2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2018 - 7 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity w ith the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide

EC Number:

600-519-8

Cas Number:

1040873-93-5

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide

Test material form:

solid: particulate/powder

Details on test material:

Test item: Blue Sema
Appearance: Black to brownish black, solid
CAS No: 1040873-93-5
EC No: 600-519-8

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 80 – 83 days (males and females)
- Weight at study initiation: 302 – 366 g for male animals; 205 – 250 g for female animals
- Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals/ cage
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally, by gavage. The route of application is selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at 5 +/- 3 °C until use.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 23, 69 and 230 mg/mL)
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 mL of each formulation (20, 60 and 200 mg/mL) to be administered to the animals and five aliquots of 5 mL control substance (vehicle) were taken. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 95 % at ca. 1 mg/mL and 103 % at ca. 200 mg/mL).
Homogeneity: the maximal difference between the measured concentrations was ≤ 9 % (9-10 replicates), therefore the Blue Sema in distilled water formulations were considered to be homogeneous.

Duration of treatment / exposure:

Male animals were dosed for 50 days (14 days pre-mating and 1-6 days mating in animals plus 30-35 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-6 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

corresponding to 0 mg dye/kg bw/d

Doses / concentrations 2

Dose / conc.:

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

Remarks:

corresponding to 100 mg dye/kg bw/d

Doses / concentrations 3

Dose / conc.:

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

Remarks:

corresponding to 300 mg dye/kg bw/d

Doses / concentrations 4

Dose / conc.:

1 150 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 1000 mg dye/kg bw/d

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary dose range finding study. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Parameters checked in table No. 1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals were additionally weighed on gestational day 10 in order to give accurate treatment volumes. Body weight was measured on day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

FOOD CONSUMPTION: Yes
- The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also be determined by weekly interval during post-mating period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the terminal necropsy
- Anaesthetic used for blood collection: Yes, Isofluran
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table No. 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the terminal necropsy
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES:
- Time schedule for collection: Blood samples were collected for determination of serum levels of thyroid hormones (T4, TSH) from all male animals at termination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.

HISTOPATHOLOGY: Yes
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, adrenal glands and pituitary and all organs showing macroscopic lesions of all adult animals were preserved. All organs showing macroscopic lesions and the the organs listed in Table No. 4. were preserved for five male and five female animals randomly selected from each group.

Other examinations:

Organ Weight
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight were reported. Relative organ weight (to body and brain weight) were calculated and reported. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed. The thyroid weight was determined – if relevant – after fixation. Paired organs were weighed together; absolute organ weight will be reported. Relative organ weight (to body and brain weight) were calculated and reported.

Statistics:

The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia on the head was noted for one female control animal (1/12) on Days 7-13 in the pre-mating period and on gestation days 0-12. This observation was detected at the weekly detailed clinical observations on Days 7 and 13 and on gestation days 0 and 7. Clinical signs were noted for one male animal (1/12) at 100 mg/kg bw/day as follows: moderately decreased activity between Days 13 and 19, abnormal limb position on Days 14-19, swelling on the chest (~1cm in diameter) on Days 14-21 and a scar at the same place on the chest between Days 17-33. This animal was symptom-free from Day 34 until the end of the study. At 300 mg/kg bw/day, piloerection was observed in one female animal on gestation days 22-23 until lactation day 1. In this group alopecia on the abdomen was observed for one female on gestation days 19-21 and on lactation days 0-14, until termination. Alopecia and dermal/ subcutaneous alterations (swelling, wound) in single animals were individual findings observed in non-treated rats and were independent from the treatment. Piloerection was due to the elaborated delivery of one female animal in the mid dose group and was considered not to be test item related. There were no clinical signs in animals administered 1000 mg/kg bw/day at the daily or at the detailed weekly clinical observations during the entire study.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality in the 100, 300 or 1000 mg/kg bw/day treatment groups during the course of study (male and female).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slight but statistically significant difference with respect to the control was only detected at the higher mean body weight in female animals at 1000 mg/kg bw/day on lactation day 13. This slight change was considered to be indicative of biological variation and not related to the test item.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significance with respect to the control was detected in female animals at 100 mg/kg bw/day at the slightly higher mean daily food consumption between gestation days 0-7 and 14-21.
The mean daily food consumption was comparable in the control and test item treated animals at 300 or 1000 mg/kg bw/day during pre-mating and post mating periods in male animals and during the course of pre-mating, gestation and lactation periods in female animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All examined hematology parameters were comparable in male animals in the control and in all test item treated groups. Slight but statistically significant differences with respect to the controls were noted for two parameters in female animals at 100 and 1000 mg/kg bw/day: lower mean corpuscular (erythrocyte) volume (MCV) and higher mean red blood cell (erythrocyte) count. These differences were considered to be toxicologically not relevant due to the minor degree and the lack of dose dependency.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

