Oils, animal, sulfated, sodium salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL (repro) = 1000 mg/kg/day (test item)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

From 2013, August 16 to 2014, April 2

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across from a GLP Guideline Study (OECD 422)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

yes

Remarks:

Devations are described below, The study integrity was not adversely affected by the deviations.

Principles of method if other than guideline:

List of protocol deviations:
1. On Day 4 of lactation, no clinical observations were registered in the computer for pup 5 of litter
56 (Group 2).
Evaluation: Sufficient information available. Moreover, no findings were noted on Day 3 and 5,
and its body weight was determined on Day 4.
2. Analysis of stability of the test substance under test conditions was not performed as part of
this study, since Total Organic Carbon (TOC) analysis is a general method that does not
distinguish original substance from hydrolysis product(s).
Evaluation: Data from the sponsor indicated stability in water for >24 hours at a concentration
of 5%. As all formulations (2, 6 and 20%) were prepared daily within 4 hours prior to dosing, it
can be assumed that the formulations under the current study conditions were also sufficiently
stable.
3. Carbon analysis of formulations were recorded electronically using Shimadzu TOC-Control V
version 2.10 (Shimadzu, Kyoto, Japan).
Evaluation: omission from the protocol. Appropriate method was used.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

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

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

Source F0: Charles River Deutschland, Sulzfeld, Germany.

Age at start F0-treatment: Approximately 11 weeks.

Number of F0-animals 40 females and 40 males.

Acclimatization F0: At least 5 days prior to start of treatment.

Health inspection F0: Upon receipt of the animals.

Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Identification F0: Earmark and tattoo.

Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes
and placentas cleaned up, nest build up and/or feeding of pups started).
Females that were littering were left undisturbed.

Number of pups: 430 pups.

Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air
changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on
the outcome of the study.

Accommodation
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).

Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).

Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were
individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams
the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm.
Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x
20.3 cm) without cage-enrichment, bedding material, food and water.
Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Spezialdiäten GmbH, Soest, Germany).

Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started).
Females that were littering were left undisturbed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As the validated method was not finalized yet, formulation samples were collected during in-life (31 October 2013) and stored in the freezer at ≤-15°C until analyses. However, as the NPOC method was not suited to measure the test substance in these frozen samples, additional samples were collected
after the in-life phase (19 December 2013). These formulations were prepared in the same manner as used during the study.
For quantitative analysis of the test substance in formulation, Total Organic Carbon (TOC) analysis was used (see project 503334). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability could not be determined since TOC analysis is a general method that does not distinguish original substance from hydrolysis product(s); therefore results on stability assessment were not reported.
Accuracy of preparation was considered acceptable if mean measured concentrations were 90-110% of target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female no. 47 (Group 1) was not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animal per sex per dose

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Main observation and examinations have been described in the repeated-dose study.
General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any
deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Not reported

Sperm parameters (parental animals):

From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Litter observations:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were
evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

See repeated dose toxicity

Postmortem examinations (offspring):

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed tofollow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%)
Fertility index (%)
Conception index (%)
Gestation index (%)
Duration of gestation Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check
Percentage live females at First Litter Check
Percentage of postnatal loss Number of dead pups before planned necropsy
Viability index

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically significant clinical signs were noted during the observation period.
Incidental findings that were noted included piloerection and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted.
The statistically significant changes in body weight gain noted for males at 100 mg/kg were not considered toxicologically relevant as no dose response relationship was observed and the individual values were within normal limits.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The statistically significantly decreased prothrombin time for males treated at 300 mg/kg was considered to be of no toxicological relevance as this occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment up to 1000 mg/kg.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
The statistically significant increase noted for number of ambulations for males at 100 mg/kg was not considered toxicologically relevant as the individual values were within normal limits and no dose response relationship was seen.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Possible treatment-related findings were recorded in the heart of males and adrenal glands of females.
Heart, males:
- an increase in severity (slight degree) of mononuclear inflammation in 1/5 males treated at 1000 mg/kg, compared to minimal degrees in 1/5 males treated at 0 mg/kg, in 1/5 males at 100 mg/kg and in 1/5 males at 300 mg/kg.
- an increase in incidence and/or severity of myofiber degeneration (2/5, up to slight) in males treated at 1000 mg/kg, compared to minimal degree in 1/5 males treated at 300 mg/kg and absence of this finding in control males and males treated at 100 mg/kg.
Adrenal glands, females: an increase in incidence of vacuolation of the zona glomerulosa in 4/5 females treated at 1000 mg/kg, compared to 1/5 females treated at 0 mg/kg, 1/5 females at 100 mg/kg and 1/5 females at 300 mg/kg.
The remainder of microscopic findings recorded, were within the normal range of background pathology encountered in Wistar (Han) rats of this age.
In each group one or two females were not pregnant. No treatment-related findings were recorded in the reproductive organs of those females and their matching males.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

