Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 26, 2013 to June 26, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride

EC Number:

629-716-7

Cas Number:

1211950-04-7

Molecular formula:

No molecular formula

IUPAC Name:

N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride

Constituent 2

Reference substance name:

N,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free sample

IUPAC Name:

N,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free sample

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): N,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free sample
- Substance type: Light yellow waxy powder
- Physical state: Powder
- Purity: 96.0 %m/m
- Lot/batch No.: 20130125JB
- Expiration date of the lot/batch: 25 January 2023
- Storage condition of test material: At room temperature in the dark under nitrogen
- Purity/composition correction factor required: No
- Hygroscopic: Yes, store in well-sealed container
- Volatile: No
- Reactivity: Reactive to oxygen
- Test substance handling: Flush container with nitrogen after handling
- Stability at higher temperatures: Yes, maximum temperature: 80°C, maximum duration: 60 min
- Stability in water: Stability for at least 5 hours at room temperature under normal laboratory light conditions and 8 days in the refrigerator in the dark under nitrogen is confirmed over the concentration range 1 to 10 mg/mL.

Test animals

Species:

rat

Strain:

other: Wistar (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old).
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 153 grams; females: 122 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 26 March - 26 June 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
From Day 1 to 30, formulations (w/w) were prepared daily within 5 hours prior to dosing. From Day 31 onwards, formulations (w/w) were prepared for a maximum of 8 days prior to dosing. Formulations were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

DOSE VOLUME:
10 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations in Weeks 1, 6 and 13). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and stability over 8 days in the refrigerator in the dark were also determined (highest and lowest concentration, in week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, 7 d/w.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 28-day oral study with the test substance by daily gavage in the rat (information provided by the sponsor)

Positive control:

Not required.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: at pretest: all animals (including spare animals), at Week 13: Groups 1 and 4

ESTROUS CYCLE DETERMINATION
- Time schedule for examinations: Day 69 up to and including Day 90 of treatment.
- How many animals: All females, except for one female at 0 mg/kg (no vaginal lavage was possible, since the vagina was not open)

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines .

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: during Week 12/13 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.

Sacrifice and pathology:

GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
No mortality occurred that was considered to be related to treatment with the test substance. One animal at 100 mg/kg died at blood sampling at the end of treatment. This was considered not to be related to treatment.

CLINICAL SIGNS
Treatment at 100 mg/kg resulted in the following clinical signs:
- Rales in 5/10 males from Week 8 onwards and in 4/10 females from Week 4 onwards,
- Hunched posture in 1/10 males in Weeks 8 to 10, in 2/10 females in Weeks 7 to 10 and in all females from Week 13 onwards.
Lethargy, flat posture, shallow respiration and ptosis shown by one male at 100 mg/kg on a single day only in Week 4 of treatment was considered unrelated to the test substance, given the incidental occurrence and absence of similar findings among all other animals of this dose group.
Salivation seen after dosing among animals of the 100 mg/kg dose group from Week 3 of the treatment period onwards was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance and/or may be related to the microscopic observation of prominent intercalated ducts in the mandibular salivary glands.
No other treatment related clinical signs were observed. Incidental findings that were noted included scabs (neck), chromodacryorrhoea, rales (females at 30 mg/kg) and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
At 100 mg/kg, body weights and body weight gain were lower than controls in males and females achieving a level of statistical significance on most occasions. Body weight gain was lower from Week 2 onwards and in females from Week 4 onwards. Group mean body weight after 13 weeks of treatment was approximately 20 and 12% lower than control mean weight for males and females, respectively. At 10 and 30 mg/kg, body weights were similar to controls throughout treatment.

FOOD CONSUMPTION
At 100 mg/kg, a trend towards slightly lower food consumption was noted for males. When corrected for body weight, food intake values were similar to control levels. Food consumption of males and females at 100 mg/kg remained within normal levels for rats of this age and strain. At 10 and 30 mg/kg, food intake was similar to control levels.