All examined clinical chemistry parameters were comparable in male animals in the control and in all test item treated groups. In the female animals, statistically significant difference with respect to the control was observed at the slightly higher mean concentration of sodium (Na+) and albumin (ALB) at 300 mg/kg bw/day and at the lower mean Urea concentration (UREA) at 100 mg/kg bw/day. These slight, but statistically significant differences were considered to have no toxicological relevance because all these changes were with low degree and there was no dose dependency.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In the male animals, there were no statistically significant or biologically relevant differences between the control and the 100, 300 or 1000 mg/kg bw/day groups in the weights of the examined organs at the end of the treatment period.
In the female animals at 300 mg/kg bw/day, the mean liver weights (absolute and relative to body weight) were slightly higher than in the control group.
In the female animals at 300 and 1000 mg/kg bw/day, statistical significance was noted for the slightly higher mean kidney weights (absolute only) when compared to the control group. A dose-dependent increase was observed; however, the increased kidney weight was not correlated with any histopathological findings.
These slight differences with respect to the control were considered to have no toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings in the organs or tissues in male animals in the control group (12/12).
In one male animal dosed with 100 mg/kg bw/day (1/12), hemorrhage in the thymus was observed.
At 300 mg/kg bw/day, in one male animal (1/12), the left side seminal vesicle was smaller than normal. Pea-size hemorrhage on the right lung and dark blue content in the cecum and colon were observed in one male (1/12) of this dose.
In five male animals at 1000 mg/kg bw/day (5/12), dark blue content in the cecum and colon was observed. One of these males (1/12) had pea-size hemorrhage on one right lobe of lungs. There were no macroscopic findings in the organs or tissues in female animals in the control and 100 mg/kg bw/day groups (12/12 in each).
In one female animal at 300 mg/kg bw/day, marked hydrometra was observed (1/12). Alopecia on the abdomen was noted for single female animals at 300 mg/kg bw/day (1/12) and at 1000 mg/kg bw/day (1/12). The cecum was smaller than normal and its wall was thickened at 300 and 1000 mg/kg bw/day (1/12, both).
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats.
The smaller than normal seminal vesicle had decreased amount of secrete, without any other pathological lesion.
Due to the lack of related histopathological alterations (inflammatory or other pathological lesion) these findings were considered to be toxicologically not relevant.
The macroscopically blue discoloration of intestinal content and the thickened cecum wall were not correlated with abnormal histological finding in the mucous membrane, therefore it was considered to have no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (1000 mg/kg bw/day). One male animal at 300 mg/kg bw/day had smaller than normal seminal vesicle (one side). Histological examination revealed decreased amount of secrete in the ductuli, without any other pathological lesion (inflammation, fibrosis etc. (1/1). This finding was considered as individual disorder without toxicological relevance. In one female animal (1/1) at 300 mg/kg bw/day, dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was associated with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 control male, 2/5 males and 2/5 females at 1000 mg/kg bw/day), acute hemorrhage in the lungs (1/1 male at 300 mg/kg bw/day; 1/5 male at 1000 mg/kg bw/day; lungs were evaluated histologically in one male animal in mid dose group on the basis of macroscopic finding) and in the thymus (1/1 male at 100 mg/kg bw/day; the thymus was evaluated histologically in one male animal in low dose group on the basis of macroscopic finding) were detected sporadically. The pulmonary emphysema and hemorrhages in the lungs and thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 control male; 1/5 male at 1000 mg/kg bw/day; 1/5 control female) is a physiological immune-morphological phenomenon, occurring also in non-treated rats and has no toxicological relevance.
The macroscopic findings in the intestines (blue discoloration of content in cecum and colon in male animals – 1/12 at 300 mg/kg bw/day; 5/12 at 1000 mg/kg bw/day – and thickened wall of the smaller than normal caecum in two female animals – 1/12 at 300 mg/kg bw/day; 1/12 at 1000 mg/kg bw/day) were not correlated with abnormal histological finding in the mucous membrane of the affected large intestines.
Hypoplasia of hair follicles was observed in one female animal at 1000 mg/kg bw/day (1/12) in accordance with focal alopecia on the abdominal region. In the lack of inflammatory lesions, Trichophyton or Microsporum bodies or Demodex mites, this finding was considered as an individual, non-infectious lesion, having no toxicological relevance.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in the animals.
The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no significant differences with respect to the control in the thyroid hormone (free T4 and TSH) levels in parental male animals or in offspring sampled on postnatal day 13 at any dose level.

Offspring:
There was no test item related effect on offspring’s extra uterine mortality. Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13. The anogenital distances (in male or female offspring) or nipple retention (male) were similar in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day). The body weight development of the offspring was unaffected by the test item. Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of test item related adverse effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Blue Sema administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; respectively to uncorrected doses of 115, 345 and 1149 mg/kg bw/day) by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats.

Executive summary:

A study according OECD TG 422 was performed to obtain initial information on the toxic potential of the test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally to rats. Blue Sema was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 20, 60 and 200 mg/mL calculated by the active ingredient content and corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control. Blue Sema was stable in distilled water in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day and in a refrigerator for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Blue Sema concentrations in the dosing formulations varied within the range between 89% and 109% of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 50 days). Females were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-57 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/ post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides of adult male animals were determined. Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and full histopathology examination.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

No mortality was observed at 100, 300 or 1000 mg/kg bw/day groups during the course of study (male and female). Clinical signs of systemic toxicity were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations nor at the functional observations. The behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period. Test item related changes in the body weight or body weight gain were not detected. The body weight development was not affected and it was comparable in the control and test item treated groups. The mean daily food consumption was similar in male or female animals in control and at 100, 300 and 1000 mg/kg bw/day during the entire study. A test item influence on the estrous cycle was not found at any dose level (100, 300 and 1000 mg/kg bw/day). There were no significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (100, 300 and 1000 mg/kg bw/day).

Hematological evaluation did not reveal adverse test item related changes in the examined parameters and there were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).

There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring).

Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 300 or 1000 mg/kg bw/day. Dark blue content was observed in the cecum and colon of some male animals at 1000 mg/kg bw/day and of one male at 300 mg/kg bw/day.

The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 or 1000 mg/kg bw/day groups at the end of the treatment period.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 1000 mg/kg bw/day groups.

No adverse effect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Based on these observations the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats was 1000 mg/kg bw/day and the NOAEL for F1 Offspring was 1000 mg/kg bw/day.