1-ethyl-3-methyl-3H-imidazolium dicyanidoamide(1-)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

toxicity to reproduction

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2015

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

- Chemical name: EMIM Dicyanamide
- Purity: 99.81 area-% (HPLC, 210 nm); 100.0 area-% (HPLC, 225 nm)
- Test item No.: 12/0071-2
- Batch identification: 0013141983
- Content: 97.4 g/100 g determined by 1H-NMR spectroscopy
- Homogeneity: given (visually)
- Storage stability: the stability of the test item under storage conditions over the study period was guaranteed by the sponsor
- Expiry date: February 19, 2017
- Storage conditions: room temperature, under N2
- Physical state / color: liquid / colorless to brown, clear

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: about 12 weeks; females: about 9 weeks
- Weight at study initiation: (P) Males: 415.1 - 419.0 g; Females: 209.8 - 214.9 g
- Housing: during the study period, the rats were housed individually in polycarbonate cages type III (floor area of about 800 cm²), with the following exceptions:
1. During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany
2. During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
3. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study. Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, was added for environmental enrichment. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: yes (duration not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h)

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced twice weekly, at least.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks maximum, daily from 16.00 h until 06.30 - 09.00 h
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water over a period of 7 days at room temperature was proven. At the beginning (during pre-mating), twice during gestation and once during lactation of the study samples of all concentrations were taken. Given that the test substance is completely miscible with drinking water, solutions were considered to be homogenous and no homogeneity analyses were carried out. The samples collected at the beginning of the administration period and during the lactation period were analyzed. An automated HPLC System with autosampler, pump and UV/VIS-detector was used. The column used was a Primesep 200, 5 µm, 250 mm x 3.2 mm (SIELC).

Duration of treatment / exposure:

2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals (males 28 days, females 55 days)

Frequency of treatment:

Daily

Dose / conc.:

500 ppm

Remarks:

Test group 1;
- Reduced for females to 250 ppm during the lactation period.
- Mean dose value: 32 and 54 mg/kg bw/day for males and females, respectively

Dose / conc.:

2 000 ppm

Remarks:

- Test group 2;
- Reduced for females to 1000 mg/kg bw/day during the lactation period.
- Mean dose value: 94 and 177 mg/kg bw/day for males and females, respectively

Dose / conc.:

8 000 ppm

Remarks:

Test group 3;
- Reduced from 8000 ppm [152 and 396 mg/kg bw/d (m,f)] to 6000 ppm [401 and 439/494 mg/kg bw/d (m,f: 2. week of premating/gestation)] from study day 7 onwards. Test dose for females was 3000 ppm (511 mg/kg bw) during the lactation period.
- Mean dose value: 339 and 476 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a test study was performed in male and female Wistar rats (BASF SE, 2016: 01R0071/12N143). Each 4 male and female animals were treated via the drinking water at concentrations of 0, 4000 and 12000 ppm. Due to reduced food and water consumption and lower body weight/body weight change values in animals of the high-dose group, the concentration for this test group was reduced to 8000 ppm from study day 3 onwards. On study day 9, one male animal had to be sacrificed in a moribund condition. Additional findings did not occur in any of the other animals. The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioural changes and/or signs of overt toxicity. The parturition and lactation behaviour of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. On weekdays (except Saturdays, Sundays and public holidays) the parturition behaviour of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examinations included: abnormal behaviour in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the faeces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The following exceptions were notable for female animals:
1. During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
2. Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
3. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males
4. Females without litter and after weaning (PND 13) were weighed once a week

FOOD CONSUMPTION
Time schedule for examinations: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
1. Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
2. Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
3. Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examination: generally, water consumption was determined once a week (over a period of max. 4 days) for male and female parental animals, with the following exceptions:
1. Water consumption was not determined during the mating period (male and female F0 animals)
2. Water consumption of the F0 females with evidence of sperm was determined on GD 6-7, 13-14 and 19-20.
3. Water consumption of F0 females, which gave birth to a litter was determined for PND 3–4, 6-7, 9-10 and 12-13.
Water consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.
- Calculation of intake: to calculate the mean values of entire administration period for each test group, the values of each study period, i.e. premating (14 days), gestation (20 days) and lactation (13 days), were assessed according to days of treatment.

HAEMATOLOGY
- Time schedule for collection of blood: at termination for males and in the morning at PND 14 for females
- Anaesthetic used for blood collection: Yes, isolfurane
- Animals fasted: Yes
- How many animals: first 5 surviving parental males per group and the first 5 females with litters (in order of delivery) per group.
- Parameters checked: Leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), prothrombin time

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at termination for males and in the morning at PND 14 for females
- Anaesthetic used for blood collection: Yes, isolfurane
- Animals fasted: Yes
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), ypsilon-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), Total thyroxine (T4) and the amount of thyroid stimulating hormone (TSH).

NEUROBEHAVIOURAL EXAMINATION
- Functional observation battery: A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
1. Home cage observations: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
2. Open field observations (the animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes): behaviour on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait abnormalities, activity/arousal level, urine excreted within 2 minutes (amount/colour), rearing within 2 minutes, other findings
3. Sensory motor tests/reflexes (the animals were then removed from the open field and subjected to following sensory motor or reflex tests): reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behaviour during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
- Motor activity assessment: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group.

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Sperm parameters (parental animals):

Parameters examined in male parental generation: epididymides, glans penis, stages of spermatogenesis in the testes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, maximum of 4 pups/litter (4/sex/litter as nearly as possible); at necropsy on PNDs 4 and 13 all pups were sacrificed

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths and live births, clinical observations including gross-morphological findings, body weights, presence of nipples/areolae in male pups, anongenital distance/index.

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

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

ORGAN WEIGHTS
- Weights determined from all animals sacrificed on schedule: terminal body weight, Bulbourethral gland (Cowper’s gland), epididymides, glans penis, M. levator ani together with musc. bulbocavernosus (male animals), prostate, seminal vesicle with coagulating glands, testes, thyroid glands
- The following weights were determined in 5 animals per sex/ test group sacrificed on schedule (females with litters only, same animals as used for clinical pathology examinations): adrenal glands, brain, heart, kidneys, liver, spleen, thymus

HISTOPATHOLOGY (also see table under 'Any other information on materials and methods incl. tables'.
- Examinations included: all gross lesions, adrenal glands, brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), ovaries, oviducts, Peyer’s patches, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cords), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Postmortem examinations (offspring):

- All pups with scheduled sacrifice on PND 4, resp. PND 13 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Pathology Laboratory.
- All stillborn pups and all pups that died before day 13 were examined externally, eviscerated and their organs were assessed macroscopically.
- All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

- Water and food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days: simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
- Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
- Mating days until day 0 p.c., %postimplantation loss, pups stillborn, %perinatal loss: pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
- Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
- % live male day x, %live female day x: comparison of the dose group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.
- Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.
- Blood parameters: one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians

Reproductive indices:

Mating and fertility index: the pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. Additionally, gestation index, live birth index, implantations and post-implantation losses were recorded.

