Alcohols, C16-18 and C18-unsatd., ethoxylated, sulfates, sodium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun - 12 Sep 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

adopted in 2016

Deviations:

yes

Remarks:

- maximum humidity not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C16-18 and C18-unsatd., ethoxylated, sulfates, sodium salts

EC Number:

500-345-1

EC Name:

Alcohols, C16-18 and C18-unsatd., ethoxylated, sulfates, sodium salts

Cas Number:

157627-95-7

Molecular formula:

UVCB

IUPAC Name:

Sulfuric acid, monoesters with alcohols, C16-18 and C18-unsatd., (even numbered), ethoxylated, sodium salts (>1 < 2,5 mol EO)

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, Saint-Germain-Nuelles, France
- Females (if applicable) nulliparous and non-pregnant: not reported
- Age at study initiation: 11 weeks
- Weight at study initiation: 187 - 365 g
- Fasting period before study: no
- Housing: Males and females were housed separately in groups of 5 during the pre-mating period. During mating, 1:1 male and female were housed together. During post-mating, males were group-housed with other males in groups of 5, while females were housed individually. Housing was in plastic cages containing appropriate bedding. For psychological/environmental enrichment, animals were provided with items such as a wooden gnaw block and shredded paper, except when interrupted by study procedures/activities. It is considered that there are no known contaminants in the enrichment that could interfere with the outcome of the study.
- Diet: pelleted diet (SAFE Reference No. A04C-10); ad libitum (except during designated procedures)
- Water: softened and filtered (9.2 µm) municipal drinking water; ad libitum (except during designated procedures)
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): ≥ 35%
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 /12 (except during any designated procedures)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared on a twice weekly basis and divided into aliquots to be dispensed on each dosing occasion. Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. Test item formulations were stirred for at least 30 minutes. The dosing volume was
5 mL/kg bw/day, adjusted to the most recent body weight measurements.

VEHICLE
- Concentration in vehicle: 18.6, 55.6 and 186 mg/mL for the 93, 278 and 930 mg/kg bw/day dose levels, respectively.
- Amount of vehicle: 5 mL/kg bw/day, adjusted to the most recent body weight measurements
- Lot/batch no.: C0272 (Expiry date: 31 May 2022); C0405 (Expiry date: 30 Sep 2022)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method: GC/FID (gas chromatography with flame-ionized detection; validated analytical procedure: 48866VAS.MET)
Time: first and last week of the study
Results: All formulations at 20, 60 and 200 mg/mL used on the first day of treatment were in agreement with acceptance criteria. The formulations were homogenous. No test item was found in the control dose formulation.

Duration of treatment / exposure:

males: 50 days (2 weeks before mating, during mating period, until the day before necropsy)
females: 2 weeks before mating, throughout mating, gestation, and lactation until the day before necropsy

Frequency of treatment:

once daily, 7 days / week

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a 14-day dose-range finding study in the rat conducted at Charles River Lyon (Study No. 20292894). In this study, the test item, was administered daily by the oral route (gavage) at dose levels of 300 and 1000 mg/kg bw/day to groups of 4 male and 4 female Wistar rats for 14 days. The test item was well tolerated and only induced minor changes in biochemistry and/or hematology parameters for both sexes as well as a minor increase in kidney weight at 1000 mg/kg bw/day for males. The dose of 1000 mg/kg bw/day was considered suitable as high dose for this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test.
- Rationale for animal assignment: After arrival, animals were randomly assigned to groups. Males and females were randomized separately. Before the initiation of dosing, one female at 278 mg/kg bw/day and one female at 930 mg/kg bw/day were replaced by spare animals due to irregular estrous cycle and lameness, respectively.
- Fasting period before blood sampling for clinical biochemistry: 16 h

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for clinical signs and at least twice daily for mortality (except on days of receipt and study termination where frequency was at least once daily); additionally once after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on each weighing day
- Twice after dosing and every 2 weeks, a specific individual out-of-cage examination for post-dosing hypersalivation and any associated clinical changes was performed.
- Animals were observed for specific clinical signs in a standard arena once before the first administration and at least weekly throughout the study (except during pairing or delivering).

