Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

according to OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2017 to 20 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

EC Number:

269-284-3

EC Name:

Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Cas Number:

68214-04-0

Molecular formula:

C44H24Cl2N14Na6O20S6

IUPAC Name:

hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Test material form:

solid: particulate/powder

Details on test material:

Appearance Red powder
Storage at room temperature (20 ± 5°C).

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats are commonly used and recommended to assess toxicity. A large number of publications on this subject are available as well as historic data and first-hand experience at the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

EST ANIMALS ,
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Wistar Han (IGS) (Crl:WI(Han))
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 weeks
- Weight at study initiation: Males: 384-452 g; Females: 220-262 g
- Fasting period before study: none
- Housing: Males up to 3 in open macrolon cages type 2000P; Females (individually), paired animals
and single dams with their litters in open macrolon cages type III.
- Diet: Maintenance diet for rats and mice, No. 1324 TPF, ad libitum; Dams: Breeding diet for rats and mice No. 1314 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage) ad libitum
- Water: sterilized tap water ad libitum)
- Acclimation period: 6-9 days (pre-treatment period for females 14 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): ca 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

autoclaved community tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The substance was dissolved in water at concentrations of 256,8 (high dose until study day 28), 128,4 (high dose from study day 29), 64,2 and 16,1 mg/mL, corresponding to 200, 100, 50 and 12,5 mg/mL of Cmain. Since the test item is stable for 14 days at 5 ± 3°C , the test item solutions were prepared for up to 14 days in advance and were stored at 5 ± 3°C until day of application.

- VEHICLE : autoclaved community tap water
- dosing volume: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: minimum 7 days (based on one animal conceiving on day 7)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings: 1 additional mating period for 4 females in the high dose group
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol:
The reproductive-success of the high dose group was below the minimal acceptable number of 8 pregnant females per group according to the guideline OECD422. Thus, 4 animals of the high dose group were included into a second mating phase with proven sires from the first mating period. For three animals a sperm plug was detected, all four animals achieved pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the substance in the respective solutions was determined once within pre-mating phase (02.10.2017), at the beginning of the gestation phase (16.10.2017) and at the end of gestation/ beginning of lactation phase (06.11.2017). Samples were analysed by HPLC/UV after storange and shipment at 5 ± 3°C.

Identification: HPLC_3
Components: Degasser G1379A; Quaternary pump G1311A; Autosampler G1313A; Column compartment G1316A; UV/VIS-Detector MWD G1365B
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR11d, Build 3302


Solvent for stock solution: Demineralised water
Solvent for calibration: Demineralised water
Column: Dr. Maisch Reprosil Pur 120 C18-AQ, 5 µm, 150 x 4.6 mm
with precolumn Phenomenex SecurityGuard C18, 4 x 3 mm
Eluents: A: 50 mM ammonium acetate; B: Acetonitrile
Gradient: Time (min) % A % B
0.0 85 1 5
3.5 30 70
5 30 70
5.5 85 15
7.5 85 15
Flow rate: 1.5 mL/min
Column temperature: 40 °C
Detection: 512 nm
Injection volume: 10 µL
Retention time: 2.3 – 2.6 min

Calibration: 5 – 100 mg/L: y=0.17487x+0.00069 (R2=0.99998)
Recovery QC samples: 60 mg/L 99.8-100.4%; 50 mg/L 97.2-97.5%; 50 mg/L: 98.5-98.7% of nominal
LOQ 5 mg/L

Duration of treatment / exposure:

Males:14 days prior to mating; during mating and 3-4 weeks post mating (total at least 47 days)
Females: 14 days prior to mating; during mating, during gestation and until day 13 of lactation (total at least 51 days)
For 4 pairs of the 1000/500 mg/kg bw group a second mating period was included.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Remarks:

Based on content of the chromophore (Cmain)
During day 1-28 1000 mg/kg bw was administered. This dose was lowered due to mortality of two animals in this group and one animal in the 250 mg/kg bw group showing signs of aspiration

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

Based on content of the chromophore (Cmain)

