; the dose of 1000 mg/kg/day would be expected to be tolerated for 13-weeks by females.
In combination with the knowledge from a Combined 28-day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of DYTEK® DCH-99 in Rats by Oral Gavage 28 (oral gavage; NOTOX B. V. Project No. 479003, 2007), the following dose levels were selected: 0, 50, 150 and 500 mg/kg/day.

- Fasting period before blood sampling for clinical biochemistry:
overnight withdrawal of food

- Dose range finding studies:
yes, see above, Labcorp Study Number: 8439542

Positive control:

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

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 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 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 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 animals

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 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 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 section 'Additional information on materials and methods incl tables')

HISTOPATHOLOGY: Yes (see table 1in section 'Additional information on materials and methods incl tables')

- Full List: All animals of Groups 1 and 4
- Abnormalities, spleen, mesenteric lymph nodes, left axillary lymph nodes, Peyer’s patches and lungs: All animals of Groups 2 and 3
- Epididymides, prostate, seminal vesicles with coagulating gland, testes, heart and adrenal glands: All terminal males of Groups 2 and 3

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, thyroid hormones 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
For more details please see also the attachment '8439543-statistical methods' in section 'overall remarks, attachments'
The study monitor considers the analyses used to be appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse clinical signs during the study.
At the weekly clinical observation, there was a higher incidence of salivation, compared to
controls, in females receiving 500 mg/kg/day 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 further details on the results of the detailed physical examinations and arena observations - group distribution of observations presented in tabular form please refer to table 12.1 in the attached file type "full results tables".

Mortality:

no mortality observed

Description (incidence):

no deaths occurred during the study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a reduction of body weight gain in males receiving 500 mg/kg/day, compared with
control, which occurred from Week 2 of dosing onwards. From Week 2-13, males receiving
500 mg/kg/day gained 26% less body weight than the controls, which resulted in overall gain
during the treatment period (Week 0 to 13) being 21% lower than control and statistically
significant.
Body weight gain was unaffected in males receiving 50 or 150 mg/kg/day and at all dose
levels in females.
For further details on the results of body weight - group mean values measurements presented 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 further details on the results of food consumption - group mean values evaluation presented 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):

effects observed, treatment-related

Description (incidence and severity):

Water intake was consistently higher than control in males receiving 150 or 500 mg/kg/day,
with the overall (Week 1 to 13) intake being 22 or 23% higher than controls, respectively.
There was no similar finding in males receiving 50 mg/kg/day, or at any dose level in
females.
For further details on the results of water consumption - group mean values evaluation presented 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 analysis performed during Week 13 revealed, compared with controls,
low lymphocyte, eosinophil, basophil and large unstained cell counts, resulting in low total
leucocyte counts, in both sexes receiving 500 mg/kg/day. In addition, monocyte count was
slightly lower than control in females receiving 500 mg/kg/day. In males, all individual total
leucocyte and lymphocyte counts were below the historical control data (98-percentile range:
total leucocyte count 3.41x109 to 10.92x109/L; lymphocyte count 2.60x109 to 8.65x109/L
(n=236)), with the exception of the total leucocyte count for one animal. In both sexes,
several individual eosinophil, large unstained cell and monocyte counts (females only) were
below the historical control data (98-percentile range: eosinophil count 0.02x109 to
0.23x109/L in males (n=236) and 0.01x109 to 0.18x109/L in females (n=239); large unstained
cell count: 0.01x109 to 0.14x109/L in males (n=236) and 0.00x109 to 0.12x109/L in females
(n=239); monocyte count: 0.03x109 to 0.28x109/L in females (n=239)).
There were statistically significant decreases of hematocrit and hemoglobin concentration,
compared to control, in females receiving 500 mg/kg/day which associated with slightly low
mean cell hemoglobin and mean cell hemoglobin concentration and high reticulocyte count
and red cell distribution width. The majority of 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; mean cell hemoglobin 17.3 to 21.3 pg; mean cell
hemoglobin concentration 29.2 to 38.1 g/dL; reticulocyte count: 0.096x1012 to 0.237x1012/L;
red cell distribution width: 9.4 to 12.1% (n=241)), with the exception of one or two females
with marginally lower hemoglobin concentration or mean cell hemoglobin and slightly higher
reticulocyte count. There were no similar findings in males.
All other inter-group differences from control, including those attaining statistical
significance, were minor or lacked dose response, and were therefore attributed to normal
biological variation. Such differences included the statistically significantly shorter
prothrombin time in females receiving 500 mg/kg/day, where there was no effect on activated
partial thromboplastin times and such small decreases are considered to be of no toxicological importance. They also included the statistically significantly low reticulocyte
count in males receiving 50 mg/kg/day, where there was no similar finding in the higher dose
groups and the slightly high mean cell hemoglobin concentration in males receiving
150 mg/kg/day, where there was no effect on hematocrit, hemoglobin concentration or
erythrocyte count.
For further details on the results of hematology - group mean values during week 13 of treatment presented 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 in Week 13 revealed, compared
with control, statistically significantly high alanine and aspartate amino-transferase activities
in both sexes receiving 500 mg/kg/day, with alanine amino-transferase activity also being
marginally high in males receiving 150 mg/kg/day. In males receiving 500 mg/kg/day, the
majority of individual alanine amino-transferase activities were above the historical control
data (98-percentile range: 21 to 84 U/L (n=245)) but in females at this dose level all values
were within the historical control data (98-percentile range: 16 to 72 U/L (n=243)). Several
males receiving 500 mg/kg/day also had aspartate amino-transferase activities that were
above the historical control data (98-percentile range: 51 to 156 U/L (n=245)) but for
females, all individual values were within the historical control data (98-percentile range:
49 to 169 U/L (n=243)).
Bile acid concentrations were higher than control in both sexes receiving 500 mg/kg/day,
with the difference from control being statistically significant in males. There was, however,
a high degree of individual variation in both sexes, but particularly in females. With the
exception of one male and two females, 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)).
In males receiving 500 mg/kg/day, there was a statistically significant reduction of creatinine
concentration, compared to controls, but with the exception of one individual with an
unusually low value, all individual values were within the historical control data
(98-percentile range: 21 to 55 µmol/L (n=245)).
Total cholesterol and low- and high-density lipoprotein concentrations were statistically
significantly high in males receiving 500 mg/kg/day and total cholesterol and high-density
lipoprotein concentrations were also slightly high in males receiving 150 mg/kg/day, but
statistical significance was not attained. All individual values, with the exception of two
individuals, 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); low-density lipoprotein concentration 0.11 to 0.60 mmol/L (n=120)).
In females, triglyceride concentrations were statistically significantly high at 500 mg/kg/day,
but the other plasma lipids were unaffected and all individual triglyceride values were within
the historical control data (98-percentile range: 0.20 to 1.28 mmol/L (n=114)).
Plasma albumin concentrations were higher than control in males receiving 500 mg/kg/day,
although all individual values were within the historical control data (98-percentile range:
32 to 42 g/L (n=242)), resulting in the albumin to globulin ratio being statistically
significantly higher in these animals, but also within the historical control data (98-percentile
range: 1.06 to 1.73 (n=245)). There was no similar trend in females.
There were some variations of plasma electrolyte concentrations in both sexes, compared to
controls, the majority of which were statistically significant. In both sexes, sodium
concentrations were low at 500 mg/kg/day and chloride concentration was low at 150 or
500 mg/kg/day. In males, calcium and phosphorus concentrations were high at 150 or
500 mg/kg/day and in females, calcium and potassium concentrations were high at
500 mg/kg/day. For the variations of sodium, potassium, chloride and phosphorus
concentrations, the majority of individual values were within the historical control data
(98-percentile range: sodium concentration 135 to 147 mmol/L in males (n=245) and 134 to
147 mmol/L in females (n=243); potassium concentration 3.04-5.26 mmol/L in females
(n=243); chloride concentration 97.0 to 104.0 mmol/L (n=105) in males and 94.0 to
105.0 mmol/L in females (n=104); phosphorus concentration 1.31 to 2.96 in males (n=105)
and 1.09 to 2.11 mmol/L in females (n=79)), except for one male receiving 500 mg/kg/day
which had a chloride value below this range (No. 11; 94.4 mmol/L). For the variations of
calcium concentration, four males and one female had values that were above the historical
control data (98-percentile range: 2.48 to 2.87 mmol/L in males and 2.46 to 2.94 mmol/L in
females (n=104)).
All other inter-group differences from control were minor or lacked dose response and were
therefore attributed to normal biological variation. Such differences included the statistically
significantly high alkaline phosphatase activities in males receiving 150 or 500 mg/kg/day,
where there was no dose-response and all values were well within the background range (44
to 135 U/L; n=245).
For further details on the results of blood chemistry - group mean values during week 13 of treatment presented 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):