Offspring viability indices:

Pup number and status at delivery, viability index (ratio of number of live pups on day 4 vs day 0 after birth), survival index (ratio of number of live pups on day 13 after birth vs day 4 after birth) and sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Males and females (except gestation and lactation periods): during the pre-mating period, a single treatment-related finding was observed in test group 3, i.e. reduced nutritional condition was observed for a female of test group 3 on study days 7 to 9. The finding was assessed to be related to impaired water and food consumption during the first week of treatment at a concentration of 8000 ppm. The animal recovered after reducing the test substance’s concentration in drinking water to 6000 ppm from study day 7 onwards. No additional clinical findings of toxicological concern occurred in pre-mating, mating and post-mating (males only) periods. Between pre-mating days 7 to 9, one male of test group 3 showed black encrusted nose. The finding was assessed to be incidental and not related to treatment. One male of test group 1 showed piloerection between mating days 7 to 10. The same was true for another male animal, which showed piloerection on mating day 7 only. The findings were assessed to be incidental and not related to treatment.
- Females during gestation: no clinical signs were observed in female animals of any test group during the gestation period.
- Females during lactation: during the lactation period, incidental findings occurred in individual animals of test groups 1-3: one female of test group 1 showed bloody/reddish vaginal discharge in combination with piloerection on 1 to 2 days after delivery. The animal recovered during the lactation period on PND 3. One animal of test group 2 showed semiclosed eyelids, slightly reduced general and nutritional conditions, piloerection and 2 injuries, i.e. at the anogenital region as well as at the left flank. An animal of test group 2 lost its complete litter 1 day after delivery. Until sacrifice, no other findings became evident. An animal of test group 3 showed moderately reduced general and slightly nutritional conditions, piloerection and abnormal flexibility of the right forelimb during the last 2 days before sacrifice. All findings were assessed to be incidental as no picture of a definitive toxicological mode of action became evident and a clear dose-response relationship did not occur.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died ahead of schedule.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A continuous decrease of mean body weight was observed in male animals of test group 3 (8000 ppm) during the first week of application (pre-mating period). Since the test substance’s concentration in drinking water was reduced to 6000 ppm from study day 7 onwards, the animals started to gain weight. However, mean body weight of these animals was significantly lower during the entire pre-mating (-16% to -10%) and mating (-10%) periods. No significant changes in mean body weights were observed for male animals of test groups 1 and 2 (500 and 2000 ppm).
In female animals of test group 3 (8000 ppm), a body weight loss could be observed on study day 3 (pre-mating period), but the animals recovered already between study days 3 and 7 and gained weight. Thus, no significant differences could be observed during the late pre-mating and the early gestation periods. Mean body weight values became significantly lower again on GDs 14 (-6.2%) and 20 (-8.5%). The significance continued during the entire lactation period, except on PND 4 because of the high standard deviation. No significant changes in mean body weights were observed for female animals of test groups 1 and 2 during the entire application period.

With regard to body weight change values, a similar picture could be drawn. In male animals of test group 3 (8000 ppm) a significant body weight loss was observed between study days 0-3 and 3-7 as well as 0-14 (pre-mating period). The body weight change value was only significantly higher between study days 7-10 after reducing the test substance’s concentration in drinking water. No significant difference was observed during the mating period. No significant changes in mean body weight change values were observed for male animals of test groups 1 and 2 (500 and 2000 ppm). In female animals of test group 3, mean body weight change value was significantly lower after 3 days of application (loss of weight) but significantly higher during the following 4 days. No significant differences could be observed during the late pre-mating and the early gestation periods. Mean body weight change values became significantly lower again between GDs 14-20 resulting in a lower value between GDs 0-20. No significant differences were observed during the lactation period. No significant changes in mean body weight change values were observed for female animals of test groups 1 and 2 during the entire application period.

The impaired body parameter development of male and female animals of test group 3 over nearly the entire administration period were most likely related to reduced water and food consumption as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in food consumption were observed for male and female animals of test group 3 (8000 ppm) during the first week of treatment, showing a maximum deviation to the control animals between study days 3 and 7 in males (-55%) and between study days 0 and 3 in females (-42%). Food consumption values turned to normal from study day 7 onwards clearly showing a relation to reduced test substance concentrations in drinking water.

During gestation, food consumption values were slightly, but significantly lower between GD 7-14 and 14-20. As the finding correlated to slightly impaired body weight increases in these animals, a relation to treatment was assumed. During lactation, no changes were observable for these animals. These changes were most likely related to reduced water consumption as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

No impairment of food consumption was observable for male and female animals of test groups 1 and 2 during the entire administration period.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was significantly decreased in both sexes of test group 3 (8000 ppm) during the first application week, showing a maximum deviation to the control animals between study days 0 and 3, i.e. by -81% in males and by -72% in females. As the water consumption in both sexes was still very low during the following days and impaired food consumption as well as body weight loss were manifest, the concentration of the test substance in the vehicle was reduced to 6000 ppm from study day 7 onwards. Although the animals recovered with regard to body weight development, water consumption was still significantly lower in male animals and female animals towards the end of pre-mating period as well as in female animals during the entire gestation period. During lactation, water consumption in female animals of test group 3 was only marginally affected as the concentration in drinking water was reduced by 50%. This adjustment maintained the dams at the desired target doses of the test substance during this period of increased water intake. Taken together, these changes were most likely related to palatability problems as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Water consumption values were also significantly reduced in male and female animals of test group 2 on several days of the study. However, these changes were still within the normal range for this rat strain and an impact on body weight development could not be observed. Thus, these changes in water consumption were without any toxicological relevance.

Water consumption values of male and female animals in test group 1 were once increased between pre-mating days 7 to 10. A relation to treatment was not assumed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among haematological parameters were observed.