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; from Week -1, on Day 1 and throughout the study. Mated females: GD 0, 3, 6, 9, 12, 15, 18 and 21; LD 1, 4, 7, and 13

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

FUNCTIONAL TESTS
- performed on 5 animals/sex/group during the last days of treatment
- Locomotor activity: The motor activity in a novel environment was monitored by a photobeam analysis system (Motor Monitor supplied by Kinder Scientific LLC, Poway, USA) for 1 h. The arena was divided by 7 and 15 equally spaced infrared beams in width and length, respectively; the time and the number of beams interrupted by the animal were recorded. Motor activity is divided into 2 types: Ambulatory activity (large animal movements such as walking) and fine movements (including grooming and head movements). The proportion of beams blocked by the rat for each type of activity was calculated.
- Functional Observation Battery (FOB): Behavioural and functional tests including startle response, pupillary and righting reflex tests and fore-limb grip strength test.
- Assessed parameters for motor activity: Horizontal and vertical movements were recorded by an automated infrared sensor over a 60-min period.

HEMATOLOGY, COAGULATION AND CLINICAL CHEMISTRY
- performed on 5 animals/sex/group at scheduled euthanasia
- Examined parameters for hematology (incl. coagulation): red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, reticulocyte count, platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count, large unstained cells count, activated partial thromboplastin time and prothrombin time.
- Examined parameters for clinical chemistry: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bile acids, total bilirubin, urea nitrogen, creatinine, calcium, phosphorus, total protein, albumin, globulin, albumin/globulin ratio, glucose, cholesterol, triglycerides, sodium, potassium, and chloride.

THYROID HORMONES (to be revised)
- Thyroid hormone levels (TSH and T4): determined at ??? (to be updated) in males and on LD14 for females, and additionally in 2 culled pups/litter on PND4. (did they or did they not? to be updated)

PREGNANCY OBSERVATIONS
- Examined parameters in pregnant females: date of mating, date of parturition (LD0), duration of gestation, and abnormalities of nesting or nursing behaviour.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily and used to determine the stage of the estrous cycle for each female from 2 weeks before treatment until identification of mating or separation from the male and at necropsy.

Sperm parameters (parental animals):

Parameters examined in the male parental generation:
- testis weight, epididymis weight
- other: For the testes from all adult males of Groups 1 (control) and 4 (high dose) and all males suspected to be infertile or which died before mating, in addition to the hematoxylin and eosin- stained slide, an additional slide was prepared stained with PAS and examined. A detailed, qualitative evaluation of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings were recorded, where appropriate.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes: 4/sex/litter as nearly as possible; excess pups were killed

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups (live and dead), external abnormalities, number, weight and sex of pups on PND 1, PND4, PND7, PND10 and PND13, Anogenital Distance (AGD) on PND1 (normalized with the cube root of body weight), presence of areola/nipples on PND13 for males, external and necropsy findings of dead pups

GROSS EXAMINATION OF DEAD PUPS:
not reported

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not examined

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals euthanized after completion of mating period (Days 50/51); maternal animals: all surviving animals euthanized on LD14; scheduled euthanasia was performed after overnight fasting; maternal animals who did not deliver were sacrificed on post-coitum Day 26 or 25 days after the last day of the mating period in case of no evidence of mating.

GROSS NECROPSY
For all scheduled necropsies, adult males and females had their terminal body weight recorded. A full macroscopic post-mortem examination was performed on all F0 animals (unscheduled or scheduled euthanasia). This included examination of the external surfaces and all orifices; the cranial cavity; the thoracic and abdominal cavities and organs and their contents; and the carcass. Special attention was paid to the reproductive organs. For females, the number of implantation sites was recorded additionally. The uterus of apparently non-gravid females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's fixative). Tissues identified were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. For details on the list of tissues weighed, collected, processed and evaluated, please refer to attachments under section "attached background material".

