Registration Dossier

Administrative data

Description of key information

Oral: The 28d NOAEL (No Observed Adverse Effect Level) = 40 mg/kg/day for rat
Oral: The extrapolated 90d NOAEL (No Observed Adverse Effect Level) = 13 mg/kg/day for rat
Dermal: The 28d NOAEL (No Observed Adverse Effect Level) for MALES and FEMALES = 150 mg/kg/day for rat

Oral: 14 days and 90 days NTP 1993 study on analogue substance Mn2+ ( MnSO4)

Weight if evidence: several published literature on repeat dose toxicity from manganese exposure

Inhalation: Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals administered the read-across substance was determined to be 20 µg/L.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20.3.-22.9.2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, BioTest, s. r. o., 28125 Konárovice, Czech Republic
- Age at study initiation: 6 - 7 weeks
- Fasting period before study: no
- Housing: in SPF (specific pathogen free) animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood
- Diet: ad libitum, complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN), producer: Mill Kocanda, Jesenice by Prague.
Diet was sterilised before using.

- Water: ad libitum, free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law,
Health Ministry. Water was sterilised before using.
- Acclimation period: minimally 6 days
- Identification: Identification of animals was made by colour marks on fur (system 1 – 6), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group.

All the study proceeded in SPF (Specifiedc Pathogen Free) animal house of CETA in conditions according to SOP No. 12.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Study time schedule:
Test substance delivery: 3. 3. 2006
Dose-range finding study: 20. 3. – 12. 4. 2006
Main study: 25. 4. – 25. 5. 2006
Date of animal arrival: 18. 4. 2006
Start of administration: 25. 4. 2006
End of administration: 25. 5. 2006
Observation: 25. 4. – 25. 5. 2006 – main groups
25. 4. – 6. 6. 2006 – satellite groups
Urinalysis: 22. – 25. 5. 2006 - main groups
5. – 6. 6. 2006 - satellite groups
Blood taking and necropsies: 23.- 26. 5. 2006 - main groups
6. – 7. 6. 2006 - satellite groups
Histopathological examination: 6. 7. - 22. 9. 2006

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The concentrations of solutions in all three dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form (test substance solution in water for injectione) was prepared daily just before administration. The vehicle control group was administered by water for injectione in the same volume. The application form of the test substance was prepared daily before administration; it
was mixed 10 minutes by magnetic stirrer. The procedure was based on the results of analyses of test substance application form homogeneity
and stability (see ANNEX 2). These results showed that the test substance at defined laboratory conditions (laboratory temperature, mixing of
solutions by defined manner) is homogenously dissolved in water and the solution is stable at last for 120 minutes.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua, the test substance was administered dissolved in water for injectione.
- Manufacturer: Infusia a.s., Hořátev, Czech Republic
- Batch number: 276805
- Attest No.: 2768/05
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of test substance was performed by a spectrophotometry.
Test substance stability and homogeneity were determined by measuring an absorbance of its water solution in visible range of spectrum.
Duration of treatment / exposure:
28 days (4 weeks)
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 - 10.00 a.m.)
Remarks:
Doses / Concentrations:
40 mg/kg bw/day (m/f)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day (m/f)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day (m/f)
Basis:
actual ingested
No. of animals per sex per dose:
Main groups:
1. group - control with water for injectione 6 males + 6 females
2. group - the lowest dose 40 mg/kg/day 6 males + 6 females
3. group - the intermediate dose 100 mg/kg/day 6 males + 6 females
4. group - the highest dose 250 mg/kg/day 6 males + 6 females
Satellite groups:
5. group - satellite control with water for injectione 6 males + 6 females
6. group - satellite the highest dose 258 mg/kg/d 6 males + 6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose-range finding study with 14-day application period was performed. The dose levels 30, 60, 120 and 240 mg/kg/day (in water for injections) were chosen with respect to the acute toxicity study results. In the dose-range finding study, the dose-dependent decrease of body weight
increments were observed in the males. Clinical observation did not detect the impact of the test substance on the health condition of animals.
Result of haematology examination showed that the test substance does not have an impact to basic blood parameters. Macroscopic changes of
organs (marked structure of liver, changed colour of liver and kidney, change of stomach mucosa) were found in dose levels 60, 120 and 240
mg/kg/day.
- Rationale for animal assignment: Animals were randomly divided into the control and test groups and marked individually.
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 14 days after the end of application
- Section schedule rationale: all animals were sectioned
Positive control:
no
Observations and examinations performed and frequency:
HEALTH CONDITIONS: Yes
- Time shedule: daily

MORTALITY CONTROL: Yes
- Time shedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed before the first application and then weekly.
- In the first part of observation, the behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerec tion, stereotypes or bizarre behaviour of animals.
- The second part was the observation during the removal from the cage. These parameters were observed: reaction to handling, elasticity of skin, col our of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- The body weight of animals was recorded on automatic balances with group average computing module.
First weighing was performed before the first application and then weekly. All animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per week (in grams).


FOOD CONSUMPTION AND COMPOUND INTAKE:
-In the given day of every week the remainder of pellets was weighed in each cage, the new food was weighted out and the food consumption for last week was computed.
The average values in groups were calculated for each week of the study.

FOOD EFFICIENCY:
-Food consumption for animal/day was calculated of average values of each group.
From growth increments and food consumption the food conversion was calculated:food conversion (%)=weight increment/food consumption x 100


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice a week
The drinking water requirement consumption was recorded twice a week. The average values in groups (water consumption per animal per day) were calculated for each week of the study.

OPHTHALMOSCOPIC EXAMINATION: Not performed

HAEMATOLOGY: Yes
- Time schedule for collection of blood: : 29th (main groups) and 42nd day of study (satellite groups)
- Anaesthetic used for blood collection: Yes, light diethyl ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 29th and 42nd day of study
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: 28th and 41st day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.3] were examined.


FUNCTIONAL OBSERVATION: Yes
- Time schedule for examinations: fourth week
- Dose groups that were examined: all animals
- Battery of functions tested: the sensory reactivity on auditory, papillary reflex, visual and proprioceptive stimuli were evaluated and motor activity
assessment was conducted.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities was carried out.
Organs for subsequent histopatological examination were taken out and stored in containers with fixative (neutral 9 % formaldehyde).

HISTOPATHOLOGY: Yes (see table No. 4)
- Tissue specimens were fixed in 4% neutral formaldehyde processed by routine paraffin technique and stained by hematoxyline-eosine.
Cryotome sections of liver and kidneys were stained by oil red for neutral lipids.

At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epidydimides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes (SI) were computed according to the following formula: SI = weight of organ x 100 / body weight (= relative weight of organ).
Statistics:
Data processing
All the primary data (body weight, food consumption, water consumption, health condition control, general observations, clinical observational battery, functional observations, haematological examinations, biochemistry, urinalysis, biometry of organs, necropsy findings, histopathological examinations) were recorded in protocols. These primary data were used for calculations and processed to tables.

Statistical analysis
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry and biometry of organs. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with the satellite highest dose group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
In control and treatment animals no clinical changes were observed during the whole study. No mortality occured during the study period.

HEALTH CONDITION CONTROL
Main groups – MALES and FEMALES
The health condition of all animals was controlled daily during the check-in, acclimatisation and application period. No signs of diseases were found out.
Satellite groups – MALES and FEMALES
The health condition of all animals was controlled daily during the check-in, acclimatisation, application period and during the recovery period. No signs of diseases were found out.


BODY WEIGHT AND WEIGHT GAIN
Average body weight:
MALES
Main groups
Since the 1st week the growth curves of all treated groups proceeded slightly under control growth curve. Maximal difference was in dose level 250 mg/kg/day. In this dose level, difference of body weight compared to control increased from 17 grams (in the 1st week application) till 33 grams (in the4th week).
Satellite groups
The growth curves of satellite treated group were slightly below the control one, as in corresponding main group. This weight difference against control group persisted also after the end of application.

FEMALES
Main groups
During the 1st to the 3rd week of study, the growth curves of all treated groups were well-balanced with the control group. In the 4th week, the growth curve of the highest dose level 250 mg/kg/day runs slightly below then in the control group.
Satellite groups
The body weights of females in the dose level 250 mg/kg/day were slightly lower then in the control group. During recovery period after application the growth curve of the treated group had slightly rising trend. In the end of recovery period the body weight of treated animals was slightly higher then in control group.

Body weight increment:
MALES
Main groups
During the 1st week of application, average body increments were lower in all treated males against control group. In the highest dose level marked decrease of increments was recorded and continued during all period of application.
Satellite groups
Average body weight increments of treatment satellite group of males were lower then in control animals. In the 6th week of study (the 2nd week of recovery period) the body weight increments of the highest dose level were higher then in control group.

FEMALES
Main groups
Average body weight increments of females in dose level 250 mg/kg/day were lower then in control group in the 1st week of study. Higher increments of all treated groups were recorded in the 2nd week. During 3rd- 4th week of study increment of animals were lower against control group in the dose levels 100 and 250 mg/kg/day.
Satellite groups
During the 1st – 2nd week of study, average body weight increments were lower in dose level 250 mg/kg/day. In the 3rd week the increment was higher against control group. In the highest dose level decrease of body weight (negative increase) was recorded in the 4th week of study. In the recovery period average body weight increment in treatment group was higher then in control group in 5th week of study and lower in the 6th week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption
MALES
Main groups
Food consumption of all treatment groups of males was lower in comparison the control group, mostly in the dose level 250 mg/kg/day. Consumption was decreased depending on dose level in the1st and 4th week of application.
Satellite groups
Food consumption of treatment group was lower than in control group during the whole study (except the 3rd week).

FEMALES
Main groups
Food consumption of treatment groups of females was analogous with food consumption of control group. Only in the dose level 250 mg/kg/day, food consumption was slightly lower during whole application period.
Satellite groups
Food consumption of treated females was lower than in control during whole application period. The consumption increased during the recovery period.


FOOD EFFICIENCY
Food conversion
MALES
Main groups
In the 1st week of application, food conversion was decreased in the dose level 250 mg/kg/day; in the 2nd week of study conversion was decreased in the dose levels 100 and 250 mg/kg/day; in the 3rd week of study food conversation was decreased only in the highest dose level; in the 4th week food conversion was decreased in all treated groups. In the dose level 250 mg/kg/day the food conversion was decreased against the control group during the whole study.
Satellite groups
In treated group the food conversion was degreased against the control group during whole application period (except the 2nd week). During the 5th week the food conversion of satellite groups were balanced and in the 6th week the conversion in treated group was higher than in the control group.

FEMALES
Main groups
Food conversion of females of all treated groups was analogous with the control group during the 1st week. On the contrary food conversion of all treated groups was higher against the control group in the 2nd week of study. Since the 3rd week the food conversion was lower in dose levels 100 and 250 mg/kg/day, markedly in the highest dose level. In the dose level 40 mg/kg/day the food conversion was lower then in the control group only in the 3rd week of application.
Satellite groups
The food conversion of the dose level 250 mg/kg/day was lower since the 1st week of study; the 3rd week of study the conversion was higher against the control group, but next week was lower again. During 1st week of recovery period (the 5th week of study) the food conversion of treated group was higher and in the 2nd week of study (the 6th week of study) conversion was lower against the control group again.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
MALES
Main groups
During the 1st week of study, the water consumption was decreased depending on dose level. Since 2nd week of study, water consumption was markedly lower only in males of the dose level 250 mg/kg/day. In the dose levels 40 and 100 mg/kg/day the water consumption was analogous to the control group.
Satellite groups
Water consumption of treated group was markedly lower against the control group during whole study (application and recovery period).

FEMALES
Main groups
Water consumption was lower in females of the dose level 250 mg/kg/day. In the dose levels 40 and 100 mg/kg/day the water consumptions were also slightly lower since the 2nd week of application.
Satellite groups
Water consumption of treated group was lower against control group during whole study (application and recovery period).


HAEMATOLOGY
MALES
Main groups
Slight decrease of total erythrocyte count was found in the dose level 100 mg/kg/day. Slight increase of total erythrocyte count and accompanying increase of haemoglobin concentration and haematocrit were recorded in the dose level 40 mg/kg/day. In this dose level, increased value of haemoglobin concentration was statistically significantly different against control group. Value of leucocyte count increased depending on dose levels. APTT was slightly decreased only in the dose level 40 mg/kg/day.
Other measured parameters in the dose level 40 and 100 mg/kg/day and all parameters in the dose level 250 mg/kg/day were similar compared with animals in the control group.

Satellite groups
Statistically significant increases of total erythrocyte count, haematocrite and haemoglobine concentration were recorded in treated group. Value of total leucocyte count was also higher than in the control group. PT was statistically significantly increased against the control group.
Other measured parameters were similar compared with animals in the control group.

FEMALES
Main groups
Decrease of MCV was recorded in the dose level 100 mg/kg/day. Values of leucocyte count and trombocyte count were slightly increased in all treated groups. Decreased counts of monocytes and granulocytes and increased count of lymphocytes were measured in all treated groups; these changes were marked in the dose levels 40 and 100 mg/kg/day.
Other measured parameters were similar compared with animals in the control group.
Satellite groups
Values of total erythrocyte count, leucocyte count ant trombocyte count were slightly higher against the control group in females of treated group. Statistically significantly increased value of monocytes and accompanying decreased value of lymphocytes were recorded in dose level 250 mg/kg/day.PT was also significantly increased against the control group.
Other measured parameters were similar compared with animals in the control group.

BIOCHEMICAL EXAMINATION
MALES
Main groups
Decreased values of total protein and albumine were recorded in the dose levels 100 and 250 mg/kg/day. The changes were statistically significant in the highest dose level. Values of total urea and ALP were increased in dose level 250 mg/kg/day. Increased value of total urea and creatinine were recorded also in the dose level 40 mg/kg/day.
Other measured parameters were similar with the control group.
Satellite groups
Statistically significantly increased value of calcium was recorded in the treated group of animals. Rising trends were detected also in parameters total protein and ALP.
Other measured parameters were similar with the control group.


FEMALES
Main groups
Decreased values of total cholesterol were recorded in all treated groups. Because these decreased values were under of detection limit, statistical evaluation was not possible to perform. Decreased values of total protein and albumine were recorded in the dose levels 100 and 250 mg/kg/day. Value of bilirubine was lower then in the control group in dose level 100 mg/kg/day. Slightly decreased values of total urea were detected in all treated groups.
Other measured parameters were similar with the control group.
Satellite groups
Statistically significant increased value of creatinine was recorded in treated group of females. Decrease was recorded also in value of total urea.


URINALYSIS
MALES
Main groups
Decrease of urine volume; increase of pH, specific weight and content of protein were recorded in all treated group against the control group. These changes were significant in the dose level 250 mg/kg/day. The content of leucocytes, ketones and urobilinogen, in urea were recorded only in the highest dose level.
Satellite groups
Decrease of urine volume, increase of pH and content of protein were recorded in treated satellite group against the control group. The content of leucocytes was also recorded in this dose level.

FEMALES
Main groups
Urine volume was decreased against the control group in the dose levels 40 and 250 mg/kg/day. Increase of pH and specific weight were recorded in all treated groups. The content of protein, ketones and urobilinogen in urea were recorded also in the highest dose level.

Satellite groups
Increase of pH and specific weight were recorded in the satellite treated group.


ORGAN WEIGHTS
MALES
Main groups
In the highest dose level (250mg/kg/day), decreased absolute weight of liver was found out. On the contrary, the relative weights of liver were slightly increased against the control group in all treated groups. Increased relative weight of testes and epididymides were recorded in the dose levels 100 and 250 mg/kg/day. Absolute weights of testes were slightly increased in males of all treated groups. Slightly increased absolute weights of epididymides were found only in dose levels 100 and 250 mg/kg/day. Increased weight (relative and absolute) of the spleen was recorded in exposed groups 250 mg/kg/day.
Satellite groups
Increased weight (relative and absolute) of the spleen was recorded in males of the treated satellite groups. The relative weight of the spleen was statistically significantly increased against the control group. Absolute weight of liver was slightly lower and relative weight was slightly higher then the in control animals.
Absolute and relative weight of testes and epididymides were similar with the control group.

FEMALES
Main groups
Slightly increased weight (relative and absolute) of kidneys and spleen were recorded in the highest dose level 250 mg/kg/day. In the dose level 100 mg/kg/day, absolute and relative weight of spleen were slightly decreased. Increase of absolute and relative weight of uterus was found out in all treated dose levels.
In all treated groups, the weights of next organs were similar with the control group.
Satellite groups
Statistically significantly increased weights (relative and absolute) of uterus were recorded in animals of treated satellite group. In the exposed females, absolute and relative weights of spleen were slightly decreased against control group.
In all treated groups, the weights of other organs were similar with the control group.


GROSS PATHOLOGY
MALES
Main groups
During the macroscopic examination of males no important pathological changes were found out. In 5-4-1-0 males no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity petechiae on thymus in 0-0-2-0 males, and focal changes on lung in 0-0-1-1 males were diagnosed.
In abdominal cavity marked structure of liver in 1-2-0-3 males, irregular ochre colour or discolouration of liver in 1-2-0-2 males was found out. The macroscopic changes were often found in stomach: oedema of mucosa in 0-0-4-0 animals, local changes (erosion) in 0-0-1-2 males, irregular colour 0-0-1-1 and irregular structure of mucosa in 0-0-0-3. Incidental findings included focal change on kidneys in 0-0-0-1 male, atrophy of testes in 1-0-0-0 male, and inflammation of epididymides in 1-0-0-0 male.
In cranial cavity no changes were diagnosed.
Satellite groups
During the macroscopic examination of satellite males no important pathological changes were found out. In 5-3 males no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity focal changes on lung in 1-1 males were only found out.
In abdominal cavity irregular colour of liver in 0-1 males and atrophy of testes in 0-1 male were recorded.
In cranial cavity no changes were diagnosed.

FEMALES
Main groups
During the macroscopic examination of females no important pathological changes were found out. In 6-4-0-1 females no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity petechiae and irregular colour of thymus in 0-0-1-0 females, focal changes on lung in 0-0-1-1 females, and irregular colour of lung in 0-0-1-0 female were recorded.
In abdominal cavity marked structure of liver in 0-1-3-1 females, irregular ochre colour or discolouration of liver in 0-2-5-3 females was found out. Atrophy of spleen in 0-0-2-1 females and irregular colour of spleen in 0-0-4-1 females were diagnosed. The macroscopic changes were often found in stomach and forestomach: oedema of mucosa in 0-0-2-3 animals, local changes (erosion) in 0-0-1-4 males, mucosa with mucin in 0-0-1-1 females and irregular structure of mucosa in 0-0-0-3 females. Irregular colour of forestomach was only found out in 0-0-0-1 female. Dilatation of uterus with fluid was diagnosed in 0-0-2-1 females.
In cranial cavity no changes were diagnosed.


Satellite groups
During the macroscopic examination of satellite females no important pathological changes were found out. In 4-3 females no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity petechiae on thymus in 1-0 females were only found out.
In abdominal cavity marked structure and irregular ochre colour of liver in 0-1 female, in stomach: oedema of mucosa in 0-1 female, mucosa with mucin 0-1 female and swelling of Peyer´s patches in 1-0 female were recorded. Dilatation of uterus with fluid was diagnosed in 0-2 females.
In cranial cavity no changes were diagnosed.


