2-(methylamino)ethanol, compound with sulphur dioxide

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: 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 (as cited in study report): Methylaminoethanol (Test item No.07/0540-2)
- Substance type: organic
- Physical state: colorless liquid
- Analytical purity: 99.7 area-% (Analytical Report: 07L00323)

- Purity test date: November 06-07, 2007
- Lot/batch No.: from continuous production
- Expiration date of the lot/batch: 24 Oct 2007


- Stability under test conditions: The stability of the test item under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: 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
- 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 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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0 ] of pregnancy

- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged ( Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.):
- Any other deviations from standard protocol: no

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)

Details on study schedule:

- Age at mating of the mated animals in the study: [13-14] weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, 450 mg/kg bw/day
Basis:
nominal conc.

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
- Rationale for animal assignment (if not random): randomized

- 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

Parental animals: Observations and examinations:

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
- Time schedule for examinations: a functional observation battery was performed at the end of the administration period starting at about 10:00 h.
- Dose groups that were examined: the first 5 animals/sex and group
- Battery of functions tested: the FOB consisted of home cage observations, open field observations and sensorimotor tests/assessment of reflexes. Motor activity was assessed on the same day as the FOB.

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

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no data]
- If yes, maximum of [all] pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:] viability index was calculated as follows: (number of live pups on PND4/number of liveborn pups on the day of birth)x100. The same for sex ratio: (number of live male or female pups on day 0/ 4/number of live male and female pups on day 0/ 4)x100
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [after approximately 1 week post-mating period]
- Maternal animals: All surviving animals [after PND 4]

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)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and were sacrificed at [4] days of age. All surviving pups (after sacrifice on PND 4 by means of CO2), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic
evaluation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: all gross lesions, lungs and spinal cord (cervical, thoracic and lumbar cord) were preserved in neutrally buffered 4 % formaldehyde solution and then analyzed.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

Food consumption, body weight and body weight change (parental animals and pups (for the pup weights,
the litter means were used) number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss: DUNNETT-test (two-sided)
Reproduction indices and urinalysis, except color, turbidity, volume and specific gravity, 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 : FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test (one-sided)
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters, urine volume,urine specific gravity and organ weights : KRUSKAL-WALLIS test (two-sided).

Reproductive indices:

Male mating index %: (number of males with confirmed mating* /number of males placed with females)x100; *- defined by a female with vaginal sperm or with implants in utero;
Male fertility index (%): (number of males proving their fertility */number of males placed with females)x100; * - defined by a female with implants in utero;
Female mating index (%): (number of females mated */ number of females placed with males)x100; * - defined as the number of females with vaginal sperm or with implants in utero;
Female fertility index (%): (number of females pregnant */number of females mated **)x100; * defined as the number of females with implants in utero; ** defined as the number of females with vaginal sperm or with implants in utero.
Gestation index (%): (number of females with live pups on the day of birth/number of females pregnant *); * - defined as the number of females with implants in utero;
Live birth index(%): (number of liveborn pups at birth/total number of pups born)x100;
Post implantation loss (%): (number of implantations number of pups delivered/number of implantations)x100

Offspring viability indices:

Viability index (%): (number of live pups on PND4/number of liveborn pups on the day of birth)x100. The same for sex ratio: (number of live male or female pups on day 0/ 4/number of live male and female pups on day 0/ 4)x100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

In test group 3 (450 mg/kg bw/d) one male animal (animal no. 37) was found dead within the first week of the study. One male animal (animal no. 32) of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, one female animal (animal no. 126) of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in one male animal (animal no. 36) and in study weeks 1, 6 and 7 in six female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in two male animals (animal nos. 32 and 36) and in study weeks 1, 6 and 7 in two female animals (animal nos. 132 and 135). In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in one male animal (animal no. 32). Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in one
male animal (animal no. 39).

The detailed clinical observations on study days 0, 7, 13, 21, 28 in males and females and
additionally day 35, 42 and 49 in female animals did not reveal any additional abnormalities
in animals of test groups 0-3 (0, 50, 150 and 450 mg/kg bw/d).


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

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 (see section 4.4.1.1. Absolute organ weights) this change
was assessed as incidental and not related to treatment.

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.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) not applicable

