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]

Endpoint summary

Description of key information

In a test according to OECD 422 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.

Three animals (two of the high dose groups and one of the medium dose group) were found dead during premating or first mating phase. Animals died soon after dosing. Therefore, a gavage-related reflux and, as a consequence, regurgitation and aspiration of test item into trachea and lung are assumed as cause of death. Two animals of the high dose groups were found dead and two animals of the medium dose groups were sacrificed due to technical gavage errors.

Animal behavioral signs of salivation after dose application were observed in the test item-treated male and female animal groups, but the findings were predominantly observed at the high dose.

Regarding the body weight and the body weight gain, no significant differences were observed between all substance-treated animal groups (male and female) and their respective vehicle control groups. The monitoring of food consumption revealed no test item-related difference between all female and male experimental dose groups and the respective vehicle control groups. The monitoring of water consumption revealed a dose dependent increased water consumption in males, when compared to vehicle animals.

The T4-hormone analysis in blood plasma from experimental animal groups did not reveal a substance-related significant difference in absolute T4-hormone content between test groups and respective control groups.

No adverse effects of the test item were observed in hematology and clinical biochemistry of selected animals from all test item-treated groups. Effects seen were within historical control ranges.

The necropsy of males and females did not reveal any findings, which could be regarded as adverse or toxicological relevant for animal reproduction or development. None of the findings could be associated with the administration of the test item.

Male dose groups showed higher liver weights than control animals. This finding was considered substance related but toxicological irrelevant.

There were no macroscopic observations attributed to the substance. There were no substance-related microscopic observations in male and female reproductive organs. Substance-related microscopic observations were only found in the kidney and were characterized by minimal to moderate mononuclear inflammatory cell infiltrates in the renal interstitium.

Daily administration of the substance 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.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

test 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

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

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:

TEST 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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

autoclaved community tap water

Details on oral 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

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 storage 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

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

Dose / conc.:

500 mg/kg bw/day (nominal)

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

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Based on content of the chromophore (Cmain)

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

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 graduated (1 in 4) serial dilutions thereof (250 mg/kg, 62,5 mg/kg) were assigned to be medium and low dose levels, respectively.

Observations and examinations performed and frequency:

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 dose group females were not analysed)
- Dose groups that were examined: all
- Parameters checked: T4 hormone (thyroxin)

Sacrifice and pathology:

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/kg bw group were investigated histopathologically.

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.

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
1 at 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):

see attached document
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):

see attached document
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):

see attached document
Male dose groups showed substance-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 substance. 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):

see attachment
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 (see attached document).

Immunological findings:

no effects observed

Description (incidence and severity):

No test item-related changes in total T4-hormone content in blood plasma could be detected in the experimental animals (see attached document)

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males at 1000/500 mg/kg bw: significantly increased liver weight (+11%)
Males at 62.5 mg/kg bw: significantly increased liver weight (+11%)
Females at 62.5 mg/kg bw: significantly decreased spleen weight (-18%)
No relationship with dose and no correlation with histopathological findings.
All other effects were non-significant (see overview table)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Red staining in organs associated with oral gavage (gastrointestinal tract), elimination (urinary tract), the male reproductive system but also selected peripheral organs (e.g. skin) and tracheal-tissue were stained by the test item.
Incicental findings included: reddening of the lymphnodes that could be related to inflammation (see attachment)

Neuropathological findings:

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

A female of the vehicle control group revealed nine cysts of different size at the left uterus-horn, histopathologically described as implantation sites with decidual alterations.

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.

Details on results:

Three animals (two of the high dose groups and one of the medium dose group) were found dead during premating or first mating phase. Animals died soon after dosing. Therefore, a gavage-related reflux and, as a consequence, regurgitation and aspiration of test item into trachea and lung are assumed as cause of death. Two animals of the high dose groups were found dead and two animals of the medium dose groups were sacrificed due to technical gavage errors.
There were no treatment related effects on body weight (gain) and food consumption. Water intake was increased in males of the mid and high dose groups.
No abnormal clinical signs or substance related changes in animal behavior were detected over the course of the study. The main clinical effect that was reported was salivation and this effect increased in incidence with increasing doses. Grip-strength and beam-walking tests returned no abnormal findings. No toxicological effects of the test item were observed in hematology and clinical biochemistry of selected animals from all treatment groups. In addition no effect on thyroxine levels in males and females was reported. Macroscopy revealed red staining of the GI-tract and urinary tract. Incidental findings included red/inflammatory effects on the lymphnodes, that could not be related to treatment. Liver weights were increased in the low and high dose group, but without a dose-response relationship. Substance-related microscopic observations were only found in the kidney and were characterized by minimal to moderate mononuclear inflammatory cell infiltrates in the renal interstitium (non-adverse).

