2-(methylamino)ethanol, compound with sulphur dioxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylaminoethanol

EC Number:

203-710-0

EC Name:

2-methylaminoethanol

Cas Number:

109-83-1

IUPAC Name:

2-(methylamino)ethanol

Details on test material:

- Name of test material: Methylaminoethanol (2-(Methylamino)ethanol)
- Analytical purity: 99.7 area-%
- Batch No.: from continuous production
- Physical state: colourless liquid
- Date of production: 24 Oct 2007
- Expiration date of the batch: 24 Oct 2009
- Storage conditions: room temperature, under N2

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: the weight variation of the animals did not exceed 20 % of the mean weight of each sex.
- Housing: individually, in type M III polycarbonate cages
- Diet: ground Kliba maintenance diet mouse/rat (GLP), meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10 air changes per hour
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

highly deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (highly deionized water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The test substance solutions were prepared in such intervals that the stability was guaranteed.

VEHICLE
- Vehicle: highly deionized water
- Concentration in vehicle: 5, 15, 45 mg/mL
- Amount of vehicle: 10 mL/kg bw (dose volume)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in highly deionized water at room temperature for a period of 10 days was proven before the start of the administration period (Project No.: 01Y0540/078008). Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and at the end of the administration period. The concentrations ranged from 90.1 to 102.2 % of the nominal concentrations.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

Frequency of treatment:

once daily (at the same time in the morning)

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, 450 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected by the Sponsor

- Other
After the acclimatization period, at least 13 days after the beignning of treatment, males and females from the same test group were mated overnight in a ratio of 1:1 or 1:2.
On study day 32, a functional observation battery and motor activity measurement were carried out in the first 5 male animals per group.
The females were allowed to litter and rear their pups until day 4 after parturition. On postnatal day 4, all pups were sacrificed and examined.
On study day 53, a functional observation battery and motor activity measurement were carried out in the first 5 female animals (with litter) per group.
From the first 5 male animals and the first 5 female animals (with litter) urinalyses were carried out on study days 34 (males) and 50 (females). Hematological and clinico-chemical examinations were carried out on study days 35 (males) and 55 (females).
At the end of the study (study day 35 for males, study day 55 for females), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

F0-GENERATION:

MORTALITY
- Time schedule: a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
- Time schedule: a cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena.

BODY WEIGHT
- Time schedule: once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Females after weaning (PND 4) until sacrifice were weighed once a week (for the calculation of the administration volume only)

FOOD CONSUMPTION
- Time schedule: once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

HEMATOLOGY
Parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore differential blood smears were prepared and stained according to WRIGHT without being evaluated. The clotting analyses were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: yes (Isoflurane)
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time.

CLINICAL CHEMISTRY
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinico chemical parameters.
- Time schedule for collection of blood: in the morning
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), alkaline Phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), inorganic Phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), total Bilirubin (TBIL), total protein (TPROT), Albumin (ALB), globulins (GLOB), triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Parameters examined: pH, protein, Glucose, ketones, Urobilinogen, Bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes. Functional Observation Battery
- Time schedule for examinations: A functional observational battery was performed at the end of the administration period starting at about 10:00 h.
- Dose groups that were examined: in the first five animals per sex and group.
- Battery of functions tested:
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching
the cage or rack, noise) were avoided during these examinations in order not to influence the
behavior of the animals. Attention was paid to:
1. posture
2. tremors
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings
- Open field observations: the parameters examined are listed in the Table 2.
- Sensory activity / grip strength are listed in the Table 3
-Motor activity:
The motor activity (MA) was measured on the same day as FOB was performed in 5 parental
males and females (with litter) per group. The examinations were performed using the Multi-
Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose,
the animals were placed in cages for the time of measurement. Four beams were allocated
per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes in each
case. The sequence at which the animals were placed in the cages was selected at random.
The measurement was started at about 14:00 h. On account of the measuring variant
"staggered", the starting time was varied by the time needed to place the animals in the
cages. For each animal, measurement was started individually when the 1st beam was
interrupted and ended exactly 1 hour later. The animals received no food or water during the
measurements. After the transfer of the last animal in each case, the room where the
measurements were carried out was darkened.

