Oils, fish, sulfated, sodium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: Read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read Across from a GLP well conducted study on the supporting structural related substance

Justification for type of information:

The justification for the use of the similar substance is detailed in the Read Across document attached in section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Castor oil, sulfated, sodium salt

EC Number:

269-123-7

EC Name:

Castor oil, sulfated, sodium salt

Cas Number:

68187-76-8

Molecular formula:

not applicable

IUPAC Name:

not applicable

Test material form:

liquid

Details on test material:

Test item name: SOLFORICINATO DI SODIO 80V

Test animals

Species:

rat

Strain:

other: Crl:WI(Han) from Charles River Laboratories, Margate, UK

Details on species / strain selection:

Known to be accepted by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at the start of dosiung: 6 to 8 weeks old;
female conidtions: nulliparous female
Housing Cages: conforming to the 'Code of practice for the housing and care of animals bred, supplied or used for scientific purposes' (Home Office, London, 2014).
Housing density: Groups of up to 5 of the same sex
Rooms: exclusive to study
Acceptable temperature range: 22°C +/- 3°C
Acceptable humidity range: 40 to 70 %.
Air changes: A minimum of 15 air changes/hour.
Photo-period: 12 hours nominal.
Exceptions Where experimental procedures dictate, they will be documented in the study record.
Diet: Ad libitum access to 5LF2 EU Rodent Diet.
Water: Ad libitum access to water bottles (mains water supply).
Bedding: Suitable wood bedding (Aspen); changed weekly.
Environmental enrichment: Wooden aspen chew blocks (minimum). Other approved methods of enrichment may be used.
Analysis and certification: Diet and bedding – per batch (reviewed prior to use).
Water – periodic analysis.
Environmental enrichment – as available.
Central records maintained.
Food / water restriction: As required by experimental procedures.
Assessment on arrival All animals: clinical inspection for ill-health.
Duration of pre-dosing period: At least 2 weeks.
Animal health assessment: Performed prior to start of dosing.
Method of assigning to treatment groups: Total randomisation procedure.
Randomisation body weight check: Prior to start of dosing (animal assignment may be amended if unacceptable body weight differences occur). At the commencement of the study the weight variation of animals used should be minimal and not exceed 20% of the mean weight of each sex.
Treatment group positions in the cage battery: Animals will be positioned on batteries to avoid potential for cross contamination, To enable exposure of each cage/battery to similar environmental conditions
Identification of the test system: Subcutaneous electronic transponder. Cages will be identified with study information including study number and animal number/s.
Identification for FOB: To facilitate the functional observations the lowest numbered animal in each cage-pair will be identified by permanent ink circle drawn on the dorsum of each appropriate animal. The identification mark will be replaced as necessary.
Procedure acclimatisation None.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Route Oral: gavage.
Justification Possible route of human exposure.

Dose volume 20 mL/kg. Dose volumes will be calculated from the most recent body weight for each animal.
Storage conditions in the animal facility prior to dose administration
Formulation receipt area: 15 to 25°C
Animal room: 15 to 25°C
Stirred before dosing Yes: continuously for at least 15 minutes before dosing commences (excluding vehicle control
group).
Stirred during dosing Yes: continuously (excluding vehicle control group).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see attached report on section 8

justification for dose selection:
High dose Based on the findings of the previous OECD 422 study, a high dose level of 450 mg/kg/day was selected, as a dose level of 300 and 1000 mg/kg/day showed non-adverse kidney changes. Therefore this dose level is anticipated to show some test article related effects without causing overt toxicity. Intermediate dose Selected as an appropriate intermediate dose level based on guideline requirements of two to four fold increase in dose level. Low dose Anticipated to be a no observed effect level (NOEL). Background information In a previous OECD 422 combined repeat dose and reproduction/developmental toxicity screening test (Will Research Project Number 503342) on this test article was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days and females were exposed for up to 47 days. Adult males administered 1000 mg/kg/day showed a subtle increase in severity of mononuclear inflammation and in incidence and/or severity of myofiber degeneration in the heart. Increased seminal vesicles weights (absolute and relative) of were also noted at this dose level. In addition, relative kidneys weights were higher for males treated at 300 and 1000 mg/kg/day, although no correlations with blood parameters and histopathological examination were observed, therefore these changes were considered to be of no toxicologically significant. Adult females administered 1000 mg/kg/day showed an increased incidence of vacuolation of the zona glomerulosa of the adrenal glands. However, this was considered non-adverse. Increased uterus weights (absolute and relative) were noted following 1000 mg/kg/day. No reproductive toxicity was observed up to 1000 mg/kg/day and no developmental toxicity was observed in the offspring following maternal administration up to 1000 mg/kg/day. Based on these results, the No Observed Adverse Effect Levels (NOAEL) was derived as 300 mg/kg/day for males and 1000 mg/kg/day for females. A NOAEL for reproductive and offspring development was considered as 1000 mg/kg/day.

Duration of treatment / exposure:

90 d

Frequency of treatment:

daily, excluding day of necropsy

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day (nominal)

No. of animals per sex per dose:

main groups 10 males and 10 females
recovery groups 5 males and 5 females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Health monitoring: Observe animal in cage to monitor health status. Any animal which shows marked signs of ill health or overt toxicity may be isolated and may be killed and subject to the relevant terminal procedures.
Animal cohort: All Animals
Frequency of observation: Twice daily, beginning and end (nominal) of the working day.

Clinical examinations: Remove from cage and perform physical examination.Individual animal record maintained.
Animal cohort: All animals
Frequency of observation: Once daily from the start of dosing and on the day of terminal necropsy.