HISTOPATHOLOGY: NON-NEOPLASTIC
MALES
Main groups
The affections were often diagnosed in stomach and liver. Focal atrophy of phundal glands was observed in stomach of 1-3-1-0 males and eosinophile infiltration was recorded in stomach of 0-0-1-2 animals. Various alterations were registered in liver (in all groups). Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 2-4-4-3 males. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 4-2-2-3 males. Biliary proliferation was found out in 0-1-3-0 males and oval cells proliferation was recorded in 0-1-1-0 males. Extramedullary haemopoiesis was found out in liver of 0-0-0-1 males. Focal inflammation of liver was detected in 4-3-3-0 males. The changes of intestine were found sporadically: hyperplasia of MALT (MALT – mucosa associated lymphoid tissue) in large intestine of 1-0-0-0 males, eosinophiles infiltration in small intestine of 0-1-0-0 males, and hyperplasia of MALT in small intestine of 1-0-0-0 males. Areactive necrosis of the mucosa was diagnosed in rectum of 0-0-0-3 males.
Steatosis of kidneys was found in 5-4-6-6 males. Next sporadic changes were recorded in kidneys: tubular atrophy in 0-0-1-0 males, hydronephrosis in 0-0-0-1 males, intersticial nephritis in 0-1-0-0 males, proteinuria in 0-0-2-0 animals.
In lymph nodes the following pathological changes were diagnosed: hyperplasia in 2-0-0-0 males, reactive histiocytosis in 0-2-2-0 males, activation in 1-0-0-0 males. Extramedullary haemopoiesis was found in spleen of all males (6-6-6-6). Intensity of haematopoiesis was similar in all groups of males.
Nodular hyperplasia of adrenal gland cortex was detected in 1-0-0-0 males. Active haematopoiesis were kept in bone marrow of all males (6-6-6-6).
Inflammation of larynx was recorded in 0-2-1-2 males. Sporadic affections were also diagnosed in next organs: inflammation in treachea of 0-1-0-0 males, inflammation of lung in 0-0-1-1 males, focal proliferation of glial cells of brain in 0-0-0-1 males.
Incidence of pathological affections in male genital tract was sporadic. Atrophy of germinal epithelium of testes was found in 1-0-0-0 males and inflammation of epididymis in 1-0-0-0 males. Histopathological findings in prostate gland were also sporadic: focal intersticial inflammation in 3-0-0-0 males. The affections in mammary gland were more often: lactating mammary gland in 3-2-1-0 males and involution in 0-2-0-0 males.
Satellite groups
The affections in stomach and intestine were only sporadic. Focal atrophy of phundal glands was observed in stomach of 0-1 males. Focal inflammation of small intestine was recorded in 0-1 males. More often alterations were registered in liver. Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 1-1 males. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 5-5 males. Extramedullary haemopoiesis was found in liver of 0-1 males. Focal inflammation of liver was detected in 4-1 males.
Steatosis of kidneys was found in 6-5 males. Extramedullary haemopoiesis was found in spleen of all satellite males 6-6 males. Intensity of haematopoiesis was similar in both groups of males. Active haematopoiesis were kept in bone marrow of all satellite males (6-6).
Sporadic affections were also diagnosed in next organs: nodular hyperplasia of adrenal gland cortex in 2-0 males, focal inflammation of the myocardium of heart in 2-0 males, and focal oedema of medulla spinalis in 1-0 males.
Inflammation of larynx was recorded in 4-2 males. The affections were also diagnosed in lungs: inflammation in 2-3 males, dystelectasis in 1-0 males.
Focal interstitial inflammation in prostate gland was recorded in 4-1 males. Proliferation of epithelium in prostate gland was found out in 1-0 males. Histopathological findings in mammary gland were sporadic: lactating mammary gland in 2-0 males.

FEMALES
Main groups
The affections were often diagnosed in stomach and liver. Focal atrophy of phundal glands of stomach was diagnosed in 3-2-0-0 females. Eosinophile infiltration was recorded in stomach of 0-0-0-6 animals and oedema of mucosa of stomach in 0-0-0-6 animals. Various alterations were registered in liver (in all groups). Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 3-3-2-3 females. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 3-3-4-3 males. Biliary proliferation was found in 0-0-3-0 females and oval cells proliferation in 0-0-3-0 females. Extramedullary haemopoiesis was found in liver of 0-0-1-0 females. Focal inflammation of liver was detected in 1-1-1-2 females. The changes were found in small intestine: hyperplasia of MALT (MALT – mucosa associated lymphoid tissue) in 1-0-0-0 animals, and eosinophile infiltration in 1-3-0-0 females.
Steatosis of kidneys was found in 5-5-6-6 females. Metastatic mineralization was recorded only in 0-0-1-0 females.
Sporadic affections were also diagnosed in next organs: reactive histiocytosis in lymph node of 1-0-0-0 females, cyst in thyroid gland of 1-0-0-0 male and focal proliferation of ependymal cells in brain of 0-0-0-1 females.
Extramedullary haemopoiesis was found in spleen of all females (6-6-6-6). Intensity of haematopoiesis was similar in all groups of females.
Also active haematopoiesis were kept in bone marrow of all females (6-6-6-6).
Inflammation of lung was recorded in 1-1-0-4 females and dystelectasis in lung of 1-0-0-0 female. Sporadic affections were also diagnosed in larynx: inflammation in 0-0-2-0 females
Female genital tract: Hydrometra of uterus in 0-0-1-0 females. The affections in mammary gland were more often: involution in 5-5-3-3 females.
Satellite groups
The affections were sporadically diagnosed in stomach. Eosinophile infiltration was recorded in stomach of 1-0 animals, oedema of mucosa in stomach of 1-0 females and muscular dystrophy of stomach of 1-0 animals. Various alterations were registered in liver (in all satellite groups). Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 1-2 females. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 5-4 females. Focal inflammation of liver was detected in 3-4 females.
Steatosis of kidneys was found in 6-5 females. Metastatic mineralization was recorded only in 0-1females.
Sporadic affections were also diagnosed in next organs: inflammation of larynx in 1-3 females, inflammation of trachea in 0-1 females, histiocytosis in lung of 0-1 female, inflammation of lung in 0-2 female, oedema of brain in 0-1 animal, and focal proliferation of ependymal cells in brain of 0-1 female.
Extramedullary haemopoiesis was found in spleen of all females (6-6). Intensity of haematopoiesis was similar in both groups of females.
Also active haematopoiesis were kept in bone marrow of all females (6-6).

Hydrometra of uterus was diagnosed in 0-2 females and fibrosis of endometrium was recorded in uterus of 0-4 females. The affections in mammary gland were more often: involution in 3-4 females.




Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxic effect observed
Critical effects observed:
not specified

DISCUSSION OF RESULTS

   From the results of laboratory examination of rats strain Wistar, the negative effect of 28-day oral application of the test substance Potassium permanganate is apparent in animals of both sexes.

   Stress for organism was manifested by lower food consumption and food conversion, what caused lower growth increment of treated animals in comparison with control animals. This effect was recorded since 1st week of application and was markedly in males.

   Negative effect of the test substance on digestive tract was confirmed by macroscopic examination. In the middle and highest dose level, numerous changes of stomach mucosa, changes of structure and irregular colour of liver were recorded. By histological examination the inflammation changes of stomach mucosa (eosinophile infiltration) and oedema of mucosa were diagnosed in females of the highest dose level. In males these changes were recorded only sporadically.         

  Increased relative weight of liver was measured in males of all treated groups. Biliary proliferations were diagnosed in liver of males and females in the middle dose level. In both sexes (markedly in females) of the middle dose level the proliferation of oval cells in liver was found out. These cells are considered to be liver stem cells. Peripheral or irregular steatosis was diagnosed with similar frequency in all animals of treated and control groups. The changes in hepatic function were confirmed by biochemical examination of blood. In the highest dose level, increased value of ALP (alkaline phosphatase) and decreased values of total protein and albumin were recorded in males. Also the presence of urobilinogen in urea might have been influenced by dysfunction of liver. Biochemical examination of blood showed markedly decreased concentration of total cholesterol in all treated females. In males the decrease of this value lead to the levels under limits of measurability.

   The histopathological changes of the other organs in gastro-intestinal tract were found out only sporadically. Only in males of the highest dose level presence of areactive necrosis of the mucosa in rectum was recorded in half of animals. This change was not recorded in any other group of treated males and females or control animals. Dysfunction of digestion might have been a cause of impairment of the physical condition of animals. The grip strength (one of parameters of functional observation battery) decreased in both sexes of the highest dose level

The damage of gastro-intestinal tract was reversible. After 14-day period without application the food consumption, growth increments and food conversion were comparable with control animals. Macroscopic changes of liver were still recorded in satellite animals, but their frequency was lower in comparison with corresponding main group. Accompanying biochemical parameters were similar to the control groups or even lower.

  The haematology parameters of red blood cells were the second sphere of serious damage. Decreased value of total erythrocyte count and accompanying decrease of haematocrite and haemoglobine concentration in peripheral blood were recorded in males of the highest and middle dose level and females of the highest dose level. At the end of period without test substance application the contrary effect was recorded, which bears evidence of compensation process. Increased values of total erythrocyte count, haematocrite and haemoglobine concentration were measured in both sexes. The effect was statistically significant only in males.

  The changes in blood coagulation were the third sphere of damage. Increased value of trombocyte count was recorded in all treated groups of females. After 14-day observation period this increase was significant. Slightly increased value of trombocyte count was recorded also in satellite treated males. Also prolongation of protrombine time detected in all treated groups might have been a symptom of disbalance among components of haemocoagulation. The effect of the test substance on this blood component was protracted – the effect manifested itself even after period without application.

 The application of the test substance had also effect on urine excretion system. Urinalysis showed decrease of urine volume depending on increasing dose level. On the contrary specific weight of urine and pH urine were increased depending of dose level. Presence of protein in urine was recorded in all groups of males (also in control males), but the protein content was increased depending on dose level. In females the presence of protein in urine was recorded only in the highest dose level. The presence of leucocytes in urine was recorded only in males of the highest dose level. These changes in urine were irreversible, but expected damage of kidney was not approved histologicaly. Only sporadic findings of proteinuria and hydronephrosis in kidney could be associated with above-mentioned findings. Statistically significantly increased value of creatinine in blood in satellite treated females also could signalise the kidney damage.

   Accompanying symptom of the above-mentioned changes could be the changes in proportion of white blood cells - increased value of total leucocyte count and increased portion of monocytes. These changes might have been caused by defensive action of organism against toxicity of the test substance. Alternatively they could by attributed to inflammation changes in respiratory air-ways. Histopathological examination showed these inflammation changes in organs of males and females of all treated main groups and also in satellite treated groups.

       Histopathological changes in brain (focal proliferation of glial cells, proliferation of ependymal cells and oedema) were not numerous, but were unusual. These changes were recorded only in animals of the highest dose level (both males and females). The 28-day study is not long enough for determination, if these changes are really caused by application of the test substance.   

       Histopathological findings in genital organs showed rather changes caused by senility of organism. Only hydrometra of uterus in females of treated groups (also in satellite animals) might have been caused by activation of estral cycle by application of the test substance. In males slightly increased weights of testes and epididymis were recorded in treated animals.

Conclusions:
The dose level 40 mg/kg/day could be regarded as the NOAEL (no adverse effect level). Sporadically finded changes at this dose level are considered to be a result of normal variation for rats of the strain and age used and they has no toxicological importance.
Executive summary:

 Following effects were detected after repeated 28-day repeated oral application of the test substance,Potassium permanganate

 

Dose level 250 mg/kg/day

    reversible changes:

-          decrease of body weight increment in both sexes

-          decrease of food consumption in both sexes, markedly in males

-          decrease of food conversion in both sexes

-          decreased values of total protein and albumine in both sexes, statistically significant in males

-          increased content of protein in urea – in both sexes, markedly in males

-          occurrence of urobiline and ketones in urea – in both sexes

-          slightly increased weight of testes and epididymides

 irreversible changes:

-         decrease of water consumption – in both sexes

-         increased value of total leucocyte count – in both sexes, markedly in males

-         slightly increased value of thrombocyte count - in females

-         slightly increased value of ALP – in males

-         decrease of urine volume – in both sexes

-         increased value of pHand specific weight of urine – in both sexes

-         increased weight of spleen – statistically significant in males (this change was reversible in females)

-         increased weight of uterus – statistically significant in females

protractedchanges:

-         increased value of total erythrocyte count, haematocrite and concentration of haemoglobine – in both sexes, statistical significant in males

-         increased value of monocyte fraction – statistically significant in females

-         prolongation of PT - protrombine time - statistically significant in both sexes

-         increased value of calcium in blood – statistically significant in males

-         increased value of creatinine in blood – statistically significant in females

 

 

 

Dose level 100 mg/kg/day

-         increased value of pHand specific weight of urine – in both sexes (not so marked as in the dose level 250 mg/kg/day)

-         slightly increased weight of testes and epididymides

 

Dose level 40 mg/kg/day

 -   no toxic effect observed

 

    Overall assessment showed, that the test substance Potassium permanganate after 28-day oral application cased decrease of growth increments, food consumption and food conversion. Slightly damage of gastro- intestinal tract was confirmed by the other examination.

    Haematological examination showed negative effect on red blood component. Increased values of total erythrocytes, concentration of haemoglobin and haematocrit were recorded after end of observation period. These changes have been accompanied by increased weight of spleen. Prolongation of protrombine time was pronounced disbalance among components of haematocoagulation.

    Increased specific weight and pH urine (together with decreased urine volume) was connected with decreased water consumption. Also decreasedvalue of total protein and albumin in blood showed negative effect on urinary tract. But histopathological changes of kidney were recorded only sporadically.

  Accompanying symptoms of changes was increase value of leucocyte count and increased portion of monocytes. These changes might have been caused by defensive of organism against toxicity of the test substance but also by inflammation changes in respiratory air-ways.

The findings of genital organs showed rather changes caused by senility of organism. Only hydmometra of uterus in females of treated groups (also in satellite animals) and fibrosis in endometrium (only in satellite treated animals) might have been caused by activation of oestral cycle after application of the test substance.

 

    

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
29 March to 14 April 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
See read-across justification - section 13
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
range-finding study not conducted according to GLP
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
The animals were male and female B6C3F1 mice, obtained from Charles River Breeding Laboratories (MI). At receipt, the mice were an average of 35 days old, and they were quarantined for 20 days prior to exposure. Before the beginning of the studies, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease.
Mice were housed 5 per cage, individuals were identified by ear punch/notch and toe clip. Feed (NIH-07 open formula meal rat and mouse diet, Ziegler Brothers, Inc.) and water were available ad libitum. The mice were housed in polycarbonate cages (Lab Products, Inc.) on stainless steel racks, with heat-treated hardwood chips as bedding.
The temperature of the animal room was 23.3±2°C, the relative humidity was 40-80%. Fluorescent lighting was provided for 12 hours/day, and there were aproximately 12 air changes per hour.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
A premix with manganese (II) sulfate monohydrate and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a
Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes. Dose formulations were prepared once.
Groups of five male and five female mice were fed diets containing 0, 3,130, 6,250, 12,500, 25,000, or 50,000 ppm manganese (II) sulfate monohydrate. The level of manganese in the diet received by controls was approximately 92 ppm. The appropriate feed was supplied twice weekly and was available ad libitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability analyses of the dose formulations were conducted by the analytical chemistry laboratory using a spectrophotometric method. Homogeneity was confirmed; stability of the dose formulations was established for 2 weeks in the dark at room temperature and for 1 week exposed to air and light. A subsequent study confirmed the stability of the dose formulations for 3 weeks under the conditions listed above. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings strongly support the conclusion that the manganese remained in the divalent state. The dose formulations were prepared once. Dose formulations were discarded 21 days after the date of preparation.
Periodic analyses of the dose formulations of manganese (II) sulfate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analyzed once. All dose formulations were within the specified 10% of the target concentration.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily - ad libitum in diet
Remarks:
Doses / Concentrations:
0, 3130, 6250, 12500, 25000, or 50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Five mice/sex/dose
Control animals:
yes, plain diet
yes, historical
Details on study design:
Animals were assigned to treatment groups by weight intervals. Animals from each interval were randomized and proportionately assigned to cages, then the cages were assigned to dose groups using an appropriate table of random numbers.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Clinical findings were recorded twice daily. Feed consumption was recorded weekly by cage. The animals were weighed at study initiation, on day 7, and at the end of the studies.
At the end of the study, blood from the vena cava of all animals was collected for haematology analyses (haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, and leukocyte count and differential).
Sacrifice and pathology:
A gross necropsy was performed on all animals. The brain, heart, right kidney, liver, lungs, left testicle, and thymus were weighed. Tissue samples of the livers from high-dose and control rats were collected for manganese concentration analyses. Histopathologic examinations were not conducted.
Other examinations:
The managanese concentration in tissue was determined.
Statistics:
Pairwise comparisons and were used to identify differences between control and treated groups. The significance of pairwise comparisons was determined according to the methods of Dunnett (1955), Williams (1971, 1972), Shirley (1977), Dunn (1964), Jonckheeres test (1954) or the Mann-Whitney U test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
One female mouse in the 25000 ppm group died of unknown causes on day 1; all other mice survived to the end of the study (Table 1). No significant evidence of toxicity was observed except possible body weight effects in both sexes (Table 1). However, the report stated that no conclusions could be made regarding the body weight data because of poor randomization of animals at study initiation. No organ weight differences were attributed to manganese (II) sulfate monohydrate exposure. Absolute or relative organ weight differences in some of the exposure groups were probably related to body weight differences between exposed and control groups. No chemical-related differences in haematology parameters were observed. Manganese concentrations in the livers of 50000 ppm mice were 8 to 15 times higher than those found in controls (males: control, 0.966 µg/g; 50000 ppm, 8.020 μg/g; females: 0.708 µg/g; 10.300 µg/g).
Dose descriptor:
NOAEL
Effect level:
50 000 ppm
Sex:
male/female
Critical effects observed:
not specified

Table 1. Survival, body weights and feed consumption.

Concentration (ppm)

Survivala

Mean body weight and Weight Changesb(g)

Final Weight Relative to Controls (%)

Feed Consumptionc

Initial

Final

Change

Week 1

Week 2

Male

0

5/5

21.4±0.6

25.6±1.2

4.2±0.8

 

4.2

4.2

3130

5/5

23.8±0.4*

26.8±0.7

3.0±0.5

105

2.8

3.2

6250

5/5

24.4±0.5**

26.0±1.0

1.6±0.5**

102

3.0

3.5

12500

5/5

24.6±0.7**

24.0±0.7

0.6±0.5**

94

3.7

4.3

25000

5/5

24.8±0.4**

24.4±0.2

0.4±0.2**

95

5.1

4.6

50000

5/5

19.2±0.4*

21.8±0.7*

2.6±0.5**

85

3.2

4.9

Female

0

5/5

15.6±0.6

21.0±1.0

5.4±0.8

 

3.3

4.2

3130

5/5

18.4±0.2**

18.0±0.3**

0.4±0.2**

86

3.8

4.8

6250

5/5

17.8±0.2**

17.2±0.4**

0.6±0.4**

82

4.1

4.3

12500

5/5

18.6±0.7**

16.8±0.6**

1.8±0.4**

80

4.1

5.2

25000

4/5d

18.2±0.4**

17.0±0.4**

1.3±0.3**

81

4.8

6.0

50000

5/5

18.6±0.4**

15.2±0.5**

3.4±0.2**

72

3.5

3.69

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** P 0.01

aNumber of animals surviving at 14 days/number initially in group

bWeights given as mean ± standard error.

cFeed consumption is expressed as grams per animal per day.

dDay of death: 1

Conclusions:
The 14 day NOAEL was considered to be 50000 ppm.
Executive summary:

Groups of five male and five female mice received diets containing 0, 3130, 6250, 12500, 25000, or 50000 ppm manganese (II) sulfate monohydrate for 14 days. One female mouse in the 25,000 ppm group died on day 1 of unknown causes; all other mice survived to the end of the study. Differences in body weights between exposed and control mice could not be attributed to chemical adminsitration. Therefore the NOAEL can be considered to be 50000 ppm.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
30 August to 30 November 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
See readacross justification- section 13
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Remarks:
FDA
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
The animals were male and female B6C3F1 mice, obtained from Simonsen Labs, Inc. (CA). At receipt, the mice were an average of 43 days old, and they were quarantined for 20 days prior to exposure. Before the beginning of the studies, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. At the end of the studies, serologic analyses were performed on five male and five female controls using the protocols of the NTP Sentinel Animal Program.
Mice were housed 5 per cage, individuals were identified by ear clip/notch and toe clip. Feed (NIH-07 open formula meal rat and mouse diet, Ziegler Brothers, Inc.) and water were available ad libitum. The mice were housed in polycarbonate cages (Lab Products, Inc.) on stainless steel racks, with heat-treated hardwood chips as bedding.
The temperature of the animal room was 23.3±2°C, the relative humidity was 40-80%. Fluorescent lighting was provided for 12 hours/day, and there were aproximately 12 air changes per hour.
Details on oral exposure:
A premix with manganese (II) sulfate monohydrate and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a
Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes. Dose formulations were prepared once.
Groups of 10 male and 10 female mice were fed diets containing 0, 3130, 6250, 12500, 25000 or 50000 ppm manganese (II) sulfate monohydrate. The level of manganese in the diet received by controls was approximately 92 ppm. The appropriate feed was supplied twice weekly and was available ad libitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability analyses of the dose formulations were conducted by the analytical chemistry laboratory using a spectrophotometric method. Homogeneity was confirmed; stability of the dose formulations was established for 2 weeks in the dark at room temperature and for 1 week exposed to air and light. A subsequent study confirmed the stability of the dose formulations for 3 weeks under the conditions listed above. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings strongly support the conclusion that the manganese remained in the divalent state. The dose formulations were prepared weekly. Dose formulations were discarded 21 days after the date of preparation.
Periodic analyses of the dose formulations of manganese (II) sulfate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analyzed three times during the study. All dose formulations were within the specified 10% of the target concentration.
Duration of treatment / exposure:
90-91 days (13 weeks)
Frequency of treatment:
Daily - ad libitum in diet
Remarks:
Doses / Concentrations:
0, 3130, 6250, 12500, 25000 or 50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 mice/sex/dose
Control animals:
yes, plain diet
yes, historical
Details on study design:
Animals were assigned to treatment groups by weight intervals. Animals from each interval were randomized and proportionately assigned to cages, then the cages were assigned to dose groups using an appropriate table of random numbers.
Dose levels were chosen for this study based on the results obtained in the 14 day repeat dose toxicity test: there were no overt signs of toxicity therefore the dose levels remained the same.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Clinical findings were recorded weekly. Feed consumption was recorded weekly by cage. The mice were weighed at the beginning of the studies and twice weekly thereafter. At the end of the study, blood was collected from the vena cava of all animals for haematology analyses (haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, and leukocyte count and differential).
Sacrifice and pathology:
A necropsy was performed on all animals. The brain, heart, right kidney, liver, lungs, left testicle, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 µm, and stained with haematoxylin and eosin.
A complete histopathologic examination was performed on all control and high-dose animals. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, blood, bone marrow (sternum), brain, colon, duodenum, oesophagus, gallbladder, heart, kidney, liver, lung, mammary gland, mandibular lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, spleen, stomach, testes/epididymis, thyroid, gland, trachea, thymus, urinary bladder, and uterus.
Other examinations:
No other examinations reported.
Statistics:
Pairwise comparisons and were used to identify differences between control and treated groups. The significance of pairwise comparisons was determined according to the methods of Dunnett (1955), Williams (1971, 1972), Shirley (1977), Dunn (1964), Jonckheeres test (1954) or the Mann-Whitney U test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, 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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
One control male mouse and one female mouse receiving 3130 ppm died of unknown causes during this study (Table 1). Mean body weight gains of all exposed males were significantly lower than that of the control group, and the final mean body weight of the 50000 ppm group was 13% lower than that of the controls. The mean body weight gain of 50000 ppm females was significantly lower than that of the controls. Feed consumption by exposed male and female mice was similar to that by the controls (Table 1). Mean daily ingestion of manganese (II) sulfate monohydrate ranged from 330 to 7400 mg/kg body weight in males and 390 to 6900 ppm in females. The absolute and relative liver weights of 50000 ppm male mice were significantly lower than those of the controls; absolute and relative liver weights of females were similar to those of the controls. The percent haematocrit, haemoglobin concentrations, and mean erythrocyte volumes of 50000 ppm male and female mice were significantly lower than those of the controls. These findings suggest microcytic anemia and may be related to a sequestration or deficiency of iron. Although the total leukocyte counts in the two highest male exposure groups were significantly lower than that in the control group, this may not be related to manganese (II) sulfate monohydrate ingestion. A few mice in the male and female exposure groups exhibited fight wounds. Three 50000 ppm males had mild epithelial hyperplasia and hyperkeratosis
of the forestomach.
Dose descriptor:
NOAEL
Sex:
male
Basis for effect level:
other: Reduced body weight gain was seen in all exposed male groups
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
25 000 ppm
Sex:
female
Critical effects observed:
not specified

Table 1. Survival, body weights and feed consumption

Concentration (ppm)

Survivala

Mean body weight and Weight Changesb(g)

Final Weight Relative to Controls (%)

Feed Consumptionc

Initial

Final

Change

Week 1

Week 13

Male

0

9/10d

25.0±0.5

31.4±0.6

6.6±0.5

 

3.4

3.0

3130

10/10

25.7±0.3

30.5±0.5

4.8±0.4**

97

3.0

3.0

6250

10/10

26.3±0.2*

31.0±0.3

4.7±0.4**

99

3.4

3.3

12500

10/10

26.0±0.3

30.9±0.4

4.9±0.4**

98

3.4

3.8

25000

10/10

25.9±0.2

30.6±0.5

4.7±0.5**

97

2.6

3.3

50000

10/10

25.1±0.4

27.4±0.3**

2.3±0.4**

87

3.0

4.7

Female

0

10/10

20.0±0.2

24.2±0.3

4.2±0.3

 

2.4

2.3

3130

9/10e

20.0±0.3

24.2±0.5

4.1±0.3

100

3.3

2.4

6250

10/10

20.5±0.2

24.3±0.3

3.8±0.3

100

2.8

2.2

12500

10/10

21.0±0.3

24.5±0.3

3.5±0.3

101

2.7

3.0

25000

10/10

20.3±0.3

24.2±0.4

3.9±0.4

100

3.1

3.4

50000

10/10

20.1±0.2

22.8±0.3**

2.7±0.2**

94

2.8

3.0

* Significantly different (P≤0.05) from the control group by Williams' or Dunnett's test.

** P≤0.01

a Number of animals surviving at 14 days/number initially in group

b Weights given as mean ± standard error.

c Feed consumption is expressed as grams per animal per day.

d Week of death: 11

e Week of death: 6

Conclusions:
No NOAEL could be identified for males on the basis of reduced body weight gain in all treated groups. The NOAEL for females can be considered to be 25000 ppm, on the basis that body weight gain and and haematology parameters were affected in the 50000 ppm group.
Executive summary:

Groups of 10 male and 10 female mice received diets containing 0, 3130, 6250, 12500, 25000, or 50000 ppm manganese (II) sulfate monohydrate for 90 days. Mean daily ingestion of manganese (II) sulfate monohydrate ranged from 330 to 7400 mg/kg body weight in males and 390 to 6900 mg/kg body weight in females. No deaths were chemical related. The mean body weight gains of exposed male mice and of 50000 ppm female mice were significantly lower than those of controls. The absolute and relative liver weights of 50000 ppm males were significantly lower than those of controls. The percent haematocrit and haemoglobin concentration of males and females exposed to 50000 ppm were lower than those of the controls, and the mean erythrocyte volumes were significantly lower than those of the controls. The total leukocyte counts of males in the 25000 and 50000 ppm groups were significantly lower than that of the controls. No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion. Epithelial hyperplasia and hyperkeratosis of the forestomach occurred in three 50000 ppm males.

No NOAEL could be identified for males on the basis of reduced body weight gain in all treated groups. The NOAEL for females can be considered to be 25000 ppm, on the basis that body weight gain and and haematology parameters were affected in the 50000 ppm group.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1 to 15 February 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
See the read-across report attched in section 13
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
range-finding study not conducted according to GLP
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
The animals were male and female F344/N rats obtained from Charles River Breeding Laboratories (MI). At receipt, the rats were an average of 31 days old. They were quarantined for 19 days prior to exposure. Before the beginning of the studies, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease.
Rats were housed 5 per cage, individuals were identified by ear punch/notch and toe clip. Feed (NIH-07 open formula meal rat and mouse diet, Ziegler Brothers, Inc.) and water were available ad libitum. The rats were housed in polycarbonate cages (Lab Products, Inc.) on stainless steel racks, with heat-treated hardwood chips as bedding.
The temperature of the animal room was 23.3±2°C, the relative humidity was 40-80%. Fluorescent lighting was provided for 12 hours/day, and there were aproximately 12 air changes per hour.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
A premix with manganese (II) sulfate monohydrate and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a
Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes. Dose formulations were prepared once.
Groups of five male and five female rats were fed diets containing 0, 3130, 6250, 12500, 25000, or 50000 ppm manganese (II) sulfate monohydrate. The level of manganese in the diet received by controls was approximately 92 ppm. The appropriate feed was supplied twice weekly and was available ad libitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability analyses of the dose formulations were conducted by the analytical chemistry laboratory using a spectrophotometric method. Homogeneity was confirmed; stability of the dose formulations was established for 2 weeks in the dark at room temperature and for 1 week exposed to air and light. A subsequent study confirmed the stability of the dose formulations for 3 weeks under the conditions listed above. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings strongly support the conclusion that the manganese remained in the divalent state. The dose formulations were prepared once. Dose formulations were discarded 21 days after the date of preparation.
Periodic analyses of the dose formulations of manganese (II) sulfate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analyzed once. All dose formulations were within the specified 10% of the target concentration.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily -ad libitum in diet
Remarks:
Doses / Concentrations:
0, 3130, 6250, 12500, 25000, or 50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Five rats/sex/dose
Control animals:
yes, plain diet
yes, historical
Details on study design:
Animals were assigned to treatment groups by weight intervals. Animals from each interval were randomized and proportionately assigned to cages, then the cages were assigned to dose groups using an appropriate table of random numbers.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Clinical findings were recorded daily days 1 to 8, then twice daily days 9 to 14. Feed consumption was recorded weekly by cage. The animals were weighed at study initiation, on day 7, and at the end of the studies.
At the end of the study, blood from the vena cava of all animals was collected for haematology analyses (haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, and leukocyte count and differential).
Sacrifice and pathology:
A gross necropsy was performed on all animals. The brain, heart, right kidney, liver, lungs, left testicle, and thymus were weighed. Tissue samples of the livers from high-dose and control rats were collected for manganese concentration analyses. Histopathologic examinations were not conducted.
Other examinations:
The managanese concentration in tissue was determined.
Statistics:
Pairwise comparisons and were used to identify differences between control and treated groups. The significance of pairwise comparisons was determined according to the methods of Dunnett (1955), Williams (1971, 1972), Shirley (1977), Dunn (1964), Jonckheeres test (1954) or the Mann-Whitney U test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
All rats survived to the end of the study. The mean body weight gain of the male 50000 ppm group at the end of the 14-day study was 57% less than that of the control group, and the final mean body weight of this group was 13% lower than that of the controls. The mean body weight gain of 50000 ppm females was 20% less than that of the controls and the final mean body weight was 7% lower than that of the controls. Males and females in each exposure group consumed approximately equal amounts of manganese (II) sulfate monohydrate per body weight (25 to 370 mg/kg). During the first week, feed consumption by 50000 ppm males was 19% lower than controls, whereas that by 50000 ppm females was 15% lower. During the second week, however, feed consumption by both male and female 50000 ppm groups was similar to that by controls. Data are shown in Table 1.
Males exposed to 50000 ppm and all exposed groups of females exhibited diarrhoea during the second week. In the haematology evaluation, the total leukocyte and neutrophil counts were significantly increased in 50000 ppm groups, particularly males. Other slight changes in haematology parameters were not considered related to chemical ingestion. At necropsy, the absolute and relative liver weights of 50000 ppm male rats were significantly lower than those of the controls. Manganese concentrations in the livers of 50000 ppm males and females were more than twice those of controls (males: control, 2.80 µg/g; 50000 ppm, 5.92 µg/g; females: 2.40 µg/g, 6.82 µg/g).
Dose descriptor:
NOAEL
Effect level:
25 000 ppm
Sex:
male/female
Critical effects observed:
not specified

Table 1. Survivial, body weights, and feed consumption

Concentration (ppm)

Survivala

Mean body weight and Weight Changesb(g)

Final Weight Relative to Controls (%)

Feed Consumptionc

Initial

Final

Change

Week 1

Week 2

Male

0

5/5

183±14

241±12

58±2

 

17.2

17.2

3130

5/5

176±6

235±5

59±3

98

16.6

17.2

6250

5/5

182±15

242±13

60±3

101

16.7

17.7

12500

5/5

176±7

235±6

58±3

98

16.7

17.7

25000

5/5

186±7

243±6

57±1

101

17.0

18.1

50000

5/5

185±5

210±6*

25±2**

87

13.9

16.8

Female

0

5/5

140±3

165±4

25±2

 

12.2

11.4

3130

5/5

144±5

171±5

27±3

104

14.2

11.8

6250

5/5

134±4

157±3

23±2

95

11.8

11.0

12500

5/5

136±5

163±6

27±1

99

13.2

11.9

25000

5/5

139±5

166±4

27±1

101

13.3

12.0

50000

5/5

134±5

153±5

20±1

93

10.4

12.1

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** P 0.01

aNumber of animals surviving at 14 days/number initially in group

bWeights given as mean ± standard error.

cFeed consumption is expressed as grams per animal per day.

Conclusions:
The 14 day NOAEL was 25000 ppm.
Executive summary:

Groups of five male and five female rats received diets containing 0, 3130, 6250, 12500, 25000, or 50000 ppm manganese (II) sulfate monohydrate for 14 days. All rats survived to the end of the study. Male rats exposed to 50000 ppm had a mean body weight gain 57% lower and a final mean body weight 13% lower than those of the controls. The mean body weight gain of 50000 ppm females was 20% lower and the final mean body weight was 7% lower than those of the controls. During the second week, 50000 ppm males and females exhibited diarrhoea. Manganese concentrations in the livers of 50000 ppm males and females were more than twice those of controls (males: control, 2.80 µg/g; 50000 ppm, 5.92 µg/g; females: 2.40 µg/g, 6.82 µg/g).

The 14 day NOAEL was considered to be 25000 ppm.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
30 August to 2 December 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
See readacross justification section 13
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Remarks:
FDA
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
The animals were male and female F344/N rats obtained from Charles River Breeding Laboratories (NY). At receipt, the rats were an average of 31 days old. They were quarantined for 19 days prior to exposure. Before the beginning of the studies, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. At the end of the studies, serologic analyses were performed on 5 control animals/sex using the protocols of the NTP Sentinel Animal Program.
Rats were housed 5 per cage, individuals were identified by ear clip/notch and toe clip. Feed (NIH-07 open formula meal rat and mouse diet, Ziegler Brothers, Inc.) and water were available ad libitum. The rats were housed in polycarbonate cages (Lab Products, Inc.) on stainless steel racks, with heat-treated hardwood chips as bedding.
The temperature of the animal room was 23.3±2°C, the relative humidity was 40-80%. Fluorescent lighting was provided for 12 hours/day, and there were aproximately 12 air changes per hour.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
A premix with manganese (II) sulfate monohydrate and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a
Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes. Dose formulations were prepared once.
Groups of 10 male and 10 female rats were fed diets containing 0, 1600, 3130, 6250, 12500, or 25000 ppm manganese (II) sulfate monohydrate. The level of manganese in the diet received by controls was approximately 92 ppm. The appropriate feed was supplied twice weekly and was available ad libitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability analyses of the dose formulations were conducted by the analytical chemistry laboratory using a spectrophotometric method. Homogeneity was confirmed; stability of the dose formulations was established for 2 weeks in the dark at room temperature and for 1 week exposed to air and light. A subsequent study confirmed the stability of the dose formulations for 3 weeks under the conditions listed above. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings strongly support the conclusion that the manganese remained in the divalent state. The dose formulations were prepared weekly. Dose formulations were discarded 21 days after the date of preparation.
Periodic analyses of the dose formulations of manganese (II) sulfate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analyzed three times during the study. All dose formulations were within the specified 10% of the target concentration.
Duration of treatment / exposure:
93-94 days (13 weeks)
Frequency of treatment:
Daily -ad libitum in diet
Remarks:
Doses / Concentrations:
0, 1600, 3130, 6250, 12500 or 25000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes, plain diet
yes, historical
Details on study design:
Animals were assigned to treatment groups by weight intervals. Animals from each interval were randomized and proportionately assigned to cages, then the cages were assigned to dose groups using an appropriate table of random numbers.
Dose levels were chosen for this study based on the results obtained in the 14 day repeat dose toxicity test: 25000 ppm was chosen as the highest dose for the 90 day study based on a reduction in mean body weight gain in the 50000 ppm male and female rats.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
Clinical findings were recorded weekly. Feed consumption was recorded weekly by cage. The rats were weighed at the beginning of the studies and weekly thereafter. At the end of the study, blood was collected from the vena cava of all animals for haematology analyses (haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, and leukocyte count and differential).
Sacrifice and pathology:
A necropsy was performed on all animals. The brain, heart, right kidney, liver, lungs, left testicle, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 µm, and stained with haematoxylin and eosin.
A complete histopathologic examination was performed on all control and high-dose animals. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, blood, bone marrow (sternum), brain, cecum, colon, duodenum, oesophagus, heart, kidney, liver, lung, mammary gland, mandibular lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial or clitoral gland, prostate gland, salivary gland, spleen, stomach, testes/epididymis, thyroid, gland, trachea, thymus, urinary bladder, and uterus.
Other examinations:
No other examinations reported.
Statistics:
Pairwise comparisons and were used to identify differences between control and treated groups. The significance of pairwise comparisons was determined according to the methods of Dunnett (1955), Williams (1971, 1972), Shirley (1977), Dunn (1964), Jonckheeres test (1954) or the Mann-Whitney U test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
No rats died during the study. The mean body weight gain in males receiving 3130 ppm was marginally lower than that of the controls and was significantly lower in the three highest female dose groups than the controls (Table 1). Final mean body weights of all exposed animals were within 5% of those of the controls. Feed consumption by exposed rats was similar to that by the controls (Table 3). Mean daily ingestion of manganese (II) sulfate monohydrate ranged from 110 to 1700 mg/kg body weight in males and 115 to 2000 mg/kg in females. Females ingested an average of 20% more manganese (II) sulfate monohydrate than males in the corresponding exposure groups.
Absolute and relative liver weights of all exposed males and of the female 25000 ppm group were significantly lower than those of the controls. The absolute and relative lung weights of all exposed females were also significantly lower than those of controls. No other biologically significant organ weight differences were observed between exposed and control animals. Although the total leukocyte counts were similar in exposed and control males, neutrophil counts were significantly higher in all exposed male groups, whereas lymphocyte counts were significantly lower in the 6250, 12500, and 25000 ppm groups. In contrast, the total leukocyte counts of 6250, 12500, and 25000 ppm females were significantly lower, primarily because of lower lymphocyte counts. A marginal but significant increase in percent hematocrit and erythrocyte counts occurred in males exposed to 6250, 12500, or 25 000 ppm. The relationship between these differences and the ingestion of manganese (II) sulfate monohydrate is not clear. No clinical or histopathologic findings were attributed to administration.
Dose descriptor:
NOAEL
Sex:
male
Basis for effect level:
other: All exposed males exhibited absolute and relative liver weights that were lower than controls
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
3 130 ppm
Sex:
female
Basis for effect level:
other: Effects on body weight gain were seen at doses of 6250 ppm and above
Critical effects observed:
not specified

Table 1. Survival, body weights and feed consumption

Concentration (ppm)

Survivala

Mean body weight and Weight Changesb(g)

Final Weight Relative to Controls (%)

Feed Consumptionc

Initial

Final

Change

Week 1

Week 13

Male

0

10/10

136±5

291±4

155±4

 

14.9

13.1

1600

10/10

142±4

294±5

152±4

101

14.5

13.5

3130

10/10

149±3

291±4

141±4

100

14.8

13.6

6250

10/10

148±2

294±3

146±3

101

15.0

9.6

12500

10/10

150±11

290±6

140±11

99

14.9

14.9

25000

10/10

140±4

284±6

144±4

97

14.1

14.4

Female

0

10/10

99±1

184±2

84±2

 

10.7

9.2

1600

10/10

103±1

181±2

79±2

99

10.8

9.3

3130

10/10

96±1

175±2*

80±3

95

10.9

9.2

6250

10/10

101±1

176±2*

75±1**

96

10.7

14.3

12500

10/10

106±1**

178±1*

73±2**

97

10.7

10.5

25000

10/10

104±1**

174±3**

70±2**

95

12.1

10.3

* Significantly different (P≤0.05) from the control group by Williams' or Dunnett's test.

** P≤0.01

a Number of animals surviving at 14 days/number initially in group

b Weights given as mean ± standard error.

c Feed consumption is expressed as grams per animal per day.