Estrous cyclicity was unaffected by treatment.
For further details on the results of stage of estrous cycle at termination - group values evaluation presented in tabular form please refer to table 12.10 in the attached file type 'full results tables'.

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 revealed, compared with control, slightly low thyroid stimulating hormone concentrations in both sexes receiving 500 mg/kg/day, though this was statistically significant only in males.
The T4 levels were significantly increased in male animals receiving 150 mg/kg/day. However, as no dose dependency is observed and there were no associated findings in the respective organs in macro- and micropathology this is thought of representing biological variance. Also body weight adjusted thyroid weights were unaffected. None of the other parameters showed a statistical difference from control.
The slight decrease in T3 in females (mid and high dose) is likely due to biological variation since, there was no effect on organ weight, no associated histopathological changes and there was no increase of TSH concentration and no similar decrease of T3 in males or of T4 in either sex.

Generally it should be stressed that fluctuation in hormone levels are well known and 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.).

For details on results please refer to table 2 in 'Any other information on results incl. tables'.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The analysis of urine performed during Week 13 revealed, when compared with control, low
urinary volume and high urinary specific gravity in males receiving 500 mg/kg/day which
resulted in a consequentially high incidence of dark yellow urine.
Please refer to table 3 in 'Any other information on results incl. tables' for details.

Creatinine, sodium and potassium outputs were low and glucose output was slightly high in
males receiving 500 mg/kg/day whilst in females there was an increase of protein output at
500 mg/kg/day and a dose-related increase of glucose and chloride output at 150 and
500 mg/kg/day.

All other inter-group differences from control were minor or lacked dose response and were
therefore attributed to normal biological variation.
For further details on the results of the urinalnalysis group mean values during week 13 of treatment presented in tabular form please refer to table 12.9 in the attached file type "full results tables".

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength were unaffected by treatment.
Motor activity was unaffected by treatment.
Statistical significance was attained for slightly low cage-floor beam breaks at the 12- and
18-minute timepoints at all doses in males but this was a transient variation that did not affect
the total scores and was not evident in females. Consequently, it was attributed to normal
variation.
For further details on the results of sensory reactivity observations and grip strength - summary of findings during week 12 of treatment as well as motor activity - group mean scores (beam breaks) during week 12 of treatment presented 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):

The analysis of organ weights performed after 13 weeks of treatment revealed, compared
with control, low epididymides and combined seminal vesicles, prostate and coagulating
gland weights in males receiving 500 mg/kg/day, with the extent of the decrease being
statistically significant at this dose level and with all individual values being below the
historical control data (98-percentile range: left epididymis 0.499 to 1.107 g (n=80); seminal
vesicles/prostate/coagulating gland 1.883 to 3.019 g (n=50)). In addition, the combined
prostate, seminal vesicles and coagulating gland weights were also low in males receiving
150 mg/kg/day, with two individual values being below the background range.

Adrenal gland weights were statistically significantly high in males receiving 500 mg/kg/day.
but all individual values were within the historical control data (98-percentile range: 0.040 to
0.083 g (n=180)) with the exception of one male receiving 500 mg/kg/day which had a value
that was slightly above the range (No. 19; 0.098 g).

Liver weights were high in both sexes receiving 150 or 500 mg/kg/day and were statistically
significant at 500 mg/kg/day and in males receiving 150 mg/kg/day. All individual values
were, however, within the historical control data (98-percentile range: 9.241 to 19.304 g in
males and 6.400 to 11.435 g in females (n=180)).

Kidney weights were high in both sexes receiving 500 mg/kg/day and in males receiving
150 mg/kg/day, with the extent of the increase being broadly dose-related and were
statistically significant in the same sexes and dose levels. However all individual values remained within the historical control data (98-percentile range: 1.599 to 3.096 g in males
and 1.059 to 1.946 g in females (n=180)).

Heart weights were high in both sexes receiving 150 or 500 mg/kg/day, with the extent of the
increase being broadly dose-related and statistically significant. All individual values were
within the historical control data (98-percentile range: 0.786 to 1.742 g in males and 0.618 to
1.105 g in females (n=180)), although in the females, two control animals, one animal
receiving 50 mg/kg/day and one animal receiving 150 mg/kg/day had values that were
slightly below this range.

Thymus weights were lower than control in both sexes receiving 150 or 500 mg/kg/day and
in males receiving 50 mg/kg/day, with the extent of the decrease being broadly dose-related
and statistically significant. In males, the majority of individual values at 500 mg/kg/day
were below the historical control data (98-percentile range: 0.177 to 0.478 g (n=180)) but in
females, all individual values were within the historical control data (98-percentile range:
0.161 to 0.397 g (n=180)) except for two control animals and two females receiving
500 mg/kg/day.

Ovary weights were high at all dose levels in females, with the extent of the increase being
dose-related and statistically significant at 150 or 500 mg/kg/day, although all individual
values were within the historical control data (98-percentile range: 0.064 to 0.135 g (n=180)).

All other 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; presence only in absolute or relative to
body weight ratios but not all; lack of a dose relationship or correlative findings; and/or the
magnitude was considered small. This included slightly high body weight-relative brain,
pituitary gland and thyroid with parathyroid gland weights in males.
Please refer to table 4 in 'Any other information on results incl. tables' for details.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the macroscopic examination, pale areas were observed on the lungs in both sexes receiving 500 mg/kg/day. Although this finding was also observed in one male receiving 50 mg/kg/day, the microscopic evaluation revealed a different pattern from that seen at 500 mg/kg/day and the finding in this animal was therefore unlikely to have been related to treatment.
Please refer to table 5 in 'Any other information on results incl. tables'.

