Tris(2-ethylhexyl) phosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL (reproductive, rat) = 1000 mg/kg/day (OECD 443, Meijer, 2020)

NOAEL (general toxicity, rat) = 1000 mg/kg/day (OECD 443, Meijer, 2020)

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Sep 2019 - 02 Jul 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance to international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

yes

Remarks:

Only one sample collected for formulation analysis, F0-Female No.175, received a 10% higher dose on Lactation Day 9. Female No. 558 vaginal smears late collection and other slight deviation on observation, measurements & necropsy procedures.

GLP compliance:

yes (incl. QA statement)

Justification for study design:

Defined in ECHA Compliance Check Decision CCH-D-2114392241-54-O1/F

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han rats

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. the testing facility has general and reproduction/developmental/neurological 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, neurological toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: Not stated.
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: Males: 152 and 194 g & Females: 104 and 143 g
- Fasting period before study: No
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed in in polycarbonate cage (up to 5 animals of same sex, dosing groups and Cohort) except F0-Male No. 73 (Group 3) was housed in the wrong cage for one night from Day 40 until Day 41 of treatment (during pre-mating). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21). The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labelled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
-Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 12 days prior to the commencement of treatment.
DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 ºC (actual mean daily temperature was 20 to 21 ºC)
- Humidity (%): 40 to 70 % (actual mean daily Humidity was 42 -70%)
- Air changes (per hr): =>10 air changes/hour with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 04 Sep 2019 To 03 Apr 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. Detection of mating was not confirmed in first instance for Female Nos. 130, 135, 156, 175, 179 and 192. Evidence of mating was obtained indirectly by delivery of a litter.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further mattings after two unsuccessful attempts: yes
- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated.
- Any other deviations from standard protocol: No

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. 20173310). Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20173310) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

F0-female = 16-18 weeks
F0-female which failed to deliver : 13-14 weeks
F0-male = 11-12 weeks
F1- Cohort 1A = 9-10 weeks
F1- Cohort 1B= 11-12weeks
F1- Cohort 1C = 2-6 weeks

Frequency of treatment:

Once daily seven days a week.

Details on study schedule:

The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced).
F0-males and females were treated up to and including the day before scheduled necropsy.
F0-female Nos. 104, 107, 115, 117, 121, 123, 126, 127, 130, 135, 136, 148, 162, 172, 174, 176, 179, 181, 182, 193, 196, 199 (Groups 1, 2, 3 and 4), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C, were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed.

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1

No. of animals per sex per dose:

F0 = 25
F1 Cohort 1A -C = 20
F1 Cohort surplus = 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with oral exposure of Tris(2-ethylhexyl) phosphate in rats (Test Facility Study No. 20173303) and in an attempt to produce graded responses to the test item. In this study, out of 8 females one female was not mated and two females were not pregnant which could be an indication of a possible test-item related effect on fertility. No other toxicologically relevant effects were noted. Therefore, based on the results of this study in combination with no oxicologically relevant effects observed, the limit dose of 1000 mg/kg/day was selected as high dose for the Main Study. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Rationale for animal assignment (if not random): NA
 - Other: The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week. The dose volume for each animal was based on the most recent body weight measurement exception • F0-Female No. 175 (Group 3), received a 10% higher dose than required on Lactation Day 9 of treatment. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced). A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

 - Time schedule: Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.
- Cage side observations checked in table [No.?] were included: Yes


 DETAILED CLINICAL OBSERVATIONS: Yes / No / No data

-Time schedule: Observations conducted prior to dosing and 0-30 minutes after dosing then twice daily up to the day prior to necropsy.


 BODY WEIGHT: Yes 

- Time schedule for examinations: 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, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy. For Female No. 524 (control, Cohort 1C), body weight was collected one day after attainment of vaginal opening.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. 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, 14 and 21. No food consumption was determined for F0-Female Nos. 105, 106 (control), 143 (Group 2) and 178 (Group 4) during Lactation Days 1-4. In addition, no body weight was determined for F0-Female Nos. 143 (Group 2) and 178 (Group 4) on Lactation Day 1.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

 - Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

 OTHER: Not specified

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating 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 scheduled necropsy, a vaginal lavage was also taken. During the second period, daily vaginal lavage was performed from PND 75 to 88. Vaginal smears to determine first estrus were not started one day after attainment of vaginal opening but after 6 days for Female No. 558 (Group 2, Cohort 1A). After 7 days it was confirmed that sexual maturity was reached for this animal. No vaginal smears were collected on the day of scheduled necropsy for F0-Female Nos. 104 (Group 1) and 199 (Group 4).

