Alcohols, C9-11, branched and linear, ethoxylated, sulfates, sodium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun - 25 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun - 25 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Les Oncins, Saint-Germain-Nuelles, France
- Females nulliparous and non-pregnant: not reported
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 275 - 369 g; Females: 181 - 248 g
- Fasting period before study: No
- Housing: Single housed in polycarbonate cages containing appropriate bedding
- Diet: SSNIFF rat/mouse pelleted maintenance diet, batch No. 62874737 (SSNIFF Spezialdiäten GmbH, Soest, Germany), sterilized by irradiation; ad libitum (except during designated procedures)
- Water: Municipal tap water filtered with a 0.22 µm filter; ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 8 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: Water for injection

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The control and test item dose formulations were stirred continuously just before dosing and during dosing and were maintained under delivery conditions (at room temperature).

VEHICLE
- Concentration in vehicle: 7.5, 25.0 and 75.0 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: 0F607, 0F629, 0F632, 0F657

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were analysed by Gas Chromatography with FID detection (GC/FID) for the determination of Alcohols, C9-11(branched and linear), ethoxylated (1-2.5 EO), sulfates, sodium salts.
The test item concentrations in the administered dose formulations for groups 3 (100 mg/kg bw/day) and 4 (300 mg/kg bw/day) analysed in Weeks 1 and 4 remained within an acceptable range of variations (-3.4% to +4.0%) when compared to the nominal values (acceptance criterion: ± 10% of the nominal concentrations).
No test item was observed in the control dose formulations.
The lowest concentration (7.5 mg/mL) used for group 2 (30 mg/kg bw/day) was below the expected range of concentrations reported in the analytical method (i.e. 10 to 250 mg/mL in study No. 48807 VAS). Therefore, the results (-1.3% and +10.5%) are presented for information only.
Nevertheless, as described in the corresponding analytical study report, precision/accuracy was demonstrated for the analysis of dose formulations from 8 mg/mL to 300 mg/mL. As 7.5 mg/mL is very close to 8 mg/mL, the results (-1.3% and +10.5% in Weeks 1 and 4, respectively) were considered to be scientifically relevant.

Duration of treatment / exposure:

Males: At least 28 days (beginning during 14 days of pre-mating period)
Females: Maximum period of 63 days (14 days pre-mating until post-natal day 13)

Frequency of treatment:

Daily, 7 days/week

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study (Study No. 48808 TSR (Papineau, 2021)) performed in the same species in which the test item was administered daily by gavage to female Wistar-Han rats at the dose levels of 500 or 1000 mg/kg bw/day in water for injection for 10 days. The dosing volume was 4 mL/kg bw/day.
Under the experimental conditions of the study, adverse clinical signs, body weight loss and reduced food consumption were observed from 500 mg/kg bw/day associated with premature death at 1000 mg/kg bw/day. Based on the results of this study, the doses of 500 and 1000 mg/kg bw/day were considered to exceed the Maximum Tolerated Dose. Therefore, 300 mg/kg bw/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 100 mg/kg bw/day).

- Fasting period before blood sampling for clinical biochemistry: Yes. Min. of 14 hrs., max. of 18 hrs. before blood sampling.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily beginning upon arrival through termination. In addition to the daily cage side observation, animals were observed between 1 to 3 hours post-dose on the days of dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed on each weighing day

BODY WEIGHT: Yes
- Time schedule for examinations:
All animals (including all females prior to mating): Weekly; from at least Week -1, Day 1 and throughout the study
Mated females: GD 0, 4, 7, 11, 14, 17 and 20; PND 1, 4, 7, 10 and 13
Animals were weighed more often when necessary in order to monitor health status

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations:
All animals (including all females prior to mating): Weekly; from Day 1 until the start of the mating period
Mated females: For the periods: GD 0-4, GD 4-7, GD 7-11, GD 11-14, GD 14-17 and GD 17-20; PND 1-4, PND 4-7 and PND 7-13

WATER CONSUMPTION: A visual assessment of water consumption was made at each change of water bottle

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the orbital sinus)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (Min. of 14 hrs., max. of 18 hrs)
- How many animals: 5 animals/sex/group
- Parameters checked:
Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count, Platelet count, White blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count, Basophil count, Large unstained cells count. Coagulation parameters: Activated partial thromboplastin time, Fibrinogen, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the orbital sinus)
- Animals fasted: Yes (Min. of 14 hrs., max. of 18 hrs)
- How many animals: 5 animals/sex/group
- Parameters checked:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, Total bilirubin, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride

PLASMA/SERUM HORMONES: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the orbital sinus)
- Animals fasted: Yes (Min. of 14 hrs., max. of 18 hrs)
- How many animals: All parental animals
- The level of the thyroid hormone (T4) was determined by LC-MS/MS technology for males sampled at termination. Remaining plasma was kept at -80°C pending possible Thyroid Stimulating Hormone (TSH) analysis. Plasma samples obtained on PND 14 from parental females was kept at -80°C pending possible analysis.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations / Dose groups that were examined: First 5 males and females of each group at the end of the dosing period. For females this was performed on Day 13 post partum, after euthanasia of pups.
- Parameters evaluated:

Detailed Clinical Examination:
- In the cage: "touch escape" or ease of removal from the cage
- In the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis)
- In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia

Reactivity to Manipulation and Different Stimuli:
Touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.

