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REACH

2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

EC number: 229-419-9 | CAS number: 6528-34-3

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

In a subacute repeated oral-gavage study (OECD 422) the test item was administered at dosages of 100, 300, and 1000 mg/kg body weight/day. Test item was administered to male rats for 32 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.

A subchronic (90-day) toxicity study in Fischer F344 rats was performed with a close analogue of the test item. Groups of 10 male and 10 female rats received 0, 50, 200 and 1000 mg/kg/day by oral gavage in corn oil for 90 days. The study included additional control and high dose groups of 6 males and 6 females each analyzed after a 4-week recovery period. All animals survived until scheduled termination without any toxic signs in life. In haematology, clinical biochemistry and urinalysis some scattered significant differences, altogether without a clear dose response and all only minor in severity, were noted. Post mortem examination including histopathology did not reveal any test substance related toxic alterations. The test substance caused elevated liver weights in the high dosed females. As there were no corresponding histopathological or clinical-biochemical alterations found, the most likely interpretation is an adaptive response by enzyme induction. No parallel trend was present in the males. The effects did not persist until the end of the recovery period. No other test substance-related effect was noted. The No-observed-adverse-effect-level (NOAEL) of the test item was 1000 mg/kg bw/day in both sexes.

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Study period:

From 09 FEB 2012 and 03 JUL 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422) and in compliance with GLP.

Justification for type of information:

See Chapter 13.2: Read Across Justification Document

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 315 to 369 g (males) and 187 to 243 g (females)
- Identification: parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink: on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was a 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) batch/lot nos. 02105111001, 02105111201, 100099 and S211008A63972). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the report as an Appendix.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the report as an Appendix.

Route of administration:

oral: gavage

Vehicle:

other: 1.0% CMC / 0.05% Tween 80 in highly purified water

Details on oral exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

The test item was weighed into a glass beaker on a tared precision balance. Appropriate amount of the vehicle was weighted and added to the test item (w/w). Dose formulations were mixed on a magnetic stirrer for approximately 2 hours until mixtures were homogeneous. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.

Based upon the results of stability analyses performed within the non GLP Harlan Laboratories study 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat, dose formulations are stable for at least 7 days if stored at room temperature.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg bw/day (control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (control group), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)
- Duration of Acclimatization Period: 7 days
- Duration of Treatment Period: 32 days males and approximately 7 weeks females

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle only of each aliquot used on day 7 of the treatment. During the sixth week of the treatment samples of about 0.5 g were taken to repeat the measurement of concentration, homogeneity and stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and stored at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS measurement following an analytical procedure developed by Harlan Laboratories. The test item was used as the analytical standard.

The following results were obtained:

Blank samples showed no absorbance and, therefore, it was confirmed that only 1.0% CMC / 0.05% Tween 80 in highly purified water applied within the control experiment.

The application formulations investigated during the study were found to comprise the tets item in the range of 83.0% to 111.3% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of tets item in the preparations was approved because single results found did not deviate more than 9.8% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept seven days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean, except for group 2 and 3 prepared on 16-Feb-2012 that exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 11.8%, and in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 35.8%. However, there are some hints of what could be the cause. On the first and 7th treatment day as well as backup samples (date of preparation 16-Feb-2012), erroneously high amount of samples (about 2 g instead 0.5 g) were taken for analytical measurements making it difficult to work up. That was considered to probably be the reason for the differences as a second time using small amount of sample (0.5 g) shows the homogeneity and stability of the application formulation.

In conclusion, the results indicate the accurate use of the test item and 1.0% CMC / 0.05% Tween 80 in highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

MALES

32 days

FEMALES

about 7 weeks

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment End: On day before sacrifice
- Blood Sampling: At Termination
- Necropsy: After 32 days of treatment, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 4 post partum
- Blood Sampling: On day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)

Positive control:

not required

Observations and examinations performed and frequency:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Males: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period and weekly during after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY

At one time during the study (males shortly before the scheduled sacrifice and females on day 4 post partum) relevant parameters were performed with 5 P generation males and 5 P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained on the day of the scheduled necropsy from 5 males of each group. Blood samples from 5 lactating females of each group were obtained at the end of the pre-pairing period. Blood samples were drawn from the retro-orbital plexus of all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

URINALYSIS

The following urinalysis parameters were determined in five males of each group, which are allocated to the blood analysis, during the last week of the study using timed urine volume collection:
- Volume (18 hours)
- Specific gravity (relative density)
- Color
- Appearance
- pH
- Nitrite
- Osmolality
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood/Blood cells

Sacrifice and pathology:

TERMINATION AND NECROPSY

Males were sacrificed after treatment for 32 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes. For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

SEMINOLOGY AND SPERMATID COUNT

Sperm analysis was performed on the first 5 males per group.

Motility:
At necropsy of adult males an epididymal sperm sample was obtained from the left cauda epididymidis of each male. The sample was diluted with a pre-warmed (about 35 °C) physiological medium, and shortly after being obtained, one hundred sperm was counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

Morphology:
A second sperm sample from the left cauda epididymidis was used for morphological assessment after fixation and Eosin staining. 500 sperm per sample was evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head only (tail detached)
C: Complete sperm, misshapen hook
D: Complete sperm, abnormally curved hook
E: Complete sperm, reversed head
F: Abnormal head only (tail detached)

Morphological sperm evaluation was performed only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

Sperm, Spermatid Count:
The left caudal epididymis and left testis was taken for determination of homogenization-resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 ± 5°C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

ORGAN WEIGHTS

At the scheduled sacrifice, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator for histopathology phase were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.

A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator for inclusion in the main report as an appendix.

Other examinations:

MATING, GESTATION, LACTATION

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if: the daily vaginal smear was sperm positive or a copulation plug was observed. The day on which positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

REPRODUCTIVE AND OFFSPRING VIABILITY INDICES

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

LITTER OBSERVATIONS

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

POSTMORTEM EXAMINATION OF OFFSPRING

Pups were sacrificed on day 4 post partum. All animals were sacrificedby by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups dead during the study, except those excessively cannibalized, were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction data, grip strength, landing foot splay, body temperature, locomotor activity, hematology and biochemistry parameters, urinalysis, sperm analyses and organ weights:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables can be dichotomized without loss of information.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow stained faeces was noted in all males and females in dose groups; this was due to the staining properties of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Yellow stained faeces was noted in all males and females in dose groups; this was due to the staining properties of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Transient reduction in body weight gain was noted in males at the dose levels of 1000 and 300 mg/kg bw/day .