Postmortem examinations (offspring):

SACRIFICE
Unselected pups were euthanized on PND4; selected pups were euthanized on PND13.

GROSS NECROPSY
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. Pups were euthanized and submitted to macroscopic examination of the abdominal and thoracic cavities. Any abnormalities observed were recorded but not preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
Thyroid glands were preserved for 1 PND13 pup/sex/litter for subsequent histopathological examination.

Statistics:

Data are expressed as means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
Parametric/Non-Parametric evaluations: Levene’s test was used to assess the homogeneity of group variances. Groups were then compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if at least 1 of these tests was significant. If the overall F-test or Kruskal- Wallis test was found significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Non-Parametric evaluations: The groups were compared using an overall Kruskal-Wallis test. If no trend was found and the overall Kruskal-Wallis test was found significant, then the above pairwise comparisons were conducted using Dunn’s test.
Incidence evaluations: Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Data were generated with Provantis 10, apart from locomotor activity and thyroid hormone data, which were anlayzed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analyzed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least 1 group were analyzed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.

Reproductive indices:

Gestation index (in %)= (Number of females with live pups / Number of pregnant females) x 100

Pre-birth loss (in %, presented as Post-implantation loss/litter) = ((Number of implantations- Number of pups born) / (Number of implantations)) x 100

Post-implantation loss = ((Number of implantation sites - Number of live pups) / Number of implantation sites) x 100

Pre-coital interval (in days): (Sum of days until successful insemination / number of inseminated females)

Copulation (Mating) index (in %) = (Number of inseminated females / Number of paired animals) x 100

Fertility index (in %) = (Number of pregnant female partners / Number of inseminated females) x 100

Offspring viability indices:

Live birth index (in %) = (Number of pups born alive / Number of pups born) x 100

Viability index (in %) = (Number of pups alive on PND4 / (Number of pups alive at birth) x 100

Lactation index (in %, presented as Survival index) = (Number of pups alive on PND13 / Number of pups alive on PND4 (after adjustment of litter size)) x 100

Sex ratio (proportion of male pups) = (Number of males / number of pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Transient slight to moderate hypersalivation and associated clinical signs (pedalling and ploughing) were noted in all treated groups just after dosing (+5 minutes), throughout the dosing period and in a dose-related manner for males and at higher incidence at 278 and 930 mg/kg bw/day, during gestation and lactations period for females. The increased incidence of hypersalivation in the treated groups for both sexes was considered to be a physiological response rather than a sign of systemic toxicity considering the minor severity of the finding and its time of occurrence (i.e., just after dosing). This sign may be related to irritancy/taste of the test item and was considered not as adverse in the absence of any effect on the general health status of the rats. Isolated clinical signs were noted amongst treated and control groups during the dosing period including abnormal breathing, bent tail, skin lesions, scabs, broken teeth and thin covered fur and were considered incidental, related to the dosing procedure or to the reproductive phase of the females.
For details on clinical signs, please refer to attachments under section "attached background material".