Motor Activity:
A motor activity test was performed in an actimeter in order to assess motor activity in a novel environment.
Activity was monitored by an automated infra-red sensor equipment recording individual animal activity over a 60-minute period.
The following parameters were reported: Horizontal movements, vertical movements.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals, on completion of the mating period (a minimum of 28 days adminstration), after at least 14 hours fasting (maximum 18 hours)
- Maternal animals: All surviving animals on PND 14
Animals were euthanized by an intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all parental animals. This included examination of the external surfaces and all orifices; the cranial cavity and the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
A vaginal smear was made on the day of necropsy to allow correlation with histopathology in reproductive organs. The smears were stained with Harris Schorr.

ORGAN WEIGHTS
Adrenals, Brain (including medulla/pons cerebellar and cerebral cortex), Cowper’s (i.e. bulbo-urethral) glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbo cavernous muscle complex, Liver, Ovaries (with oviducts), Pituitary gland, Prostate (dorso-lateral and ventral), Seminal vesicles (including coagulating glands), Spleen, Testes, Thymus, Thyroid with parathyroids (after fixation), Uterus (horns and cervix)

HISTOPATHOLOGY
- The tissues identified below were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
- Histopathological evaluation was performed on the first five euthanized males and females of the control and high dose animals (gross lesions of all dose groups were evaluated)
- Histopathological evaluation was also performed on the reproductive organs and thyroid from any animal that did not conceive, or from pregnant females that did not deliver, to investigate possible causes: i.e. from males 1006, 1007, 3003, 3007, 4001 and 4010 and females 1508, 1509, 3503, 3509, 4501 and 4512

Adrenals, Brain (including medulla/pons cerebellar and cerebral cortex), Caecum, Colon, Cowper’s (i.e. bulbo-urethral) glands, Duodenom, Epididymides (Preserved in Modified Davidson’s fixative), Oesophagus, Eyes with Harderian glands (Preserved in Modified Davidson’s fixative), Femoral bone with articulation, Glans penis, Gut-associated lymphoid tissue (GALT), Heart, Ileum, Jejunum, Kidneys, Levator ani plus bulbo cavernous muscle complex, Liver, Lungs with bronchi, Lymph nodes (mandibular and mesenteric), Mammary gland area, Macroscopic lesions, Ovaries (with oviducts), Pituitary gland, Prostate (dorso-lateral and ventral), Rectum, Sciatic nerve, Seminal vesicles (including coagulating glands), Skeletal muscle, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with bone marrow, Stomach with forestomach, Testes (Preserved in Modified Davidson’s fixative ), Thymus, Thyroids with parathyroids, Trachea, Urinary bladder, Uterus (horns and cervix), Vagina

Statistics:

See Attachment 1

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Hypersalivation was noted during the premating period (1/10 females at 300 mg/kg bw/day) and the gestation and lactation periods (1/8 females and 3/8 females at 100 and 300 mg/kg bw/day, respectively).
- Loud breathing was noted in 1/10 males at 100 mg/kg/day. This finding was also noted in females during the premating period (1/10 females at 30 mg/kg bw/day) and the gestation and lactation periods (3/8 females at 300 mg/kg bw/day).
- Dull coat was also observed in 1/8 females showing hypersalivation and loud breathing at 300 mg/kg bw/day during the gestation period.

These findings were considered to be test item-related but non-adverse as they were recorded on a few occasions and did not impact the overall sanitary status of these animals.

All the other clinical signs (i.e. alopecia, thin fur, scabs, nodule on tail, nodules on the neck corresponding to lactating mammary glands, liquid discharge from the vagina, soft faeces, reflux at dosing, pallor of extremities, running in circles and/or short/broken teeth) were considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals, resulted from the gavage procedure and/or are commonly observed findings in this species and strain.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no test-item related effects on mean body weights or mean body weight changes during premating periods in males and females and during the post-mating period for males.
For further details, please refer to Table 1 under section "Any other information on results, incl. tables".
There were also no test-item related effects on mean body weights or mean body weight changes during the gestation and period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day and when compared with controls, a slightly higher water consumption was noted in females from Week 5 (end of gestation period) of the study. As the differences that affected 5/8 females (i.e. 62.5%) were not accompanied by other findings, they were not considered to be adverse. For further details, please refer to Table 2 under section "Any other information on results, incl. tables".