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

1. IN-LIVE DATA OF PARENTAL ANIMALS

VIABILITY / MORTALITY

All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS

Feces stained yellow were noted in all males and females in all dose groups starting from day 2 or 3 of the treatment until completion of the study. This observation was due to staining properties of the yellow coloured test item.

No further test item-related clinical signs or observations were noted in males or females at any dose level.

Incidentally, scabs on the neck were noted in one male in the control group.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

No test item-related findings were noted during weekly detailed clinical observations.

Incidentally, in one female at the dose level of 1000 mg/kg bw/day missing upper incisors on day 6 and 13 of the gestation period were recorded.

No further findings were noted in males or females in any dose group.

FUNCTIONAL OBSERVATIONAL BATTERY

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

All findings recorded during the tests were considered not to be test item-related. Decreased or increased rearings, increased faeces balls and vocalization were noted in individual animals without dose dependency. At the dose level of 1000 mg/kg bw/day, one female was noted with multiple findings: reduced activity, ruffled fur, decreased rearings and salivation. Because of isolated occurrence, this observation was considered not to be related to the treatment.

Remaining parameters under investigation were similar in all groups.

LOCOMOTOR ACTIVITY

No effects on locomotor activity were noted in males or females at any dose level.

Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1079, 1198, 1252 and 1207 in males and 965, 917, 853 and 867 in females.

FOOD CONSUMPTION OF MALES

No effects on food consumption were noted in males at any dose level.

Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: ±0.0%, -1.2% and -4.6% during the pre-pairing period and -2.0%, -2.0% and -0.8% during the after pairing period (percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES

No effects on food consumption were noted in females at any dose level.

Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day on days 8 - 11 of the pre-pairing period and at the dose level of 300 mg/kg bw/day on days 7 - 14 of the gestation period. In the absence of any dose dependency, these differences were not related to the treatment.

Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +8.0%, ±0.0%, and +3.4% during the pre-pairing period, +1.8%, +6.3% and +3.1% during the gestation period and +11.7%, -3.0% and +3.8% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES

At the dose levels of 1000 and 300 mg/kg bw/day, reduction in body weight gain was noted during the pre-pairing period; mean body weight gain within this period was +10% and +12% at the high- and mid-dose level respectively, compared to +14% in the control group. Reduction in body weight gain was statistically significant at the high-dose level starting from day 3 until the end of pre-pairing period and at the mid-dose level on days 7, 9, 11, 12 and 14 of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.
Body weights of males in all dose groups were similar to the respective control values during the entire study period.
Because the reduction in body weight gain at the high- and mid-dose levels was reversible and did not result in any significant differences in body weights, this observation was considered not to be adverse.

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +14%, +13%, +12% and +10% during the pre-pairing period, +3%, +2%, +3% and 3% during the pairing period and +1%, +1%, +1% and +2% during the after pairing period (percentages refer to the body weight change within the respective period).

BODY WEIGHTS OF FEMALES

Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 8%, 9%, 8% and 8% during the pre-pairing period, 53%, 53%, 59% and 57% during the gestation period and 6%, 7%, 6% and 6% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No test item-related effects on hematology parameters were noted in males or females at any dose level.

At the dose level of 300 mg/kg bw/day in males, amount of large unstained cells (LUC) was statistically significantly lower when compared to the control value. No reduction of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

CLINICAL BIOCHEMISTRY

No test item-related effects on biochemistry parameters were noted in males or females at any dose level.

At the dose level of 300 mg/kg bw/day in males, potassium concentration was statistically significantly higher when compared to the control value. No increase of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

In females following parameters were statistically significantly lower if compared to the respective control values: concentration of albumin at the dose level of 100 mg/kg bw/day, concentration of albumin and albumin to globulin ratio at the dose level of 300 mg/kg bw/day and concentration of total protein and albumin at the dose level of 1000 mg/kg bw/day. Changes of these values were not dose depended and all values were within historical control range. Therefore these changes were considered not to be test item-related.

URINALYSIS

No changes in urine parameters were noted in males at any dose level.

3. TERMINAL FINDINGS - PARENTAL ANIMALS

SEMINOLOGY AND SPERMATID COUNT

At the dose levels of 1000 and 300 mg/kg bw/day, statistically significant changes in motility of sperms were noted. Mean count of progressive sperms was reduced at the high dose level and it was 60.4% compared to 75.6% in the control group. This value is within the range of a limited pool of historical controls for OECD 422 (n=4) extended by OECD 416 data (n=5). Mean count of not motile sperms was increased and was 35.5% and 34.2% at the high- and mid-dose dose levels, respectively, compared to 20.2% in the control group. These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares). In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar (testis) or slightly higher (epididymidis) when compared to the respective control values.

ORGAN WEIGHTS

No test item-related changes in organ weights were noted in males or females at any dose level.

At the dose level of 100 mg/kg bw/day in males, statistically significantly lower weight of testis was noted. This effect was due to reduced testis weights of one male (no. 15, malformation of testis in this male correlated with histopathological findings). No significant changes of testis weights were noted at the mid- and high dose levels and therefore these changes were not related to the treatment with the test item.

No further significant differences in absolute or relative organ weights were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS

Type and distribution of findings noted during macroscopical examination of males and females did not indicate any test item related effect.

HISTOPATHOLOGY FINDINGS

Under the conditions of this experiment, the test item did not induce histopathological lesions. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

During examination of reproduction organs of infertile males and females, bilateral total tubular degeneration of testes was found in male no. 15 at the dose level of 100 mg/kg bw/day. This lesion was considered to be the reason for infertility of male no.15. No further findings correlated to the infertility were noted in reproduction organs of the remaining infertile males or females.

Key result

Dose descriptor:

NOAEL

Remarks:

for general toxicity (P)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Transiently lower body weight gain noted at the dose levels of 1000 and 300 mg/kg bw/day was considered not to be adverse.

Key result

Dose descriptor:

NOAEL

Remarks:

for general toxicity (P)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: No toxicologically relevant findings were noted in females up to the dose level of 1000 mg/kg bw/day, the highest dose level used.

Dose descriptor:

NOAEL

Remarks:

for reproduction (P)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Reduced motility of sperms noted at the dose levels of 1000 and 300 mg/kg bw/day was considered not to be adverse.

Dose descriptor:

NOAEL

Remarks:

for development (F1)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Under the condition of the study, post-implantation loss, body weights of pups and results of examination of pups did not indicate developmental effects up to the dose level of 1000 mg/kg bw/day, the highest dose level used.