Post dosing observations: Observations related to time of dosing. Character and timing of reactions to treatment will be recorded. All observations, including absence of observations, will be recorded. The start time of the observations will be based on the last animal dosed per group/sex. Additional observations may be carried out by technicians; the Study Director should be informed / involved in discussions.
Animal cohort: All animals
Frequency of observation: Week 1: once daily at pre-dose and 1 hour postdose.

Functional observational battery (FOB): Where possible, the observations will be performed at the same time on each occasion. At the time of testing, the observer will be unaware of each animal’s dose level.

Detailed Clinical Observational measurements: Only abnormal or noteworthy signs will be documented in the individual and summary tables in the report. Observations will be recorded by exception.
Animals will be observed within the home cage, in the hand and in the arena (2-minute duration).
Animals will be observed for potential effects on: Behaviour, Gait, Posture, Respiration, Secretion, Excretion, Involuntary movements, Skin, Tail, Eyes, Pelage, Activity, In addition, animals will be subjected to elicited responses in order to assess their
reaction to a stimulus/manipulation. Quantitative measures of latency to first step, number of rears, faecal boli and urine pools will be recorded during the arena observation.
Animal cohort : dosed animals , Frequency of observation: Pre-treatment- Dosing Phase: Once weekly from week 1
Animal cohort: Recovery; Frequency of observation: Pre-treatment- Dosing Phase: Once weekly from week 1-Recovery phase: Weekly

Motor Activity
Locomotor activity will be recorded for 60 minutes and reported in 10 minute bins.
Locomotor activity will be recorded with the room lights turned off and white noise switched on. The white noise generator will be maintained (level recorded at the beginning and end of each session) at an appropriate level so that a sound meter registers between 60 and 80 dB (Decibels), during the course of the scheduled sessions. Animals will be allowed to acclimatise in their holding cages to the white noise for at least 10 minutes prior to any procedure taking place in the room. Recordings of activity will be made using the Kinder Motor Monitor system with the following parameters reported:
Basic movement: Simple tally of all horizontal beam breaks
Fine Movement: Measure of small movements such as grooming and head movements. Animal remains in a fixed point e.g. a single beam break with no other beams affected.
Total Ambulation: Measure of large movements. When a new beam is blocked and the anchor beam is broken i.e animal has relocated its whole body (e.g. a step forward).
Total Ambulation = X Ambulation + Y Ambulation
Total distance travelled Record of distance travelled around the cage.
Total Distance Travelled = HD Periphery Distance + Center Distance
Rearing (Event) Beam break on top grid.
Animal cohort: dosed animals Frequency of observation: Week 12
Animal cohort: recovery group Frequency of observation: Week 12; End of recovery period

Quantitative Assessments
The following parameters will be measured:
- Quantitative forelimb and hind limb grip strength
- Hind limb foot splay
Full details are outlined in the Covance Standard Operating procedure (SOP) HARRT R5/132.
Animal cohort: dosed animals Frequency of observation: Week 12
Animal cohort: Recovery group; Frequency of observation: Week 12; End of recovery period

Body weight
Individual body weight (Reported by Study Day Number).
The terminal body weight is recorded in Necropsy, after overnight fasting, immediately prior to necropsy.
Sporadic/decedent animals may not be fasted prior to terminal body weight.
Animal cohort: all animals; Frequency of observation: Pre-study: (randomisation body weight check); Day –4 (males) and Day -5 (Females); Once weekly from Day 1 and on the day prior to terminal necropsy (unfasted). A fasted terminal body weight will also be recorded immediately prior to necropsy.

Food consumption
Recorded in g; calculated as g/animal/day.
Animal cohort: dosed animals: Frequency of observation: At least once weekly from Week 1.
Animal cohort: Recovery group; Frequency of observation: At least once weekly from Week 1.

Ophthalmic examinations
Mydriatic agent administered prior to indirect ophthalmoscopy examination.
Animal cohort/ Frequnecy
Group 1, 2, 3, 4/Preatreatment
Group 1 and 4/ Week12
Group 3 and then 2/ If deemed necessary
Recovery:
Group 1 and 4/ Pre-treatment.
Group 1 and 4/ Week 12
Group 1 and 4 /Week 17

Oestrous cycles
The stage of the oestrous cycle will be recorded prior to necropsy.
Animal cohort/ Frequency of observation:
Females/ The stage of the oestrous cycle recorded on the day of necropsy within 1 hour of blood sample collection for thyroid hormones.

Sacrifice and pathology:

see attached study plan

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observation Battery (FOBS) No test article-related effects were noted following weekly open field arena observations, or Motor activity, grip strength or foot splay during Week 12, for test article-treated animals compared with controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A marginal increase in absolute epididymis weights was noted for 450 mg/kg/day males compared with controls, although this was not evident for recovery group males following a 4-week treatment-free period .
Thyroid weights were marginally increased for recovery group females previously administered 450 mg/kg/day, compared with concurrent controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The most notable macroscopic finding was the dose-related increase in incidence of mottled or pale liver observed for males from each test article treated group compared with controls. Mottled liver was only observed for one 450 mg/kg/day female. This was not evident following the 4-week recovery period.

Description (incidence and severity):

in progress

Details on results:

Thyroid hormones in progress
Pathology in progress

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

ca. 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

food consumption and compound intake

haematology

mortality

ophthalmological examination

urinalysis

2

Dose descriptor:

NOAEL

Effect level:

ca. 288 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

food consumption and compound intake

haematology

mortality

ophthalmological examination

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The substance was tested for OECD 408, 90 day in rats, oral. Some final evaluations are still ongoing (see attached study plan and declaration), however based on the information currently available the NOAEL is estabihsed at 450 mg/kg. bw/day (test item), 288 mg/kg bw/day (acive ingredient).