Conclusions:
No NOAEL could be identified for male rats on the basis of liver weight differences. A NOAEL of 3130 ppm for female rats can be assigned on the basis of reduced body weight gain at doses of 6250 ppm and higher.
Executive summary:

Groups of 10 male and 10 female rats received diets containing 0, 1600, 3130, 6250, 12500, or 25000 ppm manganese (II) sulfate monohydrate for 90 days. Mean daily ingestion of manganese (II) sulfate monohydrate ranged from 110 to 1700 mg/kg body weight in males and 115 to 2000 mg/kg in females. All rats survived to the end of the study. Mean body weight gains were marginally lower than that of controls in males exposed to 3130 ppm or more; mean body weight gains were significantly lower than that of the controls in females exposed to 6250, 12500, or 25000 ppm. At the end of the study, absolute and relative liver weights of all exposed male rats and of 25000 ppm female rats were significantly lower than those of controls. The total leukocyte count in males was similar between exposed and control rats; however, neutrophil counts of all exposed groups were greater than those of the controls, whereas lymphocyte counts of the 6250, 12500, and 25000 ppm groups were significantly lower than those of the controls. Total leukocyte counts in 6250, 12500, and 25000 ppm females were significantly decreased because of a decrease in lymphocytes. Male rats also demonstrated marginal but significant increases in percent haematocrit and erythrocyte count in the 6250, 12500, and 25000 ppm groups. No clinical or histopathologic findings in rats were chemical related.

No NOAEL could be identified for male rats on the basis of liver weight differences. A NOAEL of 3130 ppm for female rats can be assigned on the basis of reduced body weight gain at doses of 6250 ppm and higher.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 July 2012 to 04 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
other: Target Substance
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals. Bedding material was sterilised white wood shavings.
- Diet: ad libitum
- Water: Water taken from the public supply was available ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing periods of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 26 °C
- Humidity: 30 - 69 %
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES:
From: 02 July 2012
To: 04 March 2013
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

- Dose formulation Preparation and analysis
Test material formulation was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh size of 100 μm prior to use, except on one occasion where a sieve mesh of 180 μm was used.

- Preliminary Aerosol Characterisation Investigations
Characterisation of the aerosol generating/exposure system was undertaken prior to commencement of the animal exposures to demonstrate satisfactory performance. Preliminary aerosol characterisation investigations demonstrated that aerosol concentrations were stable spatially within the exposure system and over time and the particle size distribution investigations showed that test formulation particles for Groups 2 to 4 were respirable for the rat.

- Aerosol Generation
Test material aerosols were generated using a Wright Dust Feed generator device. Prior to the commencement of aerosol generation, a reservoir canister was packed with the test material powder formulation. The powder cake was slowly advanced into the scraper blade at an appropriate speed and scraped powder carried in a pressurised air stream.
The Wright Dust Feed generator device was operated at an appropriate target scraper speed, and air flow rate identified during the preliminary aerosol characterisation investigations. The generated test aerosols were then delivered to the flow past exposure chamber via a connecting tube manifold and mixed with dilution air to achieve the target aerosol concentration. A vacuum pump system was used to continuously exhaust test aerosols from the exposure chamber. Each aerosol generation system was operated to sustain a dynamic airflow sufficient to ensure an evenly distributed exposure aerosol.

- Inhalation Exposure (see Figure 1)
Exposure to the test aerosols was performed using an appropriately sized modular nose only stainless steel flow past exposure chamber (in-house design). Separate inhalation exposure systems were used for the delivery of test aerosol to each treatment group. Each inhalation exposure system was located in an extract booth (to prevent cross-group contamination). This exposure technique allowed a continuous supply of test aerosol to be delivered to each animal; the biased flow created using the flow-past chamber design ensured that there was no re-breathing of the test atmosphere.
For all inhalation exposures, the rats were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ noses protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push fit through rubber ‘o’ rings in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes. The exposure system was operated at an appropriate target total airflow. All flow rates (delivered and extracted) were monitored visually using calibrated flow meters. Exposure chamber flow rates, temperature and relative humidity were monitored and recorded at appropriate intervals during each daily exposure period.

TEST ATMOSPHERE
The aerosol concentration of test material formulation (Groups 2 to 4) or air (Group 1) in the animals’ breathing zone was measured gravimetrically for all groups at regular intervals throughout each daily exposure period.
The test aerosols were sampled using glass-fibre filters (47 mm Whatman GF/B) contained in a stainless steel filter holder in-line with a sampling system comprising a vacuum pump, flow meter and gas meter. Filter samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate to ensure that there was no overloading of the filter which would cause a reduction in sampling flow rate. The filters were weighed before and after sampling and the aerosol concentration calculated using the weight of formulation collected and the volume of air sampled.
In addition to the aerosol chamber concentration assessment, blank filter samples were taken to assess background levels of test material and retained for analysis.
All retained filters from Groups 1 to 4 were placed in amber glass jars and stored in a refrigerator set to maintain 4 °C prior to analysis for the determination of the aerosol concentration of test material.
A real time aerosol monitor (Casella Microdust, Casella Measurements, UK) was used to assist in monitoring/ assessing the target concentrations at the start of generation each day and provided a continuous overview of any fluctuations in aerosol concentration.

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The substrate collection plates (34 mm stainless steel) and back up filter (34 mm Westech) were weighed before and after sampling to determine the total amount of test and/or vehicle aerosol collected in each particle size range.
After weighing, the substrate collection plates and back up filters of were retained in amber glass jars and stored in a refrigerator set to maintain 4 °C.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at the testing facility.
Duration of treatment / exposure:
F0 animals were dosed for 10 weeks prior to mating; for F0 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F0 females were dosed throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning; for F1 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F1 females were dosed throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.
Frequency of treatment:
Daily (ca. 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of gestation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca. 1 hour per day
From Day 3-4 of lactation: ca. 2 hours per day
From Days 5-20 of lactation until prior to termination (ca. Day 21 of lactation): ca. 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.
Dose / conc.:
5 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 6 µg/L air
analytical conc. F1 generation: 4 µg/L air
Dose / conc.:
10 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 15 µg/L air
analytical conc. F1 generation: 10 µg/L air
Dose / conc.:
20 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 25 µg/L air
analytical conc. F1 generation: 17 µg/L air
No. of animals per sex per dose:
- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected for use based on results from a preliminary reproduction study in rats. In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values (TLVs) proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Weights of F0 animals were recorded one week prior to the first day of dosing, then weekly thereafter until the start of the mating period. Males continued to be weighed weekly until termination; but for females, weighing resumed on Day 0 of gestation (the day of detection of a positive mating sign), and then on Days 7, 14 and 20 of gestation and Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the majority of the litters and continued until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. Females that did not show a positive mating sign were weighed weekly until parturition or termination. Females who had a positive mating sign but failed to litter reverted to the weekly weighing regimen following their theoretical Day 24 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was quantitatively measured for both sexes weekly, starting one week before treatment commenced (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until placement of males in individual cages prior to mating. Weekly measurements continued after the 14 day mating period. For females, following a clear indication of mating, food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: Yes
- Time schedule: Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHER:
- Bioanalytical Sample Collection
Blood (1 mL) was collected from the tail vein of all animals, after careful cleaning of the sample site to avoid any possible contamination, into tubes containing lithium heparin. Samples were collected at the following time-points:
F0 generation: samples were collected from all animals pre-dose, prior to mating and prior to weaning/necropsy
F1 generation: samples were collected from all animals after selection (timing and volume dependent on weight of animal), prior to mating and at necropsy (or shortly prior to, as appropriate).
The samples were stored at -80 °C prior to analysis of manganese in whole blood performed with a validated ICP-MS method.

- Observation of Females with Litters during Lactation
The females were allowed to litter normally. If any animal suffered from a difficult or prolonged parturition, this was recorded. The day of birth of the litter (day on which the first pups are born) was designated Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

- Sexual Maturation in F1 Animals
Commencing at 28 days of age, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day. Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

- Oestrous Cyclicity
Over a 2 week period prior to the initiation of mating, vaginal lavages were taken early each morning and the stages of oestrous observed were recorded.

- Sperm Parameters
The tip of the cauda epididymis was placed in Medium 199 containing 0.2 % BSA and HEPES. The sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was examined using a Hamilton Thorne sperm motility analyser; sufficient replicates to provide 200 motile sperm were assessed (except where it was obvious that motility was compromised for that animal).
The remaining portion of the cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From a sample of the sperm suspension described above, a sperm smear was prepared and stained with eosin. From the Control and High dose animals, two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce.
One testis was decapsulated and homogenised. The homogenate may have been sonicated to remove tissue debris etc., as required. The number of homogenisation-resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

LITTER OBSERVATIONS
- The number of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually.
- Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.
Sacrifice and pathology:
SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

UNSCHEDULED DEATHS
These animals, including those killed or found dead, had a terminal body weight recorded and were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. The tissues list for animals at scheduled necropsy along with representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral 10 % formalin. The reproductive tracts of all females were examined for signs of implantation (if they had been paired for mating prior to necropsy), the number of any implantation sites being recorded.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of external surfaces and orifices, cranial, thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal gland, pituitary gland, prostate gland, thyroid glands, kidneys, liver, lungs, ovaries, spleen, testes and uterus.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents examined. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the following tissues were collected from all animals and preserved in 10 % neutral buffered formalin: brain, epididymides, adrenal glands, pituitary gland, prostate gland, seminal vesicle gland, thyroid glands, kidneys, larynx, liver, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, spleen, testes (preserved in modified Davidson’s fixative), anterior and posterior trachea, uterus and vagina.

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix) and vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Other examinations:
SACRIFICE / GROSS NECROPSY of offspring
Pups that were not selected for post-weaning assessments were killed at the same time as their mother.
Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

- Offspring found dead or killed (prematurely) before Day 14 of lactation
Where practicable, these animals were sexed, then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10 % formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal decedents were discarded.

- Offspring (pre-weaning) found dead or killed (prematurely) on or after Day 14 of lactation
These animals were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10 % formalin. These carcasses were then discarded.

- F1 and F2 Weanlings at scheduled termination
From each litter, 3 male and 3 female pups (where they were available – if a litter only had females or males, then up to 6 of the relevant sex were selected) were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10 % formalin. From one of the 3 pups of each sex, the weights of the brain, spleen and thymus were recorded, and these organs were preserved. Representative samples of any abnormal tissues from any of the 6 pups were also preserved. The carcasses were then discarded.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality were necropsied as described in the preceding paragraph. The remaining carcasses were discarded.

HISTOPATHOLOGY
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test, i.e. pairwise comparison was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 422 had this sign recorded on only one day (Day 14 of the study) and Animal 424 had wheezing recorded on 5 occasions (Days 91 and 112-115 of the study). Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca. Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental.
Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina (the animal gave birth to one live pup). The uterus also contained live foetuses and one late death. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition and had given birth to 3 live pups. One dead foetus was found in the right uterine horn at necropsy. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejunum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 752 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there was a decrease in body weight gain in males over Days 0-21 of the study. From Day 21 of the study, the body weight gains were generally comparable to the controls but the group mean weights remained lower than the controls throughout the study. At target 20 μg/L, there was a group mean body weight gain in females prior to mating were similar to the controls, however body weight gains over Days 0-20 of gestation were slightly lower than the controls. Gains over lactation were similar to the controls.
- F1 animals
At target 20 μg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study, however gains over the following week were greater than the controls and then remained comparable with the controls throughout the remainder of the treatment period. Slight intergroup differences in group mean body weight gains in the F1 females prior to mating were too small to be attributed to treatment. At 20 μg/L, there was a slight reduction in body weight gains throughout gestation compared to the controls.
There were no effects of treatment noted in the lactation females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 and 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 and 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In all treated groups of the F0 generation, the levels of test material in the blood increased significantly on commencement of dosing in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups, which did not indicate any obvious accumulation over the dosing period.
In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test material in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values, indicating an increased exposure throughout the dosing period.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, reduced brain weights in males achieved statistical significance (P<0.05) compared with controls. However, the lower body weight was also statistically significant (P<0.05); following covariance analysis, brain weight did not achieve significance and therefore was not positively attributed to treatment. In all treated females, there was a statistically significant increase in lung weights, compared with the controls; these increases were still present following covariance analysis (P<0.01 at target 5 μg/L and P<0.001 at target 10 and 20 μg/L). Other slight differences in organ weights such as an increased thyroid weight in males at target 5 μg/L and an increase in kidney weights of females at target 10 μg/L were not attributed to treatment.
- F1 animals
At target 5 and 10 μg/L, kidney weights in males were statistically higher than the control, however there was no dose relationship to this increase and following covariance analysis, these findings were no longer evident. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights in females (P<0.05 at target 10 μg/L and P<0.001 at target 20 μg/L) following covariance analysis. Other slight differences in organ weights such as an increased adrenal weight in females at target 20 μg/L were not attributed to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

- F0 animals
In the larynx there was a broadly dosage-related minimal to moderate squamous metaplasia with minimal to moderate submucosal inflammation. Minimal to marked ulceration of the laryngeal epithelium was associated with the squamous metaplasia in several animals from all treated groups. Occasional incidences of mineralisation, intraluminal necrotic debris or intra-epithelial pustules were seen in some of the treated animals.
In the lungs the principal test material related change was seen in centroacinar regions where there was minimal or mild inflammation and focal or diffuse minimal or mild bronchoalveolar hyperplasia. This latter finding was considered reactive. Minimal or mild goblet cell hyperplasia in the bronchial or bronchiolar epithelium was present in animals exposed to target 20 μg/L together with occasional incidences of degeneration and/or squamous metaplasia of the bronchiolar epithelium. Minimal inflammatory findings (inflammatory cell foci and perivascular inflammatory cell infiltration) were also present with a greater incidence in animals exposed to the test material than in controls.
In the nasal cavity, minimal or mild goblet cell hyperplasia and minimal to moderate eosinophilic globules in the olfactory epithelium were observed in all the male treated groups and in females exposed to target 10 or 20 μg/L. At all dose levels, there was a greater incidence of minimal or mild submucosal inflammatory cell infiltration compared to controls.
In males, inflammation of the nasolacrimal duct and squamous metaplasia of the ductal epithelium was seen in most animals exposed to target 10 or 20 μg/L. In addition to these changes, incidences of minimal or mild focal degeneration of the olfactory, respiratory or transitional epithelia, minimal or mild atrophy of the olfactory epithelium, ulceration and focal squamous metaplasia were observed, mainly in animals exposed to target 10 or 20 μg/L, but occasionally in animals at target 5 μg/L. Deposits of crystalline material, presumed to be test material, was seen in the nasolacrimal ducts of a few animals in the treated groups.
Minimal goblet cell hyperplasia was observed in the pharynx of most males exposed to target 20 μg/L and there were occasional incidences of minimal or mild focal epithelial degeneration, focal inflammation and focal squamous metaplasia in males exposed to target 10 or 20 μg/L.
In the trachea, minimal or mild focal squamous metaplasia and inflammation at the carina and minimal or mild focal epithelial degeneration at sites other than the carina were observed at all dose levels.
Other microscopic findings observed were considered incidental, or of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
- F1 animals
Crystals were occasionally observed in the nasal cavity and in the pharynx from animals exposed to target 10 or 20 μg/L. They consisted of small amounts of needle-shaped crystals either deposited on the olfactory epithelium in the nasal cavity, or free in the lumen of the pharynx. These crystals were considered to result from deposition of test material in some parts of the respiratory tract.
Squamous metaplasia in the nasal cavity was observed mainly in the nasolacrimal duct and to a lesser extent in the transitional and respiratory epithelia.
In the trachea, findings such as epithelial degeneration, squamous metaplasia and submucosal inflammation were observed predominantly in the carina.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
The stages of the oestrus cycles and their mean duration were similar in all groups for both generations.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects on the sperm motility, count or morphology at any of the dose levels applied, in either generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on mating performance, fertility or duration of gestation in either generation.

OTHER FINDINGS (PARENTAL ANIMALS)
- Sexual Maturation
The age and body weight at preputial separation or vaginal opening of the F1 generation animals in all treated groups was similar to the controls.

LITTER SIZE AND PUP MORTALITY
- F0 generation, F1 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 20 μg/L, there was an increase in the number of animals losing more than 2 pups at birth (total pups born/no. of implantation sites). However, the mean birth index (%) was well within the background range and these increases were considered to be incidental.
- F1 generation, F2 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 10 and 20 μg/L, pup survival (no. losing >3 pups) over Days 0-4 of lactation was slightly lower than the controls. However, the number of animals losing the entire litter was comparable with controls and the remaining animals generally lost 4 pups. In addition, there was no clear dose related response to these reductions and these were considered not to be an effect of treatment.

LITTER AND PUP WEIGHTS
- F0 Generation
In all treated groups, group mean litter and pup weights were comparable to the controls.
- F1 Generation
At target 20 μg/L, group mean litter weights were slightly lower than the controls which reflected the smaller litter size at this level. However, although the litter weights were slightly lower than the controls, the mean pup weights in both males and females were comparable to the controls.

ABNORMALITIES AMONG PUPS
The type and distribution of observations amongst pups did not indicate any association with treatment.

ORGAN WEIGHTS
- F0 generation, F1 production
At target 20 μg/L, there was a reduction in thymus weight of the females, compared with the controls (P<0.01). Following covariance analysis, this reduction did not achieve statistical significance. There were no effects on organ weights at target 5 and 10 μg/L.
- F1 generation, F2 production
Slight intergroup differences in organ weights did not achieve statistical significance and were not attributed to treatment.

GROSS PATHOLOGY
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to treatment with the test material.

HISTOPATHOLOGY
There were no treatment related findings observed in the tissues examined of the F1 or F2 weanlings.
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Table 1: F0 Blood Analysis Results

F0 Males

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

6

Prior to mating

6

13

23

27

Prior to Necropsy

6

19

27

29

F0 Females

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

7

Prior to mating

6

16

28

39

Prior to Necropsy

7

16

24

33

Control animals were exposed via the food and water.

At target 20 μg/L, manganese levels prior to mating were 350 % higher than controls in males and 550 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 383 and 371 % higher for males and females, respectively.

At target 10 μg/L, manganese levels prior to mating were 283 % higher than controls in males and 367 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 350 and 243 % higher for males and females, respectively.

At target 5 μg/L, manganese levels prior to mating were 117 % higher than controls in males and 167 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 217 and 129 % higher for males and females, respectively.

Table 2: F1 Blood Analysis Results

F1 Males

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

12

16

16

17

Prior to mating

6

9

13

19

Prior to Necropsy

6

9

14

21

F1 Females

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

13

12

15

15

Prior to mating

6

10

16

23

Prior to Necropsy

7

10

16

21

At target 20 μg/L, manganese levels prior to mating were 217 % higher than controls in males and 283 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 250 and 200 % higher for males and females, respectively.

At target 10 μg/L, manganese levels prior to mating were 117 % higher than controls in males and 167 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 133 and 129 % higher for males and females, respectively.

