Alcohols, C12-13, 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:

24 Dec 2019 - 26 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:

24 Dec 2019 - 26 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 (incl. QA statement)

Remarks:

Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

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
- Age at study initiation: males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: males 278-352 g, females 198-239 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 45 - 53
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

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:
- 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure (UPLC-MS based on ammonium adduct).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 15% 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. 20218719).

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 Finding Study with oral gavage administration of Alcohols, C12-13, branched and linear, ethoxylated (2 EO) in rats, and in an attempt to produce graded responses to the test item (Test Facility Reference No. 20218720).

- 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 once 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, coagulation, 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 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):

Test item-related clinical signs of toxicity were observed in treated animals during daily detailed clinical observations.

Group 2 (100 mg/kg bw/day): In one male red staining of the nose was recorded on six consecutive days during the mating period, after which it recovered. No clinical signs of toxicity were observed in females at 100 mg/kg bw/day.

Group 3 (300 mg/kg bw/day): In three males and two females clinical signs of toxicity were observed on 1-3 days of treatment, at the beginning of Week 2 or during post-coitum, in one male red staining of the mouth was recorded on Days 8-9 of treatment. No other clinical signs were recorded for this animal. Two males and one female showed slight breathing rales on Day 10 of treatment. In the same female ventro-lateral recumbency and pon Days 9-10 post-coitum. One female had breathing rales on three consecutive days during Week 2 of lactation.

Group 4 (1000 mg/kg bw/day): Males and females at 1000 mg/kg bw/day showed clinical signs of toxicity at the end of Week 1/beginning of Week 2 of treatment, which lasted for several days and then recovered. In females, clinicals signs were also recorded during lactation. In three males at 1000 mg/kg bw/day flat and/or hunched posture, lethargy, piloerection and/or ptosis of the eyes were recorded on Days 7-8 of treatment. Red staining of the mouth was recorded in one male on Day 8 of treatment. Breathing rales were observed in six males on one or two days during Week 2 of treatment, and in one male on two days in Week 4 of treatment. In all females at 1000 mg/kg bw/day flat and hunched posture, breathing rales (slight to moderate) and piloerection were observed on two or more occasions from Day 6 up to Day 13 of treatment and during lactation. Slight ptosis of the eyes was recorded on two or more occasions from Day 9 up to Day 13 of treatment. Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg bw/day dose groups starting at Week 1 or 2 of the treatment period was considered not toxicologically relevant, taking into account the nature and limited severity of the effect and its time of occurrence (i.e. after dosing). This observation was considered to be a physiological response rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes in body weights and body weight gain were observed in females up to 1000 mg/kg bw/day.

In males receiving 300 and 1000 mg/kg bw/day test material, body weight gain was decreased from Day 8 of treatment onwards (statistically significant on some occasions for animals receiving 1000 mg/kg bw/day only). Decreased body weight gain resulted in body weights up to 0.95x (300 mg/kg bw/day) and 0.94x (1000 mg/kg bw/day) of control (not statistically significant). These differences were considered to be test item-related.

Statistically significant changes in body weight gain in females at 100 and 300 mg/kg bw/day during lactation were considered to be unrelated to treatment since all values were within the normal range and no dose-related trend was apparent.

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 up to 1000 mg/kg bw/day.
Food consumption in males and females at 1000 mg/kg bw/day was decreased in Week 1 of treatment (up to 0.87x of control for relative food consumption, not statistically significant). As this observation was transient, it 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):

Decreased mean corpuscular volume (MCV) was seen in males and females at 1000 mg/kg bw/day (0.96x of control, not statistically significant). Mean values for males were just below the historical control range; for females mean values remained within the normal range. Considering the magnitude of change, this difference was considered not toxicologically relevant.

Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. Increased reticulocyte concentrations in females starting at 100 mg/kg bw/day were considered the result of relatively low control values and not related to treatment with the test item.

Decreased activated partial thromboplastin time (APTT) in females starting at 100 mg/kg bw/day were considered the result of relatively high control values and not related to treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased urea levels were seen in males at 1000 mg/kg bw/day (1.49x of control). Mean values were outside the historical control range. This change was considered test item-related.

Increased creatinine concentrations were seen in males and decreased creatinine concentrations in females at 1000 mg/kg bw/day (1.09x and 0.91x of control, respectively). Mean values were outside the historical control range. This change was considered test item-related.

