Alcohols, C12-15, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 MAR 2020 to

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:

From 18 MAR 2020 to

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 (incl. QA statement)

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.

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
- Age at study initiation: males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: males 266-317 g, females 201-271 g
- 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: 7 days

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): 20-21
- Humidity (%): 46-56
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES
From: 18 MAR 2020 to 20 MAY 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- 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 (Test Facility Study No. 20219687).
- Concentration in vehicle: 4 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 (Test Facility Study No. 20219687).
- 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 less than or equal to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-64 days.
Females which failed to deliver or had a total litter loss 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 Finding Study with oral gavage administration of the test material in rats (Test Facility Reference No. 20219688), and in an attempt to produce graded responses to the test item.
- 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.

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.

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.

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 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.

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 were 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 was significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among all animals of the 300 and 1000 mg/kg/day dose groups and some animals of the 100 mg/kg/day dose group 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 rather than a sign of systemic toxicity.
Incidental findings that were noted included rales, alopecia and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item up to 1000 mg/kg/day.
One female of the control group and one female at 300 mg/kg/day were euthanized on Lactation Days 7 and 3, respectively, as they had a total litter loss.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered not affected by treatment with the test item up to 1000 mg/kg/day.
At 1000 mg/kg/day, mean relative food consumption was increased (1.27x of control; statistically significant) in females over lactation Days 1-4. No toxicologically relevance was attached to this finding, as food intake had returned to control levels in the subsequent intervals.
Any statistically significant changes in food consumption before or after correction for body weight observed in Groups 2 and 3 were considered to be unrelated to treatment since no trend was apparent regarding dose.

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 up to 1000 mg/kg/day.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
The statistically significant increase in mean activated partial thromboplastin time (APTT) in males at 100 and 300 mg/kg/day was considered unrelated to administration of the test item due to absence of a dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered affected by treatment with the test item at 300 and 1000 mg/kg/day.
The following statistically significant changes distinguished treated from control animals:
• Mean albumin concentration was slightly increased (both 1.06x; above historical control range) in males at 300 and 1000 mg/kg/day.
• Mean urea concentration was increased (1.37x; within historical control range) in males at 1000 mg/kg/day.
• Mean cholesterol concentration was decreased (0.74 and 0.65x, respectively; both below historical control range) in males at 300 and 1000 mg/kg/day.
• Mean bile acid concentration was increased (2.90x; within historical control range) in females at 1000 mg/kg/day.
These changes were considered as non-adverse since they were not associated with any adverse pathological alterations.

Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

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

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional tests were inadvertently not performed in females.
Functional observation parameters were considered not affected by treatment with the test item up to 1000 mg/kg/day in males.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined males up to 1000 mg/kg/day. Grip strength was similar between control and high dose males.
Motor activity was similar between treated and control male groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight changes were noted in the liver of males and females, in the thyroid glands of males and in the adrenal glands of females. These changes (which were considered to be non-adverse) are summarized in Table 1.
Any other differences, including those that reached statistical significance (i.e. absolute and relative to body weight uterus weight at 100 mg/kg/day, and heart- and kidney weight relative to body weight in females at 1000 mg/kg/day) were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, test item-related macroscopic enlarged liver was observed in a single male and female and enlarged adrenal glands were observed in a single female. These changes were considered 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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver, jejunum and forestomach of males and females at 1000 mg/kg/day, in the thyroid gland of males starting at 100 mg/kg/day and in the adrenal gland of females at 1000 mg/kg/day. These changes are summarized in Table 2, 3 and 4.
Liver: Centrilobular hepatocellular hypertrophy was noted at 1000 mg/kg/day in all males up to slight degree and in 2/6 females at minimal degree.
Jejunum: Vacuolation in the lamina propria was noted at 1000 mg/kg/day in 4/5 males up to slight degree and in 3/5 females at minimal degree.
Forestomach: Minimal squamous cell hypertrophy was noted at 1000 mg/kg/day in 4/5 males and in a single female.
Thyroid glands: An increased incidence and/or severity of follicular cell hypertrophy was noted starting at 100 mg/kg/day in males up to slight degree.
Adrenal glands: Diffuse cortical hypertrophy was noted at 1000 mg/kg/day in 3/5 females up to slight degree.
All of these changes were considered non-adverse.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Critical effects observed:

no

Table 1. Mean Percent Organ Weight Differences from Control Group

		Males			Females
Dose level (mg/kg/day):	100	300	1000	100	300	1000
LIVER
Absolute	14*	18**	33**	0	27**	39**
Relative to body weight	8*	14**	36**	3	20**	36**
THYROID GLANDS
Absolute	5	2	15	na	na	na
Relative to body weight	2	0	18*	na	na	na
ADRENAL GLANDS
Absolute	na	na	na	4	16	35**
Relative to body weight	na	na	na	13	17	39**

na = not applicable

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

Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
LIVER ^a	5	5	5	6	6	5	6	6
Centrilobular hypertrophy
Minimal	-	-	-	1	-	-	-	2
Slight	-	-	-	5	-	-	-	-
JEJUNUM ^a	5	5	5	5	5	5	5	5
Vacuolation
Minimal	-	-	-	3	-	-	-	3
Slight	-	-	-	1	-	-	-	-
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia
Minimal	-	-	-	4	-	-	-	1

^a = Number of tissues examined from each group.

