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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-14 to 2018-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
- Basis for dose level selection
A high-dose level of 500 mg/kg bw/day was selected based on the findings of a previous rat study with alpha-iso-methyl ionone, which is the main component of the test substance (Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat, SPL Project Number 2153-002, 2006). Although effects were seen in the 90-day study at the high dosage of 500 mg/kg bw/day, it was considered a suitable high dosage for the present study.

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one
EC Number:
204-846-3
EC Name:
3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one
Cas Number:
127-51-5
Molecular formula:
C14H22O
IUPAC Name:
3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one
Test material form:
other: Liquid
Details on test material:
- Name of test material : Alpha-isomethylionone
- Molecular formula: C14-H22-O
- Molecular weight : 206.327
- Substance type: Organic
- Physical state:Liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: UT16050005
- Expiration date of the lot/batch: 28 May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable
- Vehicle: The control article (vehicle) was Corn Oil supplied by Sigma-Aldrich (Batch Number MKB29899V).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulations were prepared daily for the first 14 days, then weekly thereafter. Formulations were stored refrigerated (2 to 8°C) for the first 14 days, then room temperature (15 to 25°C) thereafter, in a sealed container, protected from the light.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Females nulliparous and non-pregnant: yes
- Age at study initiation: at least 12 weeks
- Weight at study initiation: males weighed between 308.3 and 405.0 g; females weighed between 184.4 and 256.5 g
- Housing: During the pre-pairing phase, animals were housed in groups of up to four (by sex and dose group). During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group housing after the pairing phase.
- Diet: Animals had ad libitum access to VRFI Rat and Mouse Breeder Diet (Special Diets Services Ltd, Witham, United Kingdom).
- Water: tap water ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30-70
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a suspension in Corn Oil.

Details on mating procedure:
- M/F ratio per cage: 1 / 1
- Females without evidence of mating by Pairing Day 10 were paired for up to an additional 5 days with proven males of the same dose group.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations prepared for use during Week 1 and 6 of the dosing phase were analyzed by HPLC to determine the achieved concentration.
Duration of treatment / exposure:
Males were dosed for up to 42 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]). Females were dosed 2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until LD 13, inclusive). Dosing was omitted for any female in or near parturition at the time of daily dosing.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 90 day toxicity study in rats, repeated oral (gavage) administration of the test article at dose levels of 5, 30, or 500 mg/kg bw/day resulted in changes in liver and spleen weights for males administered 500 mg/kg bw/day.
Furthermore, increases in blood creatinine, total protein, and cholesterol were noted for both sexes administered 500 mg/kg bw/day. Males also showed an increase in plasma albumin levels.
Microscopic examination showed hepatocyte enlargements (centrilobular and generalized) for both sexes administered 500 mg/kg bw/day. Follicular cell hypertrophy of thyroid and bone marrow hyperplasia was also noted for some males administered 500 mg/kg bw/day. Other changes were also apparent in the kidneys of males. As such, the no observed adverse effect level (NOAEL) was considered to the intermediate dose of 30 mg/kg bw/day.
However, no clinical observations or changes in body weight or food or water intake were noted which were considered indicative of toxicity. Based on the finding of this study, and given the shorter dosing period, the high dose level of 500 mg/kg bw/day was considered acceptable for use in this study. The oral route of administration was chosen because it is an acceptable and commonly used route of exposure for regulatory studies of this type.

Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded once during the predose phase, on the first day of dosing, and at weekly intervals thereafter and on the day of necropsy. Female body weights were recorded once during predose; on the first day of dosing; weekly prior to pairing and until confirmation of mating; on GD 0, 7, 14, and 20; and on LD 0, 1, 4, 7, 13, and 14 (prior to necropsy).

FOOD CONSUMPTION AND COMPOUND INTAKE
-The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.

Other:
Blood samples of adult animals for thyroid hormone analysis (2 x 0.6 mL [Serum Separator Tube], nominal) were withdrawn from the jugular vein on the day of necropsy; sampling was performed at a similar time on each occasion.
Pups culled on PND 4 had blood samples (1 x 0.6 mL [Serum Separator Tube], nominal) for thyroid hormone analysis withdrawn via decapitation to provide one pooled sample for each litter, where possible.
Pups sacrificed on PND 13 had blood samples (2 x 0.6 mL [Serum Separator Tube], nominal) for thyroid hormone analysis withdrawn by cardiac puncture from two male and two female pups from each litter.


