Alcohols, C9-11, branched and linear, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan - 12 Jun 2020

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan - 12 Jun 2020

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:

adopted 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 205-242 g
- Fasting period before study: NA
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days for males and 10 days for females

DETAILS OF FOOD AND WATER QUALITY:
No known contaminants in the feed or water at levels that would interfere with the objectives of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 35-53
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 23 JAN 2020 to 30 MAR 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected following a Compliance Check Final Decision (CCH-D-2114493190-51-01/F). Since the registered substance is a liquid, ECHA concluded that testing should be performed by the oral route.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Trial preparations were performed to select the vehicle (corn oil) and to establish a suitable formulation procedure.
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until dosing.
- Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure.
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20219836).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 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: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of Alcohols, C9-11, branched and linear, ethoxylated in rats and in an attempt to produce graded responses to the test item (Test Facility Study No. 20219837).
- F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0- females were not fasted overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

BODY WEIGHT: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

CLINICAL CHEMISTRY AND HAEMATOLOGY: Blood of all F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthasia (isoflurane). F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

SERUM HORMONES: Measurement of total T4 was conducted for F0-males.

NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor activity.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals were sacrificed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

GROSS PATHOLOGY AND ORGAN WEIGHTS: All animals were subjected to a full post mortem examination and the following organs weighed; Brain, cervix, epididymis, adrenal, coagulation gland, parathyroid, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus.

HISTOPATHOLOGY: Histopathology was conducted for the following tissues: Aorta, nasopharynx, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal, c, harderian, lacrimal, mammary, parathyroid, pituitary, prostate, salivary, seminal vesicle, thyroid,
gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea, urinary bladder, uterus, vagina.

Other examinations:

Measurement of total T4 was conducted for F0-males. Assessment of T4 in F0-females and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males.

Statistics:

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% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For all females treated at 1000 mg/kg/day, piloerection and slight to severe lethargy were noted during the entire study period. In the beginning of the study, the majority of the animals were affected and this was accompanied by abnormal (7/10), flat (9/10) and hunched posture (10/10) and/or uncoordinated movements (9/10). At the end of study, these clinical signs were noted in individual animals and (ventro-)lateral recumbency (1/10), loss of righting reflex (1/10), decreased locomotor activity (2/10) and/or abnormal gait (1/10) were also noted in individual animals. Rales (6/10) was noted for the majority of females during the entire study period, which was incidentally accompanied with slow breathing (2/10), labored (1/10), deep (1/10) and/or shallow respiration (2/10) for individual animals. Other clinical signs that were incidentally noted for individual animals included chromodacryorrhoea (1/10), lean appearance (2/10) and ptosis (3/10). For males treated at 1000 mg/kg/day, rales (8/10) was noted for the majority of the animals during the last two weeks of the study, which was accompanied by labored respiration (2/10) in individual animals. Clinical signs noted in a majority of the animals were considered toxicologically relevant. Piloerection (1/10) was also incidentally noted for individual animals. At the incidence observed, these clinical signs were considered not toxicologically relevant. At 300 mg/kg/day, rales and chromodacryorrhoea were incidentally noted in males and piloerection was incidentally noted in females, based on the incidence observed, these clinical signs were considered not toxicologically relevant. Salivation seen after dosing for males and females at 1000 mg/kg/day and males at 300 mg/kg/day was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. No clinical signs were noted for the control, animals treated at 100 mg/kg/day and during weekly arena observations.