At target 5 μg/L, manganese levels prior to mating were 50 % higher than controls in males and 66 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 50 and 43 % higher for males and females, respectively.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

Table 3: F0 Group Mean Body Weight Values (g)

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

-7

212

212

207

204

123

128

130

124

0

253

255

246

245

152

156

156

151

7

285

287

275

267*

176

179

178

172

14

314

316

304

288***

195

201

198

191

21

337

343

330

305***

212

218

220

213

28

347

354

339

315***

216

223

229

221

35

366

371

360

338**

232

241

246

237

42

375

382

373

350*

240

252

256

247

49

388

402

384

363*

249

262*

266**

256

56

401

415

397

374*

257

268

271

260

63

419

430

410

389**

262

272

274

265

70

428

441

422

395**

265

276

278

267

77

434

445

433

402**

-

-

-

-

84

442

457

441

414*

-

-

-

-

91

448

465

447

417*

-

-

-

-

98

455

473

453

427*

-

-

-

-

105

463

483

459

436*

-

-

-

-

112

468

485

462

435*

-

-

-

-

119

458

488

478

451

-

-

-

-

Change 0 - 70

-

-

-

-

113

120

122

116

Change 0 - 112

215

231

215

190*

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

***Significantly different from Group 1: p<0.001

Day 0 = first day of treatment

 

Table 4:F0 Females Group Mean Body Weight Values (g) During Gestation and Lactation

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

269

271

281

266

7

294

297

306

290

14

326

327

334

318

20

379

377

388

366

Weight Gain² (% of control)

110 (-)

106 (96)

107 (97)

100 (91)

Day of Lactation³

1

323

273

282

271

7

321

323

326

314

14

344

345

349

336

21

329

330

337

331

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

 

Table 5: F0 Group Mean Body Weight Values (g)

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

0

59

62

60

63

56

58

58

60

5

110

121*

98*

84***

76

79

77

76

12

123

135

126

123

106

110

110

106

19

166

174

167

164

135

137

139

135

26

206

221

215

206

157

161

163

157

33

246

263

254

241

178

180

183

177

40

277

295

285

268

193

197

197

193

47

302

323*

311

291

206

209

211

207

54

319

341*

327

308

216

217

219

214

61

340

364*

344

324

226

225

226

224

68

346

369

351

332

231

231

232

228

75

353

376

358

341

-

-

-

-

82

365

390*

374

256

-

-

-

-

89

374

399

385

365

-

-

-

-

96

376

404*

390

369

-

-

-

-

103

384

415*

399

380

-

-

-

-

110

382

418**

402

384

-

-

-

-

Change 0 - 68

-

-

-

-

175

172

174

168

Change 0 - 110

322

356**

343

322

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

***Significantly different from Group 1: p<0.001

Day 0 = first day of treatment

 

Table 6:F0 Females Group Mean Body Weight Values (g) During Gestation and Lactation

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

232

230

229

229

7

259

257

258

253

14

289

287

289

281

20

339

337

339

329

Weight Gain² (% of control)

107 (-)

107 (100)

110 (103)

100 (93)

Day of Lactation³

1

243

243

237

240

7

290

287

282

280

14

321

317

307

307

21

315

311

304

304

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

Conclusions:
Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L.
Executive summary:

The long term toxicity of the test material was investigated in a two generation reproductive toxicity study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. F0 animals were randomised into 4 test groups, each containing 28 males and 28 females. These animals were dosed with the test material for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls; however this reflected a slightly smaller litter size at this level and this accounts for the lower litter weights. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation. No test material-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations.

Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L, in the nasal cavity, pharynx, larynx and lung in all dose groups of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups, which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test material in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, a No Observed Effect Level (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material.

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See "Remark"
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
20 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two-generation reproductive toxicity study on a read-across substance.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 July 2012 to 04 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
other: Target Substance
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals. Bedding material was sterilised white wood shavings.
- Diet: ad libitum
- Water: Water taken from the public supply was available ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing periods of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 26 °C
- Humidity: 30 - 69 %
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES:
From: 02 July 2012
To: 04 March 2013
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

- Dose formulation Preparation and analysis
Test material formulation was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh size of 100 μm prior to use, except on one occasion where a sieve mesh of 180 μm was used.

- Preliminary Aerosol Characterisation Investigations
Characterisation of the aerosol generating/exposure system was undertaken prior to commencement of the animal exposures to demonstrate satisfactory performance. Preliminary aerosol characterisation investigations demonstrated that aerosol concentrations were stable spatially within the exposure system and over time and the particle size distribution investigations showed that test formulation particles for Groups 2 to 4 were respirable for the rat.

- Aerosol Generation
Test material aerosols were generated using a Wright Dust Feed generator device. Prior to the commencement of aerosol generation, a reservoir canister was packed with the test material powder formulation. The powder cake was slowly advanced into the scraper blade at an appropriate speed and scraped powder carried in a pressurised air stream.
The Wright Dust Feed generator device was operated at an appropriate target scraper speed, and air flow rate identified during the preliminary aerosol characterisation investigations. The generated test aerosols were then delivered to the flow past exposure chamber via a connecting tube manifold and mixed with dilution air to achieve the target aerosol concentration. A vacuum pump system was used to continuously exhaust test aerosols from the exposure chamber. Each aerosol generation system was operated to sustain a dynamic airflow sufficient to ensure an evenly distributed exposure aerosol.

- Inhalation Exposure (see Figure 1)
Exposure to the test aerosols was performed using an appropriately sized modular nose only stainless steel flow past exposure chamber (in-house design). Separate inhalation exposure systems were used for the delivery of test aerosol to each treatment group. Each inhalation exposure system was located in an extract booth (to prevent cross-group contamination). This exposure technique allowed a continuous supply of test aerosol to be delivered to each animal; the biased flow created using the flow-past chamber design ensured that there was no re-breathing of the test atmosphere.
For all inhalation exposures, the rats were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ noses protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push fit through rubber ‘o’ rings in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes. The exposure system was operated at an appropriate target total airflow. All flow rates (delivered and extracted) were monitored visually using calibrated flow meters. Exposure chamber flow rates, temperature and relative humidity were monitored and recorded at appropriate intervals during each daily exposure period.

TEST ATMOSPHERE
The aerosol concentration of test material formulation (Groups 2 to 4) or air (Group 1) in the animals’ breathing zone was measured gravimetrically for all groups at regular intervals throughout each daily exposure period.
The test aerosols were sampled using glass-fibre filters (47 mm Whatman GF/B) contained in a stainless steel filter holder in-line with a sampling system comprising a vacuum pump, flow meter and gas meter. Filter samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate to ensure that there was no overloading of the filter which would cause a reduction in sampling flow rate. The filters were weighed before and after sampling and the aerosol concentration calculated using the weight of formulation collected and the volume of air sampled.
In addition to the aerosol chamber concentration assessment, blank filter samples were taken to assess background levels of test material and retained for analysis.
All retained filters from Groups 1 to 4 were placed in amber glass jars and stored in a refrigerator set to maintain 4 °C prior to analysis for the determination of the aerosol concentration of test material.
A real time aerosol monitor (Casella Microdust, Casella Measurements, UK) was used to assist in monitoring/ assessing the target concentrations at the start of generation each day and provided a continuous overview of any fluctuations in aerosol concentration.

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The substrate collection plates (34 mm stainless steel) and back up filter (34 mm Westech) were weighed before and after sampling to determine the total amount of test and/or vehicle aerosol collected in each particle size range.
After weighing, the substrate collection plates and back up filters of were retained in amber glass jars and stored in a refrigerator set to maintain 4 °C.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at the testing facility.
Duration of treatment / exposure:
F0 animals were dosed for 10 weeks prior to mating; for F0 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F0 females were dosed throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning; for F1 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F1 females were dosed throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.
Frequency of treatment:
Daily (ca. 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of gestation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca. 1 hour per day
From Day 3-4 of lactation: ca. 2 hours per day
From Days 5-20 of lactation until prior to termination (ca. Day 21 of lactation): ca. 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.
Dose / conc.:
5 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 6 µg/L air
analytical conc. F1 generation: 4 µg/L air
Dose / conc.:
10 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 15 µg/L air
analytical conc. F1 generation: 10 µg/L air
Dose / conc.:
20 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 25 µg/L air
analytical conc. F1 generation: 17 µg/L air
No. of animals per sex per dose:
- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected for use based on results from a preliminary reproduction study in rats. In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values (TLVs) proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Weights of F0 animals were recorded one week prior to the first day of dosing, then weekly thereafter until the start of the mating period. Males continued to be weighed weekly until termination; but for females, weighing resumed on Day 0 of gestation (the day of detection of a positive mating sign), and then on Days 7, 14 and 20 of gestation and Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the majority of the litters and continued until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. Females that did not show a positive mating sign were weighed weekly until parturition or termination. Females who had a positive mating sign but failed to litter reverted to the weekly weighing regimen following their theoretical Day 24 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was quantitatively measured for both sexes weekly, starting one week before treatment commenced (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until placement of males in individual cages prior to mating. Weekly measurements continued after the 14 day mating period. For females, following a clear indication of mating, food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: Yes
- Time schedule: Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHER:
- Bioanalytical Sample Collection
Blood (1 mL) was collected from the tail vein of all animals, after careful cleaning of the sample site to avoid any possible contamination, into tubes containing lithium heparin. Samples were collected at the following time-points:
F0 generation: samples were collected from all animals pre-dose, prior to mating and prior to weaning/necropsy
F1 generation: samples were collected from all animals after selection (timing and volume dependent on weight of animal), prior to mating and at necropsy (or shortly prior to, as appropriate).
The samples were stored at -80 °C prior to analysis of manganese in whole blood performed with a validated ICP-MS method.

- Observation of Females with Litters during Lactation
The females were allowed to litter normally. If any animal suffered from a difficult or prolonged parturition, this was recorded. The day of birth of the litter (day on which the first pups are born) was designated Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

- Sexual Maturation in F1 Animals
Commencing at 28 days of age, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day. Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

- Oestrous Cyclicity
Over a 2 week period prior to the initiation of mating, vaginal lavages were taken early each morning and the stages of oestrous observed were recorded.

- Sperm Parameters
The tip of the cauda epididymis was placed in Medium 199 containing 0.2 % BSA and HEPES. The sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was examined using a Hamilton Thorne sperm motility analyser; sufficient replicates to provide 200 motile sperm were assessed (except where it was obvious that motility was compromised for that animal).
The remaining portion of the cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From a sample of the sperm suspension described above, a sperm smear was prepared and stained with eosin. From the Control and High dose animals, two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce.
One testis was decapsulated and homogenised. The homogenate may have been sonicated to remove tissue debris etc., as required. The number of homogenisation-resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

LITTER OBSERVATIONS
- The number of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually.
- Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.
Sacrifice and pathology:
SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

UNSCHEDULED DEATHS
These animals, including those killed or found dead, had a terminal body weight recorded and were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. The tissues list for animals at scheduled necropsy along with representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral 10 % formalin. The reproductive tracts of all females were examined for signs of implantation (if they had been paired for mating prior to necropsy), the number of any implantation sites being recorded.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of external surfaces and orifices, cranial, thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal gland, pituitary gland, prostate gland, thyroid glands, kidneys, liver, lungs, ovaries, spleen, testes and uterus.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents examined. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the following tissues were collected from all animals and preserved in 10 % neutral buffered formalin: brain, epididymides, adrenal glands, pituitary gland, prostate gland, seminal vesicle gland, thyroid glands, kidneys, larynx, liver, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, spleen, testes (preserved in modified Davidson’s fixative), anterior and posterior trachea, uterus and vagina.

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix) and vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Other examinations:
SACRIFICE / GROSS NECROPSY of offspring
Pups that were not selected for post-weaning assessments were killed at the same time as their mother.
Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

- Offspring found dead or killed (prematurely) before Day 14 of lactation
Where practicable, these animals were sexed, then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10 % formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal decedents were discarded.

- Offspring (pre-weaning) found dead or killed (prematurely) on or after Day 14 of lactation
These animals were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10 % formalin. These carcasses were then discarded.

- F1 and F2 Weanlings at scheduled termination
From each litter, 3 male and 3 female pups (where they were available – if a litter only had females or males, then up to 6 of the relevant sex were selected) were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10 % formalin. From one of the 3 pups of each sex, the weights of the brain, spleen and thymus were recorded, and these organs were preserved. Representative samples of any abnormal tissues from any of the 6 pups were also preserved. The carcasses were then discarded.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality were necropsied as described in the preceding paragraph. The remaining carcasses were discarded.

HISTOPATHOLOGY
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test, i.e. pairwise comparison was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 422 had this sign recorded on only one day (Day 14 of the study) and Animal 424 had wheezing recorded on 5 occasions (Days 91 and 112-115 of the study). Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca. Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental.
Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina (the animal gave birth to one live pup). The uterus also contained live foetuses and one late death. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition and had given birth to 3 live pups. One dead foetus was found in the right uterine horn at necropsy. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejunum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 752 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there was a decrease in body weight gain in males over Days 0-21 of the study. From Day 21 of the study, the body weight gains were generally comparable to the controls but the group mean weights remained lower than the controls throughout the study. At target 20 μg/L, there was a group mean body weight gain in females prior to mating were similar to the controls, however body weight gains over Days 0-20 of gestation were slightly lower than the controls. Gains over lactation were similar to the controls.
- F1 animals
At target 20 μg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study, however gains over the following week were greater than the controls and then remained comparable with the controls throughout the remainder of the treatment period. Slight intergroup differences in group mean body weight gains in the F1 females prior to mating were too small to be attributed to treatment. At 20 μg/L, there was a slight reduction in body weight gains throughout gestation compared to the controls.
There were no effects of treatment noted in the lactation females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 and 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 and 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In all treated groups of the F0 generation, the levels of test material in the blood increased significantly on commencement of dosing in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups, which did not indicate any obvious accumulation over the dosing period.
In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test material in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values, indicating an increased exposure throughout the dosing period.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, reduced brain weights in males achieved statistical significance (P<0.05) compared with controls. However, the lower body weight was also statistically significant (P<0.05); following covariance analysis, brain weight did not achieve significance and therefore was not positively attributed to treatment. In all treated females, there was a statistically significant increase in lung weights, compared with the controls; these increases were still present following covariance analysis (P<0.01 at target 5 μg/L and P<0.001 at target 10 and 20 μg/L). Other slight differences in organ weights such as an increased thyroid weight in males at target 5 μg/L and an increase in kidney weights of females at target 10 μg/L were not attributed to treatment.
- F1 animals
At target 5 and 10 μg/L, kidney weights in males were statistically higher than the control, however there was no dose relationship to this increase and following covariance analysis, these findings were no longer evident. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights in females (P<0.05 at target 10 μg/L and P<0.001 at target 20 μg/L) following covariance analysis. Other slight differences in organ weights such as an increased adrenal weight in females at target 20 μg/L were not attributed to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

- F0 animals
In the larynx there was a broadly dosage-related minimal to moderate squamous metaplasia with minimal to moderate submucosal inflammation. Minimal to marked ulceration of the laryngeal epithelium was associated with the squamous metaplasia in several animals from all treated groups. Occasional incidences of mineralisation, intraluminal necrotic debris or intra-epithelial pustules were seen in some of the treated animals.
In the lungs the principal test material related change was seen in centroacinar regions where there was minimal or mild inflammation and focal or diffuse minimal or mild bronchoalveolar hyperplasia. This latter finding was considered reactive. Minimal or mild goblet cell hyperplasia in the bronchial or bronchiolar epithelium was present in animals exposed to target 20 μg/L together with occasional incidences of degeneration and/or squamous metaplasia of the bronchiolar epithelium. Minimal inflammatory findings (inflammatory cell foci and perivascular inflammatory cell infiltration) were also present with a greater incidence in animals exposed to the test material than in controls.
In the nasal cavity, minimal or mild goblet cell hyperplasia and minimal to moderate eosinophilic globules in the olfactory epithelium were observed in all the male treated groups and in females exposed to target 10 or 20 μg/L. At all dose levels, there was a greater incidence of minimal or mild submucosal inflammatory cell infiltration compared to controls.
In males, inflammation of the nasolacrimal duct and squamous metaplasia of the ductal epithelium was seen in most animals exposed to target 10 or 20 μg/L. In addition to these changes, incidences of minimal or mild focal degeneration of the olfactory, respiratory or transitional epithelia, minimal or mild atrophy of the olfactory epithelium, ulceration and focal squamous metaplasia were observed, mainly in animals exposed to target 10 or 20 μg/L, but occasionally in animals at target 5 μg/L. Deposits of crystalline material, presumed to be test material, was seen in the nasolacrimal ducts of a few animals in the treated groups.
Minimal goblet cell hyperplasia was observed in the pharynx of most males exposed to target 20 μg/L and there were occasional incidences of minimal or mild focal epithelial degeneration, focal inflammation and focal squamous metaplasia in males exposed to target 10 or 20 μg/L.
In the trachea, minimal or mild focal squamous metaplasia and inflammation at the carina and minimal or mild focal epithelial degeneration at sites other than the carina were observed at all dose levels.
Other microscopic findings observed were considered incidental, or of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
- F1 animals
Crystals were occasionally observed in the nasal cavity and in the pharynx from animals exposed to target 10 or 20 μg/L. They consisted of small amounts of needle-shaped crystals either deposited on the olfactory epithelium in the nasal cavity, or free in the lumen of the pharynx. These crystals were considered to result from deposition of test material in some parts of the respiratory tract.
Squamous metaplasia in the nasal cavity was observed mainly in the nasolacrimal duct and to a lesser extent in the transitional and respiratory epithelia.
In the trachea, findings such as epithelial degeneration, squamous metaplasia and submucosal inflammation were observed predominantly in the carina.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
The stages of the oestrus cycles and their mean duration were similar in all groups for both generations.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects on the sperm motility, count or morphology at any of the dose levels applied, in either generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on mating performance, fertility or duration of gestation in either generation.

OTHER FINDINGS (PARENTAL ANIMALS)
- Sexual Maturation
The age and body weight at preputial separation or vaginal opening of the F1 generation animals in all treated groups was similar to the controls.

LITTER SIZE AND PUP MORTALITY
- F0 generation, F1 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 20 μg/L, there was an increase in the number of animals losing more than 2 pups at birth (total pups born/no. of implantation sites). However, the mean birth index (%) was well within the background range and these increases were considered to be incidental.
- F1 generation, F2 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 10 and 20 μg/L, pup survival (no. losing >3 pups) over Days 0-4 of lactation was slightly lower than the controls. However, the number of animals losing the entire litter was comparable with controls and the remaining animals generally lost 4 pups. In addition, there was no clear dose related response to these reductions and these were considered not to be an effect of treatment.

LITTER AND PUP WEIGHTS
- F0 Generation
In all treated groups, group mean litter and pup weights were comparable to the controls.
- F1 Generation
At target 20 μg/L, group mean litter weights were slightly lower than the controls which reflected the smaller litter size at this level. However, although the litter weights were slightly lower than the controls, the mean pup weights in both males and females were comparable to the controls.

ABNORMALITIES AMONG PUPS
The type and distribution of observations amongst pups did not indicate any association with treatment.

ORGAN WEIGHTS
- F0 generation, F1 production
At target 20 μg/L, there was a reduction in thymus weight of the females, compared with the controls (P<0.01). Following covariance analysis, this reduction did not achieve statistical significance. There were no effects on organ weights at target 5 and 10 μg/L.
- F1 generation, F2 production
Slight intergroup differences in organ weights did not achieve statistical significance and were not attributed to treatment.

GROSS PATHOLOGY
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to treatment with the test material.

HISTOPATHOLOGY
There were no treatment related findings observed in the tissues examined of the F1 or F2 weanlings.
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Table 1: F0 Blood Analysis Results

F0 Males

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

6

Prior to mating

6

13

23

27

Prior to Necropsy

6

19

27

29

F0 Females

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

7

Prior to mating

6

16

28

39

Prior to Necropsy

7

16

24

33

Control animals were exposed via the food and water.

At target 20 μg/L, manganese levels prior to mating were 350 % higher than controls in males and 550 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 383 and 371 % higher for males and females, respectively.

At target 10 μg/L, manganese levels prior to mating were 283 % higher than controls in males and 367 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 350 and 243 % higher for males and females, respectively.

At target 5 μg/L, manganese levels prior to mating were 117 % higher than controls in males and 167 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 217 and 129 % higher for males and females, respectively.

Table 2: F1 Blood Analysis Results

F1 Males

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

12

16

16

17

Prior to mating

6

9

13

19

Prior to Necropsy

6

9

14

21

F1 Females

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

13

12

15

15

Prior to mating

6

10

16

23

Prior to Necropsy

7

10

16

21

At target 20 μg/L, manganese levels prior to mating were 217 % higher than controls in males and 283 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 250 and 200 % higher for males and females, respectively.

At target 10 μg/L, manganese levels prior to mating were 117 % higher than controls in males and 167 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 133 and 129 % higher for males and females, respectively.