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The male mating index was 100% in all test groups. Fertility was proven for most of the F0 parental males of test groups 0 (control) and 1 (50
mg/kg bw/d) within the scheduled mating interval for the F1 litter. One control male and one male of test group 1 did not generate F1 pups. Furthermore, six males of test group 2 and nine males of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 11% and 90% (see Tab.4 ). For test groups 0 (control) and 1 (50 mg/kg bw/d) these findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (see PART III, Supplement). With regard to pathological findings in epididymidis and testis (see section 4.4. Pathology) the test substance did adversely affect reproduction of the F0 males in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The female mating index calculated after the mating period for F1 litter was 100% for all test groups. The mean duration until sperm was detected (GD 0) amounted to 2.4, 1.4, 2.5, and 2.9 days (0, 50, 150 and 450 mg/kg bw/d, respectively). Consequently, the differences between the test groups were assessed as being spontaneous in nature and without biological relevance. All sperm-positive rats of test groups 0 (control) and 1 (50 mg/kg bw/d) delivered pups or had implantations in utero with the following exceptions: one female (test group 0) and one female (50 mg/kg bw/d) did not become pregnant. 6 females of test group 2 (150 mg/kg bw/d), and 9 females of test group 3 (450 mg/kgbw/d) did not become pregnant. The fertility index varied between 10% and 90% (Tab. 5).
Implantation was not affected by the treatment in test group 1 (50 mg/kg bw/d) since the
mean number of implantation sites was comparable test group 0 (0 mg/kg bw/d). In test
groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) a significant reduction with only 5 and 1
implantation sites was found.The mean duration of gestation, i.e. 22.1 and 22.2 days, was similar in test groups 0 (control)
and 1 (50 mg/kg bw/d). No parturition was seen in test group 2 (150 mg/kg bw/d) except of
female No. 126 which was sacrificed on GD 23 because of an inability to deliver. Gestation
length was not calculable for test group 3 (450 mg/kg bw/d).
The gestation index varied between 89% (control group) and 100% (50 mg/kg body
weight/day). All values seen in test groups 0 (control) and 1 (50 mg/kg bw/d) reflect the normal range of
biological variation inherent in the strain of rats used for this study. All respective values were
within the range of the historical control data (PART III, Supplement) and did not show a
relation to dosing. However, a clear relation to dosing was obtained for test groups 2 (150
mg/kg bw/d) and 3 (450 mg/kg bw/d).
The mean number of F1 pups delivered per dam was not affected in test group 1 (50 mg/kg
bw/d) whereas only one pup was delivered in test group 2 (150 mg/kg bw/d) and none in test
group 3 (450 mg/kg bw/d).
The rate of liveborn pups was unaffected in test group 1 (50 mg/kg bw/d) and the live birth
index was 96%. The rate of stillborn pups was not significantly different compared to the
control group and within the range of the historical control data (PART III, Supplement).
In test group 2 (150 mg/kg bw/d) the live birth index was 0 because only one stillborn pup
was delivered.

ORGAN WEIGHTS (PARENTAL ANIMALS)

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 (PARENTAL ANIMALS)

Three males of test group 3 (450 mg/kg bw/d) showed erosions or ulcers in the glandular
stomach.
The liver was enlarged in three males and one female of test group 2 (150 mg/kg bw/d) as
well as in three males and five females of test group 3 (450 mg/kg bw/d). Four males of test
group 1 (50 mg/kg bw/d) and four 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 one female of test group 2 (150 mg/kg
bw/d) and in two 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.

HISTOPATHOLOGY (PARENTAL ANIMALS) (see Table 8)

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.

OTHER FINDINGS (PARENTAL ANIMALS) clinical chemistry, haematology, urinalysis, neurobehavioral observations (see under endpoint 7.5.1.)

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reduced fertility index, reduced number of implantation sites and no delivered pups (450 mg/kg bw/day), respectively only one stillborn pup and no liveborn pups delivered (150 mg/kg bw/day).

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were
evenly distributed among test groups 0 (control) and 1 (50 mg/kg bw/d). The respective
values reflect the normal range of biological variation inherent in the strain used in this study.

The viability index as indicator for pup mortality between PND 0-4 was 100% for test groups
0 (control) and 1 (50 mg/kg bw/d). No viable pups were observed in test group 2 (150 mg/kg
bw/d) and test group 3 (450 mg/kg bw/d).

CLINICAL SIGNS (OFFSPRING)

The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4. In one
litter (dam No. 112 of test group 1) one pup showed a papilloma-like a skin flap. This single
observation was considered to be spontaneous in nature and not to be adverse.

BODY WEIGHT (OFFSPRING)

Mean pup body weights/pup body weight changes of all pups in test group (50 mg/kg bw/d)
were comparable to the concurrent control values. The observable differences between the
groups were assessed as being spontaneous in nature and without biological relevance.
One runt of each gender was seen in test group 0 (control) and 5 female runts were seen in
test group 1 (50 mg/kg bw/d). Both values were within the range of the biological variation
inherent in the strain of rats used for this study.

SEXUAL MATURATION (OFFSPRING) not applicable

ORGAN WEIGHTS (OFFSPRING) not examined

GROSS PATHOLOGY (OFFSPRING)

One stillborn pup of test group 1 (50 mg/kg bw/d) showed post mortem autolysis. In 3 pups of
test group 1 (50 mg/kg bw/d) and in the single stillborn pup of test group 2 (150 mg/kg bw/d)
the stomach was found empty.

HISTOPATHOLOGY (OFFSPRING) no treatment -related effects in the low dose group

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 4: Reproductive performance (male animals)

	Test group 0 (0 mg/kg bw/d)	Test group 1 (50 mg/kg bw/d)	Test group 2 (150 mg/kg bw/d)	Test group 3 (450 mg/kg bw/d)
Male fertility index [%]	90	90	40	11**

Table 5: Reproductive performance (female animals)

	Test group 0 (0 mg/kg bw/d)	Test group 1 (50 mg/kg bw/d)	Test group 2 (150 mg/kg bw/d)	Test group 3 (450 mg/kg bw/d)
Female fertility index [%]	90	90	40*	10**

* p ≤ 0.05; ** p ≤ 0.01

Table 6: Absolute organ weight (parental animals)

	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%

Table 7: Relative organ weight (parental animals)

	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 8: Histopathology (parental animals)

	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