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality was noted up to 1000 mg/kg/day.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were observed up to 1000 mg/kg/day. A slightly lower body weight gain was noted for males treated at 1000 mg/kg/day during the first week of treatment. At the end of the study period, absolute body weight was of 0.97x of controls. Based on the magnitude of the change this was considered not toxicologically relevant. No effect on body weight or body weight gain was noted for females treated up to 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted up to 1000 mg/kg/day. Relative food consumption during the lactation phase was slightly lower for females treated at 1000 mg/kg/day (up to 0.92x of control; not statistically significant). Based on the magnitude of the change this was considered not toxicologically relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item. Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item. The longer prothrombin time (PT) for males treated at 100 mg/kg/day was considered to be unrelated to treatment as this occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted in clinical biochemistry parameters after treatment with the test item up to 300 mg/kg/day. For females treated at 1000 mg/kg/day, an increased bile acids concentration was noted (2.53x of control), which was slightly outside the range of historical control data. Furthermore, a decreased creatinine concentration was noted (0.89x of control), which was also slightly outside the range of historical control data. Based on the magnitude of the
change and as an opposite effect would be expected in case of target organ toxicity, the effect on creatinine was considered not toxicologically relevant. For males treated at 1000 mg/kg/day, inorganic phosphate concentrations were increased (1.18x of control), based on the magnitude of the change and as mean values were within historical control data, this was considered not toxicologically relevant. The increased potassium concentration for males of all treatment groups was considered to have arisen as a result of slightly low control values and not related to treatment with the test item. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment with the test item up to 1000 mg/kg/day. A higher hind limb grip strength was noted for males treated at 1000 mg/kg/day, which was considered to have arisen as a result of slightly low control values. Furthermore, a decreased grip strength would be expected in case of target organ toxicity. Therefore, this change was considered not related to treatment with the test item. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg/day. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver weights (absolute and relative to body weights) were noted in the 300 and 1000 mg/kg/day groups males, as shown in Table 1. Any other differences were considered not to be Alcohols, C9-11, branched and linear, ethoxylated-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values. The higher liver weight observed in the 300 and 1000 mg/kg/day group males was without microscopic correlation and without test item-related changes of liver enzymes, therefore it was considered non-adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were present in the forestomach of two 1000 mg/kg/day rats of each sex, in the form of irregular surface, combined with a thickened limiting ridge in one female. The microscopic correlates of these two macroscopic findings consisted of squamous cell hyperplasia with hyperkeratosis in both sexes and/or edema in males. Since severities of these microscopic findings were low (up to slight degree) and were present only in the superficial layers of the forestomach, they were regarded as non-adverse.
Furthermore, a reduced size of the thyroid gland was noted in two males and one female treated at 1000 mg/kg/day. In the males, this finding was unilateral observed. There was no microscopic correlation or test-item related effect on the mean thyroid gland weight. In both sexes, the individual absolute weight and relative to body weight ratios of the thyroid glands of the animals with the macroscopic finding of reduced size, were higher than the lowest concurrent control values. Therefore, this macroscopic finding was regarded as unrelated to treatment with the test item and to be non-adverse. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the forestomach of both sexes and thyroid gland of males of the 1000 mg/kg/day group and are summarized in Table 2. At 1000 mg/kg/day, signs of local irritation of the forestomach (i.e. non-glandular stomach) were present in the form of squamous cell hyperplasia with hyperkeratosis and subepidermal lymphogranulocytic infiltrate in both sexes and additional sub(mucosal) edema in males. The macroscopic correlate for the hyperplasia and edema consisted of irregular surface of the forestomach and/or thickened limiting ridge. Since severities of these microscopic findings were low (up to slight degree) and were present only in the superficial layers of the forestomach, they were regarded as non-adverse. The test item-related increased incidence of follicular cell hypertrophy of the thyroid gland of 1000 mg/kg/day males was at the observed minimal severity and in absence of other test-item related thyroid gland changes regarded non-adverse.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. This included the requested liver of males, showing incidences of one control, one 100 mg/kg/day, zero 300 mg/kg/day and two 1000 mg/kg/day group males with minimal hepatocellular hypertrophy.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

no

Table 1
Mean Percent Liver Weight Differences from Control Groups

	Males
Dose level (mg/kg/day):	100	300	1000
LIVER
Absolute	1	15	15
Relative to body weight	-2	11*	19**

*: P<0.05, **: P<0.01

Table 2
Summary Test Item-Related Microscopic Findings – Forestomach and Thyroid Gland

	Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia, with hyperkeratosis
Minimal	-	-	-	2	-	-	-	1
Slight	-	-	-	2	-	-	-	2
Edema
Minimal	-	-	-	1	-	-	-	-
Slight	-	-	-	1	-	-	-	-
Infiltrate, lymphogranulocytic
Minimal	-	-	-	3	-	-	-	1
THYROID GLAND ^a	5	5	5	6	6	0	0	5
Follicular cell hypertrophy
Minimal	1	-	1	3	-	n.a.	n.a.	-
Slight	-	1	-	1	-	n.a.	n.a.	-

^a  =  Number of tissues examined from each group; n.a.= not applicable

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C9-11, branched and linear, ethoxylated

EC Number:

500-446-0

EC Name:

Alcohols, C9-11, branched and linear, ethoxylated

Cas Number:

160901-09-7

Molecular formula:

n.a.