All other macroscopic findings were considered spontaneous and/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. They were, therefore, considered not test item related. This included dark area(s) in the pituitary as well as small epididymides, small testes and testicular subtunical fluid in one male receiving 500 mg/kg/day. The change in the epididymides correlated with reduced luminal sperm, which was a consequence of a grade 5 degeneration/atrophy seen in the testis of one male. At this severity, degeneration/atrophy in the testis was likely to be a background change (as per Historical Control Data Appendix) although it is not possible to exclude a partial contribution of the test item. There were no macroscopic changes associated with the test item related histological changes in the epididymides and testes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Male reproductive system
A high incidence and/or severity of minimal tubular vacuolation of the Sertoli cells and/or tubular degeneration/atrophy was observed in the testes of males receiving 500 mg/kg/day. In addition, a high incidence of minimal to moderate epithelial vacuolation of the epididymal body was observed in animals receiving 150 or 500 mg/kg/day and this was frequently associated with reduced luminal sperm and increased cellular debris at 500 mg/kg/day. Furthermore, a high incidence of minimal to slight acinar atrophy of the accessory sex glands (prostate, seminal vesicles and coagulating glands) was seen at 500 mg/kg/day.
The evaluation of the testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Please refer to table 6 in 'Any other information on results incl. tables'.

Hemolymphoreticular system
A high incidence of decreased cellularity was observed in the mesenteric and axillary lymph nodes, as well as in the Peyer’s patches and splenic periarteriolar lymphoid sheaths, in both sexes receiving 500 mg/kg/day. In addition, extramedullary hemopoiesis in the spleen and intrasinusoidal erythrocytes in the mesenteric lymph node were observed in both sexes at the same dose.
Please refer to table 7 in 'Any other information on results incl. tables'.

Other organs
A high incidence of alveolar aggregations of macrophages, associated with an interstitial infiltrate of mixed inflammatory cells, was observed in the lungs of both sexes receiving 500 mg/kg/day. In addition, a higher incidence of minimal rodent progressive cardiomyopathy was seen in the heart of males receiving 500 mg/kg/day when compared with that in the controls. Furthermore, a higher incidence and severity of cortical diffuse vacuolation, compared to concurrent controls, was observed in the adrenal glands of males receiving 500 mg/kg/day. The cortical vacuolation was seen mostly in the zona fasciculata but also in the zona reticularis.
Please refer to table 8 in 'Any other information on results incl. tables'

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SPERM ANALYSIS
The morphological examination of the sperm revealed, compared with control, a statistically significant decrease of motile sperm, progressively motile sperm and epididymal and testicular sperm count in males receiving 500 mg/kg/day, which were below the historical control data (range: motile sperm 90-95%; progressively motile sperm 26-45%; epididymal sperm count 473-609 million/g; total epididymal sperm count 108-141 million; testicular spermatid count 84-101 million/g; total testicular sperm count 167-199 million (n=58)). At this dose there was also a statistically significant increase of abnormal sperm (head, neck, midpiece and tail abnormalities), an associated decrease of normal sperm and an increase of decapitate sperm which were also outside of the historical control data. There was also a reduction of epididymal sperm count in males receiving 150 mg/kg/day and although statistical significance was not attained and this did not associate with any alteration of sperm morphology or motility, values were below the historical control data.
The was no effect on sperm morphology, motility or testicular sperm count at 50 or 150 mg/kg/day.
For further details on the results of sperm analysis - group mean values, sperm motion data - group mean values and sperm morphology data - group mean values presented in tabular form please refer to tables 12.11, 12.12 and 12.13 in the attached file type "full results tables".

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

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

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

cauda epididymis

coagulating gland

dorsolateral prostate gland

seminal vesicle

testes

ventral prostate gland

Treatment related:

yes

Dose response relationship:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

other: hemolymphoreticular system

Organ:

mesenteric lymph node

spleen

other: Peyer's patches, axillary lymph node

Treatment related:

yes

Dose response relationship:

not specified

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

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

TSH: Dosed groups compared to the Control group using Williams’ test.

T3/T4: Dosed groups compared to the Control group using Dunnett’s test.

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	50	150	500	0	50	150	500
Thyroxine (pg/mL) (T4)	36780	41180	47670**	34320	31770	34522	28520	31000
Triiodothyronine (pg/mL) (T3)	666	688	798	654	1005	1030	892	873
Thyroid stimulating hormone (pg/mL) (TSH)	678	625	692	332**	680	368	406	348

Table 3: Summary of Urinary Appearance in Week 13

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	50	150	500	0	50	150	500
Pale yellow	4	0	0	0	0	0	0	0
Medium yellow	6	9	8	5	10	10	9	9
Dark yellow	0	1	0	5	0	0	1	1

Table 4: Test Item-Related Effects on Organ Weights – Terminal Sacrifice After 13 Weeks of Treatment

Note: The values for the absolute and body weight-relative organ weights for dosed groups are expressed as percentage of the control mean value.

Dosed groups compared to the Control group using Williams’ test.

Body weight adjusted organ weights are presented here and in the attachment file type 'organ weights full tables' in table 6.1. Body weight relative organ weights are presented and can be assessed in the attachment file type 'organ weights full tables' in table 6.2. 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.

Sex	DYTEK DCH-99
Sex	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Epididymides
Absolute Weight (g)	1.379	97	99	65	NA	NA	NA	NA
Body Weight Adjusted (%)	1.364	97	98	70**	NA	NA	NA	NA
Prostate, seminal vesicles and coagulating glands
Absolute Weight (g)	2.251	95	94	65	NA	NA	NA	NA
Body Weight Adjusted (%)	2.214	96	91	73**	NA	NA	NA	NA
Adrenal glands
Absolute Weight (g)	0.053	113	102	134	0.067	100	101	103
Body Weight Adjusted (%)	0.052	113	100	142**	0.067	99	101	104
Liver
Absolute Weight (g)	11.867	102	109	109	7.452	104	108	123
Body Weight Adjusted (%)	11.498	103	106	125**	7.442	101	108*	126*
Kidneys
Absolute Weight (g)	2.038	99	114	109	1.330	103	103	117
Body Weight Adjusted (%)	2.007	99	112**	117**	1.329	101	103	119**
Thymus
Absolute Weight (g)	0.330	84	82	45	0.273	103	85	63
Body Weight Adjusted (%)	3.768	84	77*	63**	0.273	100	85	66**
Heart
Absolute Weight (g)	0.984	101	111	104	0.713	106	108	116
Body Weight Adjusted (%)	0.967	102	109**	113**	0.713	104	108*	118*
Ovaries
Absolute Weight (g)	NA	NA	NA	NA	0.082	116	122	124
Body Weight Adjusted (%)	NA	NA	NA	NA	0.082	113	122*	127**
Brain
Absolute Weight (g)	2.054	99	100	97	1.905	102	99	97
Body Weight Adjusted (%)	2.044	99	100	100	1.904	101	100	97
Pituitary
Absolute Weight (g)	0.009	89	100	89	0.014	107	107	107
Body Weight Adjusted (%)	0.008	100	113	113	0.014	107	107	107
Spleen
Absolute Weight (g)	0.675	90	94	80	0.488	97	88	97
Body Weight Adjusted (%)	0.656	91	90	94	0.487	95	88	99
Testes
Absolute Weight (g)	3.798	104	110	93	NA	NA	NA	NA
Body Weight Adjusted (%)	3.768	104	109	97	NA	NA	NA	NA
Thyroids and Parathyroids
Absolute Weight (g)	0.017	106	106	106	0.015	120	107	107
Body Weight Adjusted (%)	0.016	106	106	125**	0.015	120	107	107
Uterus and Cervix
Absolute Weight (g)	NA	NA	NA	NA	0.808	78	70	86
Body Weight Adjusted (%)	NA	NA	NA	NA	0.809	80	70	84

Table 5: Incidence of Test Item-Related Macroscopic Findings – Terminal Sacrifice After 13 Weeks of Treatment

The full macroscopic analysis in tabular form can be assessed in the attached file type 'macropathology data' in table 6.3.