Key result

Critical effects observed:

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45-55	56-66	67-77	78-88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of non pregnant females (A)	2	1	1	2
Numbers of pregnant females, which did not deliver any pups (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	9	10	10	8

(A) Female nos. 46,50, 59, 69, 83 and 85

(B) Female no. 87

MATING PERFORMANCE AND FERTILITY

Mating performance and fertility were not affected by the treatment at any dose level.

Percentage of mating was 100% in all groups. Mating of all females was recorded during the first seven days of the pairing period.

Mean (median) precoital times were 2.9 (3), 2.6 (3), 2.9 (3) and 2.8 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

Six females were not pregnant: two in the control group, one at each dose level 100 and 300 mg/kg bw/day and two at the dose level of 1000 mg/kg bw/day. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 81.8%, 90.9%, 90.9% and 81.8% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

One female at the high dose level had two implantation sites but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 88.9% at the high-dose level and 100% in remaining groups.

CORPORA LUTEA COUNT

No test item-related effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 13.6, 15.5, 17.4 and 14.4 in order of ascending dose levels.

At the dose level of 300 mg/kg bw/day, mean number of corpora lutea per dam was statistically significantly higher when compared to the control group. In the absence of any dose dependency, this observation was considered not to be related to the treatment.

DURATION OF GESTATION

No effects on duration of gestation were observed at any dose level.

Mean duration of gestation was 21.6, 21.8, 21.8 and 21.6 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

No effects on implantation rate and post-implantation loss were observed at any dose level.

In order of ascending dose levels, mean number of implantations was 13.0, 13.0, 14.2 and 14.4 per dam whereas mean incidence of post-implantation loss was 1.0, 1.0, 1.4 and 0.4 per dam.

LITTER SIZE AT FIRST LITTER CHECK

No effects on litter size were noted at any dose level.

During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.

Mean number of living pups per dam at first litter check was 12.0, 12.2, 12.8 and 14.1 in order of ascending dose levels.

Birth index (number of pups born alive as a percentage of implantations) was 92.3%, 92.2%, 90.1% and 97.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

No test item-related effects on postnatal loss were noted at any dose level.

In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level eight pups (from 2 litters) were missing on day 4 and at the high dose level two pups (from two litters) were missing on day 2 of the lactation period. Mean postnatal loss during four days of lactation was 0.1, 0.1, 0.8 and 0.3% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 99.1, 99.2, 93.8 and 98.2 in order of ascending dose levels.

At the dose level of 300 mg/kg bw/day, total number of pups lost was statistically significantly higher and viability index was statistically significantly lower when compared to the respective control values. These observations were due to a loss of seven pups in one litter. Because higher mortality of pups was noted only in one litter and in the absence of increased mortality of pups at the dose level of 1000 mg/kg bw/day, this observation was considered not to be related to the treatment.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

In the control group, missing tail tip was noted at first litter check and during lactation in one pup. At the dose level of 300 mg/kg bw/day, all pups in one litter had reduced temperature on day 3 and several pups from this litter had reduced temperature on day 4 of the lactation period. At the dose level of 1000 mg/kg bw/day, one pup, which was dead at first litter check had no milk in the stomach and was partially cannibalized.

No further findings were noted in pups at any dose level.

SEX RATIOS

Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 48%, 48%, 38% and 49% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

BODY WEIGHTS TO DAY 4 POST PARTUM

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

Mean body weights of pups on day 1 post partum were: 8.9 g, 9.2 g, 8.9 and 8.2 g and mean differences in body weights during lactation were +47.4%, +47.7%, 43.6% and +38.7%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

At the dose level of 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at this dose level which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was not test item-related.

MACROSCOPICAL FINDINGS

No findings were found during necropsy of pups in any dose group.

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item. The test item was administered in vehicle (1.0% CMC / 0.05% Tween 80 in highly purified water) at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 32 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

All animals survived the scheduled study period.
During the treatment, faeces stained yellow were noted in all males and females receiving test item. This observation was due to staining properties of the test item.

At the dose levels of 1000 and 300 mg/kg bw/day in males, statistically significantly reduced body weight gain was noted during the pre-pairing period. During the remaining study period body weight gain at the high- and mid-dose levels was similar to the control values. Body weights at these dose levels were similar to the control values during the entire study period. Because reduction of body weight gain was reversible and no statistically significant differences in body weights were noted at the high- and mid-dose levels, this effect was considered not to be adverse.

No further test item related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms at the dose levels of 1000 and 300 mg/kg bw/day. Statistically significant reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted at the high dose level and statistically significant increase in mean count of not motile sperms was noted at the mid-dose level. No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse.

Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The tets item was administered to male rats for 32 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (1.0% CMC / 0.05% Tween 80 in highly purified water).

The following results were obtained:

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

All animals survived the scheduled study period.

During the treatment, yellow stained faeces was noted in all males and females in dose groups. This observation was due to staining properties of the test item.

No further test item-related observations were noted during clinical daily or detailed weekly observations in males or females at any dose level.

FUNCTIONAL OBSERVATIONAL BATTERY AND LOCOMOTOR ACTIVITY OF PARENTAL ANIMALS

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

Locomotor activity was not affected by the treatment with test item in males or females at any dose level.

FOOD CONSUMPTION OF PARENTAL ANIMALS

No effects on food consumption were noted in males or females at any dose level.

BODY WEIGHT OF PARENTAL ANIMALS

In males at the dose levels of 1000 and 300 mg/kg bw/day, dose dependent and statistically significant reduction in body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high- and mid-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

Body weights and body weight gain of females were not affected by the treatment at any dose level.

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

No changes in urine parameters were noted in males at any dose level.

REPRODUCTION AND BREEDING DATA

Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL ANIMALS

At the dose level of 1000 mg/kg bw/day, reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted. At the dose level of 300 mg/kg bw/day, increase in mean count of not motile sperms was noted. A significant dose dependent trend couldn't be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

Sperm morphology and sperm count were not affected by the treatment at any dose level.

ORGAN WEIGHTS OF PARENTAL ANIMALS

No test item-related changes in organ weights were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

Type and distribution of findings noted during macroscopical examination did not indicate any test item related effect.

All findings recorded during histopathological examination were within the range of normal background lesions which may be recorded in animals of this strain and age.

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

Pups sex ratio was not affected by exposure to the test item at any dose level.

PUP WEIGHTS TO DAY 4 POST PARTUM

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

MACROSCOPICAL FINDINGS IN PUPS

No findings were found during necropsy of pups in any dose group.