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

other: mortality due to reflux at 1000/500 mg/kg

Organ:

other: test substance in the lungs

Treatment related:

yes

Dose response relationship:

not specified

Overview table

Dose	0		62.5		250		1000/500		Treatment related
Endpoint	M	F	M	F	M	F	M	F
Mortality	0/12	0/12	0/12	0/12	1/12	2/12	2/12	2/12	Yes
Clinical signs -salivation							all ((post)mating)	All (gestation)	Yes
Body weight (gain)	NTRE									No
Food consumption	NTRE									No
Water consumption					↑ (up to 28%)		↑ ( up to 39%)			Yes
Behavioral effects (beam walking + grip strength)	NTRE									No
Motoractivity	NTRE									No
Pre-coital interval	NTRE (1-5 days)*									No
Irregular oestrus cycle**		1/7		0/6		1/6		1/6
Thyroxine (T4) (males + control and HD females)							↑ (11% ns)		No
Conception rate		10/12		11/12		9/11		10/11	No
Gestation length	NTRE (22-24 days)									No
Live litters		10		11		9		10	No
Implementation loss(mean)		1.6		1.1		1.9		2.2	No
Litter size (mean)		10.4		11.4		9.7		9.0	No
Haematology			Neut↓ (15%)	Retic↓ (17%) Neut↓ (27%)	Neut↓ (27%)	Retic↓ (19%) Neut↓ (3%)	Neut↓ (27%)	Retic↓ (16%) Neut↓ (28%) WBC ↑ (10%)	Not significant Within historical controls
Clinical biochemistry			ASAT↑ (12% ns)	ALP↓ (31% ns) Chol ↑ (32%) Bile ac↓ (33% ns)	ALAT↓ (6% ns) Creat ↑ (21% ns) Bile ac ↑ (39% ns) Alb↓ (4%)	ALP↓ (20%) Chol ↑ (20%) Bile ac↓ (20%) GGT ↓ (50%)	ALP↓ (14% ns) ALAT↓ (14% ns) ASAT ↑ (10% ns) Chol↑ (19% ns) Creat ↑ (21% ns) Glucose ↓(13% ns) Bile ac ↑ (20% ns) Alb↓ (4%) A/G↓ (10%)	ALP↓ (50%) ALAT↓ (26% ns) Chol ↑ (19% ns) Creat ↓ (12% ns) Potas ↑ (16%) Bile ac ↓ (32% ns)	Within historical controls
Organ weights			Liver↑(14%)	Spleen ↓(18%) Uterus↓(29% ns)	Liver↑(6% ns)	Uterus↓(17% ns) Liver↓ (23% ns)	Liver↑(11%)	Uterus↓(27% ns) Liver↓(10% ns)	No
Marcoscopy Staining of GI/urinary tract Staining internal organs#			yes		yes yes	yes yes	yes yes	yes yes	Yes
Male/female reproductive organs	NTRE									No
Histopathology Kidney inflammatory cell infiltrates	0/5	0/5					3/5	3/5	Yes (non-adverse)

NTRE= no treatment related effects

↑/↓= increased/decreased

% compared to controls

** mean irregular of total oestrus circles during pre-exposure (14 d), premating and mating period

*for 3 pairs at 1000/500 mg/kg bw a second mating was necessary, all females achieved pregnancy; for 2 pairs at 250 mg/kg bw the pre-coital interval was > 6 days

# lymphnodes, thymus, spleen, skin and incidentally other

Conclusions:

The NOAEL is 250 mg/kg bw, based on the mortality seen that could be related to aspiration of the substance.

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.

Three animals (two of the high dose groups and one of the medium dose group) were found dead during premating or first mating phase. Animals died soon after dosing. Therefore, a gavage-related reflux and, as a consequence, regurgitation and aspiration of test item into trachea and lung are assumed as cause of death. Two animals of the high dose groups were found dead and two animals of the medium dose groups were sacrificed due to technical gavage errors.

Animal behavioral signs of salivation after dose application were observed in the test item-treated male and female animal groups, but the findings were predominantly observed at the high dose.

Regarding the body weight and the body weight gain, no significant differences were observed between all substance-treated animal groups (male and female) and their respective vehicle control groups. The monitoring of food consumption revealed no test item-related difference between all female and male experimental dose groups and the respective vehicle control groups. The monitoring of water consumption revealed a dose dependent increased water consumption in males, when compared to vehicle animals.

The T4-hormone analysis in blood plasma from experimental animal groups did not reveal a substance-related significant difference in absolute T4-hormone content between test groups and respective control groups.

No adverse effects of the test item were observed in hematology and clinical biochemistry of selected animals from all test item-treated groups. Effects seen were within historical control ranges.

The necropsy of males and females did not reveal any findings, which could be regarded as adverse or toxicological relevant for animal reproduction or development. None of the findings could be associated with the administration of the test item.

Male dose groups showed higher liver weights than control animals. This finding was considered substance related but toxicological irrelevant.

There were no macroscopic observations attributed to the substance. There were no substance-related microscopic observations in male and female reproductive organs. Substance-related microscopic observations were only found in the kidney and were characterized by minimal to moderate mononuclear inflammatory cell infiltrates in the renal interstitium.

Daily administration of the substance 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.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: fertility, no definitive conclusion can be drawn for the effects observed in the high dose groups, therefore a worst case approach has been taken

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

Justification for classification or non-classification

Based on the information available, classification of the substance according to Regulation (EC) No 1272/2008 (CLP) for toxicity after repeated exposure is not considered necessary.