At target 5 μg/L, manganese levels prior to mating were 50 % higher than controls in males and 66 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 50 and 43 % higher for males and females, respectively.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

Table 3: F0 Group Mean Body Weight Values (g)

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

-7

212

212

207

204

123

128

130

124

0

253

255

246

245

152

156

156

151

7

285

287

275

267*

176

179

178

172

14

314

316

304

288***

195

201

198

191

21

337

343

330

305***

212

218

220

213

28

347

354

339

315***

216

223

229

221

35

366

371

360

338**

232

241

246

237

42

375

382

373

350*

240

252

256

247

49

388

402

384

363*

249

262*

266**

256

56

401

415

397

374*

257

268

271

260

63

419

430

410

389**

262

272

274

265

70

428

441

422

395**

265

276

278

267

77

434

445

433

402**

-

-

-

-

84

442

457

441

414*

-

-

-

-

91

448

465

447

417*

-

-

-

-

98

455

473

453

427*

-

-

-

-

105

463

483

459

436*

-

-

-

-

112

468

485

462

435*

-

-

-

-

119

458

488

478

451

-

-

-

-

Change 0 - 70

-

-

-

-

113

120

122

116

Change 0 - 112

215

231

215

190*

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

***Significantly different from Group 1: p<0.001

Day 0 = first day of treatment

 

Table 4:F0 Females Group Mean Body Weight Values (g) During Gestation and Lactation

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

269

271

281

266

7

294

297

306

290

14

326

327

334

318

20

379

377

388

366

Weight Gain² (% of control)

110 (-)

106 (96)

107 (97)

100 (91)

Day of Lactation³

1

323

273

282

271

7

321

323

326

314

14

344

345

349

336

21

329

330

337

331

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

 

Table 5: F0 Group Mean Body Weight Values (g)

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

0

59

62

60

63

56

58

58

60

5

110

121*

98*

84***

76

79

77

76

12

123

135

126

123

106

110

110

106

19

166

174

167

164

135

137

139

135

26

206

221

215

206

157

161

163

157

33

246

263

254

241

178

180

183

177

40

277

295

285

268

193

197

197

193

47

302

323*

311

291

206

209

211

207

54

319

341*

327

308

216

217

219

214

61

340

364*

344

324

226

225

226

224

68

346

369

351

332

231

231

232

228

75

353

376

358

341

-

-

-

-

82

365

390*

374

256

-

-

-

-

89

374

399

385

365

-

-

-

-

96

376

404*

390

369

-

-

-

-

103

384

415*

399

380

-

-

-

-

110

382

418**

402

384

-

-

-

-

Change 0 - 68

-

-

-

-

175

172

174

168

Change 0 - 110

322

356**

343

322

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

***Significantly different from Group 1: p<0.001

Day 0 = first day of treatment

 

Table 6:F0 Females Group Mean Body Weight Values (g) During Gestation and Lactation

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

232

230

229

229

7

259

257

258

253

14

289

287

289

281

20

339

337

339

329

Weight Gain² (% of control)

107 (-)

107 (100)

110 (103)

100 (93)

Day of Lactation³

1

243

243

237

240

7

290

287

282

280

14

321

317

307

307

21

315

311

304

304

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

Conclusions:
Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L.
Executive summary:

The long term toxicity of the test material was investigated in a two generation reproductive toxicity study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. F0 animals were randomised into 4 test groups, each containing 28 males and 28 females. These animals were dosed with the test material for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls; however this reflected a slightly smaller litter size at this level and this accounts for the lower litter weights. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation. No test material-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations.

Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L, in the nasal cavity, pharynx, larynx and lung in all dose groups of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups, which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test material in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, a No Observed Effect Level (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material.

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See "Remark"
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
20 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two-generation reproductive toxicity study on a read-across substance.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7.1.-8.9.2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Fasting period before study: no
- Housing: Animals were housed in SPF animal room, 2-3 rats of the same sex (during acclimatization period) or individually (during treatment period) in one plastic cage (40x25x20cm) containing sterilised clean shavings of soft wood.
- Diet: ad libitum, Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using. Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%. Composition of the diet: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
- Water: ad libitum, Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 5 days
- Additional Information: The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis. Results are retained in the CETA archives. Reports of analysis of water (performed twice a year) are retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) are retained in the CETA archives. Analyses and controls mentioned above did not reveal any finding that could affect study integrity.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.


Study Time Schedule
Test substance delivery: 3. 3. 2006
Dose-range finding experiment: 7.1. – 6. 2. 2009
Main study
Date of animal arrival: 11. 2. 2009
Start of administration: 17. 2. 2009
End of administration: 19. 3. 2009
Clinical observation: 11. 2. - 19. 3. 2009 main groups
11. 2. - 31. 3. 2009 satellite groups
Urinalysis: 16. 3. - 19. 3. 2009 main groups
30. 3. - 31. 3. 2009 satellite groups
Blood taking and necropsies: 17. – 20. 3. 2009 main groups
31.3. –1. 4. 2009 satellite groups
Histopathological examination: 30. 6. – 21. 8. 2009
Evaluation of results and final report elaboration: 27. 7. – 8. 9. 2009
Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
TEST SITE
- % coverage: 10%
- Type of wrap if used: by foil and held in contact by non-irritating tape
- Time intervals for shavings or clipplings: Approximately 24 hours before testing, fur was shaved from dorsal area of the trunk of the test animals. No less than 10% of the body surface area was clear for the application of the test substance. Repeated shaving was needed at approximately weekly intervals.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test substance was removed using water
- Time after start of exposure:


TEST MATERIAL
- Amount(s) applied (volume or weight with unit):The concentrations of solutions in all dose levels were adjusted to ensure the administration of 2 mL per 100 g of body weight
- Constant volume or concentration used: yes
- The test substance was not possible to apply as solid substance because it is classified as corrosive and contact of crystallic form with skin causes skin marked irritation and full thickness destruction of skin tissue (see to the results of Acute Dermal Irritation/Corrosion Study No. 15/06/4; VUOS-CETA Report No. 0663; 2006).

VEHICLE
The test substance was administered dissolved in water for injectione.
Aqua for injections
Manufacturer: Ardeapharma, Ševětín, Czech Republic
Batch number: 0101210308, 0204190908

DOSE LEVELS AND GROUPS OF ANIMALS
- See table No. 1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of test substance was performed by a spectrophotometry.
Test substance stability and homogeneity were determined by measuring an absorbance of its water solution in visible range of spectrum.
Duration of treatment / exposure:
The treated and control groups were administered daily for the period of 28 days.
Frequency of treatment:
The animals were treated 7 days per week. The treatment starts each day at the same time (6.30 – 8.30 am).
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
600 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
20 males and 20 females - dose-range finding experiment
30 males and 30 females - main study
5 females+5 males per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Before starting of main study the dose-range finding experiment with 14-day application period was performed. The dose levels 200, 500, 750 and 1000 mg/kg/day were derived from the limit dose 1000 mg/kg/day (according to the guideline). During dose-range finding experiment the body weight and clinical observations were recorded. Basic haematological examination and gross necropsy were made at the end of experiment.

Doses for the main study - 150, 300, 600 mg/kg/day were chosen on the basis of results of dose-range finding experiment.

- Rationale for animal assignment (if not random): Animals were randomly divided into the control and test groups and marked individually.
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 14 days after the end of application
- Section schedule rationale (if not random): all animals were sectioned
Positive control:
no
Observations and examinations performed and frequency:
GENERAL CLINICAL OBSERVATION: Yes
- Time schedule: daily-during administration period
- All rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (c. from 13.30 to 15.30 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of administration, then in week intervals
- This observation was carried out before the first application and then weekly. At the first part of observation behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour. The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

FUNCTIONAL OBSERVATION: Yes
- Time schedule: in the last week of study
- This observation was done at the end of administration period at main groups or at the end of recovery period at satellite groups. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons.


HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental part
- The health condition was controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups. Pre-experimental observation of all rats was performed to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. In administration period this observation was performed before application.

MORTALITY CONTROL: Yes
- Time schedule: daily
- All rats were examined for vitality changes or mortality daily during the treatment and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- The body weight of animals was recorded on automatic balances with group average computing module. All animals were weighed immediately before euthanasia too. Weight increment was computed as an average per group per day (in grams).


FOOD CONSUMPTION: Yes
- Time schedule: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- In the specified days every week the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. Average values were calculated for each week of the study.


FOOD EFFICIENCY: Yes
- Food consumption for animal/day was calculated from average values of each group. Calculation of food conversion in %: weight increment/food consumption x 100.


WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week
- Drinking water consumption was recorded. Average values (water consumption per animal and per day) were calculated for each week of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Anaesthetic used for blood collection: Yes, light ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.


BIOCHEMICAL EXAMINATION: Yes
- Time schedule for collection of blood: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Anaesthetic used for blood collection: Yes, light ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.3] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: 28th day of study (main groups) and 42nd day of study (satellite groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.4] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde).

HISTOPATHOLOGY: Yes
- Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Parameters checked in table [No.5] were examined.

BIOMETRY OF ORGANS: Yes
-Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
- At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians or averages.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
not examined
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
- Males
The activity (poise, gait, reaction to handling) of all males at all treated groups was similar during the study and unchanging compared with activity of males at the control groups.
No changes were found out at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and respiration. Diarrhoeal was recorded in one male at the control satellite group. Excretion of other treated and control groups was without changes. Red secretion around eyes and nostrils were recorded in part of animals at all dose levels and at the control group.

- Females
The activity (poise, gait, reaction to handling) of all females at all treated groups was similar during the study and unchanging compared with activity of females at the control groups.
No changes were found out at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, excretion and respiration. Red secretion around eyes and nostrils were recorded in part of animals at all dose levels and at the control group.


MORTALITY
- Males
There were no unscheduled deaths during all the study.
- Females
There were no unscheduled deaths during all the study


BODY WEIGHT AND WEIGHT GAIN
1) AVERAGE BODY WEIGHT
- Males
During whole application period, the growth curve at all dose levels was slightly decreased in comparison to the control group but without statistical significance. Maximal difference was recorded at the highest dose level in the last week of treatment.
- Satellite males
The growth curve of treated males was relative well-balanced. Only slightly decreased in comparison to the control group (without statistical significance) was recorded in the last week of application (4th week of study).

- Females
During whole application period, the growth curves of the dose level 150 mg/kg/day was well-balanced with the control group. At the middle and highest dose levels slight decrease of body weight without statistical significance was recorded. Maximal difference was recorded at the middle dose level in the last week of treatment.
- Satellite females
The growth curve of treated females was well-balanced with the control group during whole application period. Slight increase of body weight of treated females was recorded in the last week of application.

2) BODY WEIGHT INCREMENT
- Males
After the 1st week of application the body weight increment of all treated groups was markedly decreased in comparison with the control group. At the end of the 2nd and 3rd weeks the body weight increments of all dose levels were higher than at the control group. At the end of application period the increment at all dose levels was markedly decrease again.
- Satellite males
The body weight increment of both groups was unbalanced during whole application. Markedly decreased increment of treated males was recorded in the first and last week of application period. On the contrary increased body weight increment was measured in the 2nd week of application and during the recovery period.

- Females
Decrease of body weight increment after the 1st week was recorded at the middle and highest dose levels. Slight increase of increment at the lowest and highest dose levels was measured in the end of the 2nd week. From the 3rd week to the end of application the increment of all dose levels was decreased again.
- Satellite females
The body weight increment of both groups was unbalanced during whole application. Increased increment of treated females was recorded in the 1st and 2nd week of application period. On the contrary increased body weight increment was measured in the 3rd week. From the 4th week of application and during the recovery period the body weight increment of both groups was relatively well-balanced.

FOOD CONSUMPTION
- Males
Food consumption of all treated groups was well-balanced with the control group during whole application. Slight decrease was recorded only at the highest dose level in the 1st week.
- Satellite males
The average food consumption of treated group was well-balanced with the control group during whole application and recovery period.

- Females
Food consumption of all treated groups was well-balanced with the control group during whole application.
- Satellite females
The average food consumption of treated group was well-balanced with the control group during whole application and recovery period.

FOOD CONVERSION
- Males
During the 1st week of application decreased food conversion was recorded at all dose levels. From the 2nd week to the 3rd of application the food conversion of the dose levels 150 and 300 mg/kg/day was well-balanced with the control group. At the highest dose level slightly increased food conversion was in the 3rd week. Markedly decreased conversation of all treated groups was measured at the last week of application.
- Satellite males
The food conversion of treated group was decreased in the first and last week of application. On the contrary increased conversion of treated group was recorded in the 2nd and 5th week of study. Relatively well-balanced food conversion of both groups was recorded only in the 3rd and the 6th week.
- Females
The well-balanced average food conversion of treated and control groups were recorded only at the dose level 150 mg/kg/day during the 1st and the 2nd week of study. Decreased conversion was recorded in the 2nd week of application at the dose levels 150 and during whole application period at the dose level 300 mg/kg/day. At the dose level 600 mg/kg/day decreased conversion was recorded at the 1st, 3rd and 4th week of study. Increase of food conversion was recorded only at the highest dose level in the 2nd week of application period.
- Satellite females
The average food conversion of both groups was unbalanced during application period. Increased conversion of treated group was recorded in the 1st and 2nd week of application and decreased conversion was in the 3rd week of study. In the last week of application period and during whole recovery period the food conversion of treated animals was well-balanced with the control group

WATER CONSUMPTION
- Males
The average water consumption of the treated groups was well-balanced with the control group only in the 1st week of application. Slightly increased consumption was recorded at the dose levels 150 mg/kg/day only in the 3rd week. At the dose levels 300 and 600 mg/kg/day slightly increase of water consumption was from 2nd week to the end of study.
- Satellite males

The water consumption of both groups was relatively well-balanced during whole application and recovery period.
- Females
The average water consumption of treated groups was relatively well-balanced with the control group in the first two weeks of application. Slightly increased consumption was recorded at the dose levels 150 and 600 mg/kg/day in the 3rd and 4th week. At the middle dose level the water consumption was well-balanced during whole study.
- Satellite females
In the treated group the water consumption was relatively well-balanced with the control group from the 3rd week of application to the end of recovery period. Increased consumption of treated animals was recorded in the first two weeks of application.



HAEMATOLOGY
- Males
Changes in differential leucocyte count were recorded. Value of monocytes was increased with dependence on the dose at the all dose levels. Statistical significance was found out at the middle and highest dose levels. Accompanied decreased value of lymphocytes without statistical significance was also recorded at these dose levels. Total leucocyte count was slightly increased (without statistical significance) at the dose level 150 mg/kg/day. Prolonged protrobine time was measured at the lowest and middle dose levels but statistical sinficance was recorded only at the dose level 300 mg/kg/day. Prolonged APTT (without statistical significance) was measured at the dose levels 300 and 600 mg/kg/day.
Other measured parameters were similar to the control group.
All observed parameters were within historical control range.
- Satellite males
Statistically significantly increased value of platelet count was recorded at the treated group.
Other measured parameters were similar to the control group.

- Females
Changes in differential leucocyte count were recorded. Value of monocytes was statistically significantly increased with dependence on the dose level at the all dose levels. Accompanied decreased value of lymphocytes with statistical significance was recorded at the highest dose level. Total leucocyte count was slightly increased (without statistical significance) at the dose level 150 mg/kg/day. Prolonged protrobine time was measured at the highest dose levels but without statistical significance. Prolonged APTT (without statistical significance) was measured at the dose levels 150 and 600 mg/kg/day.
Other measured parameters were similar to the control group. Statistical analysis of the data revealed no significant intergroup differences.

- Satellite females
No statistically significant changes were found out.
All measured parameters were similar to the control group.

BIOCHEMICAL EXAMINATION
- Males
All measured parameters at the dose level 150 mg/kg/day were similar to the control group. Statistical analysis of the data revealed significant intergroup differences at the dose levels 300 and 600 mg/kg/day. The sodium concentration was significantly increased in both dose levels. Significantly increased activity of ALT was recorded only at the dose level 300 mg/kg/day. Slightly increased activity of AST without statistical significance was measured also at the middle treated group. Insignificant decreased concentration of glucose was found out at the middle and highest dose levels. Slightly decreased value of creatinine and concentration of potassium were recorded at the highest dose level. Concentration of chlorides was insignificantly decreased at the lowest dose level.
- Satellite males
Increased concentration of phosphorus and decreased concentration of potassium and chloride with statistical significance was recorded at treated group. Slightly increased value of urea and decreased activity of AST were recorded at treated group without statistical significance.
All other measured parameters were similar to the control group.

- Females
Statistical analysis of the data revealed no significant intergroup differences. Slightly increased activity of AST at the highest dose level and ALT at the middle dose level were recorded. Decreased value of creatinine without statistical significance was measured at the middle and highest dose levels. All other measured parameters were similar to the control group
- Satellite females
Statistically significantly increased value of glucose and albumin was recorded at treated group. Concentration of chloride and sodium ions was significantly decreased also at this dose level. Insignificantly increased concentration of bilirubine and activity of AST was measured at the treated group. All other measured parameters were similar to the control group.


URINALYSIS
- Males
No statistically significant changes in urine were recorded at any dose levels. Slight decrease of urine volume was recorded at the dose level 600 mg/kg/day. Blood was detected in two males and nitrites in one male at the dose level 150 mg/kg/day. Urine of one animal at the middle dose level showed white cloud. Presence of leucocytes was recorded in one male at the highest dose level.
- Satellite males
Statistical analysis of the data revealed significant intergroup differences. Significantly decreased urine volume and increased pH of urine were recorded at the treated group. Urine of one treated male showed blood in urine. Also presence of leucocytes was recorded in one treated male.

- Females
There were no treatment-related changes in urinary parameters.
Statistical analysis of the data revealed no significant intergroup differences.
- Satellite females
There were no treatment-related changes in urinary parameters.
Statistical analysis of the data revealed no significant intergroup differences.


ORGAN WEIGHTS
Absolute Organ Weight
- Males
Statistical analysis of the data revealed no significant intergroup differences.
Treated animals showed slightly decreased weight of liver with dependence on the dose level. The weight of other organs was well-balanced at all groups.
- Satellite males
Absolute weight of all organs was well-balanced at both groups. Statistical analysis of the data revealed no significant intergroup differences.

- Females
Animals at the dose level 300 mg/kg/day showed statistically significantly decreased of weight of kidneys. Slightly decreased weight of kidneys was recorded also at the lowest and highest dose levels but without statistical significance. The weight of liver was slightly decreased at the middle dose level (without statistical significance). Slightly decreased absolute weight of pituitary gland and ovaries was found out at the highest dose level. Other organs was relatively well-balanced.
- Satellite females
Absolute weight of organs was well-balanced at both groups. Statistical analysis of the data revealed no significant intergroup differences.

Relative Organ Weight
- Males
Relative weight of organs was well-balanced at all dose levels. Statistical analysis of the data revealed no significant intergroup differences.
- Satellite males
Slightly insignificantly increased weight of liver was recorded at the treated group. Relative weight of other organs was well-balanced with the control group. Statistical analysis of the data revealed no significant intergroup differences.

- Females
Animals at the dose level 150 mg/kg/day showed slightly decreased weight of liver and kidneys (without statistical significance). Relative weight of other organs was relatively well-balanced. Statistical analysis of the data revealed no significant intergroup differences.
- Satellite females
The weight of liver in treated group was statistically significantly increased. Relative weight of other organs was relatively well-balanced at both groups.

PATHOLOGY
Macroscopic Findings
- Males
During the macroscopic examination no important pathologic changes were found. In 2-1-0-0 males no macroscopic changes were observed.
Examination of application area revealed changes on skin and regional lymph nodes: brown colouring of skin in 0-0-5-0 males, punctiform plagues in 0-0-2-0 males, dry and red foci of skin in 0-0-0-5, congested lymph nodes in 3-4-5-4 males and enlarged lymph nodes in 0-0-2-0 males.
In thoracic cavity focal changes (changed colour or petechial foci) in lungs were diagnosed in 0-0-0-2 males and petechie on thymus was in 0-0-0-1 male.
In abdominal cavity the irregular colour of kidneys were only found in 1-0-0-1 males.
In cranial cavity no changes were diagnosed.
- Satellite males
During the macroscopic examination of males no important pathologic changes were found. In 5-2 males no macroscopic changes were observed.
Examination of application area showed congested or enlarged regional lymph nodes only in 0-1 male.
In thoracic cavity only focal changes in lungs in 0-1 male were recorded.
In abdominal cavity no changes were recorded.In cranial cavity no changes were diagnosed.