OPHTHALMOSCOPIC EXAMINATION
There were no toxicologically relevant ophthalmology findings at pre-dose and in Week 13. Incidental ophthalmology findings consisted of (focal) corneal edema or opacity, pinpoint corneal opacities, and haemorrhage from hyaloid vessel or iris. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain.

ESTROUS CYCLE DETERMINATION
At 100 mg/kg, two females showed an acyclic estrous cycle of 11 or 14 days. All other females at 100 mg/kg, and all females of the control group and the 10 and 30 mg/kg groups showed a normal (regular) estrous cycle of 4 to 5 days during the period in which estrous cycle length was determined (Day 76 up to and including Day 90).

HAEMATOLOGY
The following statistically significant changes in haematology parameters distinguished treated animals from control animals:
- Lower relative reticulocyte counts in males at 30 and 100 mg/kg and females at 100 mg/kg,
- Lower haemoglobin and haematocrit level in females at 100 mg/kg.
The slightly higher relative neutrophils and lower relative lymphocyte counts in females at 100 mg/kg and the lower white blood cell counts (WBC) in both sexes at 10 mg/kg remained within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males and females at 100 mg/kg and in males at 30 mg/kg,
- Lower albumin and total protein level in males and females at 100 mg/kg,
- Higher urea level in males at 100 mg/kg,
- Higher glucose level in females at 100 mg/kg,
- Lower potassium level in males at 100 mg/kg,
- Lower chloride and calcium level in females at 100 mg/kg,
- Higher inorganic phosphatase level in males and females at 100 mg/kg (not statistically significant in females).
The higher alanine aminotransferase activities and lower albumin and total protein levels were generally outside the normal range for rats of this age and strain, while other changes remained within this range.
The higher sodium level in males at 10, 30 and 100 mg/kg was considered to be of no toxicological significance as these changes occurred in the absence of a dose-related trend, and remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR
Males and females at 100 mg/kg showed a reduced motor activity (both total movements and ambulations; only statistically significant for females).
One animal at 30 mg/kg showed an abnormal pupillary reflex in the right eye. As this was only one animal, this finding was considered to be of no toxicological relevance. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all other examined animals. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
The following (statistically significant) changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
- Slightly increased spleen weight and spleen to body weight ratio in males and females at 100 mg/kg (not statistically significant for absolute weights),
- Slightly lower seminal vesicles weights in males at 100 mg/kg.
Other statistically significant changes in organ weights at 100 mg/kg were attributed to the lower terminal body weight and occurred without correlating morphological findings. These changes consisted of higher brain to body weight ratio in males and females, lower heart weight and higher heart to body weight ratio in males and/or females, lower liver weight (not statistically significant in females) and higher liver to body weight ratio in males and females lower kidney weight in males and females, lower prostate weight, lower adrenal to body weight ratio in males, , higher epididymides and testes to body weight ratio, lower ovary weight and higher uterus to body weight ratio , lower thyroid and thymus weight in males .

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, anddid not show a clear dose-related incidence trend.