no effects observed

No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
At 300 mg/kg, two females (nos. 63 and 70) had a relatively low number of corpora lutea, implantation sites and/or living pups at first litter check. As this was not noted at 1000 mg/kg and these low numbers are occasionally seen historically in control data, it was not considered toxicologically
relevant.
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.
Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The statistically significantly change noted for sex ratio for pups at 300 mg/kg (71% female pups at 300 mg/kg compared to 52% female pups at the control group) was not considered toxicologically significant as this change was not noted at the higher dose of 1000 mg/kg.
Incidental findings of pups consisted of blue spot on several body parts and scabbing of the snout.
The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Two pups at 100 mg/kg and one pup at 300 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).
No toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).
Developmental results:
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).
No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition,maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).
In conclusion, treatment with Castor Oil, Sulfated, Sodium Salt, 75% by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed parental toxicity for males at 1000 mg/kg. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 300 mg/kg for males, and at least 1000 mg/kg for females
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

NOAEL (developmental) = 1000 mg/kg/day (test item)

NOAEL (developmental) = 640 mg/kg/day (active ingredient)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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 Guideline Study (OECD 414)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

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

Details on test animals or test system and environmental conditions:

Age at mating: 9 -11 weeks old and weighing at least 140 g

Mating (overnight at the supplier’s laboratory) will be confirmed by the presence of a vaginal plug in situ, or other evidence of mating if necessary. The day on which mating is confirmed will be designated as Gestation Day 0.
Delivered to Covance by Gestation Day 3, along with the Gestation Day 0 body weight data recorded at the supplier.

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: single housed
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 SDS Rat and Mouse Breeder Diet VRF1.
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.
Acclimatisation: limited to mated status
Animal health assessment: Performed prior to start of dosing.
Method of assigning to treatment groups: Randomisation procedure based on body weight and day of mating (i.e. all animals confirmed as mated on a specific day will be randomly allocated to the treatment groups).
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.
Procedure acclimatisation None.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route Oral: gavage.
Justification Possible route of human exposure
Frequency of dosing: Once daily from Gestation Days 6 to 20, inclusive.
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 from at least 15 minutes before dosing commences (excluding vehicle control group).
NB: stirring longer than half an hour is acceptable
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 information in section 8

Justification for dose levels selection:
High dose This is the limit dose and, based on the previous OECD 422 study, is considered not to cause any 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.

Details on mating procedure:

Mating (overnight at the supplier’s laboratory) will be confirmed by the presence of a vaginal plug in situ, or other evidence of mating if necessary. The day on which mating is confirmed will be designated as Gestation Day 0.
Delivered to Covance by Gestation Day 3, along with the Gestation Day 0 body weight data recorded at the supplier.

Duration of treatment / exposure:

15 days

Frequency of treatment:

Once daily from Gestation Days 6 to 20, inclusive.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Test article administration
Route Oral: gavage.
Justification Possible route of human exposure.
Dose order Group 1, 2, 3 then 4.
Groups may be dosed out of order when dosing is carried out by more than one technician.
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 from at least 15 minutes before dosing commences (excluding vehicle control group).
NB: stirring longer than half an hour is acceptable
Stirred during dosing Yes: continuously (excluding vehicle control group).
Other information None.
Method of preparation Formulated based on method supplied by Sponsor. Details of the methodology have been provided in the validation report (Study 153/19/HSII) and are retained in the study record.

Frequency of preparation
Group 1, 2 and 3: at least weekly.
Group 4: Daily and dosed within 8 hours of preparation.

Formulation analysis
Stability data: 2.5 and 22.5 mg/mL stable at ambient (approximately 26°C) for 7 days and 50 mg/mL stable at ambient for 1 day (study 153/19/HSII).
Homogeneity data: Homogeneity determined at concentration of 2.5 to 50 mg/mL (study 153/19/HSII).
Study sampling
Homogeneity Not required.
Study sampling
Achieved concentration: From formulations prepared for use on the first day of dosing (total 1 occasion). The following samples will be taken: 2 x 1 mL of each dose concentration pipetted in Eppendorf vial for analysis.
Due to the short stability for the 50 mg/mL (high dose), the formulation will be diluted down (1 in 10) to allow for accurate concentration analysis after shipment and result calculated back to the higher dose to confirm the concentration.
Residual samples: Not required in the first instance.
New Test Article batches Stability and homogeneity analyses will be performed for each batch of test article used on the study only if significant differences between the batches are advised by the Sponsor.
Storage container Uniquely labelled Eppendorf vials.
Storage of samples 15 to 25°C.
Sample shipment frequency As soon as is practicable following completion of each sampling occasion.
Sample shipment conditions 15 to 25°.