Other differences from controls were recorded at 30 and 100 mg/kg bw/day in Weeks 5 and 6. They were considered to be fortuitous as they were not dose-related and were no longer observed in Week 7 (end of lactation period).

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology:
There were no test item-related effects on haematology parameters at any dose level.
All the differences from controls, namely a trend towards a higher mean white blood cell count in males from 30 mg/kg bw/day, associated with higher mean neutrophil, lymphocyte, monocyte, eosinophil and/or basophil counts and a trend towards a lower mean white blood cell count in females from 30 mg/kg bw/day associated with lower mean neutrophil, lymphocyte, monocyte, basophil and/or large unclassified cells counts, were of low magnitude, were not statistically significant, were of opposite trends, were unrelated to microscopic findings and/or were poorly dose-related.
Therefore, these differences were considered to be unrelated to the test item treatment.

Coagulation Parameters:
There were no test item-related effects on coagulation parameters at any dose level.
All the differences from controls, namely higher mean fibrinogen concentration in females at 30 mg/kg bw/day and males and females at 100 mg/kg bw/day and/or reduced activated partial thromboplastin time in females at 300 mg/kg bw/day, were of low magnitude, were not statistically significant, were unrelated to microscopic findings and/or were poorly dose-related.
Therefore, these differences were considered to be unrelated to the test item treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical Chemistry:
There were no test item-related effects on blood biochemistry parameters at any dose level.
Differences from controls consisted of changes in mean total bilirubin in males at 30 mg/kg bw/day and in females from 30 mg/kg bw/day, mean biliary acid level in males at 100 mg/kg bw/day and females from 30 mg/kg bw/day, mean cholesterol level in males from 30 mg/kg bw/day, mean triglyceride level in males at 30 and 300 mg/kg bw/day and females at 100 mg/kg bw/day, alkaline phosphatase activity in males and females at 30 and 300 mg/kg bw/day, alanine aminotransferase activity in males at 30 mg/kg bw/day and females from 30 mg/kg bw/day.
In view of the low magnitude and the directions of the changes, the absence of related microscopic findings and/or as mean values were poorly dose-related, were not statistically significant and/or were of opposite trends, these findings were considered to be unrelated to the test item treatment.

Thyroid hormones:
There were no test item-related effects on mean T4 levels in males.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- Vaginal smears / oestrous cycle
- Genital abnormalities
- T4 levels (pups (male/female) and parental males)
- Anogenital distance
- Nipple development (males)
- Reproductive / viability parameters: gestation length, pre / post implantation loss, mating index, fertility index, gestation index, live birth index, sex ratio, pup / litter weights
- Organ weights: adrenals, brain, cowper’s (i.e. bulbo-urethral) glands, epididymides, glans penis, levator ani plus bulbo cavernous muscle complex, liver, ovaries (with oviducts), prostate, seminal vesicles (including coagulating glands), testes, thyroid with parathyroids (after fixation), uterus (horns and cervix)
- Histopathology: adrenals, cowper’s (i.e. bulbo-urethral) glands, epididymides, glans penis, levator ani plus bulbo cavernous muscle complex, liver, mammary gland area, ovaries (with oviducts), pituitary gland, prostate, seminal vesicles (including coagulating glands), testes, thyroids with parathyroids, uterus (horns and cervix), vagina
For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatement-related neurologic abnormalities.

Abnormal fur appearance at 300 mg/kg bw/day, presence of grooming at 100 and 300 mg/kg bw/day, presence of feces and urine at all dose levels, excessive quantity or abnormal appearance of feces at 100 mg/kg bw/day and low mean landing foot splay values at 30 and 300 mg/kg bw/day were considered to be of no toxicological importance as they were poorly dose-related, were not statistically significant and/or did not correlate with any other findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Key result

Critical effects observed:

no

Table 1: Body Weights and Body Weight Gains

Premating (males and females) and post-mating (males) periods:

Sex	Males				Females
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Mean body weight (g)
Day 1	309	314 (+2)	333* (+8)	310 (0)	213	214 (0)	211 (-1)	210 (-1)
Day 8	325	334 (+3)	353* (+9)	329 (+1)	218	221 (+1)	212 (-3)	214 (-2)
Day 15	340	353 (+4)	370* (+9)	348 (+2)	224	226 (+1)	215 (-4)	222 (-1)
Day 22	343	359 (+5)	370 (+8)	350 (+2)	/	/	283^a	/
Day 29	357	370 (+4)	383 (+7)	359 (+1)	/	/	307^a	/
Mean body weight change (g)
Days 1 to 8	+17	+20	+19	+19	+6	+7	+1	+3
Days 8 to 15	+14	+19	+17	+19	+5	+5	+2	+8
Days 1 to 15	+31	+39	+36	+38	+11	+12	+4*	+12
Days 15 to 22	+3	+5	+1	+2	/	/	+42^a	/
Days 22 to 29	+14	+11	+13	+9	/	/	+24^a	/
Days 1 to 29	+49	+56	+50	+49	/	/	+68^a	/

(): in brackets, percentage (%) differencevs.controls.