Increased bile acids in males and females was seen starting at 100 mg/kg bw/day (not statistically significant, up to 1.66x of control) but were considered not toxicologically relevant as mean values remained within the historical control range, no dose-response was apparent (males) or mean differences were the result of high variation (females).

Statistically significantly decreased calcium concentrations in males at 300 mg/kg bw/day were considered to be unrelated to treatment as these occurred in the absence of a dose related trend.

Endocrine findings:

effects observed, non-treatment-related

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 bw/day. At 1000 mg/kg bw/day, serum T4 levels were slightly decreased when compared to the concurrent control group (0.82x of control). However, as no statistical significance was reached, the magnitude of change was minimal, and all values were within normal limits of which the concurrent control was at the upper end, this difference was considered not related to treatment with the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment in males up to 1000 mg/kg bw/day, and in females up to 300 mg/kg bw/day. Hearing ability, pupillary reflex, static righting reflex and grip strength of the hind leg were normal in all examined animals up to 1000 mg/kg bw/day.
In females at 1000 mg/kg bw/day, grip strength of the fore leg, total movements and ambulations were decreased (0.79x, 0.59x and 0.68x of control, respectively, statistically significant for grip strength only). Mean values remained within the historical control range. These changes were considered test item-related.

In the absence of a dose-related trend, statistically significantly increased ambulations in males at 300 mg/kg bw/day were considered unrelated to treatment. 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 weights of the liver (both sexes) and adrenal gland (females) were noted, as shown in Table 1.

A higher liver weight was recorded for both sexes at 1000 mg/kg bw/day (absolute and relative to body weight) and in females at 300 mg/kg bw/day (relative to body weight). The microscopic correlate for the higher weights in males was centrilobular hepatocellular hypertrophy. There was no microscopic correlate for the higher liver weights in females. Higher adrenal gland weights were recorded for females at 1000 mg/kg bw/day (absolute and relative to body weights). The microscopic correlate for the higher adrenal gland weights was cortical hypertrophy, zona fasciculata.

Any other statistically significant changes in organ weights were considered not to be test item-related, as no dose-related trend was observed and/or control values were relatively high.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

An enlarged liver correlating with increased liver weight was recorded for a single female at 1000 mg/kg/day. At the incidence observed and in absence of a microscopic correlate, this was considered not toxicologically relevant.

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 at 1000 mg/kg/day in the liver of males and adrenal gland of females. These findings are presented in Table 2.

Liver (males): Centrilobular hepatocellular hypertrophy was noted in all males at 1000 mg/kg/day at minimal to slight degree.

Adrenals gland (females): Cortical hypertrophy of the zona fasciculata was recorded in 2/5 females at 1000 mg/kg/day at minimal degree.

In addition, in the heart of a single male at 1000 mg/kg/day multifocal necrosis of myofibres (left ventricle) at slight degree was recorded for which a relation to treatment could not be ruled out.
The remainder of the recorded microscopic findings (including hypertrophy of follicular cells in the thyroid gland) were within the range of background pathology encountered in rats of this age and strain subjected to a combined 28-Day oral toxicity study. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

1 000 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 and Adrenal Glands Weight Differences from Control Groups

	Males					Females
Dose level (mg/kg/day):	100		300	1000		100	300	1000
LIVER
Absolute	7		-4	29**		12	13	34**
Relative to body weight	5		3	35**		9	14**	34**
ADRENAL GLANDS
Absolute	-		-	-		21	9	32*
Relative to body weight	-		-	-		17	8	33*

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

Table 2: Summary Test Item-Related Microscopic Findings

Dose level (mg/kg/day):	0	100	300	1000
LIVER (males)^a	5	5	5	5
Hypertrophy hepatocellular              Centrilobular
Minimal	-	-	-	3
Slight	-	-	-	2
ADRENAL GLANDS (females)^a	5	5	5	5
Hypertrophy cortical            zona fasciculata
Minimal	-	-	-	2

^a = Number of tissues examined from each group.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C12-13, branched and linear, ethoxylated

EC Number:

500-457-0

EC Name:

Alcohols, C12-13, branched and linear, ethoxylated

Cas Number:

160901-19-9

Molecular formula:

not applicable; UVCB

IUPAC Name:

Alcohols, C12-13, branched and linear, 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: Males 10-11 weeks, females 13-14 weeks of age
- Weight at study initiation: Males 278 - 353 g, females 198 - 239 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 45 - 53
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

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.

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure (UPLC-MS based on ammonium adduct).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 15% 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. 20218719).