Doses / concentrations 3

Dose / conc.:

62.5 mg/kg bw/day

Remarks:

Based on content of the chromophore (Cmain)

No. of animals per sex per dose:

12 males + 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on a dose range finding study
In a previously performed dose range finding study (DRF), a slightly different batch of the substance was administered in escalating doses up to 1055 mg/kg body weight over a time period of 21 days. The DRF showed mild adverse test item-related effect in terms of decrease of body weight in the female test animals at dose levels of 703 and 1055 mg/kg and signs of behavioral discomfort in both, male and female animals starting from 313 mg/kg. The dose levels referred to in the DRF are based on the main constituent (Cmain) of the test item. A different (slightly purer) batch of the substance was used for the main study. Since the OECD guideline 422 states, that the highest dose level should be chosen with the aim of inducing toxic effects but not death nor obvious suffering, the maximum dose for this study was based on the DRF data described above, showing no mortality or obvious suffering at the highest dose level. In this respect, the maximum dose of 1000 mg/kg was considered an acceptable high dose for this study. Two graduate (1 in 4) serial dilutions thereof (250 mg/kg, 62,5 mg/kg) were assigned to be medium and low dose
levels, respectively.

Positive control:

NA

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moratility, daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes in a standard arena
- Time schedule: weekly
Appearance (general status, physiology, autonomic functions, neurology, tonus); Motoric / exploration behavior; Excitation and Abnormal behavior

BODY WEIGHT: Yes
- Time schedule for examinations: At least once weekly (including once before beginning of application); during pregnancy, females were weighed individually on days 0, 7, 14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9, 13 post-partum and on day of termination

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
At least once weekly (including once before beginning of application); during pregnancy, females were weighed individually on days 0, 7, 14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9, 13 post-partum and on day of termination

FOOD EFFICIENCY: not measured

WATER CONSUMPTION Yes
- Time schedule for examinations:
At least once weekly (including once before beginning of application); during pregnancy, females were weighed individually on days 0, 7, 14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9, 13 post-partum and on day of termination

OPHTHALMOSCOPIC EXAMINATION:No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males at termination; females after pre-mating
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 5 males + 5 females
- Parameters checked: Leukocytes, Erythrocytes,Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin conc. (MCHC), Thrombocytes, Reticulocytes, Neutrophil granulocytes, Lymphocytes, Monocytes, Eosinophils, Basophils, blood clotting time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males at termination; females after pre-mating
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 5 males + 5 females- Parameters checked: Alkaline phosphatase (AP), Aspartate aminotransferase (AST), Alanine
aminotransferase (ALT), Gamma-glutamyl-transferase, Cholesterol, Creatinine, Urea, Sodium, Potassium, Calcium, Chloride, Glucose, Bile acids, Albumin, Globulin, A/G ratio, Total protein

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during last week of exposure on 5 males and 5 females
- Dose groups that were examined: all
- Battery of functions tested: beam-walking and grip strength
(no separate test for motor activity see clinical signs))

IMMUNOLOGY: Yes
- Time schedule for examinations: from all adult males and high dose females (samples from other female dose groups were not analysed)
- Dose groups that were examined: all
- Parameters checked: T4 hormone (thyroxin)

Oestrous cyclicity (parental animals):

Daily vaginal smears in all females fro 14 days before test start, during pre-mating and until successull mating
At termination a vaginal smear was taken to assess the estrous stage

Sperm parameters (parental animals):

Histophatological examination of the testis was conducted with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. This comprises identifying tubules with respect to their stage in the spermatogenic cycle for evaluation of germ cell death and depletion and spermatid retention

Litter observations:

STANDARDISATION OF LITTERS : no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD, day 4), presence of nipples/areolae in male pups (day 13)

GROSS EXAMINATION OF DEAD PUPS: yes for external abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes blood sampling for thyroxine on day 4 and 13 from 2 pups/litter (only day 13 samples were analysed)

Postmortem examinations (parental animals):

ORGAN WEIGHTS: Yes (see table)
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table) in addition the parathyroid and the skin of 5 animals/sex of the control and 1000/500 mg/kgbw group were investigated histopathologically.