/: not applicable.

*: statistical significance, p<0.05.

^a: corresponding to one pregnant female for which no evidence of mating was noted.

 

 

Table 2: Water Consumption (% Full Water Bottle)

Sex	Females
Dose level (mg/kg bw/day)	0	30	100	300
Week 4	75%: 9/9	75%: 10/10	75%: 7/8^(a)	75%:7/8 50%:1/8
Week 5	75%:8/9 50%:1/9	75%:6/10 50%:4/10	75%:6/8 50%:2/8	75%:3/8 50%: 4/8 25%: 1/8
Week 6	75%:7/9 50%:2/9	75%:5/10 50%:4/10 25%:1/10	75%:5/8 50%:3/8	75%:3/8 50%: 5/8
Week 7	50%:5/8 25%:3/8	75%:1/10 50%:5/10 25%:4/10	50%:6/8 25%:2/8	75%:1/8 50%:2/8 25%: 5/8

(a): one missing value due to mating period.

In bold: test item-related findings.

Conclusions:

Administration of the test item, Alcohols, C9-11(branched and linear), ethoxylated (1-2.5 EO), sulfates, sodium salts, by once daily oral gavage was well tolerated.
The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 300 mg/kg bw/day in males and females based on the absence of adverse effects at this dose level.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C9-11, branched and linear, ethoxylated, sulfates, sodium salts

EC Number:

500-465-4

EC Name:

Alcohols, C9-11, branched and linear, ethoxylated, sulfates, sodium salts

Cas Number:

160901-28-0

Molecular formula:

CnH2n+1(OCH2CH2)mOSO3Na; n = 9 - 11, m = 1 - 2.5

IUPAC Name:

Alcohols, C9-11, branched and linear, ethoxylated, sulfates, sodium salts

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories France, Les Oncins, Saint-Germain-Nuelles, France
- Females nulliparous and non-pregnant: Not reported
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 275 - 369 g; Females: 181 - 248 g
- Fasting period before study: No
- Housing: Single housed in polycarbonate cages containing appropriate bedding

- Diet: SSNIFF rat/mouse pelleted maintenance diet, batch No. 62874737 (SSNIFF Spezialdiäten GmbH, Soest, Germany), sterilized by irradiation
. Ad libitum, except during designated procedures.
- Water: Municipal tap water filtered with a 0.22 µm filter, ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -24
- Humidity (%): 30 - 70
- Air changes (per hr): 8 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The control and test item dose formulations were stirred continuously just before dosing and during dosing and were maintained under delivery conditions (at room temperature).

VEHICLE
- Concentration in vehicle: 7.5, 25.0 and 75.0 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: 0F607, 0F629, 0F632, 0F657

Details on mating procedure:

- M/F ratio per cage: 1 / 1
- Length of cohabitation: For a maximum of 14 days
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear, referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: Single housed in polycarbonate cages containing appropriate bedding

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were analysed by Gas Chromatography with FID detection (GC/FID) for the determination of Alcohols, C9-11(branched and linear), ethoxylated (1-2.5 EO), sulfates, sodium salts.
The test item concentrations in the administered dose formulations for groups 3 (100 mg/kg bw/day) and 4 (300 mg/kg bw/day) analysed in Weeks 1 and 4 remained within an acceptable range of variations (-3.4% to +4.0%) when compared to the nominal values (acceptance criterion: ± 10% of the nominal concentrations).
No test item was observed in the control dose formulations.
The lowest concentration (7.5 mg/mL) used for group 2 (30 mg/kg bw/day) was below the expected range of concentrations reported in the analytical method (i.e. 10 to 250 mg/mL in study No. 48807 VAS). Therefore, the results (-1.3% and +10.5%) are presented for information only.
Nevertheless, as described in the corresponding analytical study report, precision/accuracy was demonstrated for the analysis of dose formulations from 8 mg/mL to 300 mg/mL. As 7.5 mg/mL is very close to 8 mg/mL, the results (-1.3% and +10.5% in Weeks 1 and 4, respectively) were considered to be scientifically relevant.

Duration of treatment / exposure:

Males: At least 28 days (beginning during 14 days of pre-mating period)
Females: Maximum period of 63 days (14 days pre-mating until post-natal day 13)

Frequency of treatment:

Daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study (Study No. 48808 TSR (Papineau, 2021)) performed in the same species in which the test item was administered daily by gavage to female Wistar-Han rats at the dose levels of 500 or 1000 mg/kg bw/day in water for injection for 10 days. The dosing volume was 4 mL/kg/day.
Under the experimental conditions of the study, adverse clinical signs, body weight loss and reduced food consumption were observed from 500 mg/kg bw/day associated with premature death at 1000 mg/kg bw/day. Based on the results of this study, the doses of 500 and 1000 mg/kg bw/day were considered to exceed the Maximum Tolerated Dose. Therefore, 300 mg/kg bw/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 100 mg/kg bw/day).