Oestrous cyclicity (parental animals):
Estrous Cycle Determination
Daily vaginal washings were conducted for all females during acclimation (predose phase), from 1 week after animal transfer until the day prior to dosing. The stage of estrous was recorded, and only females with regular 4- or 5-day cycles were included on study. Daily vaginal washings were conducted from females from the start of dosing until the confirmation of mating and on the morning of LD 14.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, macroscopically examinations
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed by isoflurane anesthesia on Post-Pairing Day 14 (Day 43 of study)
- Maternal animals: All surviving animals were sacrificed by isoflurane anesthesia on LD 14 (those that achieved pregnancy)

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose).

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera. The thyroid for one pup/sex/litter was examined macroscopically.

Statistics:
Where tables/appendices are computer-generated, rounding of individual values may have occurred during the calculation of derived values. Therefore, recalculation of derived values from the individual data, as presented in this report, may have, in some instances, yielded minor variations.
Data from test-article treated animals were compared with control data. Statistical analyses were performed where appropriate.
Data were analysed using Pristima, SAS, or Tox reporting
Reproductive indices:
Mating index, Female Fecundity Index, Male Fecundity Index, Female Fertility Index, Male Fertility Index, % Pre-implantation Loss, % Post-implantation Loss, Gestation index
Offspring viability indices:
% post implantation survival index, % live birth index, Survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were reduced during the pre-pairing phase for males administered 500 mg/kg bw/day. Slightly low body weight gains were also observed for females administered 500 mg/kg bw/day over multiple phase periods, although no specific phase for these animals showed significant losses.
All differences were considered minor and within the scope of normal intergroup variation and considered non adverse.
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:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Hormone levels were affected in adult animals administered 500 mg/kg bw/day. The T4 levels were higher than concurrent controls on LD 14 for females administered 500 mg/kg bw/day, and TSH levels were low on LD 14 for test article-treated females. Male T4 values were unaffected on Post-Pairing Day 14, but TSH values were higher than controls for males administered 500 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A minor increase in the incidence and severity of follicular cell hypertrophy was noted in the thyroid, especially in males administered 500 mg/kg bw/day. This was characterized by an increase in the height of the follicular epithelium and basally located nuclei, and correlated with an increase in thyroid/parathyroid weights. In the rat, follicular cell hypertrophy is generally considered an adaptive change due to increased thyroid hormone metabolism in the liver and is commonly associated with hepatocyte hypertrophy, as was seen in the previous rat 90-day study. Therefore the thyroid findings were probably secondary and were without any influence on reproductive function.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hormone levels were affected in adult animals administered 500 mg/kg bw/day. The T4 levels were higher, than controls, on LD 14 for females administered 500 mg/kg bw/day, and TSH levels were low on LD 14 for test article-treated females. Male T4 values were unaffected on Post-Pairing Day 14, but TSH values were higher when compared with controls for males administered 500 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No post littering, test item-related incidence of pup death occurred.
Body weight and weight changes:
no effects observed
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:
no effects observed
Description (incidence and severity):
Hormone levels in pups were not affected. Total T4 and TSH values were variable but considered to be within normal variation and control values.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Administration of the test item to adult animals caused no test article-related change in the thyroid weight of a sample of PND 13 pups, compared with concurrent controls.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Ano-genital distance and nipple/areolae appeared to be unaffected.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 2: Summary of male reproductive performance

Treatment Group                                                  Control          5 mg/kg        50 mg/kg      500 mg/kg

Total males                                                          10                 10                  10                 10

   Unscheduled Deaths Prior to Cohabitation          0                  0                   0                  0

Males Cohabitated                                               10                 10                  10                 10

   Unscheduled Deaths During Cohabitation           0                  0                   0                  0

Males mating with at least 1 female                       10                  9a                  10                10

Males impregnating at least 1 female                     10                  9a                  10                10

Mating Index (%)                                                 100                90                 100               100

Fecundity Index (%)                                             100               100                100               100

Fertility Index (%)                                                100                90                 100               100

Mating index %   = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100

Fecundity index % = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100

Fertility Index %  = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100

a = Animal R0103 (Group 2 male) repaired with a spare female; cannotconfirm pregnancy status of spare female R0795. Male not included in calculations.