HISTOPATHOLOGY
Treatment-related microscopic findings were present in:
Trachea:
- Loss of cilia/loss of mucous cells was recorded at 100 mg/kg (Group 4) in 4/10 males (2: minimal, 1: moderate, 1: marked) and in 4/10 females (1: minimal, 1: slight, 2: moderate) and in females at 30 mg/kg (Group 3) in 3/10 animals (minimal).
- Hypertrophy/hyperplasia of the epithelium was recorded at 100 mg/kg in 2/10 males at a minimal degree and in 3/10 females (2: minimal, 1: slight) and in females at 30 mg/kg in 3/10 animals (2: minimal, 1: slight).
Lung (males):
- Alveolar macrophage foci (in some animals with a foamy appearance) were recorded at an increased incidence and/or severity at 100 mg/kg (Group 4) in 9/10 males (4: minimal, 5: slight) and at 30 mg/kg (Group 3) in 1/10 males (moderate). A background level of this finding was recorded in 1/10 males and 2/10 females of the control Group 1 and in 4/10 females at 100 mg/kg (Group 4), all at a minimal degree.
Duodenum:
- Hypertrophy of the duodenal villi was recorded at 100 mg in 2/10 males (1: minimal, 1: slight) and in 2/10 females (minimal).
Seminal vesicles:
- Vacuolar degeneration of the epithelium was recorded at 100 mg/kg (Group 4) in 10/10 males (5: minimal, 5: slight) and at 30 mg/kg (Group 3) in 2/10 males (minimal).
Thyroid gland (females):
- Minimal vacuolation of the follicular epithelium was recorded in 5/10 females at 100 mg/kg (Group 4).
Adrenal glands:
- Vacuolation of the zona glomerulosa was recorded at 100 mg/kg in 2/10 males (1: minimal, 1: slight) and in 7/10 females (4: minimal, 3: slight).
Mesenteric lymph node:
- Macrophage foci were recorded at an increased incidence and severity at 100 mg/kg (Group 4). This was recorded in 10/10 males (7: slight, 3: moderate) and in 10/10 females (1: minimal, 6: slight, 3: moderate). The incidences and severities of this finding recorded in the remaining dose Groups were considered to be within background: Control Group 1: 4/10 males and 4/10 females (minimal), 10 mg/kg (Group 2): 3/10 males (1: minimal, 2: slight) and 4/10 females (minimal) and at 30 mg/kg (Group 3): 6/10 males (5: minimal, 1: slight) and 5/10 females (4: minimal, 1: slight).
Salivary glands (mandibular):
- Prominent intercalated ducts were recorded at 100 mg/kg in 5/10 males (3: minimal, 2: slight) and in 6/10 females (4: minimal, 2: slight).
Spleen:
- Congestion was recorded at an increased incidence and severity at 100 mg/kg (Group 4) in 8/10 males (4: minimal, 3: slight, 1: moderate) and in 8/10 females (3: minimal, 4: slight, 1: moderate). A background level of this finding was recorded in 1/10 males of control Group 1 and in 1/10 males and 1/10 females at 30 mg/kg (Group 3) ), all at a minimal degree.
Bone marrow (sternum):
- Increased adipocytes in the sternal bone marrow at a slightly higher incidence and/or severity was recorded at 100 mg/kg (Group 4) in 5/10 males (minimal) and 4/10 females (2: minimal, 2: slight) and at 30 mg/kg (Group 3) in 5/10 females (3: minimal, 2: slight). A background level of this finding was recorded in 2/10 females of the control Group 1, 1/10 males and 3/10 females at 10 mg/kg (Group 2) and 1/10 males at 30 mg/kg (Group 3), all at a minimal degree.
Harderian glands (females):
-An increased amount of brown (porphyrin) pigment was recorded in females at 100 mg/kg (Group 4). This was seen in 7/10 animals (5: minimal, 2: slight). A background level of this finding was recorded in 2/10 males of the control Group 1, 3/10 females at 10 mg/kg (Group 2), 3/10 females at 30 mg/kg (Group 3) and 4/10 males at 100 mg/kg (Group 4), all at a minimal degree.

The findings recorded in trachea, lung (males), seminal vesicles and mesenteric lymph nodes are considered to be adverse microscopic findings at the incidences and severities recorded in this study. For the findings in duodenum, thyroid gland (females), adrenal glands and salivary glands adversity can’t be excluded.
The findings recorded in the spleen, sternal bone marrow and Harderian glands (females) can also be present as normal background findings in rats of this age and strain. Therefore, these findings, at these incidences/severities and in absence of any other indicators of toxicity in these organs, were not considered to be adverse.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.
Spermatogenic staging profiles were normal for all males examined.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

other: Non systemic effects due to malnutrition caused by test substance

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 in Weeks 1 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The concentrations analysed in the formulations of Group 2, 3 and 4 in Week 13 showed slightly higher values than the target concentrations (i.e. the mean accuracies for Group 2, 3 and 4 were 115, 110 and 117%, respectively). No explanation was found for this. The formulations of Group 2 and 4 in Weeks 1, 6 and 13 were homogeneous (i.e. coefficient of variation ≤10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours and were found to be stable in the refrigerator in the dark for at least 8 days. The long term storage samples were stable at ≤-70°C for at least 41 days.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, 90 d NOAEL for repeated oral study is determined to be 10 mg/kg bw/day.