- Fasting period before blood sampling for clinical biochemistry: Yes. Min. of 14 hrs., max. of 18 hrs. before blood sampling.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily beginning upon arrival through termination. In addition to the daily cage side observation, animals were observed between 1 to 3 hours post-dose on the days of dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed on each weighing day

BODY WEIGHT: Yes
- Time schedule for examinations:
All animals (including all females prior to mating): Weekly; from at least Week -1, Day 1 and throughout the study
Mated females: GD 0, 4, 7, 11, 14, 17 and 20; PND 1, 4, 7, 10 and 13
Animals were weighed more often when necessary in order to monitor health status.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations:
All animals (including all females prior to mating): Weekly; from Day 1 until the start of the mating period
Mated females: For the periods: GD 0-4, GD 4-7, GD 7-11, GD 11-14, GD 14-17 and GD 17-20; PND 1-4, PND 4-7 and PND 7-13

WATER CONSUMPTION: A visual assessment of water consumption was made at each change of water bottle.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the orbital sinus)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (Min. of 14 hrs., max. of 18 hrs)
- How many animals: 5 animals/sex/group
- Parameters checked:
Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count, Platelet count, White blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count, Basophil count, Large unstained cells count. Coagulation parameters: Activated partial thromboplastin time, Fibrinogen, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the orbital sinus)
- Animals fasted: Yes (Min. of 14 hrs., max. of 18 hrs)
- How many animals: 5 animals/sex/group
- Parameters checked:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, Total bilirubin, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride

PLASMA/SERUM HORMONES: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the orbital sinus)
- Animals fasted: Yes (Min. of 14 hrs., max. of 18 hrs)
- How many animals: All parental animals
- The level of the thyroid hormone (T4) was determined by LC-MS/MS technology for males sampled at termination. Remaining plasma was kept at -80°C pending possible Thyroid Stimulating Hormone (TSH) analysis. Plasma samples obtained on PND 14 from parental females was kept at -80°C pending possible analysis.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations / Dose groups that were examined: First 5 males and females of each group at the end of the dosing period. For females this was performed on Day 13 post partum, after euthanasia of pups.
- Parameters evaluated:

Detailed Clinical Examination:
- In the cage: "touch escape" or ease of removal from the cage
- In the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis)
- In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia

Reactivity to Manipulation and Different Stimuli:
Touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.

Motor Activity:
A motor activity test was performed in an actimeter in order to assess motor activity in a novel environment.
Activity was monitored by an automated infra-red sensor equipment recording individual animal activity over a 60-minute period.
The following parameters were reported: Horizontal movements, vertical movements.

Oestrous cyclicity (parental animals):

The oestrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 am):
- During the 2 weeks of the pre-treatment period
- From the beginning of the dosing period during the pre-mating and mating periods, until the females were mated
A vaginal smear made on the day of necropsy just before euthanasia (scheduled death) to allow correlation with reproductive organs histopathology.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Testis weight, epididymis weight, stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number of pups born (live, dead and cannibalized) on PND1; Number and sex of pups alive on PND 1, 4, 7, 10 and 13; Individual Body Weight: Live pups on PND 1, 4, 7, 10 and 13; Anogenital distance (AGD) on PND 1 (normalized to the cube root of PND 1 body weight); Number of nipples and of areolae in male pups on PND13

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, on completion of the mating period (a minimum of 28 days adminstration), after at least 14 hours fasting (maximum 18 hours)
- Maternal animals: All surviving animals on PND 14
Animals were euthanized by an intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all parental animals. This included examination of the external surfaces and all orifices; the cranial cavity and the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
A vaginal smear was made on the day of necropsy to allow correlation with histopathology in reproductive organs. The smears were stained with Harris Schorr.

ORGAN WEIGHTS
Adrenals, Brain (including medulla/pons cerebellar and cerebral cortex), Cowper’s (i.e. bulbo-urethral) glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbo cavernous muscle complex, Liver, Ovaries (with oviducts), Pituitary gland, Prostate (dorso-lateral and ventral), Seminal vesicles (including coagulating glands), Spleen, Testes, Thymus, Thyroid with parathyroids (after fixation), Uterus (horns and cervix)

HISTOPATHOLOGY
- The tissues identified below were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
- Histopathological evaluation was performed on the first five euthanized males and females of the control and high dose animals (gross lesions of all dose groups were evaluated)
- Histopathological evaluation was also performed on the reproductive organs and thyroid from any animal that did not conceive, or from pregnant females that did not deliver, to investigate possible causes: i.e. from males 1006, 1007, 3003, 3007, 4001 and 4010 and females 1508, 1509, 3503, 3509, 4501 and 4512