In rats of both sexes of test groups 2 and 3 and in males of test group 1 absolute reticulocyte counts were significantly higher compared to controls. Reticulocyte counts in females of test groups 2 and 3 and in males of test groups 1 and 2 were not altered dose-dependently. Even the values in males of test group 3 were within the historical control range (absolute reticulocytes 99.5-166.1 Giga/L). In males of test group 2 relative neutrophil counts were significantly decreased and relative lymphocyte counts were significantly increased. Both parameters were not changed dose-dependently. Therefore, the mentioned changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

In females of test groups 1, 2 and 3 alanine aminotransferase (ALT) activities were significantly lower compared to controls and in females of test group 1 calcium levels were significantly decreased, but the alterations were not changed dose-dependently. Therefore, these alterations were regarded as incidental and not treatment-related.

In males of test group 3 sodium levels were significantly decreased. The values were marginally below the historical control range (sodium 140.9-146.1 mmol/L). However, this was the only changed clinical chemistry parameter in these individuals. Therefore, this change was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATION BATTERY
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
- Quantitative Parameters: in male animals, the number of rearings was significantly lower in test group 1 and significantly higher in test group 2. As no changes were observed for test group 3 animals, the findings were assessed to be incidental. No changes were observed for any female animal in test groups 1-3.
- Home cage observations: no test substance-related effects were observed.
- Open field observations: one male of test group 3 showed resistance to handling. A male of test group 1 showed piloerection. Both findings were assessed to be incidental.
- Sensorimotor tests/reflexes: no test substance-related effects were observed.

MOTOR ACTIVITY ASSESSMENT
Regarding the overall motor activity as well as individual intervals, no test substance-related deviations were noted for male and female animals of any test group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the spleen of male animals, the incidence of extramedullary haematopoiesis was minimally increased in test group 3. The severity was mostly minimal (very few, very small foci of extramedullary haematopoiesis) and there was no dose-response relationship. The increased incidence of the mostly minimal extramedullary haematopoiesis did not explain the significantly increased relative spleen weight in high-dose males. The affected males with extramedullary haematopoiesis did not show higher absolute and relative spleen weights than animals without this finding. 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. As to fertility, the stages of spermatogenesis in the testes of males of test group 3 were comparable to those of the controls. One female of test group 2, which was not pregnant, and its male mating partner did not show relevant histopathological findings. The macroscopically described red discoloration of the lungs in this male correlated with blood aspiration due to sacrifice.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONES
In F0 males of test group 3, T4 levels were significantly decreased. However, in these individuals thyroid stimulating hormone (TSH) levels were not altered. Additionally, in F0 females as well as in PND4 and PND13 pups TSH and T4 levels were not altered. Therefore, the isolated T4 decrease in F0 males of test group 3 in combination with a reduced body weight of these individuals was regarded as a consequence of a reduced food intake. This is leading to a decrease of T4 and a tendency of a decreased TSH value in contrast to hypothyroidism with decreased T4 and increased TSH levels (Germain and Galton, 1985).

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was between 3.90 days and 4.20 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of test group 3 were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Male mating index: the male mating index calculated after the mating period for F1 litter was 100% in all test groups.
- Male fertility index: Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. One male animal of test group 2, did not generate F1 pups. Thus, the male fertility index was 100% in test groups 0, 1 and 3 and 90% in test group 2. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female mating index: the female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 2.7 days for test group 0 (control), 3.1 days for test group 2, 2.5 days for test group 2 and 2.9 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female fertility index: most sperm positive rats delivered pups with the exception of one female of test group 2, which had sperm in vaginal smear but delivered no pups and showed no implants. Thus, the female fertility index was 90% in test group 2 and 100% in test groups 0, 1 and 3. These values reflected the normal range of biological variation inherent in the strain of rats. The mean duration of gestation was similar in all test groups, i.e. 22.6 days (test group 0), 22.5 days (test group 1), 22.3 days (test group 2) and 22.4 days (test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Gestation index: the gestation index was 100% in test groups 0, 1 and 2 and 90% in test group 3, since one female did only deliver a single dead female pup.
- Live birth indices: the rate live birth indices were similar in all test groups, i.e. 99.1% for test group 0, 95.7% for test group 1, 93.4% for test group 2 and 98.2% for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data
- Postimplantation loss: postimplantation loss was 8.6% in test group 0 (control), 10.8% in test group 1, 3.2% in test group 2 and 12.8% in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed; treatment-related effects most likely related to palatability problems; reduction from 8000 ppm to 6000 ppm from day 7 onwards

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed; treatment-related effects most likely related to palatability problems; reduction from 8000 ppm to 6000 ppm from day 7 onwards

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

One male pup of test group 0 (control group; 0 mg/kg bw/day) showed poor general condition on lactation days 13. This finding was spontaneous in nature. All other F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4, resp. PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during PND 0-4 was 98.8% in test group 0 (control), 92.8% in test group 1, 85.7% in test group 2 and 99.1% in test group 3. The survival indicating pup mortality during PND 4-13 was 100.0% in all test groups and, therefore, not affected by the test substance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

On PND 1, runts were found in 1 litter of test group 0 (1 female), in 2 litters of test group 1 (8 females), in 2 litters of test group 2 (1 male and 5 females) as well as in 1 litter of test group 3 (1 female). All values were within the range of the biological variation inherent in the strain of rats used for this study.

Mean pup body weights/pup body weight changes of all pups in test groups 1 and 2 were comparable to the control group.

A trend towards lower mean body weight could be observed beginning on PND 4 to 13, when mean body weights of male, female as well as male and female pups together were significantly lower, i.e. -9.6% for males, -11% for females as well as -10% for both. With regard to body weight change values, significant differences occurred in test group 3 in intervals of PNDs 1-4 (male pups), 4-7 (female pups) and 7-13 (males, females as well as both sexes together). Between PNDs 1-13, both gender gained significantly less weight. These changes might have been related to reduced food and water intake of the respective dams which in turn also gained less weight during lactation. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Pup number and status at delivery: the mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred.
- Sex ratio: the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Anogenital disttance: anogenital distance of male and female pups was not significantly changed in any test group when compared to the control pups. When corrected for body weight, the anogenital indices were significantly higher in male pups of test groups 1 and 3 as well as in female pups of test group 2. Thus, these changes were considered to be incidental and not treatment-related.
- Nipple/areola anlagen: the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

ANALYSES

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature, the homogeneous distribution of the test substance in the vehicle and the correctness of the prepared concentrations. Also drinking water, food analyses and analysis on bedding and enrichment were found to be suitable.