CONCLUSION

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 FEB 2012 and 03 JUL 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422) and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

other: 1.0% CMC / 0.05% Tween 80 in highly purified water

Details on oral exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

MALES

32 days

FEMALES

about 7 weeks

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

Positive control:

not required

Observations and examinations performed and frequency:

Sacrifice and pathology:

Other examinations:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow stained faeces was noted in all males and females in dose groups; this was due to the staining properties of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Yellow stained faeces was noted in all males and females in dose groups; this was due to the staining properties of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Transient reduction in body weight gain was noted in males at the dose levels of 1000 and 300 mg/kg bw/day .

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Key result

Dose descriptor:

NOAEL

Remarks:

for general toxicity (P)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Transiently lower body weight gain noted at the dose levels of 1000 and 300 mg/kg bw/day was considered not to be adverse.

Key result

Dose descriptor:

NOAEL

Remarks:

for general toxicity (P)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: No toxicologically relevant findings were noted in females up to the dose level of 1000 mg/kg bw/day, the highest dose level used.

Dose descriptor:

NOAEL

Remarks:

for reproduction (P)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Reduced motility of sperms noted at the dose levels of 1000 and 300 mg/kg bw/day was considered not to be adverse.

Dose descriptor:

NOAEL

Remarks:

for development (F1)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

Key result

Critical effects observed:

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45-55	56-66	67-77	78-88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of non pregnant females (A)	2	1	1	2
Numbers of pregnant females, which did not deliver any pups (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	9	10	10	8

(A) Female nos. 46,50, 59, 69, 83 and 85

(B) Female no. 87

MATING PERFORMANCE AND FERTILITY

Mating performance and fertility were not affected by the treatment at any dose level.

Percentage of mating was 100% in all groups. Mating of all females was recorded during the first seven days of the pairing period.

Mean (median) precoital times were 2.9 (3), 2.6 (3), 2.9 (3) and 2.8 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

CORPORA LUTEA COUNT

No test item-related effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 13.6, 15.5, 17.4 and 14.4 in order of ascending dose levels.

DURATION OF GESTATION

No effects on duration of gestation were observed at any dose level.

Mean duration of gestation was 21.6, 21.8, 21.8 and 21.6 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

No effects on implantation rate and post-implantation loss were observed at any dose level.

In order of ascending dose levels, mean number of implantations was 13.0, 13.0, 14.2 and 14.4 per dam whereas mean incidence of post-implantation loss was 1.0, 1.0, 1.4 and 0.4 per dam.

LITTER SIZE AT FIRST LITTER CHECK

No effects on litter size were noted at any dose level.

During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.

Mean number of living pups per dam at first litter check was 12.0, 12.2, 12.8 and 14.1 in order of ascending dose levels.

Birth index (number of pups born alive as a percentage of implantations) was 92.3%, 92.2%, 90.1% and 97.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

No test item-related effects on postnatal loss were noted at any dose level.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

No further findings were noted in pups at any dose level.

SEX RATIOS

Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 48%, 48%, 38% and 49% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

BODY WEIGHTS TO DAY 4 POST PARTUM

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

MACROSCOPICAL FINDINGS

No findings were found during necropsy of pups in any dose group.

Conclusions:

Executive summary:

The tets item was administered to male rats for 32 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

The following results were obtained:

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

All animals survived the scheduled study period.

During the treatment, yellow stained faeces was noted in all males and females in dose groups. This observation was due to staining properties of the test item.

No further test item-related observations were noted during clinical daily or detailed weekly observations in males or females at any dose level.

FUNCTIONAL OBSERVATIONAL BATTERY AND LOCOMOTOR ACTIVITY OF PARENTAL ANIMALS

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

Locomotor activity was not affected by the treatment with test item in males or females at any dose level.

FOOD CONSUMPTION OF PARENTAL ANIMALS

No effects on food consumption were noted in males or females at any dose level.

BODY WEIGHT OF PARENTAL ANIMALS

Body weights and body weight gain of females were not affected by the treatment at any dose level.

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

No changes in urine parameters were noted in males at any dose level.

REPRODUCTION AND BREEDING DATA

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL ANIMALS

Sperm morphology and sperm count were not affected by the treatment at any dose level.

ORGAN WEIGHTS OF PARENTAL ANIMALS

No test item-related changes in organ weights were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

Type and distribution of findings noted during macroscopical examination did not indicate any test item related effect.

All findings recorded during histopathological examination were within the range of normal background lesions which may be recorded in animals of this strain and age.

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

Pups sex ratio was not affected by exposure to the test item at any dose level.

PUP WEIGHTS TO DAY 4 POST PARTUM

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

MACROSCOPICAL FINDINGS IN PUPS

No findings were found during necropsy of pups in any dose group.

CONCLUSION

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

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

Deviations:

Principles of method if other than guideline:

Guideline study.

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: F344/DuCrl, SPF
- Source: Charles River Deutschland GmbH, 97633 Sulzfeld, Deutschland
- Age at first administration: ca. 7 weeks
- Weight at first administration: males: 165 g, females: 121 g
- Identification: By tattooing the individual animal number into the right pinna and cage tag.
- Housing: Optimal hygienic conditions. Makrolon cages Type IV (33 cm x 55 cm area, 20 cm height). The animals were caged in groups (3 or 4 animals of one group and one sex per cage). The first 4 animals of a group were housed together in one cage and the remaining ones in groups of 3 in two more cages.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (item V1534-3 ) ad libitum.
- Water: Tap water, acidified with HCl to pH ¿3, from an automatic watering system, ad libitum.
- Nesting material: Nesting material and nibbling wood bricks (sized 10 cm x 2 cm x 2 cm), same material as the bedding material, were offered freshly once a week.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: Average of 21.9 °C.
- Humidity: Average of 49 % .
- Air changes: 12 per hour.
- Photoperiod: Artificial light from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered after being blended with (respectively dissolved in) the vehicle. Preparations of the test substance were made freshly every day shortly before the administration to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was neither soluble in nor miscible with any of the commonly used aqueous vehicles (water, water with modified cellulose, with or without detergent). Therefore corn oil was used as vehicle, which allowed the preparation of suitable suspensions.
- Concentration in vehicle: Appropriate preparations were made to allow a uniform dose volume for all groups.
- Amount of vehicle: 5 mL per kg body weight.
- Purity: nutritional grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was analysed by a validated HPLC-method. A linear calibration line was established. The variance homogeneity was checked over the working range of 10.1 to 51.8 mg/L.
Stability: Determined on Day 1.
Test conditions: Ambient temperature, exposed to light. The concentrations of preparations for the low dosed group were checked. A loss of at most 5 % within 4 h was tolerated. The stability of higher concentrated suspensions is considered better than that of lower ones.
The test substance was found to be sufficiently stable in the preparations for at least 4 h.