Table 3. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males
Dose level (mg/kg/day):	0	100	300	1000
THYROID GLANDS ^a	5	5	5	5
Follicular cell hypertrophy
Minimal	-	3	3	4
Slight	-	-	1	1

^a = Number of tissues examined from each group.

Table 4. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Females
Dose level (mg/kg/day):	0	100	300	1000
ADRENAL GLANDS ^a	5	5	6	5
Diffuse cortical hypertrophy
Minimal	-	-	-	1
Slight	-	-	-	2

^a = Number of tissues examined from each group.

 

 

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 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C12-15, branched and linear, ethoxylated

EC Number:

500-294-5

EC Name:

Alcohols, C12-15, branched and linear, ethoxylated

Cas Number:

106232-83-1

IUPAC Name:

Alcohols, C12-15, branched and linear, ethoxylated (1-2.5 EO)

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.

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
- Age at study initiation: males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: males 266-317 g, females 201-271 g
- 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: 7 days

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): 20-21
- Humidity (%): 46-56
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES
From: 18 MAR 2020 to 20 MAY 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- 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 (Test Facility Study No. 20219687).
- Concentration in vehicle: 4 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Details on mating procedure:

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 have 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 (Test Facility Study No. 20219687).
- 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 less than or equal to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-64 days.
Females which failed to deliver or had a total litter loss 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 the test material in rats (Test Facility Reference No. 20219688), and in an attempt to produce graded responses to the test item.
- 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 F0 male parental generations: 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 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.

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
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 or three pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) 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 were 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 was 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):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among all animals of the 300 and 1000 mg/kg/day dose groups and some animals of the 100 mg/kg/day dose group 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 rather than a sign of systemic toxicity.
Incidental findings that were noted included rales, alopecia and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item up to 1000 mg/kg/day.
One female of the control group and one female at 300 mg/kg/day were euthanized on Lactation Days 7 and 3, respectively, as they had a total litter loss.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered not affected by treatment with the test item up to 1000 mg/kg/day.
At 1000 mg/kg/day, mean relative food consumption was increased (1.27x of control; statistically significant) in females over lactation Days 1-4. No toxicologically relevance was attached to this finding, as food intake had returned to control levels in the subsequent intervals.
Any statistically significant changes in food consumption before or after correction for body weight observed in Groups 2 and 3 were considered to be unrelated to treatment since no trend was apparent regarding dose.

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 up to 1000 mg/kg/day.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
The statistically significant increase in mean activated partial thromboplastin time (APTT) in males at 100 and 300 mg/kg/day was considered unrelated to administration of the test item due to absence of a dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered affected by treatment with the test item at 300 and 1000 mg/kg/day.
The following statistically significant changes distinguished treated from control animals:
• Mean albumin concentration was slightly increased (both 1.06x; above historical control range) in males at 300 and 1000 mg/kg/day.
• Mean urea concentration was increased (1.37x; within historical control range) in males at 1000 mg/kg/day.
• Mean cholesterol concentration was decreased (0.74 and 0.65x, respectively; both below historical control range) in males at 300 and 1000 mg/kg/day.
• Mean bile acid concentration was increased (2.90x; within historical control range) in females at 1000 mg/kg/day.
These changes were considered as non-adverse since they were not associated with any adverse pathological alterations.

Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.

Endocrine findings:

not specified

Description (incidence and severity):

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

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional tests were inadvertently not performed in females.
Functional observation parameters were considered not affected by treatment with the test item up to 1000 mg/kg/day in males.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined males up to 1000 mg/kg/day. Grip strength was similar between control and high dose males.
Motor activity was similar between treated and control male groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

