Amines, C12-14-tert-alkyl, compds. with 1-[2-[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]diazenyl]-2-naphthalenol 1-[2-[2-hydroxy-4(or 5)-nitrophenyl]diazenyl]-2-naphthalenol chromium complexes

Endpoint summary

Description of key information

Subacute gavage administration of the test item to male and female Wistar rats caused discoloration of skin, eyes and all organs in mid and high dose animals. Changes in clinical pathology parameters in high dose animals were indicative of an altered liver cell metabolism and intrahepatic or posthepatic cholestasis. In the lungs of all test group 3 (1000 mg/kg bw/d) animals a (multi)focal to extensive inflammation was observed and regarded as adverse.

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Apr 2014 - 07 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

other: U.S. EPA Health Effects Test Guidelines. OPPTS 870.3050; Jul 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42± 1 days
- Weight at study initiation: males: 149.5 - 167.1g; females: 123.3 - 140.9g
- Fasting period before study: no
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm^2).
- Diet: ground Kliba maintenance diet mouse/rat "GLP", ad libitum
- Water: supplied ad libitum from water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Apr 2014 To: 14 May 2014

Route of administration:

oral: gavage

Vehicle:

other: drinking water containing 0.5% Carboxymethylcellulose and Cremophor

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 0.5% Carboxymethylcellulose and Cremophor was filled up to the desired volume, subsequently mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.4; 2.0 and 10.0g/100ml
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The studies were carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test article in the drinking water containing 1% Carboxymethylcellulose and Cremophor at room temperature over a period of 4 days and at refrigerator over 7 days was proven before the start of the administration. Homogeneity analyses were performed in the highest and lowest concentration. Additionally, concentration control was performed in all concentrations at the beginning of the administration period.
The various analyses confirmed:
• the stability of the test-substance preparations for a period of 4 days at room temperature and 7 days at refrigerator conditions
• the homogeneous distribution of the test substance in the vehicle,
• the correctness of the prepared concentrations except the low dose of 40 mg/kg bw/d as only about 80% of the prepared concentrations were found.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1000, 200 and 40 (corrected to 32 mg/kg bw after concentration analysis) mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: as requested by the sponsor
- Rationale for animal assignment: Random distribution according to weight among the individual test groups

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily before the administration as well as 2 hours and within 5 hours after the administration. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed in all animals prior to the administration period and thereafter at weekly intervals. Parameters examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period and on day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION
Food consumption was determined weekly over a period of 4 days and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period, in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period, in the morning
- Animals fasted: Yes
- How many animals: all
- Parameters examined: "Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: Day 24 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.), Sediment, Color, turbidity (COL, TURB), Volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: Home cage observations / Open field observations / Sensory motor tests/ reflexes / Motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

HISTOPATHOLOGY: Yes
Organs were fixed followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.

Other examinations:

To further classify the increase in eosinophilic droplets in the kidneys of male animals in test group 3 (1000 mg/kg bw/d), the kidneys of animal Nos. 1 and 16 were immunohistochemically stained against alpha 2u globuline. The immunorelevant organs and tissues were evaluated according to the following
parameters:
Thymus:
• Increased/decreased grade of cortico-medullar ratio (related only to area)
• Increase of starry sky cells
• Changes of cellular density in the cortex
• Changes of cellular density in the medulla
Spleen:
• Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
• Altered cellular composition of follicles
• Altered number of germinal centers
Lymph nodes (mesenteric and axillary lymph nodes):
• Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
• Altered cellular composition of paracortex
• Altered number of germinal centers
• Hyperplasia of high endothelial venules
Peyer's patches (of the jejunum):
• Changes of the cellularity of follicles (including mantle zone and germinal centers)
• Changes of the cellularity of interfollicular area
Bone marrow:
• Changes of the cellularity
• Changes of the myeloid/erythroid ratio

Statistics:

• Body weight, body weight change: DUNNETT's test (two-sided)
• Feces, rearing, grip, strength forelimbs, grip strength, hindlimbs, footsplay, test, motor activity: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) if p-value was equal or less than 0.05
• Blood parameters: For parameters with bidirectional changes: KRUSKAL-WALLIS test and WILCOXON test (two-sided) if p-value was equal or less than 0.05; For parameters with unidirectional changes: WILCOXON-test (one-sided)
• Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: WILCOXON-test (one-sided)
• Urine pH, volume, specific gravity, color and turbidity: KRUSKAL-WALLIS test and WILCOXON test (two-sided) if p-value was equal or less than 0.05
• Weight parameters: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) if p-value was equal or less than 0.05