 

Table 3: Summary of female reproductive performance

Treatment Group                                                  Control          5 mg/kg        50 mg/kg      500 mg/kg

Total Females                                                   10                    10                  10                 10

   Unscheduled Deaths Prior to Cohabitation       0                     0                   0                  0

Females Cohabited                                           10                    10                  10                 10

   Unscheduled Deaths During Cohabitation        0                     0                   0                  0

Females Mated                                                 10                     9                   9                 10

Pregnant Females (including females with

   unconfirmed mating)                                     10                    10                   10                 10

      Pup(s) / Total Litter Dead                             0                     1                   0                  0

Non Pregnant Females                                       0                     0                   0                  0

Matings Per Day Periods Of Cohabitation

      Day 1                                                         3                     2                   4                  2

      Day 2                                                         1                     4                   0                  6

      Day 3                                                         3                     0                   1                  2

      Day 4                                                         2                     2                   3                  0

      Day 7                                                         1                     0                   0                  0

      Day 11                                                        0                     1                   0                  0

      Day 12                                                        0                     0                   1                  0

Mating Index %                                                100                   90                  90                100

Fecundity Index %                                            100                  100                100               100

Fertility Index %                                               100                  100                100               100

Mating index % = Mated females/females cohabited (excluding females sacrificed during Cohabitation) x 100

Fecundity Index % = Pregnant females/mated females (excluding females with an undetermined pregnancy status) x 100

Fertility Index % = Pregnant females/females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status) x 100

 

Table 4: Summary of parturition and litter data

 

Group 1

Group 2

Group 3

Group 4

Statistics

Number of dams delivering live pups

9

10

10

10

X

 

 

 

 

 

 

Number of dams with live pups on PND 13

9

9

10

10

X

 

 

 

 

 

 

Mean duration of gestation (days)

23.3

23.2

23.2

23.2

P3

 

 

 

 

 

 

Mean number of implantation sites

12.3

11.1

12.6

11.8

P3

 

 

 

 

 

 

Mean number of pups born

12.0

10.2

11.1

11.0

P3

 

 

 

 

 

 

Mean number of pups alive PND 1

11.6

10.1

10.7

10.4

P3

 

 

 

 

 

 

Mean % male pups PND 1

55.6

45.9

53.2

43.4

P3

 

 

 

 

 

 

Mean number of pups alive PND 4 before culling

11.6

11.2

10.6

10.1

P3

 

 

 

 

 

 

Mean number of pups culled PND 4

1.7

1.4

1.4

1.0

X

 

 

 

 

 

 

Mean number of pups alive PND 4 after culling

9.9

9.8

9.2

9.1

X

 

 

 

 

 

 

Mean number of pups alive PND 7

9.9

9.8

9.2

9.1

X

 

 

 

 

 

 

Mean number of pups alive PND 13

9.9

9.8

9.2

9.1

X

PND = Postnatal Day

P3 = Kruskal-Wallis and Wilcoxon

X = not analysed

 

Table 5: Summary of pup survival

 

Group 1

Group 2

Group 3

Group 4

Statistics

Number of dams delivering live pups

9

10

10

10

X

 

 

 

 

 

 

Number of dams with live pups on PND 13

9

9

10

10

X

 

 

 

 

 

 

Post-implantation survival index %

96.2a

88.0

93.5

94.0

P3

 

 

 

 

 

 

Live birth index %

96.2

90.0

96.4

94.5

P3

 

 

 

 

 

 

Survival index PND 1-4 %

100.0

100.0

99.3

95.0

P3

 

 

 

 

 

 

Survival index PND 4-7 %

100.0

100.0

100.0

100.0

X

 

 

 

 

 

 

Survival index PND 7-13 %

100.0

100.0

100.0

100.0

X

PND = Postnatal Day

a = Female R0405 excluded from calculation

P3 = Kruskal-Wallis and Wilcoxon

X = not analysed

 

Table 6: Summary of ano-genital distance (mm)

 

Number of litters examined

 

Group mean distance (mm):

 

PND 4

 

 

 

M

F

Group 1

9

Adjusted

4.2

-

 

 

(Unadjusted)

(4.1)

(2.0)

Group 2

9

Adjusted

4.0

-

 