ORGAN WEIGHTS
The following weights were determined in all parental animals sacrificed on schedule: liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart

Sacrifice and pathology:

F0 GENERATION

NECROPSY
All parental animals were sacrificed by decapitation using isoflurane anesthesia (males: study day 35, females: study day 55). The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. The animals, which died intercurrently or were sacrificed in a moribund state, were necropsied as soon as possible after their death and assessed by gross pathology.

HISTOLOGICAL ASSESSMENT
After the organs were fixed, histotechnical processing and examination by light microscopy was performed on following organs: trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, all gross lesions, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, thyroid glands/parathyroid glands, testes, epididymides, ovaries, uterus, vagina, prostate gland, seminal vesicles, coagulation glands, thymus, lymph nodes (axillary), lymph nodes (mesenteric), stomach (forestomach and glandular stomach), duodenum, jejunum (with Peyer’s patches), ileum, cecum, colon, rectum, urinary bladder, bone marrow (femur)

Other examinations:

Reproduction indices, clinical observation of litters/pups and their necropsy findings are presented in the endpoints of section 7.8.

Statistics:

- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means. Parameters analysed:
food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups per litter, implantation sites, post implantation loss

- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions. Parameters analysed:
mating index, fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy

- Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians. Parameters analysed:
feces, rearing, grip strength, landing foot-splay, motor activity, organ weights

Results and discussion

Results of examinations

Details on results:

F0 GENERATION

CLINICAL SIGNS AND MORTALITY
Mortality
In test group 3 (450 mg/kg bw/d) 1 male animal was found dead within the first week of the study. One male animal of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, 1 female animal of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

Clinical signs
In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in 1 male animal and in study weeks 1, 6 and 7 in 6 female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in 2 male animals and in study weeks 1, 6 and 7 in 2 female animals. In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in a single male animal. Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in 1 male animal. The detailed clinical observations on study days 0, 7, 13, 21 and 28 in males and females and additionally on days 35, 42 and 49 in females did not reveal any additional abnormalities in animals of all test groups.

BODY WEIGHT AND BODY WEIGHT GAIN
In test group 3 (450 mg/kg bw/d) male animals’ body weight was significantly lower in week 4 and body weight change was already significantly lower between weeks 1-2 and in summary between weeks 0-4. In test group 2 (150 mg/kg bw/d) male animals’ body weight change was significantly lower between weeks 3-4. Body weights and body weight changes of all female animals treated with 50, 150 or 450 mg/kg bw/d were not significantly changed during premating. During gestation body weights of female animals of test group 2 (150 mg/kg bw/d) were significantly lower on GD 14 and 20 and of test group 3 (450 mg/kg bw/d) body weight was even decreased on GD 20. Body weight changes of female animals during gestation were significantly lower between GD 0-7 in test group 1 (50 mg/kg bw/d) as well as between GD 0-7 and GD 7-14 in test group 2 (150 mg/kg bw/d). A body weight loss could be detected between GD 14-20 in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Consequently, the overall body weight change between GD 0-20 was also significantly lower for these test groups. Body weights and body weight changes of female animals treated with 50 mg/kg bw/d were not significantly changed during lactation. During lactation, a comparison of body weight data of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) to the control were not meaningful as only one litter consisting of one stillborn pup existed in test group 2 (150 mg/kg bw/d) and no pups were alive in test group 3 (450 mg/kg bw/d). During the post-weaning period female body weights were significantly lower in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) in study week 6 and 7. The same was true for females of test group 1 (50 mg/kg bw/d) in study week 7. As the terminal mean body weight in this test group was unaffected this change was assessed as incidental and not related to treatment.