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study. Black discolored feces were observed in all animals of test group 3 (1000 mg/kg bw/d) from study day 1 onwards and in all animals of test group 2 (200 mg/kg bw/d) from study day 9 until the end of the administration period. Black discolored skin was observed in all animals of test group 3 (1000 mg/kg bw/d) from study day 3 onwards and in all animals of test grou 2 (200 mg/kg bw/d) from study day 13 until the end of the administration period. Black discolored eyes were observed in all animals of test group 3 (1000 mg/kg bw/d) from study day 8 onwards and in all animals of test group 2 (200 mg/kg bw/d) from study day 15 until the end of the administration period. These findings were assessed as being related to treatment. Feces discoloration was not assessed to be adverse. The assessment of the discoloration of skin was not possible a an impairment of the functional integrity of this organ could not clearly be investigated in this study. The discoloration of eyes was assumed to be adverse. No signs were observed for male and female animals in test group 1 (40 mg/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN
With regard to the mean body weights, no significant changes to the control values were observed in male and female animals of test groups 1, 2 and 3 (40, 200 and 1000 mg/kg bw/d). The same was true for mean body weight change values except the body weight change values examined in male animals of test group 3 (1000 mg/kg bw/d) which were statistically significantly lower on study days 7 (-20%) and 14 (-14%). However, as the difference to the control animals did not last over the entire study period, the changes were assessed as being incidental and not related to treatment.

FOOD CONSUMPTION
No test substance-related, adverse findings were observed. All recorded values were within the biological range typical for this strain of rats.

WATER CONSUMPTION
No test substance-related changes with regard to water consumption were observed.

HAEMATOLOGY
At the end of the administration period in females of test group 3 (1000 mg/kg bw/d), platelet counts were decreased. Although total white blood cell counts were not statistically increased, an increase of absolute monocyte cell counts above the historical control range was found in females of test group 3 (1000 mg/kg bw/d). In addition, absolute large unstained cell (LUCA) counts were increased in these individuals, but the mean was within the historical control range. Therefore, this last alteration was regarded as incidental and not treatment-related. Relative neutrophil cell counts were decreased and relative lymphocyte counts were increased in males of test group 1 (40 mg/kg bw/d), but the changes were not dosedependent. Relative reticulocyte cell counts were higher in rats of both sexes of test group 3 (1000 mg/kg bw/d) and additionally in males of test group 2 (200 mg/kg bw/d). Mean corpuscular volume (MCV was decreased in males of test group 3 (1000 mg/kg bw/d). The mentioned values were within historical control ranges and therefore the changes were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
At the end of the administration period in rats of both sexes of test group 3 (1000 mg/kg bw/d), creatinine, cholesterol and globulin levels were decreased and total bilirubin levels were increased. Already in test group 2 (200 mg/kg bw/d) in males creatinine values were decreased and in females total bilirubin levels were increased. In females of test group 3 (1000 mg/kg bw/d) total protein values were decreased and in males of the same test group triglyceride levels were decreased.

URINALYSIS
In rats of both sexes of test groups 2 and 3 (200 and 1000 mg/kg bw/d), conjugated bilirubin excretion via the urine was increased. Additionally in males of test group 3 (1000 mg/kg bw/d), urine volume was increased and the specific gravity of the urine was decreased. However, these alterations per se were regarded as treatment-related, but not adverse.

NEUROBEHAVIOUR
During Functional observational battery, deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental. Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female animals of test groups 1-3 (40, 200 and 1000 mg/kg bw/d).