Homogeneity: Determined on Days 1 and 37. 3 samples of 1.0 mL each of the preparations for the three dosed groups A, B and C+CS were taken using the syringe plus probe by the technician at the beginning, in the middle and at the end of the dosing procedure for the group concerned. A deviation of the individual samples from the mean of at most ± 10 % was tolerated.
Actual concentration: Determined on Days 1 and 37. The same samples as for the determination of homogeneity were used. A deviation of the mean of the 3 samples from the target concentration of at most ± 10 % was tolerated.
The concentrations and the homogeneity of the test substance in the preparations were within the chosen limits at both sampling terms.

Duration of treatment / exposure:

Day 1 was the day of the first administration of the test substance.
The animals of all groups were treated with the test substance solutions or with the vehicle once a day on 91 (males) or 92 (females) consecutive days.
Animals of the satellite groups KS and CS were kept after cessation of dosing without a further administration for 29 (females) or 30 (males) additional days.

Frequency of treatment:

Once a day.

Remarks:

Doses / Concentrations:
0 mg/kg: control group K and also control satellite group KS
Basis:
other: nominal, gavage

Remarks:

Doses / Concentrations:
50 mg/kg: low dose, group A
Basis:
other: nominal, gavage

Remarks:

Doses / Concentrations:
200 mg/kg: mid dose, group B
Basis:
other: nominal, gavage

Remarks:

Doses / Concentrations:
1000 mg/kg: high dose, group C and also high dose satellite group CS
Basis:
other: nominal, gavage

No. of animals per sex per dose:

10 males and 10 females in the main groups K, A, B and C.
6 males and 6 females in the satellite groups KS and CS.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses chosen were derived from and based on the results of a dose range finding study. Groups of 5 male and 5 female rats each were administered 100 mg or 316 mg or 1000 mg test substance in 5 mL corn oil per kg body weight orally once a day for 14 consecutive days. Investigations performed: Animal observations, body weights and feed consumption, necropsy.
Summarized results: All animals survived until the terminal sacrifice. There were no treatment related observations in life. Body weights and feed consumption were not notably affected. All animals were found to be grossly normal at the post mortem examination.
1000 mg per kg body weight is the highest feasible dose for a subchronic toxicity study and was thus chosen as high dose. The low dose, where no toxic effects are anticipated and the mid dose were chosen by the sponsor.
- Rationale for animal assignment (if not random): randomisation procedure.
- Rationale for selecting satellite groups: A control group KS and a high dose group CS was used to investigate the development of changes after finishing of dosing.
- Post-exposure recovery period in satellite groups: 29 days for females and 30 days for males.
- Bias control: The sequence of animals for clinical observations, blood sampling, blood and urine analyses, necropsy and histopathology was randomised.

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0, 7, 14, 21, 28, 36, 42, 49, 56, 63, 70, 77, 85, 91: all animals of all groups. Days 98, 105, 112, 119: all animals of the satellite groups. Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights were determined:
Day 1 to 91: Once a week, all animals. Days 91 to 120: Satellite groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Determined per cage in weekly intervals in all animals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day 0: All animals of all groups. Day 86: All animals of all groups. Examination of the right eyes of all animals by adspection and by fundus examination, in case of the presence of lesions, and if considered as necessary, additionally by a slit lamp examination
An additional ophthalmoscopical examination at the end of the recovery period in the animals of groups KS and CS was omitted, as no test substance related alterations were noted at the end of the dosing period.
- Dose groups that were examined: each group.

HAEMATOLOGY: Yes
- Blood samples were taken from the retrobulbar vein plexus of the left eyes in the morning.
- Time schedule for collection of blood: Blood was taken on Day 92 from all male animals of groups K, A, B and C, on Day 93 from all female animals of groups K, A, B and C and of all animals of groups KS and CS on Day 121.
- Anaesthetic used for blood collection: Yes, slight ether anaesthesia
- Animals fasted: Yes. Feed was offered again immediately after the blood sampling.- Parameters determined (abbreviations, when commonly used):
¿ Red blood cell count (RBC)
¿ Haemoglobin concentration (HGB)
¿ Haematocrit (HCT)
¿ Mean corpuscular haemoglobin (MCH)
¿ Mean corpuscular haemoglobin concentration (MCHC)
¿ White blood cell count (WBC)
¿ Mean cell volume (MCV)
¿ Platelet count (PLT)
¿ Differential white blood cell count (% of the different cell species)
¿ Prothrombin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
- Blood samples were taken from the same animals and at the same time as for haematology. Plasma (with Li-heparin as anticoagulant) was obtained by centrifugation of the blood samples after incubation at 37 °C for about 1 hour.
- Parameters determined (abbreviations, when commonly used):
¿ alanin aminotransferase (ALT, GPT)
¿ albumin (ALB)
¿ alkaline phosphatase (AP)
¿ aspartate aminotransferase (AST, GOT)
¿ cholesterol (CHOL)
¿ creatinine (CREA)
¿ gamma-glutamyltransferase (GGT)
¿ glucose (GLU)
¿ potassium (K)
¿ sodium (Na)
¿ total protein (TP)
¿ urea (UREA)

URINALYSIS: Yes
- Time schedule for collection of urine:
Urine was collected overnight by placing the animals in metabolism cages and cooling the urine with ice.
Day 91/92: All males of groups K, A, B and C.
Day 92/93: All females of groups K, A, B and C.
Day 120/121: All animals of groups KS and CS.
- Parameters determined:
¿ total amount (weighed),
¿ colour, turbidity, appearance (visual),
¿ osmolarity (Osmometer)
¿ pH (pH-Meter),
¿ proteins, glucose, ketones, urobilinogen, bilirubin, erythrocytes, haemoglobin (semi-quantitative, test sticks),
¿ sediment analysis (microscopically).
- Animals fasted: Yes. Animals were given continuous access to drinking water, but no feed was offered during the urine collection time.