Conclusions:

GHS criteria not met

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2015

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

- Chemical name: EMIM Dicyanamide
- Purity: 99.81 area-% (HPLC, 210 nm); 100.0 area-% (HPLC, 225 nm)
- Test item No.: 12/0071-2
- Batch identification: 0013141983
- Content: 97.4 g/100 g determined by 1H-NMR spectroscopy
- Homogeneity: given (visually)
- Storage stability: the stability of the test item under storage conditions over the study period was guaranteed by the sponsor
- Expiry date: February 19, 2017
- Storage conditions: room temperature, under N2
- Physical state / color: liquid / colorless to brown, clear

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: about 12 weeks; females: about 9 weeks
- Weight at study initiation: Males: 415.1 - 419.0 g; Females: 209.8 - 214.9 g
- Housing: during the study period, the rats were housed individually in polycarbonate cages type III (floor area of about 800 cm²), with the following exceptions:
1. During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany
2. During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
3. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study. Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, was added for environmental enrichment. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: yes (duration not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h)

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced twice weekly, at least.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water over a period of 7 days at room temperature was proven. At the beginning (during pre-mating), twice during gestation and once during lactation of the study samples of all concentrations were taken. Given that the test substance is completely miscible with drinking water, solutions were considered to be homogenous and no homogeneity analyses were carried out. The samples collected at the beginning of the administration period and during the lactation period were analyzed. An automated HPLC System with autosampler, pump and UV/VIS-detector was used. The column used was a Primesep 200, 5 µm, 250 mm x 3.2 mm (SIELC).

Duration of treatment / exposure:

2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals (males 28 days, females 55 days)

Frequency of treatment:

Daily

Dose / conc.:

500 ppm

Remarks:

Test group 1;
- Reduced for females to 250 ppm during the lactation period.
- Mean dose value: 32 and 54 mg/kg bw/day for males and females, respectively

Dose / conc.:

2 000 ppm

Remarks:

- Test group 2;
- Reduced for females to 1000 mg/kg bw/day during the lactation period.
- Mean dose value: 94 and 177 mg/kg bw/day for males and females, respectively

Dose / conc.:

8 000 ppm

Remarks:

Test group 3;
- Reduced from 8000 ppm [152 and 396 mg/kg bw/d (m,f)] to 6000 ppm [401 and 439/494 mg/kg bw/d (m,f: 2. week of premating/gestation)] from study day 7 onwards. Test dose for females was 3000 ppm (511 mg/kg bw) during the lactation period.
- Mean dose value: 339 and 476 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a test study was performed in male and female Wistar rats (BASF SE, 2016: 01R0071/12N143). Each 4 male and female animals were treated via the drinking water at concentrations of 0, 4000 and 12000 ppm. Due to reduced food and water consumption and lower body weight/body weight change values in animals of the high-dose group, the concentration for this test group was reduced to 8000 ppm from study day 3 onwards. On study day 9, one male animal had to be sacrificed in a moribund condition. Additional findings did not occur in any of the other animals. The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioural changes and/or signs of overt toxicity. The parturition and lactation behaviour of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. On weekdays (except Saturdays, Sundays and public holidays) the parturition behaviour of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examinations included: abnormal behaviour in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the faeces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The following exceptions were notable for female animals:
1. During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
2. Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
3. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males
4. Females without litter and after weaning (PND 13) were weighed once a week

FOOD CONSUMPTION
Time schedule for examinations: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
1. Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
2. Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
3. Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examination: generally, water consumption was determined once a week (over a period of max. 4 days) for male and female parental animals, with the following exceptions:
1. Water consumption was not determined during the mating period (male and female F0 animals)
2. Water consumption of the F0 females with evidence of sperm was determined on GD 6-7, 13-14 and 19-20.
3. Water consumption of F0 females, which gave birth to a litter was determined for PND 3–4, 6-7, 9-10 and 12-13.
Water consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.
- Calculation of intake: to calculate the mean values of entire administration period for each test group, the values of each study period, i.e. premating (14 days), gestation (20 days) and lactation (13 days), were assessed according to days of treatment.

HAEMATOLOGY
- Time schedule for collection of blood: at termination for males and in the morning at PND 14 for females
- Anaesthetic used for blood collection: Yes, isolfurane
- Animals fasted: Yes
- How many animals: first 5 surviving parental males per group and the first 5 females with litters (in order of delivery) per group.
- Parameters checked: Leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), prothrombin time

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at termination for males and in the morning at PND 14 for females
- Anaesthetic used for blood collection: Yes, isolfurane
- Animals fasted: Yes
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), ypsilon-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), Total thyroxine (T4) and the amount of thyroid stimulating hormone (TSH).

NEUROBEHAVIOURAL EXAMINATION
- Functional observation battery: A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
1. Home cage observations: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
2. Open field observations (the animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes): behaviour on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait abnormalities, activity/arousal level, urine excreted within 2 minutes (amount/colour), rearing within 2 minutes, other findings
3. Sensory motor tests/reflexes (the animals were then removed from the open field and subjected to following sensory motor or reflex tests): reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behaviour during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
- Motor activity assessment: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group.

Sacrifice and pathology:

SACRIFICE
All surviving animals

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

ORGAN WEIGHTS
- Weights determined from all animals sacrificed on schedule: terminal body weight, Bulbourethral gland (Cowper’s gland), epididymides, glans penis, M. levator ani together with musc. bulbocavernosus (male animals), prostate, seminal vesicle with coagulating glands, testes, thyroid glands
- The following weights were determined in 5 animals per sex/ test group sacrificed on schedule (females with litters only, same animals as used for clinical pathology examinations): adrenal glands, brain, heart, kidneys, liver, spleen, thymus

HISTOPATHOLOGY (also see table under 'Any other information on materials and methods incl. tables'.
- Examinations included: all gross lesions, adrenal glands, brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), ovaries, oviducts, Peyer’s patches, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cords), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Other examinations:

FOOD ANALYSES
The supplier assayed the food used in the study for chemical and microbiological contaminants.

DRINKING WATER ANALYSES
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department as well as for the presence of microorganisms by a contract laboratory.