Sex	DYTEK DCH-99
Sex	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Lungs
Number Examined	10	10	10	10	10	10	10	10
Pale area(s)	0	1	0	10	0	0	0	6

Please note that tabular results of the microscopic investigations made can be found in the attached file type 'histopathology data (tables)' in table 6.4. In this section emphasis was put on the findings that were relevant for the further assessment.

Table 6: Incidence and Severity of Test Item-Related Microscopic Findings on the Male Reproductive System – Terminal Sacrifice After 13 Weeks of Treatment

Sex	DYTEK DCH-99
Sex	Males
Dose Level (mg/kg/day)	0	50	150	500
Testis, right
Number Examined	10	10	10	10
Tubular vacuolation
Minimal	0	0	0	9
Total	0	0	0	9
Tubular degeneration/atrophy
Minimal	0	1	0	4
Severe	0	0	0	1
Total	0	1	0	5

Epididymis, right
Number Examined	10	10	10	10
Epithelial vacuolation
Minimal	0	0	6	0
Slight	0	0	3	0
Moderate	0	0	0	10
Total	0	0	9	10
Reduced sperm, luminal
Minimal	0	0	0	6
Slight	0	0	0	3
Moderate	0	0	0	1
Total	0	0	0	10
Cellular debris, luminal
Minimal	0	0	0	7
Slight	0	0	0	2
Moderate	0	0	0	1
Total	0	0	0	10

Prostate
Number Examined	10	10	10	10
Acinar atrophy
Minimal	1	0	0	9
Total	1	0	0	9

Seminal vesicles
Number Examined	10	10	10	10
Acinar atrophy
Minimal	0	0	0	6
Slight	0	0	0	1
Total	0	0	0	7

Coagulating glands
Number Examined	10	10	10	10
Acinar atrophy
Minimal	0	0	0	6
Slight	0	0	0	1
Total	0	0	0	7

Table 7: Incidence and Severity of Test Item-Related Microscopic Findings on the Hemolymphoreticular System – Terminal Sacrifice After 13 Weeks of Treatment

Sex	DYTEK DCH-99
Sex	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Spleen
Number Examined	10	10	10	10	10	10	10	10
Cellularity decreased, periarteriolar lymphoid sheaths
Minimal	0	0	0	1	0	0	0	3
Slight	0	0	0	9	0	0	0	6
Total	0	0	0	10	0	0	0	9
Extramedullary Hemopoiesis, Increased
Minimal	2	1	2	3	2	2	4	0
Slight	0	0	0	6	3	2	0	6
Moderate	0	0	0	0	0	0	0	4
Total	2	1	2	9	5	4	4	10

Mesenteric lymph node
Number Examined	10	10	10	10	10	10	10	10
Cellularity decreased, generalized
Minimal	0	0	0	4	0	0	0	6
Slight	0	0	0	6	0	0	0	3
Total	0	0	0	10	0	0	0	9
Erythrocytes, intrasinusoidal
Minimal	0	0	0	4	0	1	0	3
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	5	0	1	0	3

Axillary lymph node
Number Examined	10	10	10	9	10	10	10	8
Cellularity decreased, generalized
Minimal	1	1	2	3	0	0	0	6
Slight	1	0	0	2	0	0	0	1
Moderate	0	0	0	1	0	0	0	0
Total	2	1	2	6	0	0	0	7
Peyer’s patches
Number Examined	10	10	10	10	10	10	10	10
Cellularity decreased
Minimal	0	0	0	5	0	0	0	2
Slight	0	0	0	0	0	0	0	4
Total	0	0	0	5	0	0	0	6

Table 8: Incidence and Severity of Test Item-Related Microscopic Findings in Other Organs – Terminal Sacrifice After 13 Weeks of Treatment

Sex	DYTEK DCH-99
Sex	Males				Females
Dose Level (mg/kg/day)	0	50	150	500	0	50	150	500
Lungs and bronchi
Number Examined	10	10	10	10	10	10	10	10
Alveolar macrophage aggregation
Minimal	1	1	3	6	1	2	2	5
Slight	0	0	0	4	0	0	0	5
Total	1	1	3	10	1	2	2	10
Infiltrate, Inflammatory Cell
Minimal	0	0	0	9	0	0	0	10
Total	0	0	0	9	0	0	0	10

Heart
Number Examined	10	10	10	10	10	0	0	10
Rodent progressive cardiomyopathy
Minimal	1	3	1	9	0	0	0	1
Total	1	3	1	9	0	0	0	1

Adrenal glands
Number Examined	10	10	10	10	10	0	0	10
Cortical vacuolation
Minimal	1	1	1	2	0	0	0	0
Slight	0	0	0	4	0	0	0	0
Total	1	1	1	6	0	0	0	0

Conclusions:

It is concluded that the oral administration of DYTEK DCH-99 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 and adverse histopathological findings in the reproductive organs, which were
associated with reduced sperm count, motility and increased numbers of abnormal sperm in
males receiving 500 mg/kg/day and epididymal vacuolation, associated with reduced
epididymal sperm count in males receiving 150 mg/kg/day. Adverse findings were reported
in the hemolymphoreticular system (spleen, lymph nodes and Peyer’s patches) of both sexes
receiving 500 mg/kg/day. Non-adverse findings included evidence of adaptive responses by
the liver and kidney in both sexes receiving 150 or 500 mg/kg/day and variations to some
organ weights (heart, thymus gland and ovaries) without histopathological correlate. In view
of these findings, the no-observed-adverse-effect level (NOAEL) in this study was considered
to be 50 mg/kg/day in males and 150 mg/kg/day in females.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of the submission sustance (i.e. DCH, DYTEK DCH-99; an
industrial chemical) when administered orally (by gavage) to Han Wistar rats for 13 weeks.
Three groups, each comprising ten male and ten female Han Wistar rats, received DYTEK
DCH-99 at doses of 50, 150 or 500 mg/kg/day. A similarly constituted control group received
the vehicle, water, at the same volume dose as 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, ophthalmic examination, hematology (peripheral blood), blood
chemistry, urinalysis, thyroid hormone, estrous cycles, 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 hemolymphoreticular system and male reproductive system were the targets of toxicity and there were findings in this study that were indicative of an effect on liver and kidney function.

There was no evidence that DYTEK DCH-99 affected the hormonal control of the thyroid gland, since serum TSH and T3 levels in both sexes as well as T4 levels in males were not increased, and the statistically significant increase in the males receiving 150 mg/kg/d was not considered biologically relevant particularly as there is no similar effect at the high dose gourp (lack of dose dependency). Moreover in both sexes there was no effect on thyroid gland weight and pathology. In addition, there was no effect on the female reproductive system since estrus cyclicity was
unaffected and there were no histopathological findings in the reproductive system of females
(despite the slight increase of ovary weight at all doses).