FOOD CONSUMPTION
Significantly decreased food consumption of the male animals of test group 3 (450 mg/kg bw/d) was observed during the first two study weeks. Food consumption of the female rats of test group 3 (450 mg/kg bw/d) was significantly decreased during the first study week. During gestation the food consumption in test group 2 (150 mg/kg bw/d) was significantly decreased between GD 14 and 20. During lactation food consumption in test group 2 (150 mg/kg bw/d) was significantly lower compared to the control.

HEMATOLOGY
At the end of the administration period red blood cell counts (RBC), hemoglobin concentrations and hematocrit values were decreased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Additionally, the hematocrit values were significantly decreased in females and males of test group 1 (50 mg/kg bw/d). This decrease compared to the controls was below 10 % (males: 5 %, females: 7 %), and it was the only dose-dependently changed red blood cell parameter in this test group. Therefore, the hematocrit decrease in rats of test group 1 (50 mg/kg bw/d) was regarded as treatment-related but not adverse. The mean corpuscular volume (MCV) was decreased in male rats of all treatment groups (not significantly changed in test group 3 [450 mg/kg bw/d]). The measured MCV and RBC values were used to calculate the hematocrit values. In male rats of test group 1 (50 mg/kg bw/d) the MCV reflected the decreased hematocrit value because the RBC was not changed. Therefore, the decreased MCV in these rats was regarded as treatment-related, but not adverse as mentioned above. In female rats of test group 3 (450 mg/kg bw/d) the relative reticulocyte counts were increased. No significant change was observed in the total white blood cell counts (WBC) of treated rats. However, some changes in the relative and absolute differential blood cell counts were measured (males: increased relative neutrophil counts and decreased relative eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative monocyte counts in test group 2 [150 mg/kg bw/d]; females: decreased absolute eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative neutrophil counts and increased relative lymphocyte counts in test group 2 [150 mg/kg bw/d]). These changes were regarded as being incidental and not treatment-related because they were not dose-dependently changed and not consistent in both sexes. The prothrombin time was shortened in rats of both sexes of test group 3 (450 mg/kg bw/d)
and, additionally, in females of test group 2 (150 mg/kg bw/d).

CLINICAL CHEMISTRY
Liver enzyme activity was not changed in male and female rats of any test substance-treated group. The urea levels were increased in males of test group 2 (150 mg/kg bw/d) and in rats of both sexes in test group 3 (450 mg/kg bw/d). The total Bilirubin concentrations were significantly higher in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The total protein and the Albumin levels were increased in females of test group 1 (50 mg/kg bw/d) and higher (total protein level was not significantly increased in test group 3 [450 mg/kg bw/d]), although the increases were not dose-dependent. In males the total protein levels were significantly increased in test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) and the Albumin concentrations in test group 2 (150 mg/kg bw/d), only. These parameters were not changed dose-dependently, and the deviated values were within the historical control ranges (total protein: 62.45-69.74 g/L; Albumin 36.12-39.76 g/L). Therefore, these deviations were regarded as non-adverse effects.
The Sodium concentrations were increased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) and, additionally, in males of test group 1 (50 mg/kg bw/d). The Sodium mean in males at least of the low dose group was within the historical control range (140.9-147.1 mmol/L). Apart from this, only this electrolyte level was deviated in test group 1 (50 mg/kg bw/d). Therefore, the Sodium levels increase at least in males of the low dose group was regarded as a non-adverse effect. In males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) the Cholesterol levels were decreased. The parameter was not changed dose-dependently, and such deviation was not observed in females. Therefore, the Cholesterol levels decrease in males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) was regarded as non-adverse. In treated females the Potassium concentrations were significantly higher in test group 1 (50 mg/kg bw/d), the Creatinine levels were higher in test group 2 (150 mg/kg bw/d) and the Magnesium concentrations were increased in test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d). These values were not changed dose-dependently, and the deviations of these parameters were not measured in male rats. Therefore, these changes were regarded as incidental rather than treatment related.