NEUROBEHAVIOURAL EXAMINATION: Yes.
Assessment of the behaviour, the motor activities, and the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) was performed outside the home cage in a standard arena.
On Day 87: all male animals of all groups.
On Day 91: all female animals of all groups.
On Day 119: all animals of groups KS and CS.
The eyelid and auricular reflexes (tactile and proprioceptive) were tested by slightly touching the cornea and the interior of a pinna with a nylon cord of approximately 0.6 mm diameter. Shaking of the head was considered as positive response.
Acoustic reactivity was tested by response to a moderate sound (clapping of the hands). Twitching of the body was considered as positive.
Visual reactivity was derived from the reaction to a dark sheet of paper, brought near to the animal without any physical contact. Turning towards the paper was considered as positive.
For the testing of the righting reflex, the animals were held between the front- and hind legs, turned on their backs and dropped from a height of 30 cm onto the bedding material of the standard arena. A landing on all four legs is considered to be a normal reaction.
The measurement of the forelimb grip strength was determined using a Digital Force Gauge DFIS 100.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes .
Animals were killed by inhalation of 80 % CO2 plus 20 % air and subjected to a necropsy including a gross pathological examination immediately after death on
Day 92: groups K, A, B and C (males).
Day 93: groups K, A, B and C (females).
Day 121: groups KS and CS.
The following organs / tissues were taken from all animals and fixed in Bouin's solution:
gross lesions, tissue masses or tumours
adrenal glands
aorta
brain
caecum
coagulating glands
epididymides
eyes
heart
kidneys
large intestine (colon)
liver
lungs
lymph nodes (mandibular, mesenteric)
oesophagus
ovaries
pancreas
pituitary gland
prostate
rectum
salivary glands
sciatic nerve
seminal vesicles
skeletal muscle (thigh)
skin, mammary glands
small intestine (duodenum, ileum, jejunum), prepared as "Swiss Roll"
spinal cord (cervical, thoracal, lumbar)
spleen
sternum with bone marrow
stomach
testes
thymus
thyroid and parathyroid glands
trachea.
urinary bladder
uterus

ORGAN WEIGHTS
Fresh weights of the following organs were determined of all animals at necropsy:
adrenal glands (both together)
brain
epididymides (both together)
heart
liver
kidneys (both together)
ovaries (both together)
spleen
testes (both together)
thymus

HISTOPATHOLOGY: Yes.
Groups K and C: Histopathological examination was performed in all animals of all fixed organs or tissues listed above.
Groups KS, A, B and CS: No histopathological examination was performed in groups KS, A, B and CS, as there was no indication for test substance related effects found in group C.
The tissue trimming was performed according to "Bahnemann et al.: RITA - Registry of Industrial Toxicology Animal Data - Guides for Organ Sampling and Trimming Procedures in Rats"; Exp.Toxicol.Pathol. 47 (1995), p 247 ff. with the following exceptions:
Not all possible sections, as given in the literature, were actually prepared. One section per organ (in paired organs one of each) was made with the following exceptions:
-Brain (3 sections, one at the optic chiasma, the second at the caudal border of the mammillary body, just posterior to the attachment of the pituitary and the third about 2 mm caudal to the transverse fibres of the pons). Representative regions of the brain, especially cerebrum, cerebellum and pons were included in these sections.
-Spinal cord (three sections, a cervical, a thoracal and a lumbar).
-Liver (two sections).
The small intestine (duodenum, jejunum and ileum) was fixed and trimmed to form two "Swiss Rolls" (one of the cranial and one of the caudal part), according to "Moolenbeek and Ruitenberg: The Swiss Roll, a simple technique for histological studies of rodent intestine"; Lab.Animals 15 (1981), p.57-59.
The trimmed samples of organs or tissues, as described above, were embedded in paraffin. Sections of about 5 ¿m were stained with haematoxylin and eosin (H&E). Evaluation of slides was performed using a light microscope Leica-DMRB.
The frozen liver samples were not subjected to an examination for fatty changes in frozen sections, as there was no indication for alterations noted in the H&E stained sections.
To describe the severity of lesions, the following grades were applied, if appropriate: minimal (1), mild (2), moderate (3), marked (4), severe (5).
The term "focal" together with a higher degree of severity also stands for "multifocal".

Other examinations:

None.

Statistics:

Analysis of variance followed by the Scheffé-test used for all data with means and standard deviations determined, comparison of more than two groups.
t-test used for all data with means and standard deviations determined, for comparison of two groups only.
H-test of Kruskal and Wallis followed by the test of Nemenyi used for counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled.
Chi2-test used for counted events.
Fisher's exact test used for counted events, if the Chi2-Test was not applicable.
Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used. Groups K and KS and C and CS were treated separately for statistical analysis.

Clinical signs:

effects observed, non-treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Yellow coloured faeces in most of the animals due to the colour of the test item

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Daily observation: No animal died. The faeces of all dosed animals were stained yellow. As the test substance itself is a yellow dye, the stain of the faeces is attributed to excreted test substance. One single high dosed male was found to be stained on parts of the body surface. The stain of the faeces and of the body's surface are not considered to be toxic effects.
Isolated cases of chromodakryorrhoea, alopecia and one broken incisor lack a dose relationship and are not related to the test substance.
Detailed observations: As in the daily observations, isolated cases of chromodakryorrhoea, alopecia and one broken incisor were noted, but lack a dose relationship and are not related to the test substance.

BODY WEIGHT AND WEIGHT GAIN:
There was no statistically significant difference or dose related trend noted in the body weights of both sexes.
In the body weight gain, isolated terms with significant differences in the mid or high dosed groups, with both higher and lower body weight gains, are considered to be incidental significances.

FOOD CONSUMPTION
There were no noteworthy differences or dose related trends noted in the feed consumption of both sexes. The differences between the groups are too small the give an indication for a toxic effect.

OPHTHALMOSCOPIC EXAMINATION: No indications for ocular lesions were noted at the examinations before the first dosing and at the end of the dosing period. An additional examination at the end of the recovery period was therefore omitted.

HAEMATOLOGY
There were no relevant differences during the dosing period. Small but significant changes were observed at the end of the recovery period: RBC and haemoglobin decreased in males; MCV increased in males; MCHC decreased in females. The differences are minor in dimension, were noted only at the end of the recovery period and lack corresponding effects at the end of the dosing period. Thus they are not regarded as test substance related and may rather be incidental.

CLINICAL CHEMISTRY
Alanin aminotransferase (males) lowered in the mid dosed group and elevated in the high dosed group; cholesterol (females) increased in the mid dosed group; urea (females) decreased in the recovery group.These significant group differences lack a dose related trend. Thus none of the significant group differences is attributed to the test substance.

URINALYSIS
All dosed groups of the females had a significantly lower urinary pH than the controls, but there was no dose related trend in pH. All mean urine pH data were in a range, commonly seen in control rats. Thus they are not regarded as test substance related.
In the males, there were no significant or otherwise noteworthy group differences in urinalysis results.

NEUROBEHAVIOUR: Neither test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined.
There was no significant group difference in the grip strengths.