 

(Unadjusted)

(4.0)

(1.9)

Group 3

10

Adjusted

4.2

-

 

 

(Unadjusted)

(4.3)

(2.2)

Group 4

10

Adjusted

4.0

-

 

 

(Unadjusted)

(4.1)

(2.0)

 

 

 

 

 

Statistics

 

 

P2

X

PND = Postnatal Day

P2 = ANCOVA and Dunnett’s

X = not analysed

 

Table 7: Summary of males nipples/areolae count

 

Number of litters examined

PND 13

Group 1

10

0

 

 

 

Group 2

9

0

 

 

 

Group 3

10

0

 

 

 

Group 4

10

0

PND = Postnatal Day

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) for adult animals is considered to be 500 mg/kg bw/day. The NOAEL for reproduction/fertility and for offspring development is 500 mg/kg bw/day as well.
Executive summary:

The objective of the study was to screen for effects of the test article on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition) and off-spring growth until Lactation Day (LD) 13. The study design followed OECD guideline number 421. The test article comprises predominantly of the ionone isomer, alpha-iso-methyl ionone (ca. 90%). Four groups of 10 male and 10 female Crl:WI(Han) rats were administered 0 (control article[vehicle]), 5, 50, or 500 mg/kg bw/day test item by oral gavage, at a constant dose volume of 4 mL/kg. The control article (vehicle) was corn oil.

All males survived to the end of the dosing period. No test item‑related premature deaths occurred in females. No toxicologically significant clinical observations were apparent in any group, including controls throughout the dosing period. Body weight gains were reduced during the pre-pairing phase for males administered 500 mg/kg bw/day. Slightly low body weight gains were also observed for females administered 500 mg/kg bw/day over multiple phase periods, although no specific phase for these animals showed significant losses. Food consumption values were generally unaffected, however, during the first few days of dosing, females administered 500 mg/kg bw/day did have slightly reduced food consumption, but this was back within normal expected limits after the first period (pre-pairing Day 1-4). Pre-pairing estrous cycles, fertility and reproductive performance, and parturition and litter data appeared to be unaffected. Pup weights, ano-genital distance, nipple counts, and survival were also not affected. Hormone levels were affected in adult animals administered 500 mg/kg bw/day. The T4 levels were higher than controls on LD 14 for females administered 500 mg/kg bw/day, and TSH levels were low on LD 14 for test article-treated females. Male T4 values were unaffected on Post-Pairing Day 14, but TSH values were higher when compared with controls for males administered 500 mg/kg bw/day. Hormone levels in pups were not affected. Test item-related changes in liver and thyroid/parathyroid weights were recorded for both sexes at 500 mg/kg bw/day, compared with concurrent controls. Group mean adjusted liver weights and thyroid/parathyroid weights at this dosage were higher than controls for both sexes. No macroscopic findings considered related to the treatment were recorded. A minor increase in the incidence and severity of follicular cell hypertrophy was noted in the thyroid, especially in males administered 500 mg/kg bw/day.

Administration of 500 mg/kg bw/day test item led to an effect on body weight gain for adult males prior to pairing and for females over multiple phase periods, although this change was considered not to be adverse. Pup weights and weight gains were considered unaffected. Hormone levels (T4 and TSH) were affected in adult females at this dosage during lactation, but only TSH levels were affected in males. In adults at 500 mg/kg bw/day, microscopic examination revealed a minor increase in the incidence and severity of follicular cell hypertrophy in the thyroid. The increase in adult liver weight at 500 mg/kg bw/day was consistent with data from a previous rat 90-day study with alpha-iso-methyl ionone in which liver histopathological examination showed hepatocyte hypertrophy at the high dose of 500 mg/kg bw/day. In the rat, follicular cell hypertrophy is generally considered an adaptive change due to increased thyroid hormone metabolism in the liver and is commonly associated with hepatocyte hypertrophy, as was seen in the previous rat 90-day study. Therefore the thyroid findings were probably secondary and were without any influence on reproductive function. All findings in this study were considered non adverse.

In conclusion, on the basis of these findings not being adverse in nature, the no observed adverse effect level (NOAEL) for adult animals is considered to be the high dose of 500 mg/kg bw/day. The NOAEL for reproduction/fertility as well as offspring development is 500 mg/kg bw/day.