URINALYSIS
In rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) the incidence of blood (haemoglobin) was found higher compared to the controls (in females of test group 3 not significant). Additionally, the incidence of higher leucocyte counts in the urine sediment was significantly increased in males of test group 2 (150 mg/kg bw/d). However, no significantly higher leucocyte counts were found in the urine sediment of rats of both sexes of test group 3 (450 mg/kg bw/d). In males of test group 3 (450 mg/kg bw/d), the incidence of higher transitional cell counts was increased.

The urine was discolored almost in all the males and females of test group 3 (450 mg/kg bw/d) from study week 1 onwards.

NEUROBEHAVIOUR
No test substance-related or spontaneous findings in male and female animals of all test groups during the home cage observation were observed. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. In sensorimotor test/assessment of reflexes, there were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. There were no significant deviations concerning the overall motor activity (summation of all intervals) in the male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals, in males of test groups 1 and 2 (50 and 150 mg/kg bw/d) two isolated significantly increased values were measured at interval 4. These findings were considered as being incidental since the overall motor activity was not changed and no findings were observed for female animals.

ORGAN WEIGHTS
Absolute organ weights: When compared to control group 0 (set to 100%), the mean absolute weights of the organs listed in the Table 8 were significantly increased or decreased. All other mean absolute weight parameters did not show significant differences when
compared to test group 0 (control).
Relative organ weights: The terminal body weight was significantly decreased in males of test group 3 (450 mg/kg
bw/d) and in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) resulting in
significant, secondary weight changes in various organs (Table 9)

GROSS PATHOLOGY
Three males of test group 3 (450 mg/kg bw/d) showed erosions or ulcers in the glandular stomach. The liver was enlarged in 3 males and 1 female of test group 2 (150 mg/kg bw/d) as well as in 3 males and 5 females of test group 3 (450 mg/kg bw/d). Four males of test group 1 (50 mg/kg bw/d) and 4 males of test group 2 (150 mg/kg bw/d) showed a prominent acinar pattern of the liver.
The mesenteric lymph nodes were red discolored in 1 female of test group 2 (150 mg/kg bw/d) and in 2 females of test group 3 (450 mg/kg bw/d). All other gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental.

FERTILITY
Fertility was severely impaired by test-substance administration at dose levels of 150 and 450 mg/kg bw/d. Although mating (male and female mating indices) was not influenced no lifeborn pups were delivered for both test groups.

HISTOPATHOLOGY
Kidneys: The graded severity of tubular degeneration was dose-related increased. The statistically
significant increase of the relative kidney weights in animals of test groups 2 (150 mg/kg
bw/d) and 3 (450 mg/kg bw/d) was considered to be caused by the tubular degeneration/
regeneration process.
Testes: The decrease of the absolute testes weight in males of test group 3 (450 mg/kg bw/d) was
related to the diffuse tubular degeneration.
Ovaries: In ovaries, vacuoles of different size were observed in the sex cord stroma in females of test
groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Incidence and severity was dose-related
increased (see Table 10). In addition, one female of test group 1 (50 mg/kg bw/d), one female of test group 2 (150
mg/kg bw/d) and all females of test group 3 (450 mg/kg bw/d) showed ovarian cysts. The
occurrence of cysts in females of test group 3 (450 mg/kg bw/d) was assessed as treatmentrelated.
The cysts in each one female of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg
bw/d) were considered to be rather incidental.
Although there was no clear histopathological correlate for the decreased absolute and
relative ovarian weights in females of test group 3 (450 mg/kg bw/d), a test substance-related
effect cannot be ruled out.
Spleen: Incidence and graded severity of extramedullary hematopoiesis were dose-related increased
in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The increased relative spleen weights in males of test group 3 (450 mg/kg bw/d) as well as in
females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) were associated with
these findings.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) No