There was an adverse reduction of weight gain from Week 2 in males given 500 mg/kg/day
that was indicative of a non-specific response to the toxicity of DYTEK DCH-99. Between
Week 2 and 13 the gains of these animals were reduced by 26% compared to controls during
the same period but there was no associated reduction of food intake, no clinical signs
(treatment-related clinical signs in this study were limited to salivation in a few females,
which is a common finding in studies where the test material is administered by gavage and,
as such, is of no toxicological significance) and no deaths occurred in this study. The marked
effect on the body weights of the high dose males was considered the likely cause of the
reduced plasma creatinine levels (and low urinary creatinine output) due to the
consequentially decreased muscle mass in these animals.

The hemolymphoreticular system and male reproductive system were the targets of toxicity.

In the hemolymphoreticular system there was minimal to moderately decreased cellularity in
the lymph nodes, Peyer’s patches and splenic periarteriolar lymphoid sheaths and
extramedullary hematopoiesis in the spleen and the presence of intrasinusoidal erythrocytes
in the mesenteric lymph node of both sexes receiving 500 mg/kg/day. These findings were
considered the cause of the low white blood cell counts reported at the hematological
examination and were considered adverse. Stress and nutrition may also have contributed
towards the changes seen in the hemolymphoreticular system, but they also probably
reflected a direct effect of the test item. Although decreased cellularity of specific
compartment(s) of the spleen may be caused by poor nutrition and/or stress, amongst other
factors (Willard-Mack et al. 2019), the administration of DYTEK DCH-99 was associated
with markedly lower weight gain in the high dose males only, whereas the changes in the
hemolymphoreticular system occured in both sexes. Additionally, the possibility of a stressinduced
mechanism being responsible for the low leucocyte count and decreased cellularity
in several lymphoid organs is challenged by the absence of some hallmarks of stress-induced
changes, such as hypertrophy of the cortical adrenal gland (Everds et al. 2013) leading to
increased adrenal gland weight. In the current study, high adrenal gland weight was seen in
males only and correlated with cortical vacuolation and not hypertrophy.

In the male reproductive system, there was minimal tubular vacuolation and/or tubular
degeneration/atrophy in the testes, moderate epithelial vacuolation of the epididymal body,
which often associated with reduced luminal sperm and increased cellular debris in the epididymides, and minimal to slight acinar atrophy in the accessory sex glands (prostate, seminal vesicles and coagulating glands) at 500 mg/kg/day. These findings led to a decrease of epididymides and combined seminal vesicle/prostate/coagulating gland weights and
correlated with the low sperm count, decreased motility and increased abnormal sperm at this
dose level. These findings were probable consequences of a direct effect of DYTEK DCH-99
but the possibility that the changes in the accessory sex glands may have been secondary to
reduced body weight gain and consequentially decreased testosterone, due to decreased
gonadotrophin releasing hormone (Creasey et al. 2008) cannot be discounted. In the absence
of measurement of testosterone in this study this cannot be confirmed. At 150 mg/kg/day,
there was vacuolation of the epididymis and a reduction of epididymal sperm count, and
although there was no effect on testicular sperm count and sperm motility and morphology
were unaffected at this dose, the sperm counts were below the historical control data. These findings were considered adverse.

There were findings that were indicative of an effect upon liver and kidney function.
Liver weights were high after 13 weeks in males and females given 150 or 500 mg/kg/day.
There were some findings in the blood plasma (high alanine and aspartate amino-transferase
activities at 500 mg/kg/day, with high alanine amino-transferase activity in males receiving
150 mg/kg/day, high bile acid concentrations at 500 mg/kg/day; increased total cholesterol
and reduced low- and/or high-density lipoprotein concentrations in males at 150 or
500 mg/kg/day, high triglyceride concentrations in females receiving 500 mg/kg/day and high
albumin concentrations in males receiving 500 mg/kg/day), resulting in high albumin to
globulin ratio in these animals that were indicative of an effect upon liver function. There
was, however, no associated histopathological finding, despite the increased plasma alanine
amino-transferase activities. There was also no evidence of any increased hepatocellular
vacuolation associated with the effect on cholesterol and triglyceride. Consequently, these
findings were considered non-adverse and were considered likely to be 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 males given 150 mg/kg/day and in males and
females given 500 mg/kg/day. There was an effect on water balance in males since water
intake was increased at these doses and there was consequential haemodilution, as indicated
by the decreased erythrocyte mass. However, the high dose males produced a reduced
volume of urine that had a high specific gravity and a consequentially darker colouration than
that of the controls. There were also variations of plasma and urinary electrolyte
concentrations/output in both sexes whilst females had reduced urinary glucose output and
increased urinary protein output. The increased calcium concentration in both sexes receiving
500 mg/kg/day may have been associated with the increased albumin observed, since calcium
is bound to albumin. In the absence of any associated histopathological changes in the
kidney, these findings were considered non-adverse and were likely secondary to a change in
renal function as a result of the excretion of DYTEK DCH-99 and/or its metabolites.

The increase of heart weight at 150 and 500 mg/kg/day showed a broad dose-relationship and
were considered related to treatment but in the absence of any microscopic correlate, this was
considered non-adverse. An increased incidence of rodent progressive cardiomyopathy,
compared to controls, was identified in the heart of males given 500 mg/kg/day but this
finding represents a rodent-specific change that is not relevant to humans (Berridge et al. 2016). Aggregations of alveolar macrophages associated with interstitial mixed inflammatory cell infiltrates were observed at 500 mg/kg/day, though the cause was not established in this study but may have been associated with the effect on the hemolymphoreticular system. At the minimal to slight severity observed in this study, this was considered non-adverse.

In view of these findings, the no-observed-adverse-effect level (NOAEL) in this study was considered
to be 50 mg/kg/day in males and 150 mg/kg/day in females.

Additional references used above:

Berridge BR, Mowat V, Nagai H, Nyska A, Okazaki Y, Clements PJ, Rinke M, Snyder PW, Boyle MC, Wells MY (2016) Non-proliferative and Proliferative Lesions of the Cardiovascular System of the Rat and Mouse. J Toxicol Pathol 29: 1S–47S

Creasy D, Cartwright J, Moreland S, et al (2008) Endocrine disruption: a guidance document for histologic evaluation of endocrine and reproductive tests. Internet guidance document [Online]. https://www.oecd.org/chemicalsafety/testing/40638492.pdf (accessed 28 September 2021)

Everds NE, Snyder PW, Bailey KL, Bolon B, Creasy DM, Foley GL, Rosol TJ, Sellers T (2013). Interpreting stress responses during routine toxicity studies: A review of the biology, impact and assessment. In: Toxicological Pathology (4), 560-614

Willard-Mack, C.L. et al (2019). Nonproliferative and Proliferative Lesions of the Rat and Mouse Hematolymphoid System. Toxicologic Pathology 47 (6) pp 665-783

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 50 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: One reliable subchronic study for the submission substance (Klimisch score = 1) is available. One further subacute study is at hand ( Klimisch score = 1). Overall the quality of the database is high.
System:: male reproductive system
Organ:: cauda epididymis; coagulating gland; dorsolateral prostate gland; seminal vesicle; testes; ventral prostate gland