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
thyroid glands from one male and one female pup per litter were preserved (but not examined)

Statistics:

The arithmetic mean and standard deviation were calculated for all grouped numerical data originating from monitoring the body weight, food- and water consumption and organ weights (gross pathology).
For basic analysis the respective test item groups were compared to the vehicle group by assessing of statistical significance using a two-tailed unpaired Student´s t-test. For all calculations, the significance level was set to 0,05.
In case the basic analysis returned a statistical significance additional inductive statistical analysis was applied as outlined in the main decision tree (see appendix). Except for individual blood parameters it was assumed that blood data collected in the present study were metrically scaled and normally distributed (Gaussian). Most statistical hypotheses in this study were best characterized as “many to one” – comparing a vehicle control vs. three treatment groups, respectively. Therefore the adequate analysis method was a One-Way ANOVA (Analysis of variance), followed by a post hoc Dunnett´s t-test (for organ weights), or two-way ANOVA followed by a post-hoc Bonferroni test (for body weight analysis). In case the One-Way ANOVA analysis detected a not normal distribution of residuals, the Kruskal-Wallis (H-test) analysis followed by a Dunn’s post hoc test was subsequently performed. For
all calculations, the significance level was set to 0,05.

Reproductive indices:

none reported

Offspring viability indices:

none reported

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation shortly after test item administration at 1000/500mg/kg bw and to a lesser extent at 250 mg/kg bw

Mortality:

mortality observed, treatment-related

Description (incidence):

Males:
2 at 1000/500 mg/k bw: 1 gavage error on day 5 of pre-mating, 1 regurgitation from the stomach and aspiration on day 6 of mating
1at 250 mg/kg bw: 1 gavage error on day 3 of post-mating
Females
2 at 1000/500 mg/k bw: 1 gavage error on day 4 of pre-mating, 1 regurgitation from the stomach and aspiration on day 11 of mating
2 at 250 mg/kg bw: 1 gavage error on day 7 of -mating, 1 regurgitation from the stomach and aspiration on day 10 of pre-mating

The gavage related reflux was assumed to have a physicochemical relationship with the viscous test material rather than a toxicological property of the substance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Regarding the body weight and the body weight gain, no significant differences were observed between all test item-treated animal groups (male and female) and their respective vehicle control groups. Occasional differences observed for the female animals at specific time intervals of the in life phase could be correlated with the animal’s pregnancy and lactation status and were therefore strongly assumed to be of natural origin

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The monitored time intervals did not reveal any changes of toxicological relevance in regard to food consumption between the animal test groups and their respective vehicle control groups throughout male and female in-life phases. Male animals showed some differences after the mating procedure, which could be associated with the physical stress during the mating procedure. Some variances were observed for the female animal groups, which could be associated with the specific animal condition during late pregnancy.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Male dose groups showed test item-related differences in water consumption, when compared to the respective vehicle control group. Some variances observed for the female animal groups, could be associated with the specific animal condition during phases of late pregnancy and subsequent lactation. Variations in water consumption during the complete in-life phase (males) could be related to the physical characteristics of the test item. However, a test item related effect cannot be excluded

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Effects seen (see overview table) were all not significant and considered not related to treatment

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males at 1000/500 mg/kg bw: sign decreased Albumin (-4%) and A/G ratio (-10%) within historical controls
Males at 250 mg/kg bw: sign decreased Albumin (-4%) within historical controls
Females at 1000/500 mg/kg bw: ALP sign decreased (-50%) and Potassium significantly increased (+19%) within historical controls

Other effects seen (see overview table) were all not significant and/or considered not related to treatment
No dose-resonse was observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The grip-strength and beam-walking tests did not show abnormal findings