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 liver, jejunum and forestomach of males and females at 1000 mg/kg/day, in the thyroid gland of males starting at 100 mg/kg/day and in the adrenal gland of females at 1000 mg/kg/day. These changes are summarized in Table 2, 3 and 4.
Liver: Centrilobular hepatocellular hypertrophy was noted at 1000 mg/kg/day in all males up to slight degree and in 2/6 females at minimal degree.
Jejunum: Vacuolation in the lamina propria was noted at 1000 mg/kg/day in 4/5 males up to slight degree and in 3/5 females at minimal degree.
Forestomach: Minimal squamous cell hypertrophy was noted at 1000 mg/kg/day in 4/5 males and in a single female.
Thyroid glands: An increased incidence and/or severity of follicular cell hypertrophy was noted starting at 100 mg/kg/day in males up to slight degree.
Adrenal glands: Diffuse cortical hypertrophy was noted at 1000 mg/kg/day in 3/5 females up to slight degree.
All of these changes were considered non-adverse.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
All females had regular cycles of 4 to 5 days. Extend di-estrous occurred during the mating in a number of females at 300 mg/kg/day with a regular cycle during premating. One female at 300 mg/kg/day had an inconclusive cycle determination during the pre-mating phase. Given the absence of a dose-related incidence, this finding did not indicate a relation with treatment with the test item.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed.
One male from the 1000 mg/kg/day treatment group showed moderate degeneration/atrophy of the germ cells and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This animal had no normal spermatogenic staging profile.
In all other males 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 , 1/10 couples of the 100 mg/kg/day group and 1/10 couples of the 1000 mg/kg/day group with no offspring. A single female of the control group and of the 300 mg/kg/day group had a total litter loss.
One male from the 1000 mg/kg/day treatment group showed moderate degeneration/atrophy of the germ cells and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This animal had no normal spermatogenic staging profile.
For the other couples, no abnormalities were seen in the reproductive organs, which could account for their lack of offspring or total litter loss.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
Mating index, precoital time, number of implantation sites, fertility index and gestation index was considered not to be affected by treatment with the test item up to 1000 mg/kg/day. No signs of difficult or prolonged parturition and no deficiencies in maternal care were noted among the pregnant females.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

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 clinical signs occurred among pups that were considered to be related to treatment with the test item up to 1000 mg/kg/day.
For the pups of two females in the control group and one female at 300 mg/kg/day that were found dead at PND 1, missing on PND 2 or 3, or euthanized in extremis on PND 7, absence of milk in the stomach were noted on PND 1, 2, 3 or 7. In addition, for the pups of a control female and one female at 300 mg/kg/day that were missing on PND 3 or euthanized in extremis on PND 7, a dehydrated appearance was noted on PND 1 and 7.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant.

Description (incidence and severity):

Pups of two females in the control group and one female at 300 mg/kg/day were found dead at PND 1, missing on PND 2 or 3, or euthanized in extremis on PND 7. In addition, pups of a control female and one female at 300 mg/kg/day were missing on PND 3 or euthanized in extremis on PND 7.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered to be affected by treatment with the test item at 1000 mg/kg/day.
At PND 7 and 13, mean body weights of male pups were statistically significantly decreased (0.88 and 0.86x of control, respectively) at 1000 mg/kg/day. In addition, mean body weights of female pups at 1000 mg/kg/day were decreased (0.90x of control; not statistically significant) at PND 13. At PND 13, male and female pup weights of 3/9 litters at 1000 mg/kg/day were below the historical control range.
In addition, the body weight of one pup in two litters at 100 mg/kg/day were significantly lower compared to the control mean at PND 7 and 13 indicating a growth retardation. In the absence of a dose-related trend of the same order of magnitude, these single occurrences in both litters were not considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum levels of total T4 in male and female PND 14-16 pups were affected by treatment with the test item at 100, 300 and 1000 mg/kg/day. However, under the conditions of this screening study no adverse effect was observed that could be linked to the increase of total T4 and therefore this increase was not taken into account when determining the developmental NOAEL.
At 100, 300 and 1000 mg/kg/day, mean levels of total T4 were increased to 1.28, 1.18 and 1.19x of control, respectively, in male PND 14-16 pups (statistically significant at 100 mg/kg/day only) and to 1.42, 1.26 and 1.33x of control, respectively, in female PND 14- 16 pups (statistically significant in all treatment groups). The mean values remained within the historical control range.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Mean Percent Organ Weight Differences from Control Group

		Males			Females
Dose level (mg/kg/day):	100	300	1000	100	300	1000
LIVER
Absolute	14*	18**	33**	0	27**	39**
Relative to body weight	8*	14**	36**	3	20**	36**
THYROID GLANDS
Absolute	5	2	15	na	na	na
Relative to body weight	2	0	18*	na	na	na
ADRENAL GLANDS
Absolute	na	na	na	4	16	35**
Relative to body weight	na	na	na	13	17	39**

na = not applicable

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

Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
LIVER ^a	5	5	5	6	6	5	6	6
Centrilobular hypertrophy
Minimal	-	-	-	1	-	-	-	2
Slight	-	-	-	5	-	-	-	-
JEJUNUM ^a	5	5	5	5	5	5	5	5
Vacuolation
Minimal	-	-	-	3	-	-	-	3
Slight	-	-	-	1	-	-	-	-
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia
Minimal	-	-	-	4	-	-	-	1

^a = Number of tissues examined from each group.

Table 3. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males
Dose level (mg/kg/day):	0	100	300	1000
THYROID GLANDS ^a	5	5	5	5
Follicular cell hypertrophy
Minimal	-	3	3	4
Slight	-	-	1	1

^a = Number of tissues examined from each group.

Table 4. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Females
Dose level (mg/kg/day):	0	100	300	1000
ADRENAL GLANDS ^a	5	5	6	5
Diffuse cortical hypertrophy
Minimal	-	-	-	1
Slight	-	-	-	2

^a = Number of tissues examined from each group.