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male given 278 mg/kg bw/day had abnormal breathing (laboured and abnormal sounds) noted just after dosing on Day 12. Since the signs persisted up to Day 13, the animal was subsequently euthanized for ethical reason. At necropsy, a perforated oesophagus was noted, confirming an intubation error.
There was no other unscheduled death in any group for either sex.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, although mean body weight gain was slightly lower at 930 mg/kg bw/day when compared with the control group (-19%) during the dosing period (Day 1 to Day 47), the effect was principally due to transient negligible mean weight gain during the first week of dosing (Days 1 to 8) and was considered not toxicologically significant in the absence of any impact on mean terminal body weight. There was no test item-related effect on mean body weight gain for males at 93 and 278 mg/kg bw/day.
In females, there were no test item-related effects on mean body weight gain during the premating, gestation or lactation periods.
For details on body weight, please refer to attachments under section "attached background material".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a slightly lower mean food consumption at 930 mg/kg bw/day for males from Day 1 to Day 8 when compared with the control group (-14%) and non-dose related lower mean food consumption in all treated groups for females during the premating (-8%, -6% and -9%), gestation (-6%, -4% and -13%) and lactation (-5%, -5% and -7%) periods at 93, 278 and 930 mg/kg bw/day, respectively, when compared with the control group. The differences were considered not toxicologically significant in the absence of any dose-relationship and/or any toxicologically relevant effect on absolute mean body weight for either sex.
For details on food consumption, please refer to attachments under section "attached background material".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no differences noted amongst the haematology and coagulation parameters between the control and treated male rats in any group.
There was test item-related higher white blood cells count (x1.38) including higher neutrophil (x1.54), lymphocyte (x1.23), monocyte (x1.61) and eosinophil (x1.29) counts for females.
Other differences in haematology parameters such as reticulocyte count, regardless of statistical significance, were not considered test item-related based on their small magnitude, inconsistent direction and/or absence of a dose response, general overlap of individual values with the range of control.
For details on hematology, please refer to attachments under section "attached background material".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean bile acid, ALAT and ALP concentrations were higher at 930 mg/kg bw/day for both sexes (x1.67/x2.85, x2.30/x2.00 and x1.52/1x.60, respectively, for males/females), as well as higher triglyceride concentration for females at 930 mg/kg bw/day when compared with the control group.
Other differences in clinical chemistry parameters such as lower protein concentration at 278 and 930 mg/kg/day and higher chloride at 930 mg/kg/day for males, regardless of statistical significance, were not considered test item-related based on their small magnitude, inconsistent direction and/or absence of a dose response, general overlap of individual values with the range of control.
TSH serum levels were slightly lower in F0-males at 930 mg/kg bw/day when compared with control (x0.50, respectively), but the difference did not reach statistical significance. In the absence of any increase in T4 levels for F0-males or any changes in thyroid organ weights nor microscopic findings on the thyroid glands for F0-males this was likely due to the high individual variability of this parameter rather than a test item-related effect.
For details on clinical biochemistry incl. thyroid hormones, please refer to attachments under section "attached background material".

Endocrine findings:

no effects observed

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- Vaginal smears / estrous cycle
- Genital abnormalities
- T4 levels (pups and parental animals, males/females)
- Anogenital distance
- Nipple development (males)
- Reproductive / viability parameters: gestation length, pre- / post implantation loss, number of corpora lutea, number of implantations, mating index, fertility index, gestation index, live birth index, sex ratio, pup / litter weights
- Organ weights: adrenals, brain, epididymides, liver, ovaries (with oviducts), prostate, seminal vesicles (including coagulating glands), testes, thyroid with parathyroids (fresh weight), uterus (with cervix)
- Histopathology: adrenals, epididymides, liver, mammary gland area, ovaries (with oviducts), pituitary gland, prostate, seminal vesicles (including coagulating glands), testes, thyroids with parathyroids, uterus (with cervix), vagina
For details, please refer to the respective result fields and to attachments under section "attached background material".

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Locomotor activity: There was no test item-related effect on the motor activity (ambulatory or fine movements) in any group for either sex. Males and females demonstrated habituation throughout the testing period. One male at 930 mg/kg bw/day had no or limited ambulatory movement from the initiation of the test but this was considered incidental in the absence of any impact on the mean percentage values. The percentage of habituation was comparable between 930 mg/kg bw/day and control groups and mean counts were comparable with that of the HCD range (specify).
FOB: There was no test item-related effect on the functional tests (startle response, pupillary reflex, righting reflex and grip test) in any group for either sex.
For details on the behavioural tests, please refer to attachments under section "attached background material".