Adrenals, Brain (including medulla/pons cerebellar and cerebral cortex), Caecum, Colon, Cowper’s (i.e. bulbo-urethral) glands, Duodenom, Epididymides (Preserved in Modified Davidson’s fixative), Oesophagus, Eyes with Harderian glands (Preserved in Modified Davidson’s fixative), Femoral bone with articulation, Glans penis, Gut-associated lymphoid tissue (GALT), Heart, Ileum, Jejunum, Kidneys, Levator ani plus bulbo cavernous muscle complex, Liver, Lungs with bronchi, Lymph nodes (mandibular and mesenteric), Mammary gland area, Macroscopic lesions, Ovaries (with oviducts), Pituitary gland, Prostate (dorso-lateral and ventral), Rectum, Sciatic nerve, Seminal vesicles (including coagulating glands), Skeletal muscle, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with bone marrow, Stomach with forestomach, Testes (Preserved in Modified Davidson’s fixative ), Thymus, Thyroids with parathyroids, Trachea, Urinary bladder, Uterus (horns and cervix), Vagina

Postmortem examinations (offspring):

SACRIFICE
- Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on PND 4 when blood was sampled.

PLASMA/SERUM HORMONES: Yes
- Time schedule for collection of blood: Day of euthanasia (blood was collected from the vena cava immediately after sacrifice)
- How many animals: 2 pups/litter at PND 4 and 1 pup/sex/litter at PND 13
- The level of the thyroid hormone (T4) was determined by LC-MS/MS technology for pups sampled on PND 13. Remaining plasma was kept at -80°C pending possible Thyroid Stimulating Hormone (TSH) analysis. Plasma samples obtained on PND 4 from pups was kept at -80°C pending possible analysis.

GROSS NECROPSY
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs, comprising also a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs and the internal reproductive organs.

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination: Macroscopic lesions and Thyroids with parathyroids

Statistics:

See Attachment 1

Reproductive indices:

Pre-implantation loss: ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) * 100
Post-implantation loss: ((Number of implantation sites - Number of live pups) / Number of implantation sites) * 100
Mating index: (Number of mated animals / Number of paired animals) * 100
Fertility index: (Number of pregnant female partners / Number of mated pairs) * 100
Gestation index: (Number of females with live born pups / Number of pregnant females) * 100

Offspring viability indices:

Live birth index: (Number of live born pups on Day 1 post-partum / Number of delivered pups) * 100
Viability index on Day 4 post-partum: (Number of surviving pups on Day 4 post partum (before culling) / Number of delivered pups) * 100
Lactation index on Day 13 post-partum: (Number of surviving pups on Day 13 post-partum / Number of surviving pups on Day 4 p.p. (after culling)) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Hypersalivation was noted during the premating period (1/10 females at 300 mg/kg bw/day) and the gestation and lactation periods (1/8 females and 3/8 females at 100 and 300 mg/kg bw/day, respectively).
- Loud breathing was noted in 1/10 males at 100 mg/kg/day. This finding was also noted in females during the premating period (1/10 females at 30 mg/kg bw/day) and the gestation and lactation periods (3/8 females at 300 mg/kg bw/day).
- Dull coat was also observed in 1/8 females showing hypersalivation and loud breathing at 300 mg/kg bw/day during the gestation period.

These findings were considered to be test item-related but non-adverse as they were recorded on a few occasions and did not impact the overall sanitary status of these animals.

All the other clinical signs (i.e. alopecia, thin fur, scabs, nodule on tail, nodules on the neck corresponding to lactating mammary glands, liquid discharge from the vagina, soft faeces, reflux at dosing, pallor of extremities, running in circles and/or short/broken teeth) were considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals, resulted from the gavage procedure and/or are commonly observed findings in this species and strain.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no test-item related effects on mean body weights or mean body weight changes during premating periods in males and females and during the post-mating period for males.
For further details, please refer to Table 1 under section "Any other information on results, incl. tables".
There were also no test-item related effects on mean body weights or mean body weight changes during the gestation and period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day and when compared with controls, a slightly higher water consumption was noted in females from Week 5 (end of gestation period) of the study. As the differences that affected 5/8 females (i.e. 62.5%) were not accompanied by other findings, they were not considered to be adverse. For further details, please refer to Table 2 under section "Any other information on results, incl. tables".

Other differences from controls were recorded at 30 and 100 mg/kg bw/day in Weeks 5 and 6. They were considered to be fortuitous as they were not dose-related and were no longer observed in Week 7 (end of lactation period).

