N,N”-hexane-1,6-diylbis(N’-alkylaryl)urea

Endpoint summary

Description of key information

Oral (OECD 422), rat: NOAEL ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Aug - 22 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline 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:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test), 2000

Deviations:

no

GLP compliance:

yes

Remarks:

LAUS GmbH: Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: 316-356 g (males), 205-230 g (females)
- Housing: Pre-mating: Groups of 5 animals per sex per cage in Makrolon plastic cages (MIV type, height 18 cm); Mating: 1:1 in
Macrolon plastic cages (MIII type, height 18 cm); Post-mating: Groups of 5 males per cage in Macrolon plastic cages (MIV type, height 18 cm), females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Sterilized sawdust was used as bedding material, paper as cage-enrichment/nesting material. During locomotor activity monitoring, animals were housed individually in a Hitemp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: pelleted rodent diet SM R/M-Z (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle: based on trial formulations performed at testing laboratory
- Concentration in vehicle: 1, 3 and 10 % (w/v)
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentration in formulations was in the acceptable range (100 ± 15% of nominal concentration) for all samples. Stability was shown for 5 h at room temperature at nominal concentrations 10 g/L and 100 g/L. Accurate preparation of the suspensions, homogeneous distribution of the test item in the suspensions and stability over 5 h at room temperature were demonstrated by analysis. The test item was separated with a Phenomenex Gemini-NX 2x100 mm column and detected with tandem mass spectrometry.

Duration of treatment / exposure:

males: for 29 days, starting 2 weeks before mating, during mating and up to the day prior scheduled necropsy
females: for 43-45 days, during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy)

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study, in which animals were orally exposed to 500 and 1000 mg/kg bw/day for 10 days (2014). No mortality, macroscopic findings and no effects on organ weights (liver and kidney) were observed. Uncoordinated movements on Day 4 at 500 and 1000 mg/kg bw/day and white discoloration of feces on Day 6 at 1000 mg/kg bw/day were noted. Slightly lower mean body weight gain and lower mean body weights were recorded at the highest dose level. Furthermore, lower mean absolute food consumption was observed at 500 and 1000 mg/kg bw/day. Therefore, 100, 300 and 1000 mg/kg bw were selected as the dose levels for the main study. The peak effect of occurrence of clinical signs occurred at 1 to 3 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests are to be conducted after dosing in the main study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, between 1 and 3 hours after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption was determined weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals per sex per group
- Parameters examined: white blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals per sex per group
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos), bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable), between 1 and 3 hours after dosing.
- Dose groups that were examined: all dose groups; 5 animals per sex
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex; fore- and hind-limb grip strength; locomotor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following tissues and organs were collected and fixed in 10% buffered formalin from selected 5 animals/sex/group: adrenal glands, (aorta), brain - cerebellum, mid-brain, cortex, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, (lacrimal gland, exorbital), (larynx), liver, lung, infused with formalin, lymph nodes - mandibular, mesenteric, (nasopharynx), (esophagus), ovaries, (pancreas), Peyer's patches [jejunum, ileum] if detectable, pituitary gland, preputial gland, prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, (skin), spinal cord -cervical, midthoracic, lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, (tongue), trachea, urinary bladder, uterus, vagina and all gross lesions (tissues/organs mentioned in parentheses were not examined, since no signs were noted at macroscopic examination)

The following tissues and organs were collected and fixed in 10% buffered formalin from all remaining animals and female with total litter loss: cervix, clitoral gland, coagulation gland, epididymides, female mammary gland area, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina and all gross lesions

Organ weight was determined from the following tissues/organs from selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, liver, kidneys, ovaries, spleen, testes, thymus, seminal vesicles including coagulating glands, thyroids including parathyroids, prostate and uterus

Organ weight was determined from the following tissues/organs from all remaining males: epididymides, testes

HISTOPATHOLOGY: Yes
The following organ and tissue samples were processed, embedded and cut at a thickness of 2-4 µm. Slides were stained with haematoxylin and eosin and examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of the control and high dose group were examined.
- The additional slides of the testes of the selected 5 males of the control and high dose group to examine staging of
spermatogenesis (stained with PAS/haematoxylin).
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, ovaries, uterus, and vagina) of female 42 that had a total litter loss.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Clinical signs:

no effects observed

Description (incidence and severity):

100, 300 and 1000 mg/kg bw/day, both sexes: Increase incidence of uncoordinated movements, erythema of the ears and pale feces. The signs were not considered to be adverse.

Mortality:

no mortality observed

Description (incidence):

100, 300 and 1000 mg/kg bw/day, both sexes: Increase incidence of uncoordinated movements, erythema of the ears and pale feces. The signs were not considered to be adverse.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

A dose-dependent increase in the incidence of uncoordinated movements, erythema of the ears and pale feces was noted. The incidence of uncoordinated movements was higher for animals in all treated groups, though they were also observed occasionally in the control group. Uncoordinated movements were noted for all animals at 300 and 1000 mg/kg bw/day, for 5 males and 9 females at 100 mg/kg bw/day and for 4 control males and 1 control female. This sign was not considered to be adverse as there was no toxicologically relevant effects seen for grip strength or locomotor activity.