Executive summary:

A 90-d study was conducted to determine the repeated dose oral toxicity of the test substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3 -propanediammonium chloride in rats, according to OECD Guideline 408, in compliance with GLP. Dose levels were selected by the sponsor at 0, 10, 30 and 100 mg/kg bw/day, based on the results of a 28 -day study with the test substance. The test substance, formulated in water, was administered daily for at least 90 d by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. The following parameters were evaluated at regular intervals: clinical signs, functional observation parameters, body weight, food consumption, ophthalmoscopic parameters, clinical pathology, organ weights, macroscopic examination and histopathology of a selection of tissues. No treatment-related mortality occurred in the study. Treatment at 100 mg/kg bw/day resulted in hunched posture mainly at the end of treatment, slight decrease in motor activity, a slightly lower food consumption and body weight primarily for males. Histopathological findings at 100 mg/kg bw/day consisted of loss of cilia/loss of mucous cells in the trachea, hypertrophy/hyperplasia of the epithelium in the trachea, hypertrophy of the villi of the duodenum, vacuolation of the follicular epithelium of the thyroid gland (females only), vacuolation of the zona glomerulosa of the adrenal gland, vacuolar degeneration of the epithelium of the seminal vesicles, increased incidence and severity of macrophage foci in the lungs (males only) and mesenteric lymph node and prominent intercalated ducts in the mandibular salivary glands. Changes in blood parameters at 100 mg/kg bw/day consisted of lower relative reticulocyte counts in both sexes, lower haemoglobin and haematocrit level in females, higher alanine aminotransferase activity and inorganic phosphatase level and lower albumin and total protein level in both sexes, higher urea and lower potassium level in males, and higher glucose and lower chloride and calcium level in females. Histopathological findings at 30 mg/kg bw/day consisted of loss of cilia/loss of mucous and hypertrophy/hyperplasia of the epithelium in the trachea (females only), vacuolar degeneration of the epithelium of the seminal vesicles, increased severity of macrophage foci in the lungs (in one male only). Changes in blood parameters at 30 mg/kg bw/day were confined to lower relative reticulocyte counts and higher alanine aminotransferase activity in males. Other treatment-related microscopic findings consisted of increased incidence and severity of congestion of the spleen at 100 mg/kg bw/day (correlating with increased spleen weights), increased incidence/severity of adipocytes in the sternal bone marrow at 30 (females) and 100 mg/kg bw/day and increased incidence and severity of brown (porphyrin) pigment Harderian glands in females at 100 mg/kg bw/day. These findings can also be present as normal background findings in rats of this age and strain. Therefore, at these incidences/severities and in absence of any other indicators of toxicity in these organs, these findings were not considered to be adverse. Two out of ten females at 100 mg/kg bw/day showed an abnormal (acyclic) estrous cycle, which is above the incidence reported in historical control data. Histopathological examination of the female reproductive organs did not show treatment-related lesions. The observation of an acyclic period was seen in two animals of the high dose group showing apparent toxicity, and within this group the two affected animals also showed the greatest effect on body weight. An indirect effect following non-specific systemic toxicity or malnutrition is the most likely cause for the observed acyclic estrous period in these animals. The effects of lower seminal vesicle weight seen at 100 mg (only absolute, not relative, so linked to lower BW) and vacuolar degeneration of the epithelium of the seminal vesicles at 30 and 100 mg/kg bw/day are not considered to have reproductive effects considering that spermatogenic staging profiles were normal in all dose groups. Under the study conditions, the NOAEL for repeated dose toxicity is determined to be 10 mg/kg bw/day (Wil research, 2014).