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology:
There were no test item-related effects on haematology parameters at any dose level.
All the differences from controls, namely a trend towards a higher mean white blood cell count in males from 30 mg/kg bw/day, associated with higher mean neutrophil, lymphocyte, monocyte, eosinophil and/or basophil counts and a trend towards a lower mean white blood cell count in females from 30 mg/kg bw/day associated with lower mean neutrophil, lymphocyte, monocyte, basophil and/or large unclassified cells counts, were of low magnitude, were not statistically significant, were of opposite trends, were unrelated to microscopic findings and/or were poorly dose-related.
Therefore, these differences were considered to be unrelated to the test item treatment.

Coagulation Parameters:
There were no test item-related effects on coagulation parameters at any dose level.
All the differences from controls, namely higher mean fibrinogen concentration in females at 30 mg/kg bw/day and males and females at 100 mg/kg bw/day and/or reduced activated partial thromboplastin time in females at 300 mg/kg bw/day, were of low magnitude, were not statistically significant, were unrelated to microscopic findings and/or were poorly dose-related.
Therefore, these differences were considered to be unrelated to the test item treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry:
There were no test item-related effects on blood biochemistry parameters at any dose level.
Differences from controls consisted of changes in mean total bilirubin in males at 30 mg/kg bw/day and in females from 30 mg/kg bw/day, mean biliary acid level in males at 100 mg/kg bw/day and females from 30 mg/kg bw/day, mean cholesterol level in males from 30 mg/kg bw/day, mean triglyceride level in males at 30 and 300 mg/kg bw/day and females at 100 mg/kg bw/day, alkaline phosphatase activity in males and females at 30 and 300 mg/kg bw/day, alanine aminotransferase activity in males at 30 mg/kg bw/day and females from 30 mg/kg bw/day.
In view of the low magnitude and the directions of the changes, the absence of related microscopic findings and/or as mean values were poorly dose-related, were not statistically significant and/or were of opposite trends, these findings were considered to be unrelated to the test item treatment.

Thyroid hormones:
There were no test item-related effects on mean T4 levels in F0 males at any dose level.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- Vaginal smears / oestrous cycle
- Genital abnormalities
- T4 levels (pups (male/female) and parental males)
- Anogenital distance
- Nipple development (males)
- Reproductive / viability parameters: gestation length, pre / post implantation loss, mating index, fertility index, gestation index, live birth index, sex ratio, pup / litter weights
- Organ weights: adrenals, brain, cowper’s (i.e. bulbo-urethral) glands, epididymides, glans penis, levator ani plus bulbo cavernous muscle complex, liver, ovaries (with oviducts), prostate, seminal vesicles (including coagulating glands), testes, thyroid with parathyroids (after fixation), uterus (horns and cervix)
- Histopathology: adrenals, cowper’s (i.e. bulbo-urethral) glands, epididymides, glans penis, levator ani plus bulbo cavernous muscle complex, liver, mammary gland area, ovaries (with oviducts), pituitary gland, prostate, seminal vesicles (including coagulating glands), testes, thyroids with parathyroids, uterus (horns and cervix), vagina
For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatement-related neurologic abnormalities.

Abnormal fur appearance at 300 mg/kg bw/day, presence of grooming at 100 and 300 mg/kg bw/day, presence of feces and urine at all dose levels, excessive quantity or abnormal appearance of feces at 100 mg/kg bw/day and low mean landing foot splay values at 30 and 300 mg/kg bw/day were considered to be of no toxicological importance as they were poorly dose-related, were not statistically significant and/or did not correlate with any other findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related effects on estrous sycle during the pre-mating period.
Most examined females had mucification of the vagina, as expected for lactating females (pups starting to eat solid food from PND14); other control and high dose females were in diestrus. During this transitory period, diestrus stages are also expected to be observed in a few dams that have started cycling.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The testicular morphology was similar in control and high dose animals, with no evidence of disruption of the spermatogenic cycle.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day: Two females were euthanized on Gestation Days (GD) 25 and 26, respectively, due to absence of delivery. No clinical signs were observed during the gestation period and no necropsy findings were observed at macroscopic post-mortem examination. These females were not pregnant.

100 mg/kg bw/day: Two females were euthanized on Gestation Day (GD) 26 due to absence of delivery. Loud and/or fast breathing were noted in one of these females on GDs 23 to 25. No relevant clinical signs were observed during the gestation period for the other female. No necropsy findings were observed at macroscopic post-mortem examination. These females were not pregnant.

0 mg/kg bw/day: One female was euthanized on Gestation Day 26 due to absence of delivery. No clinical signs were observed during the gestation period and no necropsy findings were observed at macroscopic post-mortem examination. This female was not pregnant. One further female was euthanized on Gestation Day 26 due to absence of delivery. Pallor of extremities was noted prior to euthanasia and the left uterine horn was thickened at macroscopic post-mortem examination. This female was pregnant (3 implantation scars in the left uterine horn).

The findings for these animals were unrelated to treatment with the test item as pregnancy status was not dose related and microscopic examination of sexual organs and thyroid glands in couples for which females were not pregnant or did not deliver did not reveal any associated cause.