Method of termination (adults) Cervical dislocation following isoflurane anaesthesia, death confirmed by exsanguination.
If urgent euthanasia of an animal in extremis is necessary the animal may be killed by intraperitoneal injection of sodium pentobarbitone (overdose) and death will be confirmed by cervical dislocation and/or exsanguination.
Method of termination (fetuses) Subcutaneous injection of sodium pentobarbitone followed by confirmation of cessation of circulation.
Food removed No.
Sequence Replicate order, where possible.
Blood sampling See section 6.
Macroscopic examination All lesions will be recorded and retained.
Tissue preservation Retained in relevant fixative.
External peer review Not required.
Additional information Tissue or blood samples remaining after completion of post life procedures of control animals may be taken for purposes unrelated to this investigation.

Maternal examinations:

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/Frequency of observation
All animals/Twice daily: beginning and end (nominal) of the working day.

Clinical examinations
Additional observations may be recorded by exception.
Animal cohort / Frequency of observation
All animals / Daily from the start of dosing until 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 all observations will be based on each individual animal/group.
Additional observations may be carried out by technicians; the Study Director should be informed / involved in discussions.
Animal cohort/Frequency of observation
All animals /Daily: at 1 hour postdose.

Body weight
Individual body weight.
Animal cohort/Frequency of observation
All animals/Gestation Days 3, 6, 9, 12, 15, 17, 20 and 21.

Food consumption
Recorded in g; calculated as g/animal/day.
Animal cohort/ Frequency of observation
All animals /Gestation Days 6, 9, 12, 15, 17, 20 and 21.

Tissue Retention Thyroid (in buffered 10% formalin)
Tissues Weight Thyroid (weighed post-fixation)
Additional information #Terminal body weight will be recorded for adjusted gravid uterus weight calculations only and will not be reported.

Histology
Tissue trimming, processing and embedding (E): Groups 1, 2, 3, 4: Thyroid, including decedents.
Microtomy and staining (E):Groups 1, 2, 3, 4: Thyroid, including decedents.

Pathology
Microscopic evaluation (E): Groups 1, 2, 3, 4: Thyroid, including decedents.
Additional information None

Ovaries and uterine content:

All lesions will be recorded and retained.
The following will be recorded as appropriate:
Caesarian data with corpora lutea:
Pregnancy status
Gravid uterine weight
Terminal body weight
Number and status of implantations
Number of corpora lutea
Uterine data:
Fetal status
Uterine horn sequence
Fetal identification
Fetal examination allocation

The uterus of any animal not apparently pregnant will be immersed in 10% ammonium sulphide solution for further examination of implantation sites.

Fetal examinations:

For each viable fetus:
Fetal weight, Placenta weight, Fetal sex, Ano-genital distance
Live fetuses: External examination
Dead fetuses: External examination and fetal sex only and then discarded.
Approximately one half of the fetuses in each litter will be examined for visceral abnormalities by microdissection. They will then be eviscerated and the carcasses will be processed for skeletal examination.
The remaining fetuses will be fixed in Bouin’s fluid and examined.
Fetal skeletal preparations: examined in 50% glycerol; retained in glycerol/propylene glycol. Fetuses fixed for visceral examination retained in relevant fixative.

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

Gross pathological findings:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

food consumption and compound intake

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Visceral malformations:

no effects observed

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

external malformations

visceral malformations

Remarks on result:

other: preliminary results

Abnormalities:

no effects observed

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

640 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in sexually adult males and females animals, as well as developmental toxicity in the offspring. However, developmental toxicity essentially means all the adverse effects induced during pregnancy that can be manifested at any point of the life span of the animal, which might in turn bring to structural abnormality, altered growth and/or organs development, functional deficiency, even death.

Table 3.7.1(a) of Annex I of EC Regulation 1272/2008 states that to classify compounds "for category 2 suspected human reproductive toxicant, reproductive effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects". To this extent the screening study performed on the substance does not provide any indication of direct adverse effect on reproduction. In fact up to the dose of 1000 mg/kg bw/day (test item) no effects were observed in both genders on reproduction, nor parental toxicity was detected. Moreover, no developmental toxic effects in the offspring were observed at all doses.

In conclusion, since no adverse effects on reproduction were observed, classification for reproductive/developmental toxicity is not warranted under Regulation 1272/2008.