- Females
During the macroscopic examination of females no important pathologic changes were found. In 3-0-0-0 females no macroscopic changes were observed.
Examination of application area revealed changes on skin and regional lymph nodes: brown colouring of skin in 0-0-5-0 females, punctiform plagues in 0-0-2-0 females, dry and red foci of skin in 0-0-0-5, crust on skin in 0-0-0-1 female, congested lymph nodes in 2-4-5-3 females and enlarged lymph nodes in 0-0-2-0 females.
In thoracic cavity only petechie on thymus in 0-1-0-0 female were found out.
In abdominal cavity irregular colour of liver in 0-0-2-0 females and marked structure in 0-0-1-0 female were observed. Dilatation of uterus with fluidwas diagnosed in 3-2-3-1 females.
In cranial cavity no changes were diagnosed.
- Satellite females
During the macroscopic examination of females no important pathologic changes were found. In 3-3 females no macroscopic changes were observed.
Examination of application area showed no changes.
In thoracic cavity only petechie on thymus in 0-1 female and focal changes in lungs (grey focus) in 1-0 female were recorded.
In abdominal cavity only irregular colour of liver in 0-1 female was recorded. Dilatation of uterus with fluid was diagnosed in 1-2 females.
In cranial cavity no changes were diagnosed.


HISTOPATHOLOGY: NON-NEOPLASTIC
- Males
Incidence of pathological affection of skin application area was increased with dependence on the dose level. Focal inflammation of skin in 0-2-3-5 males and parakeratosis or hyperkeratosis in 0-0-1-3 males were recorded. Application area without changes was found out in 5-3-1-0 males. Occurence of findings in regional lymph nodes was well-balanced at all treated and control groups: erythrocytosis in 2-4-5-4 males and hyperplasia in 5-4-5-5 males. Haemorrhage in thymus was recorded in 2-2-0-1 males.
Incidence of pathological findings in digestive system was sporadic. Atrophy of phundal glands in stomach in 2-1-2-1 males, inflammation in liver in 1-2-3-2 males and proliferation of oval cells in liver of 0-1-0-1 males were recorded. In genital tract oedema of interstitium in prostate gland of 2-5-3-0 males, inflammation of prostate gland in 0-2-2-0 males and inflammation of epididymis in 1-1-2-1 males were found out. Dystrophy in cortex of kidneys was recorded in 4-3-2-0 males. Cartilagenous metaplasia in heart was recorded in 2-0-0-1 males. Other microscopical findings were recorded only sporadically (affected only one or two males in whole study).
- Satellite males
Application area was without changes. Occurence of findings in regional lymph nodes was well-balanced at treated and control groups: erythrocytosis in 2-3 males, plasmocytosis 1-0 male and hyperplasia in 3-2 males. Atrophy of phundal glands in stomach of 1-1 males, inflammation in liver of 4-5 and foci of oval cells in liver of 0-3 males were found out in digestive system. Dystrophy in cotrtex of kidneys was recorded in 2-1 males. In genital tract oedema of interstitium in prostate gland of 4-1 males, inflammation of prostate gland in 0-2 males and inflammation of epididymis in 1-2 males were found out. Other microscopical findings were recorded only sporadically (affected only one male).

- Females
Incidence of pathological affection of skin application area was recorded only in treated groups. Focal inflammation of skin in 0-0-2-5 females, crusts on epidermis in 0-0-2-0 females and parakeratosis in 0-0-0-3 females were recorded. Application area without changes was found out in 5-5-3-0 females. Occurence of findings in regional lymph nodes was relative well-balanced at all treated and control groups: erythrocytosis in 4-4-5-5 females and hyperplasia in 5-5-5-5 females.
In digestive tract the higher incidence of pathological affections was recorded in liver. Inflammation in 5-4-4-4 females, pigmentation in 0-1-0-2 females and proliferation in 2-2-1-2 females were found out. Focal infiltration in oesophagus was found out in 1-2-0-0 females. Cartilagenous metaplasia in heart was recorded in 0-1-1-1 females. Inflammation or haemorrhages in lungs were found out in 2-2-0-0 females. In genital tract only hydrometra of uterus was recorded in 3-2-2-1 females. Other microscopical findings were recorded only sporadically (affected only one or two females in whole study).
- Satellite females
Application area was without changes. Pathologic changes were found out in digestive system. Hyperplasia in 5-2 females and erythrocytosis in 1-3 females were recorded in regional lymph nodes. In digestive system foci of oval cells in liver of 4-2 females and inflammation in liver of 5-4 females were found out. Cartilagenous metaplasia in heart was recorded in 2-1 females. Hydronephrosis in kidneys was recorded in 1-1 females Pigmentation in spleen was recorded in 2-2 females. In genital tract only hydrometra of uterus was recorded in 1-2 females. Other microscopical findings were recorded only sporadically (affected only one female).
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
urinalysis
Critical effects observed:
not specified
Conclusions:
The dermal administration of the test substance Potassium permanganate to rats for a period of twenty-eight consecutive days at dose levels 150 mg/kg/day produced no toxicologically significant changes in the parameters measured. No major functional changes in any organ systems or severe organ dysfunction were detected. Histopathological examination revealed no pathological changes, which could be related with administration of the test substance at the dose level 150 mg/kg/day. Significant changes in clinical biochemistry, haematology and urinalysis parameters, which indicate organism dysfunction, were recorded at the dose level 300 and 600 mg/kg/day.

Based on the results of laboratory investigations in clinical biochemistry, haematology and urinalysis and with respect to the results of histopathological examination the following conclusion about NOAEL can be suggested in this study:
Executive summary:

Methods

   SPF Wistar rats were used for testing. The test substance was administered dissolved in water for injections. The six groups of animals were included in the study – 4 main groups and 2 satellite groups.

   Main groups contained one control group (vehicle only) and three treated groups (doses 150, 300 and 600 mg/kg/day). Satellite groups contained one control group (vehicle only) and one treated group (600 mg/kg/day). The administration of test and control substances lasted 28 days. After that the satellite animals were observed for next 14 days without treatment.

   The stability and the homogeneity of the application form in water for injections were determined in CETA analytical laboratories. The concentrations of suspensions in all three dose levels were adjusted to ensure the administered volume of 2 mL per 100 g of body weight and over an area, which was approximately 10% of the total body surface area. 

   Before starting of main study the dose-range finding experiment with 14-day application period was performed. The dose levels 200, 500, 750 and 1000 mg/kg/day were derived from the limit dose 1000 mg/kg/day (according to the guideline). During dose-range finding experiment the body weight and clinical observations were recorded. Basic haematological examination and gross necropsy were made at the end of experiment.

 Doses for the main study - 150, 300, 600 mg/kg/day were chosen on the basis of results of dose-range finding experiment. 

   During the 28-day study clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured twice a week. Before the end of study the functional observation was accomplished. The study was finished by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The selected organs for weighing and histopathology examination were removed.

  

Results

   There were no unscheduled deaths during the test. At the middle and highest dose levels the application of the test substance caused reversible changes of application area with histopathological findings (focal inflammation of skin, erythrocytosis and hyperplasia of regional lymph nodes) which faded away after recovery period (without application).

   The animal’s health condition was very good during whole study. Clinical signs of toxicity were detected sporadically at all treated and control group. Functional observation evidenced no effect of the test substance.

   Slight effect on body weight gain was detected in both sexes which was more marked in males at the middle and highest dose levels. The body weight and body weight increment of test groups was statistically insignificantly decreased during whole application period. No adverse effect on dietary intake was detected. Unbalanced (mainly decreased) food conversion was recorded at all treated group males and females but markedly at the middle and highest dose levels. The water consumption was slightly unbalanced during whole study.

    Changes of differential leucocyte count were recorded in both sexes with statistical significance at the end of application. Increased total leucocyte count was also measured at the end of application but without statistical significance. Thehaemocoagulationexamination detected prolonged PT and APTT in both sexes but these changes achieved the statistical significance only in males at the middle dose level. These changes were not recorded in satellite treated males after 14-day recovery period.

    Statistical analysis of biochemicalparametersrevealed significant differences in males (increased sodium concentration against control at the middle and highest dose levels). Significant changes of ions concentration were recorded in both sexes at the end of recovery period.Due to presence of changed urinary parameters (decreased urine volume and increased pH of urine) this was considered to be toxicological important. Other intergroup differences considered to be adaptative response to stress without toxicological significance.

   Pathology examination revealed some but not treatment related changes in digestive system (atrophy of phundal glans in stomach, foci of oval cells or inflammation in liver), pigmentation in spleen, dystrophy in cortex of kidneys and cartilagenous metaplasia in heart. The intergroup differences were minimal, therefore they were regarded as fortuitous. Further sporadic pathologic changes were recorded in reproductive system: inflammation and oedema of interstitium in prostate gland, inflammation in epididymis, in males and hydrometra of uterus and pigmentation in spleen in females. Frequencies of these microscopic findings were similar in treated and control groups. This was considered to be of no toxicological importance. No neoplastic findings were recorded by histopathological examination.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
one guideline study with restrictions

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7.1.-8.9.2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Fasting period before study: no
- Housing: Animals were housed in SPF animal room, 2-3 rats of the same sex (during acclimatization period) or individually (during treatment period) in one plastic cage (40x25x20cm) containing sterilised clean shavings of soft wood.
- Diet: ad libitum, Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using. Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%. Composition of the diet: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
- Water: ad libitum, Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 5 days
- Additional Information: The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis. Results are retained in the CETA archives. Reports of analysis of water (performed twice a year) are retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) are retained in the CETA archives. Analyses and controls mentioned above did not reveal any finding that could affect study integrity.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.


Study Time Schedule
Test substance delivery: 3. 3. 2006
Dose-range finding experiment: 7.1. – 6. 2. 2009
Main study
Date of animal arrival: 11. 2. 2009
Start of administration: 17. 2. 2009
End of administration: 19. 3. 2009
Clinical observation: 11. 2. - 19. 3. 2009 main groups
11. 2. - 31. 3. 2009 satellite groups
Urinalysis: 16. 3. - 19. 3. 2009 main groups
30. 3. - 31. 3. 2009 satellite groups
Blood taking and necropsies: 17. – 20. 3. 2009 main groups
31.3. –1. 4. 2009 satellite groups
Histopathological examination: 30. 6. – 21. 8. 2009
Evaluation of results and final report elaboration: 27. 7. – 8. 9. 2009
Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
TEST SITE
- % coverage: 10%
- Type of wrap if used: by foil and held in contact by non-irritating tape
- Time intervals for shavings or clipplings: Approximately 24 hours before testing, fur was shaved from dorsal area of the trunk of the test animals. No less than 10% of the body surface area was clear for the application of the test substance. Repeated shaving was needed at approximately weekly intervals.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test substance was removed using water
- Time after start of exposure:


TEST MATERIAL
- Amount(s) applied (volume or weight with unit):The concentrations of solutions in all dose levels were adjusted to ensure the administration of 2 mL per 100 g of body weight
- Constant volume or concentration used: yes
- The test substance was not possible to apply as solid substance because it is classified as corrosive and contact of crystallic form with skin causes skin marked irritation and full thickness destruction of skin tissue (see to the results of Acute Dermal Irritation/Corrosion Study No. 15/06/4; VUOS-CETA Report No. 0663; 2006).

VEHICLE
The test substance was administered dissolved in water for injectione.
Aqua for injections
Manufacturer: Ardeapharma, Ševětín, Czech Republic
Batch number: 0101210308, 0204190908

DOSE LEVELS AND GROUPS OF ANIMALS
- See table No. 1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of test substance was performed by a spectrophotometry.
Test substance stability and homogeneity were determined by measuring an absorbance of its water solution in visible range of spectrum.
Duration of treatment / exposure:
The treated and control groups were administered daily for the period of 28 days.
Frequency of treatment:
The animals were treated 7 days per week. The treatment starts each day at the same time (6.30 – 8.30 am).
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
600 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
20 males and 20 females - dose-range finding experiment
30 males and 30 females - main study
5 females+5 males per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Before starting of main study the dose-range finding experiment with 14-day application period was performed. The dose levels 200, 500, 750 and 1000 mg/kg/day were derived from the limit dose 1000 mg/kg/day (according to the guideline). During dose-range finding experiment the body weight and clinical observations were recorded. Basic haematological examination and gross necropsy were made at the end of experiment.

Doses for the main study - 150, 300, 600 mg/kg/day were chosen on the basis of results of dose-range finding experiment.

- Rationale for animal assignment (if not random): Animals were randomly divided into the control and test groups and marked individually.
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 14 days after the end of application
- Section schedule rationale (if not random): all animals were sectioned
Positive control:
no
Observations and examinations performed and frequency:
GENERAL CLINICAL OBSERVATION: Yes
- Time schedule: daily-during administration period
- All rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (c. from 13.30 to 15.30 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of administration, then in week intervals
- This observation was carried out before the first application and then weekly. At the first part of observation behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour. The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

FUNCTIONAL OBSERVATION: Yes
- Time schedule: in the last week of study
- This observation was done at the end of administration period at main groups or at the end of recovery period at satellite groups. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons.


HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental part
- The health condition was controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups. Pre-experimental observation of all rats was performed to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. In administration period this observation was performed before application.

MORTALITY CONTROL: Yes
- Time schedule: daily
- All rats were examined for vitality changes or mortality daily during the treatment and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- The body weight of animals was recorded on automatic balances with group average computing module. All animals were weighed immediately before euthanasia too. Weight increment was computed as an average per group per day (in grams).


FOOD CONSUMPTION: Yes
- Time schedule: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- In the specified days every week the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. Average values were calculated for each week of the study.


FOOD EFFICIENCY: Yes
- Food consumption for animal/day was calculated from average values of each group. Calculation of food conversion in %: weight increment/food consumption x 100.


WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week
- Drinking water consumption was recorded. Average values (water consumption per animal and per day) were calculated for each week of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Anaesthetic used for blood collection: Yes, light ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.


BIOCHEMICAL EXAMINATION: Yes
- Time schedule for collection of blood: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Anaesthetic used for blood collection: Yes, light ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.3] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: 28th day of study (main groups) and 42nd day of study (satellite groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.4] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde).

HISTOPATHOLOGY: Yes
- Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
- Parameters checked in table [No.5] were examined.

BIOMETRY OF ORGANS: Yes
-Time schedule: 29th day of study (main groups) and 43rd day of study (satellite groups)
- At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians or averages.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
not examined
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
- Males
The activity (poise, gait, reaction to handling) of all males at all treated groups was similar during the study and unchanging compared with activity of males at the control groups.
No changes were found out at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and respiration. Diarrhoeal was recorded in one male at the control satellite group. Excretion of other treated and control groups was without changes. Red secretion around eyes and nostrils were recorded in part of animals at all dose levels and at the control group.

- Females
The activity (poise, gait, reaction to handling) of all females at all treated groups was similar during the study and unchanging compared with activity of females at the control groups.
No changes were found out at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, excretion and respiration. Red secretion around eyes and nostrils were recorded in part of animals at all dose levels and at the control group.


MORTALITY
- Males
There were no unscheduled deaths during all the study.
- Females
There were no unscheduled deaths during all the study


BODY WEIGHT AND WEIGHT GAIN
1) AVERAGE BODY WEIGHT
- Males
During whole application period, the growth curve at all dose levels was slightly decreased in comparison to the control group but without statistical significance. Maximal difference was recorded at the highest dose level in the last week of treatment.
- Satellite males
The growth curve of treated males was relative well-balanced. Only slightly decreased in comparison to the control group (without statistical significance) was recorded in the last week of application (4th week of study).

- Females
During whole application period, the growth curves of the dose level 150 mg/kg/day was well-balanced with the control group. At the middle and highest dose levels slight decrease of body weight without statistical significance was recorded. Maximal difference was recorded at the middle dose level in the last week of treatment.
- Satellite females
The growth curve of treated females was well-balanced with the control group during whole application period. Slight increase of body weight of treated females was recorded in the last week of application.

2) BODY WEIGHT INCREMENT
- Males
After the 1st week of application the body weight increment of all treated groups was markedly decreased in comparison with the control group. At the end of the 2nd and 3rd weeks the body weight increments of all dose levels were higher than at the control group. At the end of application period the increment at all dose levels was markedly decrease again.
- Satellite males
The body weight increment of both groups was unbalanced during whole application. Markedly decreased increment of treated males was recorded in the first and last week of application period. On the contrary increased body weight increment was measured in the 2nd week of application and during the recovery period.

- Females
Decrease of body weight increment after the 1st week was recorded at the middle and highest dose levels. Slight increase of increment at the lowest and highest dose levels was measured in the end of the 2nd week. From the 3rd week to the end of application the increment of all dose levels was decreased again.
- Satellite females
The body weight increment of both groups was unbalanced during whole application. Increased increment of treated females was recorded in the 1st and 2nd week of application period. On the contrary increased body weight increment was measured in the 3rd week. From the 4th week of application and during the recovery period the body weight increment of both groups was relatively well-balanced.

FOOD CONSUMPTION
- Males
Food consumption of all treated groups was well-balanced with the control group during whole application. Slight decrease was recorded only at the highest dose level in the 1st week.
- Satellite males
The average food consumption of treated group was well-balanced with the control group during whole application and recovery period.

- Females
Food consumption of all treated groups was well-balanced with the control group during whole application.
- Satellite females
The average food consumption of treated group was well-balanced with the control group during whole application and recovery period.

FOOD CONVERSION
- Males
During the 1st week of application decreased food conversion was recorded at all dose levels. From the 2nd week to the 3rd of application the food conversion of the dose levels 150 and 300 mg/kg/day was well-balanced with the control group. At the highest dose level slightly increased food conversion was in the 3rd week. Markedly decreased conversation of all treated groups was measured at the last week of application.
- Satellite males
The food conversion of treated group was decreased in the first and last week of application. On the contrary increased conversion of treated group was recorded in the 2nd and 5th week of study. Relatively well-balanced food conversion of both groups was recorded only in the 3rd and the 6th week.
- Females
The well-balanced average food conversion of treated and control groups were recorded only at the dose level 150 mg/kg/day during the 1st and the 2nd week of study. Decreased conversion was recorded in the 2nd week of application at the dose levels 150 and during whole application period at the dose level 300 mg/kg/day. At the dose level 600 mg/kg/day decreased conversion was recorded at the 1st, 3rd and 4th week of study. Increase of food conversion was recorded only at the highest dose level in the 2nd week of application period.
- Satellite females
The average food conversion of both groups was unbalanced during application period. Increased conversion of treated group was recorded in the 1st and 2nd week of application and decreased conversion was in the 3rd week of study. In the last week of application period and during whole recovery period the food conversion of treated animals was well-balanced with the control group

WATER CONSUMPTION
- Males
The average water consumption of the treated groups was well-balanced with the control group only in the 1st week of application. Slightly increased consumption was recorded at the dose levels 150 mg/kg/day only in the 3rd week. At the dose levels 300 and 600 mg/kg/day slightly increase of water consumption was from 2nd week to the end of study.
- Satellite males

The water consumption of both groups was relatively well-balanced during whole application and recovery period.
- Females
The average water consumption of treated groups was relatively well-balanced with the control group in the first two weeks of application. Slightly increased consumption was recorded at the dose levels 150 and 600 mg/kg/day in the 3rd and 4th week. At the middle dose level the water consumption was well-balanced during whole study.
- Satellite females
In the treated group the water consumption was relatively well-balanced with the control group from the 3rd week of application to the end of recovery period. Increased consumption of treated animals was recorded in the first two weeks of application.



HAEMATOLOGY
- Males
Changes in differential leucocyte count were recorded. Value of monocytes was increased with dependence on the dose at the all dose levels. Statistical significance was found out at the middle and highest dose levels. Accompanied decreased value of lymphocytes without statistical significance was also recorded at these dose levels. Total leucocyte count was slightly increased (without statistical significance) at the dose level 150 mg/kg/day. Prolonged protrobine time was measured at the lowest and middle dose levels but statistical sinficance was recorded only at the dose level 300 mg/kg/day. Prolonged APTT (without statistical significance) was measured at the dose levels 300 and 600 mg/kg/day.
Other measured parameters were similar to the control group.
All observed parameters were within historical control range.
- Satellite males
Statistically significantly increased value of platelet count was recorded at the treated group.
Other measured parameters were similar to the control group.

- Females
Changes in differential leucocyte count were recorded. Value of monocytes was statistically significantly increased with dependence on the dose level at the all dose levels. Accompanied decreased value of lymphocytes with statistical significance was recorded at the highest dose level. Total leucocyte count was slightly increased (without statistical significance) at the dose level 150 mg/kg/day. Prolonged protrobine time was measured at the highest dose levels but without statistical significance. Prolonged APTT (without statistical significance) was measured at the dose levels 150 and 600 mg/kg/day.
Other measured parameters were similar to the control group. Statistical analysis of the data revealed no significant intergroup differences.