ORGAN WEIGHTS
The weight changes of absolute and relative organ weights (see table below) of adrenal glands of test group 3 females (1000 mg/kg bw/d) and spleen in female animals of test groups 2 and 3 (200 and 1000 mg/kg bw/d) were regarded to be treatment-related as they were slightly above the historical control data (for test group 3). Nevertheless, no histopathologic correlates were observed. Therefore, the change was regarded to be not adverse. The increase of relative liver weight in male animals of test group 3 (1000 mg/kg bw/d) was still within historical control data and no histopathologic correlate was observed. It was regarded to be not related to treatment and not adverse.
The decrease of absolute heart weight in male animals of test group 3 (1000 mg/kg bw/d) was not regarded to be treatment-related as no histopathologic correlate was observed and the relative organ weight was not statistical significantly altered. In females of test group 3 (1000 mg/kg bw/d), the relative liver weight was significantly increased and outside historical control data. Therefore, a treatment-related effect could not be excluded but was not regarded to be adverse as no histopathologic finding was observed. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
All animals of test groups 2 and 3 (200 and 1000 mg/kg bw/d) revealed a grey to dark grey discoloration of the carcass and all organs. In some animals of test group 1 (40 mg/kg bw/d), only the content of the digestive tract revealed a grey discoloration. These findings were regarded to be treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in the lungs of male and females with incidences and grading according to the table below.
In all animals of test group 3 (1000 mg/kg bw/d) there was a (multi)focal to extensive inflammation within the lungs. Mainly areas at the brochio-alveolar transition were affected. Inflammatory foci were mainly composed of macrophages revealing a foamy cytoplasm and a lower number of neutrophils. This finding was regarded to be treatment-related. Female animal No. 26 of test group 1 (40 mg/kg bw/d) revealed a severe inflammation with single
multinucleated giant cells in the lungs. This was evidence for a foreign body reaction most likely caused by a gavage error. Therefore, this finding was application-related but not related to treatment with the test substance.
Male animals of test group 3 (1000 mg/kg bw/d) revealed a slightly higher incidence of eosinophilic droplets in the tubules of the kidneys when compared to the other test groups and control animals. An immunohistochemical staining against alpha 2µ globulin was positive in animal No. 16 of test group 3 (1000 mg/kg bw/d) and to lesser extent in control animal No. 1. Peri-/vasculitis was observed in a single control female as well as in four male animals of test group 3 (1000 mg/kg bw/d). The finding is commonly observed in the rat liver, especially in male animals (Ruben et al., 2000). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Signs of systemic toxicity were determined at the next higher dose level of 200 mg/kg bw/d and above.

Critical effects observed:

not specified

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (40)	2 (200)	3 (1000)	1 (40)	2 (200)	3 (1000)
Adrenal glands				106%	104%	131%*
Heart	98%	97%	84%*
Spleen				99%	114%*	130%*

*p ≤ 0.05; **p ≤ 0.01

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

	Male animals			Female animals
Test group	1	2	3	1	2	3
(mg/kg bw/d)	(40)	(200)	(1000)	(40)	(200)	(1000)
Adrenal glands				105%	107%	137%**
Liver	105%	105%	111%*	101%	105%	117%**
Spleen				100%	118%*	136%**

*p ≤ 0.05; **p ≤ 0.01

Gross Lesions

	Male animals				Female animals
Test group	0	1	2	3	0	1	2	3
(mg/kg bw/d)	0	(40)	(200)	(1000)	0	(40)	(200)	(1000)
No. of animals	5	5	5	5	5	5	5	5
General observation Discoloration	0	0	5	5	0	0	5	5
Cecum Discol. of contents	0	3	5	5	0	2	5	5
Colon Discol. of contents	0	1	5	5	0	1	5	5
Jejunum Discol. of contents	0	2	5	5	0	3	5	5

Histopathology

Lungs	Male animals				Female animals
Test group	0	1	2	3	0	1	2	3
(mg/kg bw/d)	0	(40)	(200)	(1000)	0	(40)	(200)	(1000)
No. of animals	5	5	5	5	5	5	5	5
Inflammation, (multi)focal	0	0	0	5	0	1	0	5
·   Grade 2				2				4
·   Grade 3				3				1
·   Grade 4						1

Kidneys	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (40)	2 (200)	3 (1000)
No. of animals	5	5	5	5
Eosinophilic droplets in tubules	5	5	5	5
·   Grade 1	5	5	5
·   Grade 2				5

Conclusions:

The oral administration of the test article by gavage to male and female Wistar rats over a period of 4 weeks resulted in signs of systemic toxicity which were determined at a dose level of 200 mg/kg bw/d and above. Therefore, the no observed adverse effect level (NOAEL) was considered to be 32 mg/kg bw/d in male and female Wistar rats (only 80% of the nominal dose level of 40 mg/kg bw/d were found during concentration control analysis).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

In a GLP-compliant subacute repeated dose toxicity study following OECD guideline 407, the test item was administered by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg body weight/day (drinking water containing 0.5% Carboxymethylcellulose and Cremophor served as vehicle control; test group 0), 40 mg/kg bw/d (test group 1), 200 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 4 weeks. Only about 80% of the nominal dose level of 40 mg/kg bw/d were found during concentration control analysis. Therefore the corrected dose level of group 3 was 32 mg/kg bw/d.