BEDDING AND ENRICHMENT ANALYSES
The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Statistics:

- Water and food consumption, body weight and body weight change: simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
- % live male day x, %live female day x: comparison of the dose group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.
- Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.
- Blood parameters: one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Males and females (except gestation and lactation periods): during the pre-mating period, a single treatment-related finding was observed in test group 3, i.e. reduced nutritional condition was observed for a female of test group 3 on study days 7 to 9. The finding was assessed to be related to impaired water and food consumption during the first week of treatment at a concentration of 8000 ppm. The animal recovered after reducing the test substance’s concentration in drinking water to 6000 ppm from study day 7 onwards. No additional clinical findings of toxicological concern occurred in pre-mating, mating and post-mating (males only) periods. Between pre-mating days 7 to 9, one male of test group 3 showed black encrusted nose. The finding was assessed to be incidental and not related to treatment. One male of test group 1 showed piloerection between mating days 7 to 10. The same was true for another male animal, which showed piloerection on mating day 7 only. The findings were assessed to be incidental and not related to treatment.
- Females during gestation: no clinical signs were observed in female animals of any test group during the gestation period.
- Females during lactation: during the lactation period, incidental findings occurred in individual animals of test groups 1-3: one female of test group 1 showed bloody/reddish vaginal discharge in combination with piloerection on 1 to 2 days after delivery. The animal recovered during the lactation period on PND 3. One animal of test group 2 showed semiclosed eyelids, slightly reduced general and nutritional conditions, piloerection and 2 injuries, i.e. at the anogenital region as well as at the left flank. An animal of test group 2 lost its complete litter 1 day after delivery. Until sacrifice, no other findings became evident. An animal of test group 3 showed moderately reduced general and slightly nutritional conditions, piloerection and abnormal flexibility of the right forelimb during the last 2 days before sacrifice. All findings were assessed to be incidental as no picture of a definitive toxicological mode of action became evident and a clear dose-response relationship did not occur.

Mortality:

no mortality observed

Description (incidence):

No animal died ahead of schedule.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A continuous decrease of mean body weight was observed in male animals of test group 3 (8000 ppm) during the first week of application (pre-mating period). Since the test substance’s concentration in drinking water was reduced to 6000 ppm from study day 7 onwards, the animals started to gain weight. However, mean body weight of these animals was significantly lower during the entire pre-mating (-16% to -10%) and mating (-10%) periods. No significant changes in mean body weights were observed for male animals of test groups 1 and 2 (500 and 2000 ppm).
In female animals of test group 3 (8000 ppm), a body weight loss could be observed on study day 3 (pre-mating period), but the animals recovered already between study days 3 and 7 and gained weight. Thus, no significant differences could be observed during the late pre-mating and the early gestation periods. Mean body weight values became significantly lower again on GDs 14 (-6.2%) and 20 (-8.5%). The significance continued during the entire lactation period, except on PND 4 because of the high standard deviation. No significant changes in mean body weights were observed for female animals of test groups 1 and 2 during the entire application period.

With regard to body weight change values, a similar picture could be drawn. In male animals of test group 3 (8000 ppm) a significant body weight loss was observed between study days 0-3 and 3-7 as well as 0-14 (pre-mating period). The body weight change value was only significantly higher between study days 7-10 after reducing the test substance’s concentration in drinking water. No significant difference was observed during the mating period. No significant changes in mean body weight change values were observed for male animals of test groups 1 and 2 (500 and 2000 ppm). In female animals of test group 3, mean body weight change value was significantly lower after 3 days of application (loss of weight) but significantly higher during the following 4 days. No significant differences could be observed during the late pre-mating and the early gestation periods. Mean body weight change values became significantly lower again between GDs 14-20 resulting in a lower value between GDs 0-20. No significant differences were observed during the lactation period. No significant changes in mean body weight change values were observed for female animals of test groups 1 and 2 during the entire application period.

The impaired body parameter development of male and female animals of test group 3 over nearly the entire administration period were most likely related to reduced water and food consumption as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in food consumption were observed for male and female animals of test group 3 (8000 ppm) during the first week of treatment, showing a maximum deviation to the control animals between study days 3 and 7 in males (-55%) and between study days 0 and 3 in females (-42%). Food consumption values turned to normal from study day 7 onwards clearly showing a relation to reduced test substance concentrations in drinking water.

During gestation, food consumption values were slightly, but significantly lower between GD 7-14 and 14-20. As the finding correlated to slightly impaired body weight increases in these animals, a relation to treatment was assumed. During lactation, no changes were observable for these animals. These changes were most likely related to reduced water consumption as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

No impairment of food consumption was observable for male and female animals of test groups 1 and 2 during the entire administration period.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was significantly decreased in both sexes of test group 3 (8000 ppm) during the first application week, showing a maximum deviation to the control animals between study days 0 and 3, i.e. by -81% in males and by -72% in females. As the water consumption in both sexes was still very low during the following days and impaired food consumption as well as body weight loss were manifest, the concentration of the test substance in the vehicle was reduced to 6000 ppm from study day 7 onwards. Although the animals recovered with regard to body weight development, water consumption was still significantly lower in male animals and female animals towards the end of pre-mating period as well as in female animals during the entire gestation period. During lactation, water consumption in female animals of test group 3 was only marginally affected as the concentration in drinking water was reduced by 50%. This adjustment maintained the dams at the desired target doses of the test substance during this period of increased water intake. Taken together, these changes were most likely related to palatability problems as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Water consumption values were also significantly reduced in male and female animals of test group 2 on several days of the study. However, these changes were still within the normal range for this rat strain and an impact on body weight development could not be observed. Thus, these changes in water consumption were without any toxicological relevance.

Water consumption values of male and female animals in test group 1 were once increased between pre-mating days 7 to 10. A relation to treatment was not assumed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among haematological parameters were observed.

In rats of both sexes of test groups 2 and 3 and in males of test group 1 absolute reticulocyte counts were significantly higher compared to controls. Reticulocyte counts in females of test groups 2 and 3 and in males of test groups 1 and 2 were not altered dose-dependently. Even the values in males of test group 3 were within the historical control range (absolute reticulocytes 99.5-166.1 Giga/L). In males of test group 2 relative neutrophil counts were significantly decreased and relative lymphocyte counts were significantly increased. Both parameters were not changed dose-dependently. Therefore, the mentioned changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

In females of test groups 1, 2 and 3 alanine aminotransferase (ALT) activities were significantly lower compared to controls and in females of test group 1 calcium levels were significantly decreased, but the alterations were not changed dose-dependently. Therefore, these alterations were regarded as incidental and not treatment-related.