Erythema of the ears was seen for 9 males and 7 females at 1000 mg/kg bw/day, 2 females each in the 100 and 300 mg/kg bw/day groups and 1 control female. This was clearly treatment-related, but in the absence of signs of ill health, it was not considered to be adverse.

Pale feces were noted for most or all animals at 300 and 1000 mg/kg bw/day. Pale feces were also noted for a few females at 100 mg/kg bw/day, though the incidence was much lower. This was likely related to the color of the cloudy, white formulations and was not adverse.

Scabbing or alopecia, rales, salivation, pale appearance (over 2 days for 1 male at 300 mg/kg bw/day), red, orange or brown staining of the neck and/or vagina, chromodacryorrhoea, piloerection (seen for a single female at 1000 mg/kg bw/day) were incidental findings seen for control and/or treated animals. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
Absolute food consumption was higher during the lactation period for animals in all treated groups (significantly higher for females at 100 and 1000 mg/kg bw/day). This was secondary to relatively low food consumption for control females. As such, these differences were not considered attributable to treatment.

HAEMATOLOGY
There were no treatment related effects on any haematology parameters with treatment up to 1000 mg/kg bw/day. Platelet counts were significantly higher for males at 300 mg/kg bw/day than controls. The value was within the range of data considered normal for this age and strain and in the absence of a dose dependent response and any other supporting changes in related parameters, it was not considered to be treatment related.

CLINICAL CHEMISTRY
There were no toxicologically relevant effects on clinical biochemistry parameters noted up to 1000 mg/kg bw/day. Glucose levels were significantly higher and bile acids were significantly lower for males at 1000 mg/kg bw/day than controls, respectively. As the differences from controls were slight (glucose), the data remained within the range considered normal for animals of this age and strain (glucose mean= 8.8, p95 = 11.9, bile acids mean = 27.6, p5= 9.70), and there were no findings from related parameters to indicate an effect of treatment, these were not considered toxicologically relevant.

NEUROBEHAVIOUR
There were no toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength observed up to 1000 mg/kg bw/day. Animals of both sexes had significantly increased forelimb grip strength at 1000 mg/kg bw/day. Grip strength was also increased for males at 100 mg/kg bw/day. This was not considered to be toxicologically relevant as the effect was at least partially attributable to relatively low values obtained for controls (females) and decreased grip strength would be expected if the differences from controls were attributable to treatment related toxicity. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
No toxicologically relevant changes in organ weights and organ to body weight ratios were seen. Absolute brain weights were significantly lower for females at 1000 mg/kg bw/day. No toxicological relevance was attributed to this, as the difference from controls was slight, no differences in brain to body ratios were seen and no corresponding microscopic findings were seen.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were attributable to treatment. Incidental findings noted for control and/or treated animals included nodule of the epididymis, reddish or dark red foci on or discoloration of the thymus, reduced size of the thymus, pelvic dilation of the kidneys, black discoloration and reduced size of the liver papillary process, thickened duodenum wall, tan focus on the clitoral gland, watery clear cysts on the kidney, enlarged mandibular lymph node, and alopecia. These were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. As such these were not considered treatment related.

HISTOPATHOLOGY
There were no test item-related microscopic findings. The histologic changes recorded were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations.

There was one female of the control group with a total litter loss. A cause for the loss of this litter could not be established from the sections examined.

The spermatogenic staging profiles were normal for all males examined.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to and including the highest dose

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (Zmarowski, 2015). Ten Sprague Dawley rats per sex and dose were treated via gavage with 100, 300, and 1000 mg/kg bw/day, respectively. The control group received the vehicle propylene glycol. Male animals were treated for 29 days, starting 2 weeks before the mating period, during mating and up to the day prior scheduled necropsy. Females were treated for 43-45 days, i.e. during 2 weeks before mating and during the mating period until successful copulation, and during gestation through at least day 4 after parturition. The doses were selected on the basis of data from a range finding study, in which 3 females per dose (500 and 1000 mg/kg bw/day) were treated orally for 10 days. No mortality, macroscopic findings and no effects on organ weights (liver and kidney) were observed. Uncoordinated movements on Day 4 at 500 and 1000 mg/kg bw/day and white discoloration of feces on Day 6 at 1000 mg/kg bw/day were noted. Slightly lower mean body weight gain and lower mean body weights were recorded at the highest dose level. Furthermore, lower mean absolute food consumption was observed at 500 and 1000 mg/kg bw/day.

In the main study, no mortality occurred during the study period that was considered to be related to treatment with the test substance. Animals in all treated groups, but particularly at 300 and 1000 mg/kg bw/day had higher incidences of clinical signs including uncoordinated movements, erythema of the ears and pale feces. As these signs were also observed for control animals (uncoordinated movements and erythema), reflected the color of the test substance formulations (pale feces) and occurred in the absence of any signs indicative of treatment related toxicity, they were considered treatment related but not adverse. No treatment-related effects were noted in functional observations, body weight, food consumption, haematology, biochemistry, or necropsy in males or females in any test substance group.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed. Therefore a NOAEL of ≥ 1000 mg/kg bw/day was derived for systemic toxicity in the OECD 422 study in male and female rats.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.