With regard to clinical examinations, clear signs of general systemic toxicity in Wistar rats of either sex were not observed. However, discoloration of eyes, skin and feces were observed in test groups 2 and 3 (200 and 1000 mg/kg bw/d). Although no other clinical findings occurred during the daily examination, the FOB and the MA measurement discoloration of the eyes was assessed to be an unwanted effect with potential influence on the function. The obviously high body burden indicated by discoloration of skin was not assessed to be adverse but the functional integrity of this organ could not clearly be investigated in this study. In addition, reversibility of these effects by a treatment-free recovery period was also not examined. No signs were observed for male and female animals in test group 1 (40mg/kg bw/d).

Regarding clinical pathology, lower globulin and cholesterol levels in rats of both sexes of test group 3 (1000 mg/kg bw/d) and decreased total protein values in females and decreased triglyceride levels in males of the same test group indicated an altered liver cell metabolism. Higher total bilirubin values in male and female rats of test group 3 as well as females of test group 2 (200 mg/kg bw/d) without any change in the red blood cell parameters and a greater excretion of conjugated bilirubin via the kidney in rats of both sexes of test groups 2 and 3 were a sign of a intrahepatic or posthepatic cholestasis. Lower creatinine values in rats of both sexes of test group 3 (1000 mg/kg bw/d) and in males of test group 2 (200 mg/kg bw/d) were due to an increased excretion of creatinine via the kidneys or a reduced metabolism in the skeletal muscle of these individuals. Higher absolute monocyte cell counts in females of test group 3 combined with reduced platelet counts may be a hint of a stress reaction in these animals affecting the coagulation system.

Regarding pathology, the lungs of males and females and kidneys of males were the target organs. The grey discoloration of the complete body of all animals of test groups 2 and 3 (200 and 1000 mg/kg bw/d) were regarded to be treatment-related. Although histopathologic correlates were not determined, the finding was assessed to be an unwanted effect. In test group 1 (40 mg/kg bw/d) only the contents of the intestine were grey discolored which was also regarded to be treatment-related but not adverse. In the lungs of all test group 3 (1000 mg/kg bw/d) animals a (multi)focal to extensive inflammation was observed. This was regarded to be caused by the test substance and adverse in nature. In the kidneys of test group 3 (1000 mg/kg bw/d) animals was a slight increase in eosinophilic droplets compared to control males. The positive result of the immunohistochemical stain against alpha 2µ globulin demonstrated these droplets to be stored alpha 2µ globulin. Eosinophilic droplets in the proximal convoluted tubules represent alpha 2u globulin, a poorly hydrolysable, low molecular weight protein characteristic for male rats. The increase of eosinophilic droplets was regarded to be treatment-related, but is not of human relevance. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Subacute oral toxicity study with 14-day recovery (RCC NOTOX B.V. 1989)

The UVCB substance was registered as a new substance in the EU in 1989. The test performed at this time caused similar, but overall less pronounced effects upon subacute oral dosing. For exemple, internal external discoloration was much less severe and no adverse effects on lung were observed up to the limit dose. The study was performed using 1% aqueous carboxymethylcellulose as vehicle and Sprague-Dawley rats. Repeated oral intake of the test substance at 1000 mg/kg/ bw/day for some animals resulted in reversible purple or blue skin/fur as well as in bluish discolouration of the pancreas and testes. The bluish discoloration of the pancreas and testes was observed in three of five males after 28 days. At the end of the recovery period, one male showed bluish discoloration in testes. For females, one of five and none of five animals showed bluish discoloration of the pancreas at the end of the treatment period and after recover, respectively. No discoloration was observed in any animal at the doses of 200 and 50 mg/kg bw.