In males of test group 3 sodium levels were significantly decreased. The values were marginally below the historical control range (sodium 140.9-146.1 mmol/L). However, this was the only changed clinical chemistry parameter in these individuals. Therefore, this change was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATION BATTERY
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
- Quantitative Parameters: in male animals, the number of rearings was significantly lower in test group 1 and significantly higher in test group 2. As no changes were observed for test group 3 animals, the findings were assessed to be incidental. No changes were observed for any female animal in test groups 1-3.
- Home cage observations: no test substance-related effects were observed.
- Open field observations: one male of test group 3 showed resistance to handling. A male of test group 1 showed piloerection. Both findings were assessed to be incidental.
- Sensorimotor tests/reflexes: no test substance-related effects were observed.

MOTOR ACTIVITY ASSESSMENT
Regarding the overall motor activity as well as individual intervals, no test substance-related deviations were noted for male and female animals of any test group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Absolute organ weights: when compared to control group 0 (set to 100%), the mean absolute weights of the terminal body weight (male) and the prostate were significantly decreased in test group 3. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights: when compared to control group 0 (set to 100%), the mean relative weights of the following organs were significantly increased in test group 3. The terminal body weight was significantly decreased in males of test group 3 resulting in significantly decreased mean absolute prostate and increased relative brain weights. The mean absolute prostate weight (0.966 g) and the mean relative brain weight (0.546%) were within the historical control data (absolute prostate weight: 0.907–1.100 g; relative brain weight: 0.494–0.639%. There were no treatment-related histopathological findings in these organs. However, the reduced terminal body weight of male animals in this test group was assumed to be treatment-related. The increased relative spleen weight in test group 3 males was also related to the decreased terminal body weight in this test group. This relative spleen weight (0.167%) as well as the absolute spleen weight (0.666 g) in test group 3 males were within the historical control range (0.132–0.196%; 0.502–0.728 g).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. In one male a red discoloration of the lungs was observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the spleen of male animals, the incidence of extramedullary haematopoiesis was minimally increased in test group 3. The severity was mostly minimal (very few, very small foci of extramedullary haematopoiesis) and there was no dose-response relationship. The increased incidence of the mostly minimal extramedullary haematopoiesis did not explain the significantly increased relative spleen weight in high-dose males. The affected males with extramedullary haematopoiesis did not show higher absolute and relative spleen weights than animals without this finding. 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. The macroscopically described red discoloration of the lungs of one male correlated with blood aspiration due to sacrifice, this male did not show relevant histopathological findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONES
In F0 males of test group 3, T4 levels were significantly decreased. However, in these individuals thyroid stimulating hormone (TSH) levels were not altered. Additionally, in F0 females as well as in PND4 and PND13 pups TSH and T4 levels were not altered. Therefore, the isolated T4 decrease in F0 males of test group 3 in combination with a reduced body weight of these individuals was regarded as a consequence of a reduced food intake. This is leading to a decrease of T4 and a tendency of a decreased TSH value in contrast to hypothyroidism with decreased T4 and increased TSH levels (Germain and Galton, 1985).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed; treatment-related effects most likely related to palatability problems; reduction from 8000 ppm to 6000 ppm from day 7 onwards

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed; treatment-related effects most likely related to palatability problems; reduction from 8000 ppm to 6000 ppm from day 7 onwards

Key result

Critical effects observed:

no

ANALYSES

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature, the homogeneous distribution of the test substance in the vehicle and the correctness of the prepared concentrations. Also drinking water, food analyses and analysis on bedding and enrichment were found to be suitable.

Conclusions:

GHS criteria not met

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-ethyl-1-methyl-1H-Imidazolium salt with N-cyanocyanamide (1:1)

EC Number:

609-330-5

Cas Number:

370865-89-7

Molecular formula:

C6 H11 N2 .C2 N3

IUPAC Name:

3-ethyl-1-methyl-1H-Imidazolium salt with N-cyanocyanamide (1:1)

Specific details on test material used for the study:

- Chemical name: EMIM Dicyanamide
- Purity: 99.81 area-% (HPLC, 210 nm); 100.0 area-% (HPLC, 225 nm)
- Test item No.: 12/0071-2
- Batch identification: 0013141983
- Content: 97.4 g/100 g determined by 1H-NMR spectroscopy
- Homogeneity: given (visually)
- Storage stability: the stability of the test item under storage conditions over the study period was guaranteed by the sponsor
- Expiry date: February 19, 2017
- Storage conditions: room temperature, under N2
- Physical state / color: liquid / colorless to brown, clear

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: about 12 weeks; females: about 9 weeks
- Weight at study initiation: (P) Males: 415.1 - 419.0 g; Females: 209.8 - 214.9 g
- Housing: during the study period, the rats were housed individually in polycarbonate cages type III (floor area of about 800 cm²), with the following exceptions:
1. During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany
2. During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
3. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study. Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, was added for environmental enrichment. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: yes (duration not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h)

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced twice weekly, at least.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water over a period of 7 days at room temperature was proven. At the beginning (during pre-mating), twice during gestation and once during lactation of the study samples of all concentrations were taken. Given that the test substance is completely miscible with drinking water, solutions were considered to be homogenous and no homogeneity analyses were carried out. The samples collected at the beginning of the administration period and during the lactation period were analyzed. An automated HPLC System with autosampler, pump and UV/VIS-detector was used. The column used was a Primesep 200, 5 µm, 250 mm x 3.2 mm (SIELC).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks maximum, daily from 16.00 h until 06.30 - 09.00 h
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals (males 28 days, females 55 days)

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

Test group 1;
- Reduced for females to 250 ppm during the lactation period.
- Mean dose value: 32 and 54 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Remarks:

- Test group 2;
- Reduced for females to 1000 mg/kg bw/day during the lactation period.
- Mean dose value: 94 and 177 mg/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

8 000 ppm

Remarks:

Test group 3;
- Reduced from 8000 ppm [152 and 396 mg/kg bw/d (m,f)] to 6000 ppm [401 and 439/494 mg/kg bw/d (m,f: 2. week of premating/gestation)] from study day 7 onwards. Test dose for females was 3000 ppm (511 mg/kg bw) during the lactation period.
- Mean dose value: 339 and 476 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a test study was performed in male and female Wistar rats (BASF SE, 2016: 01R0071/12N143). Each 4 male and female animals were treated via the drinking water at concentrations of 0, 4000 and 12000 ppm. Due to reduced food and water consumption and lower body weight/body weight change values in animals of the high-dose group, the concentration for this test group was reduced to 8000 ppm from study day 3 onwards. On study day 9, one male animal had to be sacrificed in a moribund condition. Additional findings did not occur in any of the other animals. The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioural changes and/or signs of overt toxicity. The parturition and lactation behaviour of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. On weekdays (except Saturdays, Sundays and public holidays) the parturition behaviour of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examinations included: abnormal behaviour in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the faeces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The following exceptions were notable:
1. During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
2. Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
3. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval
4. Females without litter and after weaning (PND 13) were weighed once a week

FOOD CONSUMPTION
Time schedule for examinations: Generally, food consumption was determined once a week, with the following exceptions:
1. Food consumption was not determined during the mating period.
2. Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
3. Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examination: generally, water consumption was determined once a week (over a period of max. 4 days), with the following exceptions:
1. Water consumption was not determined during the mating period (female F0 animals)
2. Water consumption of the F0 females with evidence of sperm was determined on GD 6-7, 13-14 and 19-20.
3. Water consumption of F0 females, which gave birth to a litter was determined for PND 3–4, 6-7, 9-10 and 12-13.
Water consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).
- Calculation of intake: to calculate the mean values of entire administration period for each test group, the values of each study period, i.e. premating (14 days), gestation (20 days) and lactation (13 days), were assessed according to days of treatment.

HAEMATOLOGY
- Time schedule for collection of blood: at PND 14
- Anaesthetic used for blood collection: Yes, isolfurane
- Animals fasted: Yes
- How many animals: the first 5 females with litters (in order of delivery) per group.
- Parameters checked: Leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), prothrombin time

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at PND 14
- Anaesthetic used for blood collection: Yes, isolfurane
- Animals fasted: Yes
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), ypsilon-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), Total thyroxine (T4) and the amount of thyroid stimulating hormone (TSH).

NEUROBEHAVIOURAL EXAMINATION
- Functional observation battery: A functional observational battery was performed in the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
1. Home cage observations: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
2. Open field observations (the animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes): behaviour on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait abnormalities, activity/arousal level, urine excreted within 2 minutes (amount/colour), rearing within 2 minutes, other findings
3. Sensory motor tests/reflexes (the animals were then removed from the open field and subjected to following sensory motor or reflex tests): reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behaviour during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
- Motor activity assessment: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five surviving females with litter (in order of delivery) per group.

SACRIFICE
- Maternal animals: All surviving animals

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

ORGAN WEIGHTS
- Weights determined from all animals sacrificed on schedule: terminal body weight, Bulbourethral gland (Cowper’s gland), M. levator ani, thyroid glands
- The following weights were determined in 5 animals per test group sacrificed on schedule (females with litters only, same animals as used for clinical pathology examinations): adrenal glands, brain, heart, kidneys, liver, spleen, thymus

HISTOPATHOLOGY (also see table under 'Any other information on materials and methods incl. tables'.
- Examinations included: all gross lesions, adrenal glands, brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), ovaries, oviducts, Peyer’s patches, rectum, sciatic nerve, skeletal muscle, spinal cord (cervical, thoracic and lumbar cords), spleen, sternum with marrow, stomach (forestomach and glandular stomach), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes
- Number of early resorptions: Yes

Statistics:

- Water and food consumption (parental animals), body weight and body weight change (parental animals), gestation days: simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
- Female mating indices, female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
- Mating days until day 0 p.c., %postimplantation loss, pups stillborn, %perinatal loss: pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
- Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
- % live male day x, %live female day x: comparison of the dose group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.
- Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.
- Blood parameters: one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians

Indices:

MATERNAL INDICES
Mating and fertility index: the pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. Additionally, gestation index, live birth index, implantations and post-implantation losses were recorded.

OFFSPRING VIABILITY INDICES
Pup number and status at delivery, viability index (ratio of number of live pups on day 4 vs day 0 after birth), survival index (ratio of number of live pups on day 13 after birth vs day 4 after birth) and sex ratio.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Females (except gestation and lactation periods): during the pre-mating period, a single treatment-related finding was observed in test group 3, i.e. reduced nutritional condition was observed for a female of test group 3 on study days 7 to 9. The finding was assessed to be related to impaired water and food consumption during the first week of treatment at a concentration of 8000 ppm. The animal recovered after reducing the test substance’s concentration in drinking water to 6000 ppm from study day 7 onwards.
- Females during gestation: no clinical signs were observed in female animals of any test group during the gestation period.
- Females during lactation: during the lactation period, incidental findings occurred in individual animals of test groups 1-3: one female of test group 1 showed bloody/reddish vaginal discharge in combination with piloerection on 1 to 2 days after delivery. The animal recovered during the lactation period on PND 3. One animal of test group 2 showed semiclosed eyelids, slightly reduced general and nutritional conditions, piloerection and 2 injuries, i.e. at the anogenital region as well as at the left flank. An animal of test group 2 lost its complete litter 1 day after delivery. Until sacrifice, no other findings became evident. An animal of test group 3 showed moderately reduced general and slightly nutritional conditions, piloerection and abnormal flexibility of the right forelimb during the last 2 days before sacrifice. All findings were assessed to be incidental as no picture of a definitive toxicological mode of action became evident and a clear dose-response relationship did not occur.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died ahead of schedule.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In animals of test group 3 (8000 ppm), a body weight loss could be observed on study day 3 (pre-mating period), but the animals recovered already between study days 3 and 7 and gained weight. Thus, no significant differences could be observed during the late pre-mating and the early gestation periods. Mean body weight values became significantly lower again on GDs 14 (-6.2%) and 20 (-8.5%). The significance continued during the entire lactation period, except on PND 4 because of the high standard deviation. No significant changes in mean body weights were observed for female animals of test groups 1 and 2 during the entire application period.

With regard to body weight change values, a similar picture could be drawn. In animals of test group 3, mean body weight change value was significantly lower after 3 days of application (loss of weight) but significantly higher during the following 4 days. No significant differences could be observed during the late pre-mating and the early gestation periods. Mean body weight change values became significantly lower again between GDs 14-20 resulting in a lower value between GDs 0-20. No significant differences were observed during the lactation period. No significant changes in mean body weight change values were observed for female animals of test groups 1 and 2 during the entire application period.