ORGAN WEIGHTS
The significantly altered thymus, brain and ovarian weights lack a dose relationship and are considered to be incidental significances.
Significantly elevated liver weights of the high dosed females may be possibly treatment related. As to the lack of any indications for hepatotoxicity, the changes in female liver weights may be part of an adaptive response to the vehicle.(corn oi). There was neither significant difference nor a corresponding trend in the males.

GROSS PATHOLOGY
In the vast majority of all dosed animals, the content of the gastrointestinal tract was found to be stained yellow. This is attributed to the presence of the test substance and not regarded as toxic effect.
Isolated cases of small testes, fatty tissue necrosis in mesenteric fat, ovarian cysts are considered to be parts of the background pathology without relation to the test substance.

HISTOPATHOLOGY:
There was no test substance related alteration noted histopathologically.
Some isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence.

OTHER FINDINGS: None.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

Only parameters are listed for which a significant difference between at least one treatment group and the respective control group was observed for at least one sex. All parameters not showing significant differences are omitted from the tables. A significant difference to the respective control group is indicated by a grey background.

Haematology results.

parameter (sex)	low dose (% of the negative controls)	mid dose (% of the negative controls)	high dose (% of the negative controls)	high dose after recovery (% of the negative controls)
red blood cell count(males)	100	101	101	97
haemoglobin conc.(males)	100	100	101	97
mean cell volume(males)	100	100	100	102
mean corpuscular haemoglobin conc.(females)	105	109	115	99

Clinical biochemical results.

parameter (sex)	low dose (% of the negative controls)	mid dose (% of the negative controls)	high dose (% of the negative controls)	high dose after recovery (% of the negative controls)
alanin aminotransferase(males)	84	83	88	102
cholesterol(females)	84	86	95	96
urea(females)	92	94	97	89

Urinalysis findings.

parameter (sex)	low dose	mid dose	high dose	high dose after recovery
total amount(females, % of the negative controls)	58	76	85	133
pH(females; control: pH 7.1; control recovery: pH 7.3)	6.4	6.7	6.1	7.3

Organ weights.

parameter (sex)	low dose (% of the negative controls)	mid dose (% of the negative controls)	high dose (% of the negative controls)	high dose after recovery (% of the negative controls)
thymus(males, absolute weight)	121	113	114	120
thymus(males, organ weight/body weight ratio)	121	115	114	120
thymus(males, organ weight/brain weight ratio)	120	118	115	120
brain(males, absolute weight)	100	96	99	100
liver(females, absolute weight)	105	105	110	103
liver(females, organ weight/body weight ratio)	104	103	109	99
liver(females, organ weight/brain weight ratio)	104	104	111	100
ovaries(females, absolute weight)	107	92	87	110

Conclusions:

A 90-day toxicity study in Fischer F344 rats was performed with the test item. Groups of 10 male and 10 female rats received 0, 50, 200 and 1000 mg/kg/day by oral gavage in corn oil for 90 days. The study included additional control and high dose groups of 6 males and 6 females each analyzed after a 4-week recovery period. All animals survived until scheduled termination without any toxic signs in life. In haematology, clinical biochemistry and urinalysis some scattered significant differences, altogether without a clear dose response and all only minor in severity, were noted. Post mortem examination including histopathology did not reveal any test substance related toxic alterations. The test substance caused elevated liver weights in the high dosed females. As there were no corresponding histopathological or clinical-biochemical alterations found, the most likely interpretation is an adaptive response by enzyme induction. No parallel trend was present in the males. The effects did not persist until the end of the recovery period. No other test substance-related effect was noted. The No-observed-adverse-effect-level (NOAEL) of the test item was 1000 mg/kg bw/day in both sexes.

Executive summary:

Guidelines applied

EC-Guideline 2001/59/EC, B.26.,
OECD-Guideline 408, 21 September 1998.

Dose levels

Based on the results of a 14-day dose range finding study (which was part of the present study), the dose levels for this 90-day oral gavage study were selected to be 0, 50, 200 and 1000 mg/kg/day.

Study outline

The test substance, formulated in corn oil, was administered daily for 90 days orally by gavage to SPF-bred Fischer F344/DuCrl rats. One control group (plus a control satellite group) and 3 test substance treated groups (plus a high dose satellite group) were tested, each consisting of 10 males and 10 females (6 males and 6 females in the satellite groups).

Evaluated parameters

Chemical analyses of formulations were conducted twice in the in-life phase of the study to assess concentration and homogeneity.

The following parameters were evaluated: Clinical signs (daily plus weekly in more detail), functional observations (terminally), ophthalmoscopy (initial and terminal), body weights and feed consumption (weekly), haematology plus clinical biochemistry plus urinalysis (terminal), necropsy, organ weights and histopathology.

Results

Accuracy and homogeneity of test substance preparations in corn oil were demonstrated by analyses.

All animals survived until scheduled termination without any toxic signs in life. In haematology, clinical biochemistry and urinalysis some scattered significant differences, altogether without a clear dose response and all only minor in severity, were noted. Post mortem examination including histopathology did not reveal any test substance related toxic alterations. Liver weights of the females were elevated with a dose dependency, which may indicate an adaptive response rather than a toxic effect.

A frequently noted yellow stain of the gastrointestinal contents and of the faeces is attributed to the presence of the administered test substance itself and not regarded as a toxic effect.

Discussion and Conclusion

The test substance caused elevated liver weights in the high dosed females. As there were no corresponding histopathological or clinical-biochemical alterations found, the most likely interpretation is an adaptive response by enzyme induction. No parallel trend was present in the males.

No other test substance related effect was noted.

The effects did not persist until the end of the recovery period.

The No-observed-adverse-effect-level (NOAEL) of the test substance was at 1000 mg per kg body weight in both sexes.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Justification for type of information:

See chapter 13.2: Read Across Justification Document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

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

Deviations:

Principles of method if other than guideline:

Guideline study.

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

Frequency of treatment:

Once a day.

Remarks:

Doses / Concentrations:
0 mg/kg: control group K and also control satellite group KS
Basis:
other: nominal, gavage

Remarks:

Doses / Concentrations:
50 mg/kg: low dose, group A
Basis:
other: nominal, gavage

Remarks:

Doses / Concentrations:
200 mg/kg: mid dose, group B
Basis:
other: nominal, gavage

Remarks:

Doses / Concentrations:
1000 mg/kg: high dose, group C and also high dose satellite group CS
Basis:
other: nominal, gavage

No. of animals per sex per dose:

10 males and 10 females in the main groups K, A, B and C.
6 males and 6 females in the satellite groups KS and CS.