There were no effects on mating or fertility indices and al groups had similar pre-coital time intervals (number of days taken to mate).

There were no effects on the mean duration of gestation and on reproductive or litter data.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

2

Key result

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed up to and including the highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs or relevant external abnormalities were observed at any dose level.
Findings (emaciated appearance, dehydration, haematoma, scab and/or short tail) recorded in pups during the lactation period were not dose-related, were of isolated occurrence and/or were common observations in this species and strain of rats maintained under the experimental condition of this study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no effects on the incidence of pups found dead or cannibalized.
Number of pups found dead: 0/0/2/0 (0/30/100/300 mg/kg bw/day)
Number of pups cannibalized: 1/0/0/0 (0/30/100/300 mg/kg bw/day)

There were no effects on pup viability at any dose level.

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:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormones:
In PND13 pups and when compared with controls, a minimally increase in T4 concentrations was noted in both sexes, for which a test item relationship could not be ruled out. As differences were of small magnitude, were not statistically significant, were not associated with any other findings and/or as individual values were within the range of the control values, they were not considered to be adverse. For details on thyroid hormone pup data, please refer to Table 3 under section "any other information on results, incl. tables"

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on sex ratio (mean % of males) at any dose level.

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:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

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 1: Body Weights and Body Weight Gains

Premating (males and females) and post-mating (males) periods:

Sex	Males				Females
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Mean body weight (g)
Day 1	309	314 (+2)	333* (+8)	310 (0)	213	214 (0)	211 (-1)	210 (-1)
Day 8	325	334 (+3)	353* (+9)	329 (+1)	218	221 (+1)	212 (-3)	214 (-2)
Day 15	340	353 (+4)	370* (+9)	348 (+2)	224	226 (+1)	215 (-4)	222 (-1)
Day 22	343	359 (+5)	370 (+8)	350 (+2)	/	/	283^a	/
Day 29	357	370 (+4)	383 (+7)	359 (+1)	/	/	307^a	/
Mean body weight change (g)
Days 1 to 8	+17	+20	+19	+19	+6	+7	+1	+3
Days 8 to 15	+14	+19	+17	+19	+5	+5	+2	+8
Days 1 to 15	+31	+39	+36	+38	+11	+12	+4*	+12
Days 15 to 22	+3	+5	+1	+2	/	/	+42^a	/
Days 22 to 29	+14	+11	+13	+9	/	/	+24^a	/
Days 1 to 29	+49	+56	+50	+49	/	/	+68^a	/

(): in brackets, percentage (%) differencevs.controls.

/: not applicable.

*: statistical significance, p<0.05.

^a: corresponding to one pregnant female for which no evidence of mating was noted.

 

 

Table 2: Water Consumption (% Full Water Bottle)

Sex	Females
Dose level (mg/kg bw/day)	0	30	100	300
Week 4	75%: 9/9	75%: 10/10	75%: 7/8^(a)	75%:7/8 50%:1/8
Week 5	75%:8/9 50%:1/9	75%:6/10 50%:4/10	75%:6/8 50%:2/8	75%:3/8 50%: 4/8 25%: 1/8
Week 6	75%:7/9 50%:2/9	75%:5/10 50%:4/10 25%:1/10	75%:5/8 50%:3/8	75%:3/8 50%: 5/8
Week 7	50%:5/8 25%:3/8	75%:1/10 50%:5/10 25%:4/10	50%:6/8 25%:2/8	75%:1/8 50%:2/8 25%: 5/8

(a): one missing value due to mating period.

In bold: test item-related findings.

 

Table 3: Mean T4 (ng/mL) Concentrations in PND 13 pups

Dose level (mg/kg bw/day)	0	30	100	300
T4 (ng/mL)
F0 males at termination	37.21±3.970	36.70±7.498 (-1.4)	35.32±7.433 (-5.1)	36.41±4.262 (-2.2)
PND 13 male pups	42.20±6.505	44.64±6.913 (+5.8)	42.91±6.018 (+1.7)	47.33±4.686 (+12.2)
PND 13 female pups	45.08±5.453	44.55±6.646 (-1.2)	45.82±4.111 (+1.6)	49.32±4.376 (+9.4)

(): in brackets, percentage (%) difference vs.controls.

In bold: test item-related findings. No statistical significance vs.controls.

Applicant's summary and conclusion

Conclusions:

Administration of the test item, Alcohols, C9-11(branched and linear), ethoxylated (1-2.5 EO), sulfates, sodium salts, by once daily oral gavage was well tolerated in parent animals and pups at all dose levels.
Based on these results the following conclusions were drawn:
• The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 300 mg/kg bw/day in males and females based on the absence of adverse effects at this dose level.
• The No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 300 mg/kg bw/day, based on the absence of any findings at this dose level.
• The No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 300 mg/kg bw/day, based on the absence of adverse findings at this dose level.