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Description of key information

NOAEL rat (90d) = 450 mg/Kg/day (test item)

NOAEL rat (90d)= 288 mg/kg/day (active substance 62 -64%))

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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.
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

Duration of treatment / exposure:
90 d
Frequency of treatment:
daily, excluding day of necropsy
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
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
Details on study design:
- Dose selection rationale:
Based on the findings of the previous OECD 422 study (WIL Research Europe B.V), a high dose level of 450 mg/kg/day was selected because parental toxicity was observed for males at 1000 mg/kg. This consisted of a subtle increase in severity of mononuclear inflammation and in incidence and/or severity of myofiber degeneration in the heart. Both findings were slightly above the range of background findings of Wistar (Han) rats of this age.
Therefore a possible test item-related effect cannot be excluded. The NOAEL for parental toxicity for OECD 422 was then set at 300 mg/kg bw /day

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.

- Rationale for animal assignment (if not random): The rat was selected because it is a rodent species known to be accepted by various regulatory authorities.
- Post-exposure recovery period in satellite groups: 4 week
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.

Thyroid Hormones Sample Collection and Handling
Blood samples for thyroid hormones (2 x 0.8 mL [serum separator tubes], nominal) were withdrawn from the jugular vein of toxicity and recovery animals on Day 91 of the dosing phase and Day 29 of the recovery phase, respectively. Food was removed before sample collection, and samples were collected by controlled randomization (see Protocol Deviations).
Blood samples for thyroid hormones were gently inverted several times (approximately 10) to ensure that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator. Samples were allowed to clot for at least 30 minutes at ambient temperature prior to centrifugation at 2300g and 4°C for 10 minutes. Samples were then divided into uniquely labeled amber polypropylene tubes (one primary sample and one spare sample) as soon as practicable and were protected from light until frozen at < 10°C.
Only the primary sample was analyzed in the first instance by Covance; the second sample was stored as a spare for future analysis if required.
Total Triiodothyronine (T3)
Thyroid Stimulating Hormone (TSH)
Total Thyroxine (T4)



Statistics:
Where appropriate, data evaluation and statistical analysis were performed as follows using standard laboratory methods current at time of reporting.
Data for each sex were analyzed separately. Only data collected on or after the first day of dosing were analyzed statistically.
Analysis of variance (ANOVA) and pairwise comparisons, as applicable, were usedto analyze the following:
-Absolute body weight
-Body weight change
-Clinical pathology values
-Terminal body weight
-Absolute organ weight, organ:body weight percentage, and organ:brain weight
percentage.The pairwise comparisons of interest were Groups 2, 3, and 4 versus Group 1.Prior to performing the ANOVA, Levene’s test was performed to test for equality of variances between groups. Where Levene’s test was significant (P  0.05), a rank transformation (to stabilize the variances) was applied before the ANOVA was conducted (note: Levene’s test was not applied to the rank-transformed data). Where Levene’s test was not significant (P > 0.05), ANOVA was conducted.
-For comparisons to the control; if the group effect of the ANOVA was significant
(P <= 0.05), Dunnett’s test was used for pairwise comparisons between each treated and control group.
-If the ANOVA was not significant (P > 0.05), the Dunnett’s test results were not
applicable to the evaluation and were not reported or used to interpret study data.
For other comparisons (i.e., not against a single control):
-If the group effect of the ANOVA was significant (P  0.05), t-tests were used for
pairwise comparisons.
-If the ANOVA was not significant (P  0.05), the t-test results were not applicable to the evaluation and were not reported or used to interpret study data.
Where only two groups were available for analysis, a two-sample t-test was
performed. Data containing values above/below the limit of quantitation were not analyzed, and the tables were footnoted accordingly.
see further details in the attached full study report
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
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test article-related effects on thyroid hormones were observed after the 90-day dosing phase or following a 4-week recovery phase.
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.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test article-related microscopic findings were noted.
Microscopic findings were generally consistent with the usual background pattern of findings in rats of this strain and age.
Other effects:
no effects observed
Description (incidence and severity):
No test article-related effect was noted on the stage of estrus for females following test article administration, compared with controls.
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
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
Critical effects observed:
no

for tables refer to the attached full study report

Conclusions:
The substance was tested for OECD 408, 90 day in rats, oral. Under the experimental conditions the NOAEL is estabihsed at 450 mg/kg. bw/day (test item), 288 mg/kg bw/day (acive ingredient).
Executive summary:

The objective of the study was to determine the toxicity of the test article, following once daily oral (gavage) administration to the rat for 90 days. The 4-week recovery phase enabled an assessment of reversibility or persistence of any effects.
The test article is an industrial chemical.
Male and female Crl:WI(Han) strain rats were assigned to four groups, and the test article (in formulation) was administered via oral gavage at a volume of 20 mL/kg. The control article (vehicle) was purified water.

Assessment of toxicity was based on mortality, clinical and postdose observations, functional observational battery (FOBs), locomotor activity, body weights, food consumption, ophthalmic observations, stage of estrous, thyroid hormone analysis, and clinical and anatomic pathology.
No toxicologically significant clinical observations were recorded for the terminal sacrifice animals. Clinical observations were confined to transient instances of salivation noted for males administered 450 mg/kg/day and transient instances of vocalization noted for females administered 150 or 450 mg/kg/day. Body weights and food consumption were unaffected by the test article.
Test article-related increases in locomotor activity were observed at the end of the dosing phase for males and females administered 150 or 450 mg/kg/day. These
consisted of transient increases in basis movement, fine movement, total ambulations, total rears, and total distance traveled, generally during the first 20 minutes of assessments, thereafter being generally comparable to the controls. Habituation for total ambulations and distance traveled was also lower for females administered 450 mg/kg/day, compared to controls. In the absence of any supporting data or a lasting effect over the duration of the assessments, these findings were considered to be of no toxicological significance.
Clinical pathology assessments did not reveal any test article-related changes in hematology, coagulation, clinical chemistry, or thyroid hormone assessments.

Test article-related changes in urine samples were observed for animals administered 450 mg/kg/day. A higher incidence of traces of ketones were noted for urine samples from males administered 450 mg/kg/day, and a higher incidence of paler colored urine was observed for females administered 450 mg/kg/day, compared to controls. In isolation, these findings were considered not to represent an adverse test article-related effect and were of no toxicological importance.
No overt effect on the stage of estrus was noted for test article-related females compared with controls, and no test article-related macroscopic abnormalities were noted at necropsy. Furthermore, no test article-related organ weight changes were noted and no test article-related microscopic abnormalities were noted at the highest dose levels assessed.
In conclusion, oral (gavage) administration of 0, 50, 150, or 450 mg/kg/day of the test article to rats for 90 consecutive days was well tolerated and did not result in any adverse effects. The minor changes observed in animals administered 150 or 450 mg/kg/day were considered to be of minimal toxicological significance; as such, the no observed adverse effect level is 450 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
288 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1

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

Justification for classification or non-classification

Regulation EC 1272/2008 regarding classification criteria for substances (Annex I, table 3.9.1) states that "Substances are classified in category 2 for target organ toxicity (repeated exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations".

Moreover, at paragraph 3.9.2.9.2 it reads:" In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified, dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects.

Repeated-dose studies conducted in experimental animals are designed to produce toxicity at the highest dose used in order to optimise the test objective and so most studies will reveal some toxic effect at least at this highest dose. What is therefore to be decided is not only what effects have been produced, but also at what dose/concentration they were produced and how relevant is that for humans".

These "guidance values" are provided in table 3.9.3, and refers to effects observed in a standard 90 day repeated dose study in which classification is not applicable when "significant toxic effects" are detected over a dose of 100 mg/kg/day.

Based on the results of the 90day on the test item, the substance is not classified for repeated dose oral toxicity