Control animals:

yes, concurrent vehicle

Details on study design:

Positive control:

None.

Observations and examinations performed and frequency:

Sacrifice and pathology:

Other examinations:

None.

Statistics:

Clinical signs:

effects observed, non-treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Yellow coloured faeces in most animals due to the colour of the test item.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Daily observation: No animal died. The faeces of all dosed animals were stained yellow. As the test substance itself is a yellow dye, the stain of the faeces is attributed to excreted test substance. One single high dosed male was found to be stained on parts of the body surface. The stain of the faeces and of the body's surface are not considered to be toxic effects.
Isolated cases of chromodakryorrhoea, alopecia and one broken incisor lack a dose relationship and are not related to the test substance.
Detailed observations: As in the daily observations, isolated cases of chromodakryorrhoea, alopecia and one broken incisor were noted, but lack a dose relationship and are not related to the test substance.

BODY WEIGHT AND WEIGHT GAIN:
There was no statistically significant difference or dose related trend noted in the body weights of both sexes.
In the body weight gain, isolated terms with significant differences in the mid or high dosed groups, with both higher and lower body weight gains, are considered to be incidental significances.

FOOD CONSUMPTION
There were no noteworthy differences or dose related trends noted in the feed consumption of both sexes. The differences between the groups are too small the give an indication for a toxic effect.

OPHTHALMOSCOPIC EXAMINATION: No indications for ocular lesions were noted at the examinations before the first dosing and at the end of the dosing period. An additional examination at the end of the recovery period was therefore omitted.

HAEMATOLOGY
There were no relevant differences during the dosing period. Small but significant changes were observed at the end of the recovery period: RBC and haemoglobin decreased in males; MCV increased in males; MCHC decreased in females. The differences are minor in dimension, were noted only at the end of the recovery period and lack corresponding effects at the end of the dosing period. Thus they are not regarded as test substance related and may rather be incidental.

CLINICAL CHEMISTRY
Alanin aminotransferase (males) lowered in the mid dosed group and elevated in the high dosed group; cholesterol (females) increased in the mid dosed group; urea (females) decreased in the recovery group.These significant group differences lack a dose related trend. Thus none of the significant group differences is attributed to the test substance.

URINALYSIS
In the females, the low dosed group produced significantly less urine than the controls. As there was no dose relationship in the mid and the high dosed group, this is not related to the test substance.
All dosed groups of the females had a significantly lower urinary pH than the controls, but there was no dose related trend in pH. All mean urine pH data were in a range, commonly seen in control rats. Thus they are not regarded as test substance related.
In the males, there were no significant or otherwise noteworthy group differences in urinalysis results.

NEUROBEHAVIOUR: Neither test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined.
There was no significant group difference in the grip strengths.

ORGAN WEIGHTS
The significantly altered thymus, brain and ovarian weights lack a dose relationship and are considered to be incidental significances.
Significantly elevated liver weights of the high dosed females may be possibly test substance related. As to the lack of any indications for hepatotoxicity, the changes in female liver weights may be part of an adaptive response. There was neither significant difference nor a corresponding trend in the males.

GROSS PATHOLOGY
In the vast majority of all dosed animals, the content of the gastrointestinal tract was found to be stained yellow. This is attributed to the presence of the test substance and not regarded as toxic effect.
Isolated cases of small testes, fatty tissue necrosis in mesenteric fat, ovarian cysts are considered to be parts of the background pathology without relation to the test substance.

HISTOPATHOLOGY:
There was no test substance related alteration noted histopathologically.
Some isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence.

OTHER FINDINGS: None.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

Haematology results.

parameter (sex)	low dose (% of the negative controls)	mid dose (% of the negative controls)	high dose (% of the negative controls)	high dose after recovery (% of the negative controls)
red blood cell count(males)	100	101	101	97
haemoglobin conc.(males)	100	100	101	97
mean cell volume(males)	100	100	100	102
mean corpuscular haemoglobin conc.(females)	105	109	115	99

Clinical biochemical results.

parameter (sex)	low dose (% of the negative controls)	mid dose (% of the negative controls)	high dose (% of the negative controls)	high dose after recovery (% of the negative controls)
alanin aminotransferase(males)	84	83	88	102
cholesterol(females)	84	86	95	96
urea(females)	92	94	97	89

Urinalysis findings.

parameter (sex)	low dose	mid dose	high dose	high dose after recovery
total amount(females, % of the negative controls)	58	76	85	133
pH(females; control: pH 7.1; control recovery: pH 7.3)	6.4	6.7	6.1	7.3

Organ weights.

parameter (sex)	low dose (% of the negative controls)	mid dose (% of the negative controls)	high dose (% of the negative controls)	high dose after recovery (% of the negative controls)
thymus(males, absolute weight)	121	113	114	120
thymus(males, organ weight/body weight ratio)	121	115	114	120
thymus(males, organ weight/brain weight ratio)	120	118	115	120
brain(males, absolute weight)	100	96	99	100
liver(females, absolute weight)	105	105	110	103
liver(females, organ weight/body weight ratio)	104	103	109	99
liver(females, organ weight/brain weight ratio)	104	104	111	100
ovaries(females, absolute weight)	107	92	87	110

Conclusions:

Executive summary:

Guidelines applied

EC-Guideline 2001/59/EC, B.26.,
OECD-Guideline 408, 21 September 1998.

Dose levels

Based on the results of a 14-day dose range finding study (which was part of the present study), the dose levels for this 90-day oral gavage study were selected to be 0, 50, 200 and 1000 mg/kg/day.

Study outline

Evaluated parameters

Chemical analyses of formulations were conducted twice in the in-life phase of the study to assess concentration and homogeneity.

Results

Accuracy and homogeneity of test substance preparations in corn oil were demonstrated by analyses.

A frequently noted yellow stain of the gastrointestinal contents and of the faeces is attributed to the presence of the administered test substance itself and not regarded as a toxic effect.

Discussion and Conclusion

No other test substance related effect was noted.

The effects did not persist until the end of the recovery period.

The No-observed-adverse-effect-level (NOAEL) of the test substance was at 1000 mg per kg body weight in both sexes.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subchronic
Experimental exposure time per week (hours/week):: 168
Species:: rat
Quality of whole database:: reliable with restrictions due to read across

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Justification for classification or non-classification

No classification

There were no adverse effects observed after sub-acute or sub-chronic oral exposure of rats.

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Registration Dossier

Registration Dossier

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Diss Factsheets

2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Reference 3

Reference 4

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Additional information

Justification for classification or non-classification

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