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: From 17 MAY 1990 to 1 DEC 1993
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Basic data given, comparable to current standards
Principles of method if other than guideline:: standard subacute method
GLP compliance:: yes
Remarks:: according to EPA GLP Regulations (40 CFR 792)
Limit test:: no
Species:: rat
Strain:: other: Crl:CD*BR
Sex:: male
Details on test animals or test system and environmental conditions:: - Source: Charles River Breeding Laboratories, Inc. Raleigh, North Carolina, USA
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approximately ranging from 207 to 243 grams
- Housing: individually housed in stainless steel, wire mesh cages
- Diet: ground Purinae Certified Rodent Chow #5002, ad libiitum
- Water: tap water, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C) targeted: 23 +/-2
- Humidity (%): targeted: 50 +/-10

IN-LIFE DATES: From: 8 JUN 1990 To: 6 JUL 1990
Route of administration:: other: inhalation of an aerosol/vapour mixture
Type of inhalation exposure:: nose only
Vehicle:: air
Remarks on MMAD:: MMAD / GSD: Aerosol size (MMDA): ranging from 2.6 to 5.8 micrometres
Details on inhalation exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 29 litre exposure chambers (made of glass)
- Method of holding animals in test chamber: restrainers (perforated stainless steel or polycarbonate cylinders with conical nose pieces, which were inserted into face plates to yield nose-only exposure conditions)
- System of generating particulates/aerosols: Atmospheres containing aerosol and vapour test material were generated by pumping the test material with a Harvard compact infusion pump to a glass J-tube containing 6 nm glass beads. A Goodburn Model T2 electronic torch heated the air entering the J-tube to about 60 to 70°C. The torch temperature was controlled by an Omega Model CS-6001-J temperature controller. The control chamber also had a torch at similar temperatures controlled by an Omega temperature controller. The aerosol/vapour mixture entered the chamber and was dispersed by a glass baffle to promote uniform distribution of the test material throughout the exposure chamber.
- Method of particle size determination: determined weekly for each exposure concentration with a Sierra Series 210 Cascade Impactor
- Treatment of exhaust air: Chamber atmospheres were exhausted through an emissions-abatement train consisting of a water scrubber (240 cg/ms chambers only), a cold trap, and an HSA cartridge filter prior to discharge into a fume hood.

TEST ATMOSPHERE
- Brief description of analytical method used:
The exposure chambers were monitored approximately hourly for the test material during the animal exposures. A minimum of 4 samples were
taken per exposure. For test material analysis, a measured volume of the chamber atmosphere was drawn through a glass-fiber filter in tandem with the methanol impinger. The filter trapped the test material aerosol particles, the methanol-filled impinger trapped the vapour.
The atmospheric concentration of test material aerosol was calculated from the pre- and post-sampling filter weights which were determined with a Cahn Model 26 Automatic Electrobalance. For the GC analysis, methanol trapped samples were analysed using a Hewlett-Packard Model 5890A GC equipped with a flame-ionization detector. Samples were chromatographed isothermally at 160°C on a 30 meter X 0.53 mm I.D. fused-silica column lined with polyphenylmethylsiloxane. Improved test material detection was obtained in the last 4 exposures using a Hewlett-Packard Model 5890A GC equipped with a nitrogenn-phosphorous detector. Samples were chromatographed isothermally at 160°C on a 10 meter X 0.53 mm I.D. fused-silica column lined with crosslinked 50% phenylmethylsilicone. Test material chamber concentrations were determined by comparing the GC response of the chamber samples with those of standard samples which were prepared by quantitatively diluting the test material in methanol.
Airborne particle size (reported as mass median aerodynamic diameter and percent of mass less than 10 micrometers aerodynamic diameter) was
determined weekly for each exposure concentration with a Sierra Series 210 Cascade Impactor. During each exposure, chamber temperatures were
measured continually with thermocouples during each exposure. Temperatures were averaged and recorded hourly by means of Grant 1200 Series Squirrel Meter/Logger Relative humidity was measured twice per exposure with a Belfort Instrument Co. Model 566 Electric Psychrometer, and chamber oxygen concentration was measured twice per exposure with a Biosystems Model 3100R Oxygen Monitor.
- Samples taken from breathing zone: no

VEHICLE (if applicable)
- Justification for use and choice of vehicle: due to substance characteristics and resulting aerosol/vapour mixture generation via heat-evaporation
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: see "Details on inhalation exposure"
Duration of treatment / exposure:: two weeks
Frequency of treatment:: 6 hours per day, 5 days a week, for two weeks (i.e. 10 exposures in total) followed by a 14-day recovery period
Remarks:: Doses / Concentrations:
0, 10, 49 and 240 mg per cubic metre
Basis:
analytical conc.
Remarks:: Doses / Concentrations:
0, 10, 50 and 250 mg per cubic metre
Basis:
nominal conc.
No. of animals per sex per dose:: 10 males per dose group
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: Exposure concentrations in the present study were selected based on results of rangefinding studies
- Post-exposure recovery period in satellite groups: 14 days (5 animals of each treatment group as well as control)
Statistics:: Mean body weights and body weight gains for exposed rats were compared to control rats during the exposure and recovery periods.
Data were statistically analysed by one-way analysis of variance. Exposure group values were compared to controls by the least significant difference test when the ratio of variance (F) indicated a significant among-to-within group variation. Significant differences were declared at the 0.05 probabiility level. The statistical analyses used to evaluate the clinical pathology and the pathology data are described below:
A one-way analysis of variance (ANOVA) and Bartlett's test were calculated for each sampling time. When the F-test from ANOVA was
significant, the Dunnett test was used to compare means from the control group and each of the groups exposed to the test material. When the results of
the Bartlett test were significant (p < 0.005), the Kruskal-Wallis test was employed and the Mann-Whitney U test was used to compare means from the
control group and each of the groups exposed to the test material. Significance was judged at the 5% probability level.
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: high dose group: 1 out of 10 rats showed black nasal discharge, 2 out of 10 rats showed alopecia
Mortality:: mortality observed, treatment-related
Description (incidence):: high dose group: 1 out of 10 rats showed black nasal discharge, 2 out of 10 rats showed alopecia
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: dose related lesions confined to the upper respiratory tract; fully reversible (inflammation and necrosis in the nasal mucosa and the larynx/pharynx region, of only minimal to mild severity for low and mid dose group, but moderate in the high dose group)
Details on results:: CLINICAL SIGNS AND MORTALITY
all groups: clinical signs as commonly found in restrained animals (clear nasal and ocular discharge, discoloured and wet fur)

HISTOPATHOLOGY: NON-NEOPLASTIC
all dose groups: dose dependent nasal lesions consisted of inflammation and necrosis which occurred in the nasal mucosa immediately after the two week exposure

- low dose group:
# nasal lesions, which were considered to be of minimal severity.
# minimally inflammation and necrosis in the larynx/pharynx region in 1 out of 5 rats.
- mid dose group:
# nasal lesions, which were considered to be of mild severity;
# minimally inflammation and necrosis in the larynx/pharynx region in 1 out of 5 rats.
- high dose group:
# nasal lesions, which were considered to be of moderate severity.
# minimal to moderate inflammation and necrosis in the larynx/pharynx region in 2 out of 5 rats.
#minimal to mild inflammation of the tracheal muscle in 2 out of 5 rats