- Satellite females
No statistically significant changes were found out.
All measured parameters were similar to the control group.

BIOCHEMICAL EXAMINATION
- Males
All measured parameters at the dose level 150 mg/kg/day were similar to the control group. Statistical analysis of the data revealed significant intergroup differences at the dose levels 300 and 600 mg/kg/day. The sodium concentration was significantly increased in both dose levels. Significantly increased activity of ALT was recorded only at the dose level 300 mg/kg/day. Slightly increased activity of AST without statistical significance was measured also at the middle treated group. Insignificant decreased concentration of glucose was found out at the middle and highest dose levels. Slightly decreased value of creatinine and concentration of potassium were recorded at the highest dose level. Concentration of chlorides was insignificantly decreased at the lowest dose level.
- Satellite males
Increased concentration of phosphorus and decreased concentration of potassium and chloride with statistical significance was recorded at treated group. Slightly increased value of urea and decreased activity of AST were recorded at treated group without statistical significance.
All other measured parameters were similar to the control group.

- Females
Statistical analysis of the data revealed no significant intergroup differences. Slightly increased activity of AST at the highest dose level and ALT at the middle dose level were recorded. Decreased value of creatinine without statistical significance was measured at the middle and highest dose levels. All other measured parameters were similar to the control group
- Satellite females
Statistically significantly increased value of glucose and albumin was recorded at treated group. Concentration of chloride and sodium ions was significantly decreased also at this dose level. Insignificantly increased concentration of bilirubine and activity of AST was measured at the treated group. All other measured parameters were similar to the control group.


URINALYSIS
- Males
No statistically significant changes in urine were recorded at any dose levels. Slight decrease of urine volume was recorded at the dose level 600 mg/kg/day. Blood was detected in two males and nitrites in one male at the dose level 150 mg/kg/day. Urine of one animal at the middle dose level showed white cloud. Presence of leucocytes was recorded in one male at the highest dose level.
- Satellite males
Statistical analysis of the data revealed significant intergroup differences. Significantly decreased urine volume and increased pH of urine were recorded at the treated group. Urine of one treated male showed blood in urine. Also presence of leucocytes was recorded in one treated male.

- Females
There were no treatment-related changes in urinary parameters.
Statistical analysis of the data revealed no significant intergroup differences.
- Satellite females
There were no treatment-related changes in urinary parameters.
Statistical analysis of the data revealed no significant intergroup differences.


ORGAN WEIGHTS
Absolute Organ Weight
- Males
Statistical analysis of the data revealed no significant intergroup differences.
Treated animals showed slightly decreased weight of liver with dependence on the dose level. The weight of other organs was well-balanced at all groups.
- Satellite males
Absolute weight of all organs was well-balanced at both groups. Statistical analysis of the data revealed no significant intergroup differences.

- Females
Animals at the dose level 300 mg/kg/day showed statistically significantly decreased of weight of kidneys. Slightly decreased weight of kidneys was recorded also at the lowest and highest dose levels but without statistical significance. The weight of liver was slightly decreased at the middle dose level (without statistical significance). Slightly decreased absolute weight of pituitary gland and ovaries was found out at the highest dose level. Other organs was relatively well-balanced.
- Satellite females
Absolute weight of organs was well-balanced at both groups. Statistical analysis of the data revealed no significant intergroup differences.

Relative Organ Weight
- Males
Relative weight of organs was well-balanced at all dose levels. Statistical analysis of the data revealed no significant intergroup differences.
- Satellite males
Slightly insignificantly increased weight of liver was recorded at the treated group. Relative weight of other organs was well-balanced with the control group. Statistical analysis of the data revealed no significant intergroup differences.

- Females
Animals at the dose level 150 mg/kg/day showed slightly decreased weight of liver and kidneys (without statistical significance). Relative weight of other organs was relatively well-balanced. Statistical analysis of the data revealed no significant intergroup differences.
- Satellite females
The weight of liver in treated group was statistically significantly increased. Relative weight of other organs was relatively well-balanced at both groups.

PATHOLOGY
Macroscopic Findings
- Males
During the macroscopic examination no important pathologic changes were found. In 2-1-0-0 males no macroscopic changes were observed.
Examination of application area revealed changes on skin and regional lymph nodes: brown colouring of skin in 0-0-5-0 males, punctiform plagues in 0-0-2-0 males, dry and red foci of skin in 0-0-0-5, congested lymph nodes in 3-4-5-4 males and enlarged lymph nodes in 0-0-2-0 males.
In thoracic cavity focal changes (changed colour or petechial foci) in lungs were diagnosed in 0-0-0-2 males and petechie on thymus was in 0-0-0-1 male.
In abdominal cavity the irregular colour of kidneys were only found in 1-0-0-1 males.
In cranial cavity no changes were diagnosed.
- Satellite males
During the macroscopic examination of males no important pathologic changes were found. In 5-2 males no macroscopic changes were observed.
Examination of application area showed congested or enlarged regional lymph nodes only in 0-1 male.
In thoracic cavity only focal changes in lungs in 0-1 male were recorded.
In abdominal cavity no changes were recorded.In cranial cavity no changes were diagnosed.

- Females
During the macroscopic examination of females no important pathologic changes were found. In 3-0-0-0 females no macroscopic changes were observed.
Examination of application area revealed changes on skin and regional lymph nodes: brown colouring of skin in 0-0-5-0 females, punctiform plagues in 0-0-2-0 females, dry and red foci of skin in 0-0-0-5, crust on skin in 0-0-0-1 female, congested lymph nodes in 2-4-5-3 females and enlarged lymph nodes in 0-0-2-0 females.
In thoracic cavity only petechie on thymus in 0-1-0-0 female were found out.
In abdominal cavity irregular colour of liver in 0-0-2-0 females and marked structure in 0-0-1-0 female were observed. Dilatation of uterus with fluidwas diagnosed in 3-2-3-1 females.
In cranial cavity no changes were diagnosed.
- Satellite females
During the macroscopic examination of females no important pathologic changes were found. In 3-3 females no macroscopic changes were observed.
Examination of application area showed no changes.
In thoracic cavity only petechie on thymus in 0-1 female and focal changes in lungs (grey focus) in 1-0 female were recorded.
In abdominal cavity only irregular colour of liver in 0-1 female was recorded. Dilatation of uterus with fluid was diagnosed in 1-2 females.
In cranial cavity no changes were diagnosed.


HISTOPATHOLOGY: NON-NEOPLASTIC
- Males
Incidence of pathological affection of skin application area was increased with dependence on the dose level. Focal inflammation of skin in 0-2-3-5 males and parakeratosis or hyperkeratosis in 0-0-1-3 males were recorded. Application area without changes was found out in 5-3-1-0 males. Occurence of findings in regional lymph nodes was well-balanced at all treated and control groups: erythrocytosis in 2-4-5-4 males and hyperplasia in 5-4-5-5 males. Haemorrhage in thymus was recorded in 2-2-0-1 males.
Incidence of pathological findings in digestive system was sporadic. Atrophy of phundal glands in stomach in 2-1-2-1 males, inflammation in liver in 1-2-3-2 males and proliferation of oval cells in liver of 0-1-0-1 males were recorded. In genital tract oedema of interstitium in prostate gland of 2-5-3-0 males, inflammation of prostate gland in 0-2-2-0 males and inflammation of epididymis in 1-1-2-1 males were found out. Dystrophy in cortex of kidneys was recorded in 4-3-2-0 males. Cartilagenous metaplasia in heart was recorded in 2-0-0-1 males. Other microscopical findings were recorded only sporadically (affected only one or two males in whole study).
- Satellite males
Application area was without changes. Occurence of findings in regional lymph nodes was well-balanced at treated and control groups: erythrocytosis in 2-3 males, plasmocytosis 1-0 male and hyperplasia in 3-2 males. Atrophy of phundal glands in stomach of 1-1 males, inflammation in liver of 4-5 and foci of oval cells in liver of 0-3 males were found out in digestive system. Dystrophy in cotrtex of kidneys was recorded in 2-1 males. In genital tract oedema of interstitium in prostate gland of 4-1 males, inflammation of prostate gland in 0-2 males and inflammation of epididymis in 1-2 males were found out. Other microscopical findings were recorded only sporadically (affected only one male).

- Females
Incidence of pathological affection of skin application area was recorded only in treated groups. Focal inflammation of skin in 0-0-2-5 females, crusts on epidermis in 0-0-2-0 females and parakeratosis in 0-0-0-3 females were recorded. Application area without changes was found out in 5-5-3-0 females. Occurence of findings in regional lymph nodes was relative well-balanced at all treated and control groups: erythrocytosis in 4-4-5-5 females and hyperplasia in 5-5-5-5 females.
In digestive tract the higher incidence of pathological affections was recorded in liver. Inflammation in 5-4-4-4 females, pigmentation in 0-1-0-2 females and proliferation in 2-2-1-2 females were found out. Focal infiltration in oesophagus was found out in 1-2-0-0 females. Cartilagenous metaplasia in heart was recorded in 0-1-1-1 females. Inflammation or haemorrhages in lungs were found out in 2-2-0-0 females. In genital tract only hydrometra of uterus was recorded in 3-2-2-1 females. Other microscopical findings were recorded only sporadically (affected only one or two females in whole study).
- Satellite females
Application area was without changes. Pathologic changes were found out in digestive system. Hyperplasia in 5-2 females and erythrocytosis in 1-3 females were recorded in regional lymph nodes. In digestive system foci of oval cells in liver of 4-2 females and inflammation in liver of 5-4 females were found out. Cartilagenous metaplasia in heart was recorded in 2-1 females. Hydronephrosis in kidneys was recorded in 1-1 females Pigmentation in spleen was recorded in 2-2 females. In genital tract only hydrometra of uterus was recorded in 1-2 females. Other microscopical findings were recorded only sporadically (affected only one female).
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
urinalysis
Critical effects observed:
not specified
Conclusions:
The dermal administration of the test substance Potassium permanganate to rats for a period of twenty-eight consecutive days at dose levels 150 mg/kg/day produced no toxicologically significant changes in the parameters measured. No major functional changes in any organ systems or severe organ dysfunction were detected. Histopathological examination revealed no pathological changes, which could be related with administration of the test substance at the dose level 150 mg/kg/day. Significant changes in clinical biochemistry, haematology and urinalysis parameters, which indicate organism dysfunction, were recorded at the dose level 300 and 600 mg/kg/day.

Based on the results of laboratory investigations in clinical biochemistry, haematology and urinalysis and with respect to the results of histopathological examination the following conclusion about NOAEL can be suggested in this study:
Executive summary:

Methods

   SPF Wistar rats were used for testing. The test substance was administered dissolved in water for injections. The six groups of animals were included in the study – 4 main groups and 2 satellite groups.

   Main groups contained one control group (vehicle only) and three treated groups (doses 150, 300 and 600 mg/kg/day). Satellite groups contained one control group (vehicle only) and one treated group (600 mg/kg/day). The administration of test and control substances lasted 28 days. After that the satellite animals were observed for next 14 days without treatment.

   The stability and the homogeneity of the application form in water for injections were determined in CETA analytical laboratories. The concentrations of suspensions in all three dose levels were adjusted to ensure the administered volume of 2 mL per 100 g of body weight and over an area, which was approximately 10% of the total body surface area. 

   Before starting of main study the dose-range finding experiment with 14-day application period was performed. The dose levels 200, 500, 750 and 1000 mg/kg/day were derived from the limit dose 1000 mg/kg/day (according to the guideline). During dose-range finding experiment the body weight and clinical observations were recorded. Basic haematological examination and gross necropsy were made at the end of experiment.

 Doses for the main study - 150, 300, 600 mg/kg/day were chosen on the basis of results of dose-range finding experiment. 

   During the 28-day study clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured twice a week. Before the end of study the functional observation was accomplished. The study was finished by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The selected organs for weighing and histopathology examination were removed.

  

Results

   There were no unscheduled deaths during the test. At the middle and highest dose levels the application of the test substance caused reversible changes of application area with histopathological findings (focal inflammation of skin, erythrocytosis and hyperplasia of regional lymph nodes) which faded away after recovery period (without application).

   The animal’s health condition was very good during whole study. Clinical signs of toxicity were detected sporadically at all treated and control group. Functional observation evidenced no effect of the test substance.

   Slight effect on body weight gain was detected in both sexes which was more marked in males at the middle and highest dose levels. The body weight and body weight increment of test groups was statistically insignificantly decreased during whole application period. No adverse effect on dietary intake was detected. Unbalanced (mainly decreased) food conversion was recorded at all treated group males and females but markedly at the middle and highest dose levels. The water consumption was slightly unbalanced during whole study.

    Changes of differential leucocyte count were recorded in both sexes with statistical significance at the end of application. Increased total leucocyte count was also measured at the end of application but without statistical significance. Thehaemocoagulationexamination detected prolonged PT and APTT in both sexes but these changes achieved the statistical significance only in males at the middle dose level. These changes were not recorded in satellite treated males after 14-day recovery period.

    Statistical analysis of biochemicalparametersrevealed significant differences in males (increased sodium concentration against control at the middle and highest dose levels). Significant changes of ions concentration were recorded in both sexes at the end of recovery period.Due to presence of changed urinary parameters (decreased urine volume and increased pH of urine) this was considered to be toxicological important. Other intergroup differences considered to be adaptative response to stress without toxicological significance.

   Pathology examination revealed some but not treatment related changes in digestive system (atrophy of phundal glans in stomach, foci of oval cells or inflammation in liver), pigmentation in spleen, dystrophy in cortex of kidneys and cartilagenous metaplasia in heart. The intergroup differences were minimal, therefore they were regarded as fortuitous. Further sporadic pathologic changes were recorded in reproductive system: inflammation and oedema of interstitium in prostate gland, inflammation in epididymis, in males and hydrometra of uterus and pigmentation in spleen in females. Frequencies of these microscopic findings were similar in treated and control groups. This was considered to be of no toxicological importance. No neoplastic findings were recorded by histopathological examination.

Additional information

For all the repeat dose studies on the registered substance, statistical analysis are not always reported and historical data controls are not available making these studies of limited quality. In addition, some effects are reported in most of the animals of the control and treated groups (erythrocytosis and hyperplasia in lymph nodes) therefore, these studies are considered of lower klimisch scoring and used only as supporting informtion.

Using the NTP 1993 studies (section 7.5.1) as the main studies for the eveluation of this endpoint (on multiple species, tested on analogue substance MnSO4), consistent systemic effects were reported with KMnO4 and MnSO4 when administrated orally (either by gavage or in the diet) in studies of short- or medium-term duration. They mainly consist on effects on body weight and on haematology. When exposed for longer duration to MnSO4 in the diet, additional findings were reported in various organs. Although neurological effects are well recognized as the main effects after repeated inhalation exposure manganese from human and animal data (ATSDR, 2012; Santé Canada, 2016; ANSES, 2018), the studies in section 7.5.1 do not report any effect related to neurotoxicity. Only the 28-day oral toxicity study performed with KMnO4 included clinical and functional observations, in addition to histopathology and weight of brain. Slight decrease of grip strength of pectoral legs and pelvis legs was reported in both sexes at 250 mg/kg bw/day; this effect was reversible. In addition, in females, a reversible decreased number of upstanding and increased number of emiction during 3 minutes of observation period was recorded at 250 mg/kg bw/day. The lack of evident neurotoxic effect in this study can be explained by the limited quality of the study and/or the too short duration of exposure. The studies in section 7.5.1 were not specifically designed to assess neurotoxicity that might have been reported with chronic exposure to sufficiently high doses of manganese.

The permanent neurological disorder associated with manganese is indeed known as manganism.It develops gradually, in three phases (WHO 1986 cited in MAK, 2012):

I - subclinical symptoms such as headaches, memory disorders, dizziness, weakness, tiredness; II - gait disorders, stuttering, compulsive crying and laughing, muscle dystonia (increased tonus in resting muscles);

III - fully developed disorder with symptoms like parkinsonism, that is, with severe functional disorder of the extrapyramidal brainstem ganglia; occasionally mutilating joint disorders have been described.

Manganese can enter the brain via three pathways: (1) from the nasal mucosa to the brain olfactory bulb via olfactory neural connections; (2) from the blood through capillary endothelial cells of the blood-brain barrier; and (3) from the blood through the cerebral spinal fluid via the choroid plexuses.

There is conclusive evidence from studies in humans that inhalation to high levels of manganese compounds can Lead to manganism. ATSDR (2012) considered that subclinical neurological effects (such as deficit in tests of neuromotor or cognitive funtions and altered mood states) occurred in workers chronically exposed to about 0.07 to 0.97 mg manganese/m3 (manganese in total or inhalable dust measurement) and that frank manganism have been associated with workplace inhalation exposure levels from about 2 to 22 mg manganese/m3. The MAK (2012) considered that the lowest average manganese concentration which has been shown to cause slight neurotoxic symptoms was about 0.25 mg/m3. This concentration was used as a basis for the establishment of the MAK value.

Effect of oral exposure to manganese in humans has been less investigated. Even if some publications report some associations between neurotoxicity and exposure to manganese following oral exposure, the evidence in humans is inconclusive due to several limitations noted in the studies and reports. Only one human case of neurotoxicity after accidental ingestion of KMnO4 is available. One man ingested (125 mL of 8% KMnO4 solution) for 4 weeks (total dose of 10 g) and began to notice weakness and impaired mental capacity after several weeks followed by a syndrome similar to Parkinson's disease after about 9 months. The authors speculated that the ingested MnO4– was reduced to Mn(II) or Mn(III); however, while this would be expected, it was not measured. Since MnO4– is a corrosive agent, it seems likely that it may have caused significant injury to the gastrointestinal tract (the patient did experience marked stomach pain), perhaps leading to a larger-than-normal gastrointestinal absorption of manganese (ATSDR, 2012). In the same way, some cases of neurotoxicity related to manganese exposure were also reported in patients with chronic hepatic disease (ANSES, 2018).

Effects on the central nervous system are also seen in animals after both ingestion and inhalation of high doses of manganese. For example, altered behaviour, lesions of the brain structure and modification of the level and/or activity of neurotransmitters were reported in repeated-dose toxicity studies by oral route with MnCl2 (ANSES, 2018). The principal mechanism in which manganese neurotoxicity occurs has not been clearly established. Many researchers consider that the manganese ion, Mn(II), enhances the autoxidation or turnover of various intracellular catecholamines, leading to increased production of free radicals, reactive oxygen species, and other cytotoxic metabolites, along with a depletion of cellular antioxidant defense mechanisms. It has also been suggested that the mechanism of manganese neurotoxicity may in part involve complex interactions with other minerals such as Ca(II). Most mechanistic researches on manganese neurotoxicity have focused on perturbations of the dopaminergic system, but there is evidence to suggest that early consequences of manganese neurotoxicity may involve perturbations of other neurotransmitters including GABA and glutamate in the basal ganglia and other brain regions (ATSDR, 2012).

ATSDR (2012) considered that the comparison of neurological effects across species is not straightforward due to the wide dose ranges administered, the variety of responses, and the differences in measured endpoints. In particular, it is noted that characterizing behavioral changes following basal ganglia damage in rodents was difficult. In addition, although in humans and primates, the striatum, globus pallidus, and substantia nigra are the primary neurotoxicity target for manganese, rodents do not accumulate manganese in the basal ganglia (i.e., the collection of deep regions of the brain including the striatum [comprised of the caudate and putamen]) to the same relative degree.

The long term toxicity of the test material was investigated in a two generation reproductive toxicity study which was conducted on a read-across substance under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. F0 animals were randomised into 4 test groups, each containing 28 males and 28 females. These animals were dosed with the test material for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls; however this reflected a slightly smaller litter size at this level and this accounts for the lower litter weights. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation. No test material-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations.

Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L, in the nasal cavity, pharynx, larynx and lung in all dose groups of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups, which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test material in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, a No Observed Effect Level (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material.

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L.

Justification for classification or non-classification

Based on the read-across with other manganese compounds such as MnCl2 and MnSO4, the Lead registrants proposed to change their self-classification to STOT RE 2 – H373 (brain; inhalation) considering the brain as the primary known target for manganese toxicity. This classification is based on weight of evidence - several studies on humand and animals reporting neurotoxic effects upon inhalation at varying level of exposure.