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on the mating performance or fertility in any group. All pairs of rats mated in the control, 93, 278 and 930 mg/kg bw/day groups with the exception of one female given 930 mg/kg bw/day. This isolated case was considered incidental. All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). Amongst females that mated, all became pregnant with the exception of one female given 93 mg/kg bw/day. This isolated case was considered incidental.
There were 10, 9, 10 and 9 pregnant females that successfully completed delivery in the control, 93, 278 and 930 mg/kg bw/day groups, respectively.
The mean duration of gestation was comparable in all groups (approximately 22 days).
The mean percentage of pre-birth (post-implantation) loss was comparable in all groups. The mean number of implantation sites was incidentally higher (14.1) compared with the other groups (between 11.4 and 12.3). This was principally due to 1 atypical female with 21 implantations that disproportionately influenced the group mean value. The mean number of pups delivered per litter was consequently slightly higher at 930 mg/kg bw/day (12.3) compared with the other groups (between 10.7 and 11.0) due to the incidentally higher mean number of implantations associated with the same atypical female.
For details on reproductive performance, please refer to attachments under section "attached background material".

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 930 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 930 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect 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):

There were no pup observations that suggested any association with maternal treatment. Isolated findings noted among treated and control groups including skin discoloured, bent tail, thinness, thin covered fur, pallor, absence of milk and cold body and were therefore considered incidental.
For details on clinical signs, please refer to attachments under section "attached background material".

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were 2, 1 and 8 stillborn pups from 2, 1 and 3 litters at 93, 278 and 930 mg/kg bw/day compared with none in the control group. The live birth index was consequently slightly lower at 930 mg/kg bw/day (93.8%) compared with the concurrent control group (100%) and the Historical Control Data (HCD) range (96.0% to 100%) mainly due to 1 female with 5 stillborn pups.
There were 1, 1, 1 and 8 dead, missing or cannibalized pups from 1, 1, 1 and 2 litters in the control, 93, 278 and 930 mg/kg bw/day groups, respectively, between PND1 and PND4. The viability index was consequently lower at 930 mg/kg bw/day (94.9%) compared with the control group (98.9%) and the HCD range (96.2 to 100%). This was principally due to the atypical female described in previous sections, with 7 out of 20 dead, missing or cannibalized pups prior to culling on PND4. After exclusion of the atypical female, the viability index (98.6%) was comparable with that for the concurrent control group. The lower viability index in the 930 mg/kg bw/day group was therefore considered to be incidental and not test item-related.
There was no pup found dead, missing or cannibalized between PND4 (post-culling) and PND13 so the lactation index was 100% in all groups.
For details on litter data, please refer to attachments, under section "attached background material".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a lower mean pup weight in the 930 mg/kg bw/day group between PND1 and PND4 compared with the control (-12% and -17%, respectively). Following standardization of litter size on PND4, there was statistically significantly lower mean pup weight gain through to PND13. As a consequence, terminal mean pup weight was 19% lower than in the control group (26.0 and 25.2g for males and females, respectively) compared with the concurrent control group (32.3 and 31.6g for males and females, respectively) and the HCD range
(29.0 to 32.8g and 28.0 to 32.0g for males and females, respectively).
There was no test item-related effect on mean pup body weight at 93 and 278 mg/kg bw/day.
For details on body weight, please refer to attachments under section "attached background material".

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

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:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio: There was no test item-related effect on pup sex ratio in any group. The mean value (% of males) was slightly higher at 930 mg/kg bw/day (59.8%) compared with the control group (54.2%) but this was considered incidental and probably an artifact of the small group sizes in the absence of any associated effect on anogenital distance.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed at this dose level.

2

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Applicant's summary and conclusion

Conclusions:

The present study was conducted according to GLP and OECD test guideline 422. Male and female Wistar Han rats were treated for 2 weeks before mating, during mating and, for females, throughout gestation and until PND13, at the dose levels of 93, 278 and 930 mg/kg bw/day. No test item-related adverse effects were observed for systemic parental toxicity and reproductive parameters. Thus, the NOAEL for systemic toxicity and the NOAELfor reproductive toxicity were considered to be 930 mg/kg bw/day.
At 930 mg/kg bw/day, there was a higher incidence of stillborn pups together with lower mean pup weight and postnatal pup weight development in the absence of any adverse maternal effects, indicative of a test item-related developmental effect. Therefore, the NOAEL for developmental toxicity was considered to be 278 mg/kg bw/day, and the respective LOAEL was set to 930 mg/kg bw/day.