After the two week recovery period none of the above mentioned nasal lesions were observed in any test material exposed rats.
Dose descriptor:: LOAEC
Effect level:: 10 mg/m³ air (analytical)
Based on:: test mat.
Sex:: male
Basis for effect level:: other: dose dependent effects seen on the upper respiratory tract (e.g. nasal lesions) which were from minimal to moderate severity and were fully reversible within the 14-day recovery period
Critical effects observed:: not specified
Conclusions:: In this subacute inhalation toxicity study male rats were exposed for 6 hours per day and overall 10times within two weeks to a aerosol/vapour mixture of the test material. Concentrations used were 0, 10, 49 and 240 mg/m³ (analytical). Due to the fact that effects on the respiratory tract were seen in all concentration groups tested and these effects were clearly dose related a no observed adverse effect level could not be determined. The LOAEC of this study was established to be 10 mg/m³.
Executive summary:: 10 male rats were exposed via inhalation for 6 hours per day, 5 times a week for two weeks to an aerosol/vapour mixture containing test material in concentrations of 0, 10, 49 and 240 mg/m3 (analytical concentration). There were no adverse effects observed in any of the test groups with respect to the haematologic, clinical chemical, urinalysis, and body weight parameters measured. As expected from its chemical basicity, inhaled test material produced respiratory tract irritation in exposed rats. However, the lesions (inflammation/necrosis) observed occurred only in the upper respiratory tract (mainly the nose, larynx, and pharynx), were moderate at the highest concentration tested, and were decreased in severity at the lower test concentrations. Effects observed in the 49 mg/m3 group were mainly mild and those in the 10 mg/m3 group were minimal. The observed effects were completely reversible after a two-week recovery period even at the 240 mg/m3 concentration. Although a no-adverse-effect concentration was not determined in this study, the respiratory tract effects observed at the lowest tested concentration (10 mg/m3 ) were considered minimal and reversible.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LOAEC
: 10 mg/m³
Study duration:: subacute
Species:: rat
Quality of whole database:: One reliable subacute study for the submission substance (Klimisch score = 2) is available. Overall the quality of the database is good.

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

Mode of Action Analysis / Human Relevance Framework

In an experimental study, Gacheru et al. (1989, see IUCLID section 7.12) detected that cis-1,2-diaminocyclohexane is a potent inhibitor of lysyl oxidase. So far, any influence of this interference on reproduction and general toxicity cannot be ruled out.

No other substance specific information available.

Additional information

ORAL:

It is concluded from an OECD 408 study that oral administrated (gavage) DYTEK DCH-99 to Han Wistar rats at doses of 0, 50, 150 or 500 mg/kg bw/day was generally tolerated but caused an adverse reduction of weight gain and adverse histopathological findings in the reproductive organs, which were associated with reduced sperm count, motility and increased numbers of abnormal sperm in males receiving 500 mg/kg bw/day and epididymal vacuolation, associated with reduced epididymal sperm count in males receiving 150 mg/kg bw/day. Adverse findings were reported in the hemolymphoreticular system (spleen, lymph nodes and Peyer’s patches) of both sexes receiving 500 mg/kg bw/day. Non-adverse findings included evidence of adaptive responses by
the liver and kidney in both sexes receiving 150 or 500 mg/kg bw/day and variations to some organ weights (heart, thymus gland and ovaries) without histopathological correlate. In view
of these findings, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 50 mg/kg bw/day in males and 150 mg/kg bw/day in females.

The observed effects on the male reproductive system without marked systemic toxicity in this OECD TG 408 study warrant a fertility classification (please see the section ' Toxicity for reproduction' for a detailed discussion).

In a supporting OECD 422 study (combined 28 day repeated dose toxicity study with reproductive/developmental toxicity) in rats the orally administered test material (DCH at doses of 50, 150 and 500 mg/kg bw/day) induced significant changes only in the highest dose tested; i.e. changes in clinical appearance (slight salivation), functional observations (slight hyperactivity), body weights and food consumption (decreased), clinical laboratory investigations (e.g. decreased eosinophils, ALT and AST activity increased), macroscopic and microscopic examinations (liver, lung and adrenal glands), which correlated with changes in organ weights. Furthermore, the following effects were observed:

- at 500 mg/kg bw/day (highest dose tested): reduced number of living pups, reduced gestation index.

- at all dose levels (50, 150, 500 mg/kg bw/day), but not in controls: increased postnatal loss, and, in conclusion, decreased viability index. This postnatal loss was obviously caused by cannibalism in the treatment groups.

- Reduction in viability index was however not dose-dependent.

From the results presented above a no-observed-adverse-effect level (NOAEL) for DCH of 150 mg/kg bw/day was established.

INHALATION:

In a subacute inhalation toxicity study 10 male rats were exposed for 6 hours per day and overall 10 times within two weeks to an aerosol/vapour mixture of the test material DCH. Concentrations used were 0, 10, 49 and 240 mg/m³ (analytical). Inhaled test material produced respiratory tract irritation in exposed rats. However, the lesions (inflammation/necrosis) observed occurred only in the upper respiratory tract (mainly the nose, larynx, and pharynx), were moderate at the highest concentration tested, and were decreased in severity at the lower test concentrations (Effects observed in the 49 mg/m3 group were mainly mild and those in the 10 mg/m3 group were minimal). The observed effects were completely reversible after a two-week recovery period even at the 240 mg/m3 concentration. Due to the fact that effects on the respiratory tract were seen in all concentration groups tested and these effects were clearly dose related a no observed adverse effect level could not be determined. The LOAEC of this study was established to be 10 mg/m³.

There is only this study available using DCH as test material. Thus, the LOAEC mentioned above will be used for the further assessment. However, the experimental data on related amines serves as further evidence for verifying the local irritating nature of amines in general. Especially the subchronic studies with HMD and HMD-dihydrochloride verify that the effect is not crucially time dependent either and support the respective classification.

Short summaries of these studies are provided below:

In another subacute inhalation toxicity study with the same study regimen (6 hours per day and overall 10 times within two weeks) male rats were exposed to an aerosol/vapour mixture of 2-methylpentane-1,5-diamine (MPMD, CAS 15520-10-2). MPMD is another C6-diamine but branched instead of cyclic as DCH. In this study with MPMD concentrations used were 0, 9.2, 59 and 250 mg/m³ (analytical).

The effects observed in rats exposed to MPMD ranged from severe (including death) in the 250 mg/m³ group to minimal in the 9.2 mg/m³ group. Two rats in the 250 mg/m³ group died. The body weights of the rats in the 250 mg/m³ group were significantly lower than those of the control rats during the exposure period and in the first week of the recovery period. Lung weights were increased in this group immediately after the 2-week exposure period and after the 2-week recovery period. Clinical laboratory results for rats in the 250 mg/m³ suggested the presence of dehydration/haemoconcentration indicated by increases in relative numbers of red blood cells, in haemoglobin concentration, and in haematocrit percentage, and by decreases in urine volume and increases in urine osmolality. In addition, there was a decrease in lymphocytes which was considered to be generalized reaction to stress. Gross examination at necropsy showed multifocal areas of discoloration present in the lungs of the rats exposed to 250 mg/m³ immediately after the 2-week exposure and 2 weeks later. Microscopically, exposure-related lesions were confined to the respiratory tract and were dose related. In the 59 and 250 mg/m³ groups, lesions were observed in the nose, trachea, larynx/pharynx, and lung. These lesions were mainly minimal to mild in the 59 mg/m³ group and mild to moderate in the 250 mg/m³ group. After the 2-week recovery period, the above nasal and pulmonary lesions were diminished in severity and the tracheal and laryngeal/pharyngeal lesions were no longer present. At this time, there was a slight increase in fibrous connective tissue (collagen formation) associated with pulmonary inflammatory lesions in the 250 mg/m³ group and in one rat in the 59 mg/m³ group. In rats exposed at 9.2 mg/m³ only the nose was affected. Minimal to mild inflammation of the nasal epithelium was observed in this group immediately after the 2-week exposure period. After a 2-week recovery period the nasal lesions in this group were diminished in severity and were only seen in 2 of 5 rats. Due to the fact that effects on the respiratory tract were seen in all concentration groups tested and these effects were clearly dose related a no observed adverse effect level could not be determined. The LOAEC of this study was established to be 9.2 mg/m³ (Kelly, 1991).