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 males and 10 females for each treatment group and control group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a 10-day Dose Range Finding Study with oral gavage administration of Alcohols, C12-13, branched and linear, ethoxylated (2 EO) in rats, and in an attempt to produce graded responses to the test item (Test Facility Reference No. 20218720).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted once 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 males: Testis weight, epididymis weight, prostate weight, seminal vesicle 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.
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.

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 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.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups were euthanized on PND 14-16, except for the two pups per litter selected for blood collection which were euthanized by an intraperitoneal injection of sodium pentobarbital.

The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

Pups that died before scheduled termination were examined externally and sexed (both externally and internally).

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

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.

HISTOPATHOLOGY / ORGAN WEIGTHS
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.

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 reproductive indices calculations were performed. Group mean values of precoital time and duration of gestation 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

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

Offspring viability indices:

For each group, the following calculations were performed:

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):

Test item-related clinical signs of toxicity were observed in treated animals during daily detailed clinical observations.

Group 2 (100 mg/kg bw/day): In one male red staining of the nose was recorded on six consecutive days during the mating period, after which it recovered. No clinical signs of toxicity were observed in females at 100 mg/kg bw/day.

Group 3 (300 mg/kg bw/day): In three males and two females clinical signs of toxicity were observed on 1-3 days of treatment, at the beginning of Week 2 or during post-coitum, in one male red staining of the mouth was recorded on Days 8-9 of treatment. No other clinical signs were recorded for this animal. Two males and one female showed slight breathing rales on Day 10 of treatment. In the same female ventro-lateral recumbency and pon Days 9-10 post-coitum. One female had breathing rales on three consecutive days during Week 2 of lactation.

Group 4 (1000 mg/kg bw/day): Males and females at 1000 mg/kg bw/day showed clinical signs of toxicity at the end of Week 1/beginning of Week 2 of treatment, which lasted for several days and then recovered. In females, clinicals signs were also recorded during lactation. In three males at 1000 mg/kg bw/day flat and/or hunched posture, lethargy, piloerection and/or ptosis of the eyes were recorded on Days 7-8 of treatment. Red staining of the mouth was recorded in one male on Day 8 of treatment. Breathing rales were observed in six males on one or two days during Week 2 of treatment, and in one male on two days in Week 4 of treatment. In all females at 1000 mg/kg bw/day flat and hunched posture, breathing rales (slight to moderate) and piloerection were observed on two or more occasions from Day 6 up to Day 13 of treatment and during lactation. Slight ptosis of the eyes was recorded on two or more occasions from Day 9 up to Day 13 of treatment. Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg bw/day dose groups starting at Week 1 or 2 of the treatment period was considered not toxicologically relevant, taking into account the nature and limited severity of the effect and its time of occurrence (i.e. after dosing). This observation was considered to be a physiological response rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes in body weights and body weight gain were observed in females up to 1000 mg/kg bw/day.

In males receiving 300 and 1000 mg/kg bw/day test material, body weight gain was decreased from Day 8 of treatment onwards (statistically significant on some occasions for animals receiving 1000 mg/kg bw/day only). Decreased body weight gain resulted in body weights up to 0.95x (300 mg/kg bw/day) and 0.94x (1000 mg/kg bw/day) of control (not statistically significant). These differences were considered to be test item-related.

Statistically significant changes in body weight gain in females at 100 and 300 mg/kg bw/day during lactation were considered to be unrelated to treatment since all values were within the normal range and no dose-related trend was apparent.

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 up to 1000 mg/kg bw/day.
Food consumption in males and females at 1000 mg/kg bw/day was decreased in Week 1 of treatment (up to 0.87x of control for relative food consumption, not statistically significant). As this observation was transient, it 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):

Decreased mean corpuscular volume (MCV) was seen in males and females at 1000 mg/kg bw/day (0.96x of control, not statistically significant). Mean values for males were just below the historical control range; for females mean values remained within the normal range. Considering the magnitude of change, this difference was considered not toxicologically relevant.

Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. Increased reticulocyte concentrations in females starting at 100 mg/kg bw/day were considered the result of relatively low control values and not related to treatment with the test item.

Decreased activated partial thromboplastin time (APTT) in females starting at 100 mg/kg bw/day were considered the result of relatively high control values and not related to treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased urea levels were seen in males at 1000 mg/kg bw/day (1.49x of control). Mean values were outside the historical control range. This change was considered test item-related.

Increased creatinine concentrations were seen in males and decreased creatinine concentrations in females at 1000 mg/kg bw/day (1.09x and 0.91x of control, respectively). Mean values were outside the historical control range. This change was considered test item-related.