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Males and females at 1000/500 mg/kg bw: minimal to moderate mononuclear inflammatory cell infiltrates in the renal interstitium in 3/5 animals (not considered adverse)
No effects on the male and female reproductive organs.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Analytical results (see attached table): concentrations measured were within 80-115% of nominal. The measured concentration on the 1000 mg/kg bw group fell outside the callibration range. The two additional samples at 500 mg/kg bw were within this range.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The daily monitoring of the animal’s oestrus cycles revealed at least one regular cycle for each experimental female before mating
Irregular oestrus cycles were seen at an average frequency of 1/7, 0/6, 1/6 and 1/6 in females at 0, 62.5, 250 and 1000/500 mg/kg bw (expressed as mean irregular of total oestrus circles during pre-exposure (14 d), premating and mating period)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations in testes, epididymides, prostate gland, seminal vesicles or male mammary glands.
Germ cells in the testis were evaluated by identifying tubules with respect to their stage in the spermatogenic cycle. Neither germ cell depletion nor spermatid retention were found.
The population of Sertoli cells was normal and interstitial Leydig cells did neither show atrophy or hyperplasia. Epididymides did not show desquamated germ cells, cell debris or reduced number of sperm.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

1000/500mg/kg : 6 out of 11 animals achieved pregnancy after first mating; 4 animals achieved pregnancy after second mating
250 mg/kg bw: 9 out of 11 animals achieved pregnancy after first mating; No animal was included in the second mating; 2 animals (E256/0, E257/0) conceived on day 7
62.5 mg/kg: 11 out of 12 animals achieved pregnancy after first mating; No animal was included in the second mating
Vehicle: 10 out of 12 animals achieved pregnancy after first mating; No animal was included in the second mating

Duration of the first mating period up to 14 days.

The available data do not give any indication if the initial high dose of 1000 mg/kg during the first 28 days (i.e. also during the first mating period) could be responsible for the reduced number of pregnant females, and whether the reduction to 500 mg/kg was finally decisive for the sufficient number of pregnant females after the second mating period.

Details on results (P0)

In summary, no substance-related differences in regard to evaluated mating parameters were detected between the medium and low dose groups and the vehicle control group. For the animals treated with the high dose of the substance, a reduced fertility cannot be excluded during the first mating period. However, as during the second mating period all remated females became pregnant, a final conclusion is not possible due to the changed dose level.
There were no test item-related microscopic observations in testes, epididymides, prostate gland, seminal vesicles or male mammary glands. There were no test item-related microscopic observations in ovaries, uterus, cervix or vagina.

For all female animals examined ovaries showed numerous large eosinophilic corpora lutea, typical of postparturition. Most animals had undergone a post parturition estrus cycle and were in the late metestrus, diestrus, or early proestrus phase. There was general concordance between the estrus cycle determined on vaginal mucosa by histopathology and the estrus cycle determined on vaginal smears.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: a final conclusion could not be drawn (worst case approach)

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

other: fertility could be affected at 1000/500 mg/kg bw

Organ:

other: no effects on any organ system, a final conclusion cannot be drawn

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No difference in pup development could be identified between the pups of the dose groups and the pups of the vehicle group. All pups from substance-treated dams showed a regular physical appearance, no substance-related differences in physical development were identified between dose group pups and vehicle control group pups during the course of the study.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The increased mortality rate (+2.2%) in 1000/500 mg/kg bw litters was attributed to mortality in one litter (see attachment)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The decreased body weight (gain) at 1000/500 mg/kgbw was attributed to one litter with only 2 pups (see attachment)

Gross pathological findings:

no effects observed

Description (incidence and severity):

no gross abnormalities related to the administration of the substance were detected

Description (incidence and severity):

The sex ratio (expressed as percentage males) was 48, 53, 45 and 51% at 0, 62.5,250 and 1000/500 mg/kg bw (see table)
No effects on nipple reterntion were observed (see attachment)
The anogenital distance in males and females did not differ between treatment groups and controls (see table)

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

no substance-related changes in total T4-hormone content in blood plasma could be detected (see attachment)

Details on results (F1)

In regard to evaluated parameters “no. of pups, abnormal pups and loss of offspring” no test item-related effect was observed.
There were no effects on sex ratio, thyroid hormone levels, nipple retention and anogenital distance.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Any other information on results incl. tables