In conclusion, the effects observed in Crl:CD^®BR rats exposed to MPMD ranged from severe (including death) in the 250 mg/m³ group to minimal in the 10 mg/m³ group. Determined effects diminished in severity over time but were only partially reversible within the 2-week observation period. Lesions observed after exposure to DCH were less severe, confined to the upper respiratory tract and were fully reversible within the 14-day observation period.

Except for these subacute toxicity studies, a 13-week study of the toxicity of the dihydrochloride salt of hexamethylenediamine (CAS 6055-52-3, HDDC) was conducted in male and female Fischer 344/N rats using whole body inhalation exposure (Hébert, 1993; publication and NTP report). The method followed was comparable to OECD 413 and was conducted according to the US-EPA GLP which were similar to the EU-GLP. Under the test conditions, the NOAEC (HDDC) = 16 mg/m³/day for local respiratory damage (nasal respiratory epithelium degeneration), corresponding to a NOAEC (HMD) = 10 mg/m³/day. However, this study could not be used to assess the local effects caused by the category members as only the dihydrochloride salt was used instead of the alkaline and therefore corrosive substance itself.

In a repeated- inhalation toxicity study (Ben-Dyke (Bio/dynamics), 1981), 60 S-D Rats/sex were exposed to aqueous hexamethylenediamine (HMD, CAS 124-09-4) aerosol for six hours per day, five days per week at mean analytical concentrations of 0, 12.8, 51 and 215 mg/m³ for thirteen weeks. Under the test conditions, no systemic effects were observed related to the treatment with HMD. The significant local irritation of respiratory tract, inducing clinical signs, was observed at the two highest concentrations tested. The NOAEC in male and female rats exposed by whole-body inhalation was 12.8 mg HMD/m³.

As in all these studies the main effect observed after repeated inhalation is a local effect on the respiratory tract. No adverse systemic effects were noted at concentrations were the local effects occurred. The type and the severity of the local effect observed are the same whatever the time duration the exposure is (2 weeks or 13 weeks). Therefore, the local effect observed appears to be not driven by the time of exposure.

Apart from rather systemic effects most probably due to a generalized reaction to stress only local irritating effects were observed after repeated inhalation exposure to DCH and the other amines tested. And the local effects observed are clearly due to the corrosivity of the substances. Since corrosivity is the underlying cause the classification for the corrosive properties and for STOT single exposure category 3 is considered sufficient in order to protect from long term inhalation effects - together with the risk characterisation based on a DNEL derived from the substance specific inhalation LOAEC identified in the subacute study.

In the ‘Guidance on the application of the CLP criteria’ (Version 5.0 of July 2017), page 470, paragraph 3.9.2.5.1, section irritating/corrosive substances, the following sentences were written:

‘Substance classified as corrosive may cause severe toxicological effects following repeated exposure, especially in the lungs following inhalation exposure. In such cases, it has to evaluated whether the severe effect is a reflection of true repeated exposure toxicity or whether it is in fact just acute toxicity (i.e. corrosivity). One way to distinguish between these possibilities is to consider the dose level which causes the toxicity. If the dose is more than an order of magnitude lower than that mediating the evident acute toxicity (corrosivity) then it could be considered to be repeated-dose effect distinct from the acute toxicity. In this case, classification as specific target organ (repeated exposure) would be warranted even if the substance is also classified as acutely toxic and/or corrosive.’

Since results observed in the repeated inhalation studies are clearly due to the corrosivity of the substance, we propose not to classify DCH as STOT RE Cat 1. Instead, classification of the submission substance as Corr. Cat. 1A, H314 and STOT SE Cat 3, H335 is considered suitable to protect from repeated inhalation effects.

References used, which are included in IUCLID as 'other information':

Ben-Dyke R. (1981). A 13 week inhalation toxicity study of Hexamethylene diamine in the rat. Testing laboratory: Bio/dynamics Inc. Owner company: Solutia. Study number: BD-79-7346. Report date: 1981-01-23.

Hébert C. D. (1993). NTP Technical Report on Toxicity Studies of 1,6-Hexanediamine Dihydrochloride administered by drinking water and inhalation to F344/N rats and B6C3F1 Mice. National Institutes of Health (NIH) Publication (March 1993) - United States Department of Health and Human Services. Report no.: 93-3347.

Hébert C. D., Elwell M. R., Travlos G. S., Zeiger E., French J. E. and Bucher J. R. (1993). Inhalation Toxicity of 1,6-Hexanediamine Dihydrochloride in F344/N Rats and B6C3F1 Mice. Fundamental and Applied Toxicology, 20:348-359

Kelly, DP (1991). Two-Week Inhalation Toxicity Study with Dytek A Amine in Rats. Testing laboratory: Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware 19714, USA. Report no.: HLR 45-91. Owner company: Invista. Report date: 1991-06-14.

Justification for classification or non-classification

Organ-specific effects observed after repeated inhalation exposure are confined to the respiratory tract. The effects noted in rats are clearly due to the corrosivity of the submission substance which are expected to occur even after short-term exposure. Accordingly, the classification for its corrosive properties and STOT single exposure Cat 3 is sufficient. Therefore, classification with respect to specific target organ toxicity after short-term exposure is proposed:

STOT SE Cat 3, H335, together with Corr. Cat 1

Based on findings in the oral 90-day repeated dose study, classification for reproduction toxicity after repeated dose exposure is deemed necessary according to Regulation (EC) No 1272/2008:

Reproduction toxicity, Category 2 (H361f)

(please see the section 'Toxicity for reproduction' for a detailed discussion)

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Conc. [mg/cubic metre] Mean (SD) (a)	[%] Aerosol / [%] Vapour (b)	Particle size - MMD [micrometre] (c)	Particle size - [%] < 10 micrometres (d)
Control chamber
10 (2)	33/67 (e)	4.8	79
49 (8.7)	3/97	3.6	82
240 (66)	36/64	5.1	80

a. Total test material chamber atmospheric concentration (combined aerosol and vapour). Mean and standard deviation of 10 exposures based on a minimum of 4 samples per exposure. b. Mean % of test material in the chamber atmosphere which was in the aerosol or vapour form. c. Mass median aerodynamic diameter, one measurement per week. (mean of two samples) d. Percent by weight of particles with aerodynamic diameter less than 10 micrometres. From cascade impactor determinations (mean of two samples). e. Percent aerosol for the 10 mg/m3 chamber increased dramatically during the second veek of exposures. See chamber atmosphere discussion below for further interpretation of this finding.