The impaired body parameter development of animals of test group 3 over nearly the entire administration period were most likely related to reduced water and food consumption as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in food consumption were observed for animals of test group 3 (8000 ppm) during the first week of treatment, showing a maximum deviation to the control animals between study days 0 and 3 in females (-42%). Food consumption values turned to normal from study day 7 onwards clearly showing a relation to reduced test substance concentrations in drinking water.

During gestation, food consumption values were slightly, but significantly lower between GD 7-14 and 14-20. As the finding correlated to slightly impaired body weight increases in these animals, a relation to treatment was assumed. During lactation, no changes were observable for these animals. These changes were most likely related to reduced water consumption as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

No impairment of food consumption was observable for emale animals of test groups 1 and 2 during the entire administration period.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was significantly decreased in test group 3 (8000 ppm) during the first application week, showing a maximum deviation to the control animals between study days 0 and 3, i.e. by -72%. As the water consumption was still very low during the following days and impaired food consumption as well as body weight loss were manifest, the concentration of the test substance in the vehicle was reduced to 6000 ppm from study day 7 onwards. Although the animals recovered with regard to body weight development, water consumption was still significantly lower in female animals towards the end of pre-mating period as well during the entire gestation period. During lactation, water consumption in animals of test group 3 was only marginally affected as the concentration in drinking water was reduced by 50%. This adjustment maintained the dams at the desired target doses of the test substance during this period of increased water intake. Taken together, these changes were most likely related to palatability problems as no other findings occurred which could give an explanation for the described effects. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Water consumption values were also significantly reduced in animals of test group 2 on several days of the study. However, these changes were still within the normal range for this rat strain and an impact on body weight development could not be observed. Thus, these changes in water consumption were without any toxicological relevance.

Water consumption values of animals in test group 1 were once increased between pre-mating days 7 to 10. A relation to treatment was not assumed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among haematological parameters were observed.

In rats of test groups 2 and 3 absolute reticulocyte counts were significantly higher compared to controls. Reticulocyte counts in females of test groups 2 and 3 were not altered dose-dependently. Therefore, these changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

In animals of test groups 1, 2 and 3 alanine aminotransferase (ALT) activities were significantly lower compared to controls and in animals of test group 1 calcium levels were significantly decreased, but the alterations were not changed dose-dependently. Therefore, these alterations were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATION BATTERY
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
- Quantitative Parameters: No changes were observed for any animal in test groups 1-3.
- Home cage observations: no test substance-related effects were observed.
- Open field observations: no test substance-related effects were observed.
- Sensorimotor tests/reflexes: no test substance-related effects were observed.

MOTOR ACTIVITY ASSESSMENT
Regarding the overall motor activity as well as individual intervals, no test substance-related deviations were noted for female animals of any test group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. As to fertility, one female animal of test group 2, which was not pregnant, did not show relevant gross lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All 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. One female of test group 2, which was not pregnant did not show relevant histopathological findings.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- Postimplantation loss: postimplantation loss was 8.6% in test group 0 (control), 10.8% in test group 1, 3.2% in test group 2 and 12.8% in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Total litter losses by resorption:

not examined

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

- Live birth indices: the rate live birth indices were similar in all test groups, i.e. 99.1% for test group 0, 95.7% for test group 1, 93.4% for test group 2 and 98.2% for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was similar in all test groups, i.e. 22.6 days (test group 0), 22.5 days (test group 1), 22.3 days (test group 2) and 22.4 days (test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation was similar in all test groups, i.e. 22.6 days (test group 0), 22.5 days (test group 1), 22.3 days (test group 2) and 22.4 days (test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- Female mating index: the female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 2.7 days for test group 0 (control), 3.1 days for test group 2, 2.5 days for test group 2 and 2.9 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female fertility index: most sperm positive rats delivered pups with the exception of one female of test group 2, which had sperm in vaginal smear but delivered no pups and showed no implants. Thus, the female fertility index was 90% in test group 2 and 100% in test groups 0, 1 and 3. These values reflected the normal range of biological variation inherent in the strain of rats.
- Gestation index: the gestation index was 100% in test groups 0, 1 and 2 and 90% in test group 3, since one female did only deliver a single dead female pup.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed; treatement-related effects (FC, WC, BW) most likely related to palatability problems; reduction from 8000 ppm to 6000 ppm from day 7 onwards

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): On PND 1, runts were found in 1 litter of test group 0 (1 female), in 2 litters of test group 1 (8 females), in 2 litters of test group 2 (1 male and 5 females) as well as in 1 litter of test group 3 (1 female). All values were within the range of the biological variation inherent in the strain of rats used for this study.

Mean pup body weights/pup body weight changes of all pups in test groups 1 and 2 were comparable to the control group.

A trend towards lower mean body weight could be observed beginning on PND 4 to 13, when mean body weights of male, female as well as male and female pups together were significantly lower, i.e. -9.6% for males, -11% for females as well as -10% for both. With regard to body weight change values, significant differences occurred in test group 3 in intervals of PNDs 1-4 (male pups), 4-7 (female pups) and 7-13 (males, females as well as both sexes together). Between PNDs 1-13, both gender gained significantly less weight. These changes might have been related to reduced food and water intake of the respective dams which in turn also gained less weight during lactation. A toxicological background could not be excluded completely, but was assumed to be rather unlikely.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- Live birth indices: the rate live birth indices were similar in all test groups, i.e. 99.1% for test group 0, 95.7% for test group 1, 93.4% for test group 2 and 98.2% for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- Sex ratio: the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- Pup number and status at delivery: the mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during PND 0-4 was 98.8% in test group 0 (control), 92.8% in test group 1, 85.7% in test group 2 and 99.1% in test group 3. The survival indicating pup mortality during PND 4-13 was 100.0% in all test groups and, therefore, not affected by the test substance.

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Anogenital disttance: anogenital distance of male and female pups was not significantly changed in any test group when compared to the control pups. When corrected for body weight, the anogenital indices were significantly higher in male pups of test groups 1 and 3 as well as in female pups of test group 2. Thus, these changes were considered to be incidental and not treatment-related.
- Nipple/areola anlagen: the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Effect levels (fetuses)

Key result

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

ANALYSES

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature, the homogeneous distribution of the test substance in the vehicle and the correctness of the prepared concentrations. Also drinking water, food analyses and analysis on bedding and enrichment were found to be suitable.

Applicant's summary and conclusion

Conclusions:

GHS criteria not met