Dose	0		62.5		250		1000/500		Treatment related
Endpoint	M	F	M	F	M	F	M	F
Litter size day 1 (day 13)	10.4 (9.6)		11.4 (10.1)		9.7 (8.9)		9.0 (8.3*)		no
BW litter (day 1/13)	63.8/296		72.2/316		60.4/268		58.3/258*		no
Pup weight (day 1/13)	6.1/31.2		6.4/31.5		6.4/31.4		6.6/29.2*		no
Pup mortality (%) day 0-4 Day 4-14	0.0 0.0		1.6 0.0		0.0 0.0		2.2# 0.0		no
Sex ratio (% males)	48		53		45		51		no
Thyroxine day 13 (nmol/L)	93.3		92.6		88.8		91.8		no
Anogenital distance (normalized mm)	2.2	1.3	2.2	1.3	2.1	1.2	2.2	1.3	no
Nipple count	NTRE								no
Macroscopy	NTRE								no

↑/↓= significantly increased/decreased

NTRE no treatment related effects

*Decrease compared to controls can be attributed to one litter

# Increase compared to controls can be attributed to one litter

 

Applicant's summary and conclusion

Conclusions:

Based on the information in the study report the NOAEL for reproduction is 250 mg/kg bw. The NOAEL for developmental effects is 1000/500 mg/kg bw.

Executive summary:

The substance was administered daily by oral gavage to 12 male and female adult rats in three groups at dosages of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg test item per kg body weight over a period of at least 28 days (males) and at least 49 days (females). Male and female animals were mated within their treatment group during the treatment period. Litters were used for evaluation of developmental and reproduction related parameters but did not receive test item treatment. Further 24 animals (12 males and 12 females) received the vehicle solution as control; all other parameters were identical.

Clinical signs and viability were monitored daily during the in-life phase. Individual body weight was recorded once weekly. Individual food / water consumption of female animals and group food / water consumption of male animals were recorded once weekly. Detailed clinical signs were monitored weekly from all adult animals. Grip strength and reactivity to stimuli (beam-walking test) was assessed from 5 randomly selected adult animals per sex and group during the last exposure week.

At the end of the treatment period, blood samples were taken from all adult animals to provide T4-hormone data. For assessment of additional toxicological parameters, 5 adult animals per sex and group were randomly selected for additional blood sampling (females during end of pre-mating, males at day of necropsy) to provide data on hematology and clinical biochemistry.

All adult and offspring animals were sacrificed after bleeding and examined by gross necropsy. Weights of selected organs were recorded and selected tissues and organs were preserved with special emphasis on organs of the reproductive system. From 5 randomly selected adult animals per sex and group, additional organs were preserved and examined microscopically.

Offspring parameters were assessed from each litter on day 0 post partum (pp), day 4pp, day 13pp and day of necropsy as specified by the study plan. These included numbers, stillbirth, live birth and runts, mortality, sex and presence of gross abnormalities. Blood samples from selected offspring per litter were taken on day 4pp to provide T4-hormone data.

The results of the repeated dose part can be found under repeated dose toxicity.

Regarding reproduction and development (achievement and duration of pregnancy, offspring losses, survival rate of pups and development of pups) no difference was observed for all test item treated-animals, when compared to the vehicle control animals. There was no evidence for any test item-related effect.

Conclusion:

A daily oral administration of the substance to male and female Wistar rats at dose levels of 500 mg (1000 mg during study days 1-28), 250 mg and 62,5 mg/kg body weight over a time period of 47-72 days for males and 51-91 days for females resulted in some minor animal behavioral changes observed in male and female animals predominately of the high dose groups, alterations in water consumption in male dose groups, changes in clinical biochemistry (AP lower and K higher) in the female high dose group and microscopic changes in the kidneys of male and female high dose animals. The findings were not considered adverse. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the repeated dose toxicity was set to 250 mg/kg body weight.

Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of the substance under these study conditions was set to be 250 mg/kg body weight for the female and male animals.