IUPAC Name:

Alcohols, C9-11, branched and linear, ethoxylated (1-2.5 moles ethoxylated)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch 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 ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 205-242 g
- Fasting period before study: NA
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days for males and 10 days for females

DETAILS OF FOOD AND WATER QUALITY:
No known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 35-53
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 23 JAN 2020 to 30 MAR 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Trial preparations were performed to select the vehicle (corn oil) and to establish a suitable formulation procedure.
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until dosing.
- Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within 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 had occurred, the males and females were
separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure.
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20219836).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were
treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 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: The dose levels were selected based on the results of a 10-day Dose Range Finding Study with oral gavage administration of Alcohols, C9-11, branched and linear, ethoxylated in rats, and in an attempt to produce graded responses to the test item (Test Facility Reference No. 20219837).

- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

BODY WEIGHT: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

CLINICAL CHEMISTRY AND HAEMATOLOGY: Blood of all F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthasia (isoflurane). F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

SERUM HORMONES: Measurement of total T4 was conducted for F0-males.

NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor activity

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in all F0 males: Testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia]

GROSS EXAMINATION OF DEAD PUPS
Pups found dead were examined for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the last litter of each generation was weaned.

Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

GROSS PATHOLOGY AND ORGAN WEIGHTS: All animals were subjected to a full post mortem examination and the following organs weighed; Brain, cervix, epididymis, adrenal, coagulation gland, parathyroid, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus.

HISTOPATHOLOGY: Histopathology was conducted for the following tissues: Aorta, nasopharynx, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal, coagulation gland, harderian, lacrimal, mammary, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid, gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea, urinary bladder, uterus, vagina.

Postmortem examinations (offspring):

SACRIFICE
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

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% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): Number of females mated/Number of females paired x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females/Number of females mated x 100

Offspring viability indices:

For each group, the following calculations were performed. Group mean values of duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/
Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For all females treated at 1000 mg/kg/day, piloerection and slight to severe lethargy were noted during the entire study period. In the beginning of the study, the majority of the animals were affected and this was accompanied by abnormal (7/10), flat (9/10) and hunched posture (10/10) and/or uncoordinated movements (9/10). At the end of study, these clinical signs were noted in individual animals and (ventro-)lateral recumbency (1/10), loss of righting reflex (1/10), decreased locomotor activity (2/10) and/or abnormal gait (1/10) were also noted in individual animals. Rales (6/10) was noted for the majority of females during the entire study period, which was incidentally accompanied with slow breathing (2/10), labored (1/10), deep (1/10) and/or shallow respiration (2/10) for individual animals. Other clinical signs that were incidentally noted for individual animals included chromodacryorrhoea (1/10), lean appearance (2/10) and ptosis (3/10). For males treated at 1000 mg/kg/day, rales (8/10) was noted for the majority of the animals during the last two weeks of the study, which was accompanied by labored respiration (2/10) in individual animals. Piloerection (1/10) was also incidentally noted for individual animals. At the incidence observed, these clinical signs were considered not toxicologically relevant. At 300 mg/kg/day, rales and chromodacryorrhoea were incidentally noted in males and piloerection was incidentally noted in females, based on the incidence observed, these clinical signs were considered not toxicologically relevant. Salivation seen after dosing for males and females at 1000 mg/kg/day and males at 300 mg/kg/day was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality was noted up to 1000 mg/kg/day.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were observed up to 1000 mg/kg/day. A slightly lower body weight gain was noted for males treated at 1000 mg/kg/day during the first week of treatment. At the end of the study period, absolute body weight was of 0.97x of controls. Based on the magnitude of the change this was considered not toxicologically relevant. No effect on body weight or body weight gain was noted for females treated up to 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted up to 1000 mg/kg/day. Relative food consumption during the lactation phase was slightly lower for females treated at 1000 mg/kg/day (up to 0.92x of control; not statistically significant). Based on the magnitude of the change this was considered not toxicologically relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item. Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
The longer prothrombin time (PT) for males treated at 100 mg/kg/day was considered to be unrelated to treatment as this occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes were noted in clinical biochemistry parameters after treatment with the test item up to 300 mg/kg/day. For females treated at 1000 mg/kg/day, an increased bile acids concentration was noted (2.53x of control), which was slightly outside the range of historical control data. Furthermore, a decreased creatinine concentration was noted (0.89x of control), which was also slightly outside the range of historical control data. Based on the magnitude of the
change and as an opposite effect would be expected in case of target organ toxicity, the effect on creatinine was considered not toxicologically relevant. For males treated at 1000 mg/kg/day, inorganic phosphate concentrations were increased (1.18x of control), based on the magnitude of the change and as mean values were within historical control data, this was considered not toxicologically relevant. The increased potassium concentration for males of all treatment groups was considered to have arisen as a result of slightly low control values and not related to treatment with the test item. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Endocrine findings:

not specified

Description (incidence and severity):

Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment with the test item up to 1000 mg/kg/day. A higher hind limb grip strength was noted for males treated at 1000 mg/kg/day, which was considered to have arisen as a result of slightly low control values. Furthermore, a decreased grip strength would be expected in case of target organ toxicity. Therefore, this change was considered not related to treatment with the test item. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg/day. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the forestomach of both sexes and thyroid gland of males of the 1000 mg/kg/day group and are summarized in Table 2. At 1000 mg/kg/day, signs of local irritation of the forestomach (i.e. non-glandular stomach) were present in the form of squamous cell hyperplasia with hyperkeratosis and subepidermal lymphogranulocytic infiltrate in both sexes and additional sub(mucosal) edema in males. The macroscopic correlate for the hyperplasia and edema consisted of irregular surface of the forestomach and/or thickened limiting ridge. Since severities of these microscopic findings were low (up to slight degree) and were present only in the superficial layers of the forestomach, they were regarded as non-adverse. The test item-related increased incidence of follicular cell hypertrophy of the thyroid gland of 1000 mg/kg/day males was at the observed minimal severity and in absence of other test-item related thyroid gland changes regarded non-adverse.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. This included the requested liver of males, showing incidences of one control, one 100 mg/kg/day, zero 300 mg/kg/day and two 1000 mg/kg/day group males with minimal hepatocellular hypertrophy.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Normal length and regularity of the estrous cycle was disturbed in 5/10 females at 1000 mg/kg/day. This was considered related to treatment with the test item and at this incidence considered adverse. Despite the disturbance in estrous cycle during premating, all females showed evidence of mating within 4 days after the start of the mating period. It therefore was considered that the cohabitation with a male stimulated the females to a reset of their estrous cycle. Disturbance of the estrous had no effect on the mating performance and fertility as all female delivered offspring.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 1/10 couples of the control group, 3/10 couples of the 100 mg/kg/day group and 2/10 couples of the 300 mg/kg/day group without offspring despite proof of mating. 1/10 males at 300 mg/kg/day failed to sire and 1/10 females at 100 mg/kg/day had total litter loss at lactation day 6. This lack of healthy offspring could not be explained by the microscopic examination of the reproductive organs. Mating index was not affected by treatment. All females showed evidence of mating. The mating indices were 100% for all groups. Pre-coital time was considered not to be affected by treatment. Number of implantation sites was considered not to be affected by treatment with the test item. Fertility index was considered not to be affected by treatment. The fertility indices were 90, 70, 90 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. Gestation index and duration of gestation were considered not to be affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

presumably yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of the clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not toxicologically relevantly affected. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 86, 87 and 82% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. Litter size was considered not affected by treatment.
Live litter sizes were 10.2, 10.7, 11.9 and 9.7 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively. The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. The live birth indices were 97, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. Viability indices were 100, 100, 98 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100, 98, 98 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body weights of pups at 1000 mg/kg/day were noted from PND 1 onwards (up to 0.85x and 0.87x of control in male and female pups, respectively; not always statistically significant). Other statistically significant changes in body weights of the pups were considered to be unrelated to treatment as these occurred in absence of a dose-related trend.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Histopathological findings:

not examined

Other effects:

not examined

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 (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Any other information on results incl. tables

Table 1
Mean Percent Liver Weight Differences from Control Groups

	Males
Dose level (mg/kg/day):	100	300	1000
LIVER
Absolute	1	15	15
Relative to body weight	-2	11*	19**

*: P<0.05, **: P<0.01

Table 2
Summary Test Item-Related Microscopic Findings – Forestomach and Thyroid Gland

	Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia, with hyperkeratosis
Minimal	-	-	-	2	-	-	-	1
Slight	-	-	-	2	-	-	-	2
Edema
Minimal	-	-	-	1	-	-	-	-
Slight	-	-	-	1	-	-	-	-
Infiltrate, lymphogranulocytic
Minimal	-	-	-	3	-	-	-	1
THYROID GLAND ^a	5	5	5	6	6	0	0	5
Follicular cell hypertrophy
Minimal	1	-	1	3	-	n.a.	n.a.	-
Slight	-	1	-	1	-	n.a.	n.a.	-

^a  =  Number of tissues examined from each group; n.a.= not applicable