HISTORICAL CONTROL DATA (if applicable) No

OTHER FINDINGS
- Clinical observations for females during gestation of F1 litter:
Findings were only seen in test group 3 (450 mg/kg bw/d), i.e. poor general state was
observed in 4 female animals (animal nos. 132, 135, 136 and 140) from GD 11 onwards,
salivation after treatment was observed in 2 female animals (animal nos. 136 and 139) from
GD 2 onwards and discolored urine was observed in all animals during the whole gestation
period.
- Clinical observations for females during lactation of F1 litter:
No test substance-related clinical findings occurred in the female animals. Only one pup
(dam no. 112) from test group 1 (50 mg/kg bw/d) showed a papilloma-like skin flap from day
of birth (PND 0) onwards until the end of study (Detailed findings see in the endpoint 7.8)

DECEDENTS
One male (No. 32) of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state.
Severe meningitis was observed in the brain, a spermatogenic granuloma was noted in the
right epididymis, and an erosion/ ulcer occurred in the glandular stomach. Another male (No.
37) of test group 3 (450 mg/kg bw/d) died prematurely. This male showed a severe alveolar
histiocytosis in the lungs, a moderate purulent inflammation of the trachea and an erosion/
ulcer in the glandular stomach. The premature death of these two males was considered to
be incidental.
One female of test group 2 (No. 126, 150 mg/kg bw/d) was sacrificed during parturition. One
dead fetus was found in the right uterus horn and birth channel. No more fetuses were
detected. An influence of the test substance cannot be ruled out.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on body weight impairment, reduced food consumption, clinical pathology findings, impairment of organ weights and pathological findings in affected organs seen at higher dose levels.

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

ANALYTICS

The various analyses confirmed:

- the stability of the test-substance preparations in highly deionized water over a period of up to 10 days,

- the test-substance preparation was a clear solution in highly deionized water (and thus considered to be homogenous without further analysis),

- the correctness of the prepared concentrations.

Table 8: Absolute organ weights

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150	3 (450)
Terminal body weight	101%	96%	86%**	95%	93%**	85%**
Adrenal glands				96%	90%	82%**
Brain				99%	100%	96%*
Epididymides	100%	90%	68%**
Liver	113%*	121%**	129%**	105%	123%**	124%**
Ovaries				97%	99%	74%**
Testes	103%	105%	78%**
Thymus	98%	92%	67%**	88%	83%*	69%

* : p ≤ 0.05; **: p ≤ 0.01

Table 9: Relative organ weights

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150	3 (450)
Adrenal glands	104%	102%	128%*
Brain	98%	104%	114%*	104%*	107%*	113%**
Epydidymides	94%	98%	80%**
Heart	96%	106%*	122%**	98%	105%*	116%**
Kidney	101%	110%*	126%**	108%	116%**	132%**
Liver	111%**	127%**	150%**	111%**	133%**	146%**
Ovaries				102%	106%	86%*
Seminal vesicle	104%	113%*	117%*
Spleen	102%	111%	144%**	102%	112%*	121%**
Testes	101%	110%*	91%
Thymus	97%	96%	79%*

* : p ≤ 0.05; **: p ≤ 0.01

Table 10: Histopathology

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150		3 (450)
Kidneys	Multifocal tubular degeneration				Multifocal tubular degeneration; increase of the kidney weight
		increase of the kidney weight
Testes		diffuse tubular degeneration
Epididymides		Oligospermia
Ovaries				Ovarian cysts incidental			Ovarian cysts
Spleen		extramedullary hematopoiesis			extramedullary hematopoiesis; hemosiderin storage
Liver	Fatty change of hepatocytes				enlarged livers
Fore- and glandular stomach			Erosions or ulcers			Erosions or ulcers
Mesenteric lymph node			Sinus erythrocytosis		Sinus erythrocytosis
Thymus			reduced cellularity of cortex			reduced cellularity of cortex

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 50 mg/kg bw/d for the parental rats. The NOAEL for general, systemic toxicity of the test substance was 50 mg/kg bw/d for females and less than 50 mg/kg bw/d for male animals based on the tubular degeneration in the kidneys of six males.