Increased bile acids in males and females was seen starting at 100 mg/kg bw/day (not statistically significant, up to 1.66x of control) but were considered not toxicologically relevant as mean values remained within the historical control range, no dose-response was apparent (males) or mean differences were the result of high variation (females).

Statistically significantly decreased calcium concentrations in males at 300 mg/kg bw/day were considered to be unrelated to treatment 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 bw/day. At 1000 mg/kg bw/day, serum T4 levels were slightly decreased when compared to the concurrent control group (0.82x of control). However, as no statistical significance was reached, the magnitude of change was minimal, and all values were within normal limits of which the concurrent control was at the upper end, this difference was considered not related to treatment with the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment in males up to 1000 mg/kg bw/day, and in females up to 300 mg/kg bw/day. Hearing ability, pupillary reflex, static righting reflex and grip strength of the hind leg were normal in all examined animals up to 1000 mg/kg bw/day.
In females at 1000 mg/kg bw/day, grip strength of the fore leg, total movements and ambulations were decreased (0.79x, 0.59x and 0.68x of control, respectively, statistically significant for grip strength only). Mean values remained within the historical control range. These changes were considered test item-related.

In the absence of a dose-related trend, statistically significantly increased ambulations in males at 300 mg/kg bw/day were considered unrelated to treatment. 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 at 1000 mg/kg/day in the liver of males and adrenal gland of females. These findings are presented in Table 2.

Liver (males): Centrilobular hepatocellular hypertrophy was noted in all males at 1000 mg/kg/day at minimal to slight degree.

Adrenals gland (females): Cortical hypertrophy of the zona fasciculata was recorded in 2/5 females at 1000 mg/kg/day at minimal degree.

In addition, in the heart of a single male at 1000 mg/kg/day multifocal necrosis of myofibres (left ventricle) at slight degree was recorded for which a relation to treatment could not be ruled out.
The remainder of the recorded microscopic findings (including hypertrophy of follicular cells in the thyroid gland) were within the range of background pathology encountered in rats of this age and strain subjected to a combined 28-Day oral toxicity study. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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 affected by treatment. Most females had regular cycles of 4 to 5 days. Extended di-estrous occurred in one female at 1000 mg/kg bw/day, due to which the regularity of the estrous cycle could not be determined. Given the incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was considered not to be affected by treatment with the test item. All females showed evidence of mating. Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 4 days. Number of implantation sites was considered not to be affected by treatment with the test item. Mean number of implantation sites was 11.2, 12.8, 12.2 and 11.2 in the control, 100, 300 and 1000 mg/kg/day groups, respectively. Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 90% for the control, 100 and 300 mg/kg bw/day groups and 100% for the 1000 mg/kg bw/day group. One female of the control, 100 and 300 mg/kg bw/day groups each were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

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

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:

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. The nature and incidence of 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):

One pup of the control group was found missing on PND 5. In addition, one pup at 100 mg/kg bw/day was found missing on PND 16. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, body weights of male and female pups were decreased from PND 1 onwards (statistically significant from Day 7 onwards). Body weights progressively decreased from 0.89x of control on Day 1 up to 0.80x of control on PND 13. This was considered test item-related. At 100 and 300 mg/kg bw/day male and females pup body weights were slightly lower than control as well, but not progressive (up to 0.94x of control). As differences were minimal these were considered not adverse.

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.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

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. The statistically significantly lower mean anogenital distance (normalized for body weight) of female pups at 300 mg/kg bw/day occurred without a dose related-trend and was not considered treatment-related.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg bw/day/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):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item. 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

Remarks:

development

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 Liver and Adrenal Glands Weight Differences from Control Groups

	Males					Females
Dose level (mg/kg/day):	100		300	1000		100	300	1000
LIVER
Absolute	7		-4	29**		12	13	34**
Relative to body weight	5		3	35**		9	14**	34**
ADRENAL GLANDS
Absolute	-		-	-		21	9	32*
Relative to body weight	-		-	-		17	8	33*

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

Table 2: Summary Test Item-Related Microscopic Findings

Dose level (mg/kg/day):	0	100	300	1000
LIVER (males)^a	5	5	5	5
Hypertrophy hepatocellular              Centrilobular
Minimal	-	-	-	3
Slight	-	-	-	2
ADRENAL GLANDS (females)^a	5	5	5	5
Hypertrophy cortical            zona fasciculata
Minimal	-	-	-	2

^a = Number of tissues examined from each group.