Apart from the discoloration, signs of systemic toxicity were observed at 200 and 1000 mg/kg bw. On day 29, a high number of large unstained cells was noted in males and a low number of reticulocytes was noted in males and females. The following was observed at the high dose group (1000 mg/kg bw): An increased level of total bilirubin was noted in the blood serum of males and females on day 29; slightly to moderately increase of white blood cells was noted in the urine of males only, during week 4 of treatment; microscopically observed focal cortical hypertrophy was noted in the adrenal glands of two females. The following was observed at the mid dose group (200 mg/kg bw): A low number of reticulocytes was noted in males only.

These signs do not give a clear picture of target organs and they were reversible within the 14 day recovery period.

Screening study for reproductive toxicity (BASF 2015)

In contrast to the absence of toxicity observed in the repeated dose toxicity studies (OECD 407), significant toxicity (ie mortality at 1000 mg/kg bw) was observed in parental animals in the screening study for reproductive toxicity (OECD 421, BASF 2015). The only difference between those studies were the age and accordingly the body weights of the animals. Both studies were performed with the same batch. During the OECD 407 study, rats are young and double their body weights between the beginning and the end of the study. For the OECD 421 study, the initial body weight of the rats roughly corresponds to that at the end of the OECD 407 study. For equal doses in regard to mg/kg bw, the total amount of the test material is twice as high in the OECD 421 study.

In the GLP-compliant Reproduction/Developmental Toxicity Screening Test following OECD guideline 421, the test article was given daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 0, 40, 200 and 1000/500 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (drinking water containing 0.5% Carboxymethylcellulose and Cremophor). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. During the first week of treatment impairments of food consumption and body weight development were observed for male and female animals of test group 3 (1000 mg/kg bw/d). One female animal of this test group was found dead on study day 6. Therefore, the dose level for test group 3 was reduced to 500 mg/kg bw/d from study day 7 onwards.

Regarding clinical examinations of the F0 generation, signs of general systemic toxicity were observed at a dose level of 1000 and 500 mg/kg bw/d as reduced food consumption accompanied with impaired body weight data were observed during the entire study period. These parameters were not influenced in male and female animals of test groups 1 and 2 (40 and 200 mg/kg bw/d). Discoloration of eyes, skin and feces were observed in test groups 2 and 3 (200 as well as 1000 and 500 mg/kg bw/d). The discoloration of the eyes was assessed to be an unwanted effect with potential influence on the function. No signs were observed for male and female animals in test group 1 (40 mg/kg bw/d).

Clinical examinations of the F1 generation revealed a discoloration of the skin in surviving F1 pups of test groups 1-3 from grey to black, depending on the test group. At pup necropsy, discoloration of stomach and intestinal content as well as liver, heart, lung and kidney were observed. The functional integrity of these organs could not be investigated in this study.

Regarding cytology of the bronchoalveolar lavage fluid (BAL), slightly increased neutrophil cell counts was assumed to be an indication of a mild local inflammation in the lung. Regarding pathology, no treatment-related findings in testes, epididymides or ovaries were observed. The decrease in terminal body weight of male and female animals in test group 3 (1000 and 500 mg/kg bw/d) was regarded to be treatment-related and adverse due to the fact that it was lower than historical control values for both sexes. The macroscopically observed discoloration of almost the whole body of animals of test groups 2 and 3 (200 as well as 1000 and 500 mg/kg bw/d) was thought to have been caused by the test substance. Microscopically the discoloration could not be detected. The no observed adverse effect level (NOAEL) for general systemic toxicity was 200 mg/kg bw/d for male and 40 mg/kg bw/d in female Wistar rats. Since effects were stronger in this screening study, the NOAEL of 40 mg/kg bw is used to derive the DNEL.

In conclusion, the oral administration of the test article by gavage to male and female Wistar rats over a period of 4 weeks resulted in signs of systemic toxicity which were determined at a dose level of 200 mg/kg bw/d and above. Therefore, the no observed adverse effect level (NOAEL) was considered to be 40 mg/kg bw/d in male and female Wistar rats.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP-compliant guideline study acceptable for assessment

Repeated dose toxicity: via oral route - systemic effects (target organ) respiratory: lung; urogenital: kidneys

Justification for classification or non-classification

The substance was not classified for repeated dose toxicity when it was included in Annex I of  Directive 67/548/EEC. No serious irreversible effects were observed at dose levels of less than 150 mg/kg bw upon subacute exposure.

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses of 200 mg/kg bw or less upon subacute oral exposure (OECD 407) in rats; adverse effects were observed at 1000 mg/kg bw and 500 mg/kg bw. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.