Sperm parameters (parental animals):

For all surviving males, the following assessments were performed: Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Litter observations:

STANDARDISATION OF LITTERS

- Performed on day 4 postpartum: Yes

- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. 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: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: estrous cycle determination, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

GROSS EXAMINATION OF DEAD PUPS: Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes

Postmortem examinations (parental animals):

SACRIFICE: All animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy (for exceptions F0-Female Nos. 119, 124 (control), 187, 190 and 197 (Group 4) were not fasted overnight prior to necropsy.
- Male animals: F0 Males (sired and failed to sire); After successful mating and a minimum of 10 weeks of treatment.
- F0 females which delivered: LD 23-25.
- F0 females which failed to deliver (Nos. 104, 120, 140 and 188): With evidence of mating: Post-coitum Day 27.
- F0 females with total litter loss (No. 199): Within 24 hours after the last pup was found dead or missing.
- F0 female suspected of a total litter loss: (No. 104): Within 24 hours after suspicion of total litter loss (post-coitum Day 24/Lactation Day 1). Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent). The pituitary gland of F0-Male No. 39 (Group 2) was not weighed and collected.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues were prepared for microscopic examination and weighed, respectively. Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent). For F0-Male No. 61 (Group 3) , no histopathological evaluation was required for these organs; eyes, harderian gland, testes and epididymis.

Postmortem examinations (offspring):

SACRIFICE: All animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy.
- Cohort 1A: FPND 89-101.
- Cohort 1B: ≥ PND 97.
- Cohort 1C: after positive determination of vaginal patency or balanopreputial separation.
- Cohort Surplus: PND 22-24.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent). The pituitary gland of Female No. 717 (Group 4, Cohort 1B) was not weighed and collected.
HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues were prepared for microscopic examination and weighed, respectively. Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent). For Male No. 353 (Group 3, Cohort 1A), no histopathological evaluation was required for these organs; eyes, harderian gland, testes and epididymis.

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 according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made:
Group 2 Vs. Group1
Group 3 Vs. Group1
Group 4 Vs. Group1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
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. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Reproduction and Developmental Variables (indices): Mating index males & females (%), Precoital time, Fertility index males and females (%), Gestation index (%), Duration of gestation, Post-implantation survival index (%), Live birth index (%),

Offspring viability indices:

Offspring Variables (indices): Viability index (%), Weaning index (%) and Percentage live males and females at weaning (%).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related findings were noted during the weekly arena observations in this study. For one male at 500 mg/kg/day (No. 52), lethargy was observed on one day of treatment, which was considered an isolated finding and unrelated to treatment. Test item-related salivation was observed in males and females in all treated groups from Weeks 2 until 12 of treatment. A dose-related trend towards slightly earlier onset and longer duration, as well as higher incidence was noted. This sign was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For females at 500 and 1000 mg/kg/day, a trend towards slightly higher mean body weights and body weight gains was observed throughout the pre-mating period from Day 15 onwards.Changes compared with the concurrent control group were relatively slight, reaching statistical significance for body weight gain only. At the end of the premating period, mean body weight of the 500 and 1000 mg/kg/day groups was 1.02x and 1.03x of control mean, respectively. Also during post-coitum and lactation, slightly higher mean body weights were noted at these dose levels, reaching statistical significance at 1000 mg/kg/day on all occasions, except for lactation Day 14. Body weight gain in these dams at 500 and
1000 mg/kg/day was comparable with the concurrent control, except for the means on post- coitum Day 7 and lactation Day 14 which were slightly, but statistically significantly higher or lower, respectively. Overall changes compared to the concurrent control were very minor (< 10%), with terminal body weights of 1.02x and 1.06x of control at 500 and
1000 mg/kg/day, respectively.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered affected by treatment with the test item from 500 mg/kg/day.
For males at 1000 mg/kg/day, absolute food consumption levels were normal for most weeks of treatment, with the exception of Weeks 3-6 of treatment, for which food consumption was slightly increased (1.06-1.09x of control). A trend towards higher relative food consumption levels was observed from 500 mg/kg/day. Changes were observed from Week 3 of treatment and reached statistical significance for several weeks (500 mg/kg/day) or most weeks
(1000 mg/kg/day) of treatment.
For females, absolute and relative food consumption was slightly increased at 500 and
1000 mg/kg/day for most weeks of the pre-mating period reaching statistical significance for most weeks of treatment. During the post-coitum period, absolute food consumption was slightly increased from 500 mg/kg/day reaching statistical significance for most days of treatment. Relative food consumption was considered increased at 1000 mg/kg/day only.
Any changes observed at 250 mg/kg/day or during the lactation period, were considered incidental and in absence of a dose related trend unrelated to treatment with the test item.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters were considered unaffected by treatment with the test item at
1000 mg/kg/day.
In males, mean platelets concentrations were increased at 500 and 1000 mg/kg/day (1.11x and 1.08x of control, respectively)
The following statistically significant changes were noted in treated females. Relative differences in mean values as compared to the control group are indicated between parentheses.
• A decrease in mean hemoglobin concentrations at 500 and 1000 mg/kg/day (0.94 and 0.95x, respectively)
• A decrease in mean hematocrit concentrations at 250, 500 and 1000 mg/kg/day (0.96, 0.93 and 0.96x, respectively)
• Mean corpuscular hemoglobin concentrations at 500 and 1000 mg/kg/day (0.94 and 0.95x, respectively)
All mean values remained within the historical control range and considered unrelated to treatment with the test item in absence of a clear dose response.
Any statistically significant changes observed at the end of treatment in males and females at 250, 500 and/or 1000 mg/kg/day were considered unrelated to treatment with the test item as these changes occurred in the absence of a dose-related trend.
In males, prothrombin time (PT) was increased at 250 and 500 mg/kg/day only. These changes were unrelated to treatment as they occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were considered affected by treatment with the test item at
1000 mg/kg/day. In males, mean bile acid concentration was decreased to 0.47x of control at 1000 mg/kg/day.
In females, total protein and albumin levels were increased to 1.08x of control at 1000 mg/kg/day. For both parameters, mean levels were above the upper limit of the historical control range. The decreased total bilirubin concentrations observed in females from all test item-treated groups was unrelated to treatment as no dose-related trend was evident.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Serum concentrations of TSH (thyroid stimulating hormone) and total T4 were considered not
affected by treatment with the test item.
There was a trend towards higher means of total T4 concentration with increasing doses in males starting at 250 mg/kg/day, and a lower mean concentration of total T4 in females at 1000 mg/kg/day. However, changes compared to the concurrent control group were very slight only (<10%) and did not reach statistical significance. Therefore, these changes were regarded as unrelated to treatment with the test item.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinary parameters were unaffected by treatment with the test item up to 1000 mg/kg/day.
In males, a statistically significant decrease in red blood cell sedimentation score was recorded at 500 and 1000 mg/kg/day. At both dose levels, no red blood cells were found in the urine which is completely normal.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the testes of males at 250, 500 and 1000 mg/kg/day and thyroid gland of both sexes at 1000 mg/kg/day; Thyroid gland: Follicular cell hypertrophy was noted at increased incidence and/or severity at 1000 mg/kg/day in rats of both sexes (up to slight).
Testes: All test item-treated males showed intracytoplasmic eosinophilic inclusions in most of the interstitial cells of the testes. Examination of HE stained slides, revealed no dose-response in the amount of inclusions, and therefore they were not graded but recorded as present. In general, most inclusions were rectangular, and showed a crystalline appearance. There was no evidence for double breaking light when using a polarizing filter.
The Perl’s Prussian blue staining, a staining used to detect iron, did not stain the inclusions.The PAS staining colored the inclusions very brightly, without distinction between dose levels. Based on the number and location of cells with inclusion, it was concluded that these cells represented Leydig cells. In addition, a minority of the cells within the interstitium showed PAS positive diffuse staining in the cytoplasm, comparable with the straining in the testes of control males. These PAS-positive cells were interpreted as macrophages.
Findings of note were present in the lungs of 1000 mg/kg/day group females No. 176, 179, 187 and 197 in the form of granulomatous bronchoalveolar inflammation (up to moderate degree) in one of the lung lobes of each animal and was confined to one or a few foci. This finding was considered to be related to regurgitation and/or aspiration of small amounts of test item with vehicle and was considered not to be a direct test-item related effect (Damsch et al., 2010).
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. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

not specified

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.
Note: For estrous cycle determination, vaginal lavages from the last two weeks of pre-mating (i.e. in Weeks 9 and 10) until Day 1 of mating were evaluated. Since several biologically non- plausible estrous stages were noted in Week 10, Day 7 of the pre-mating period, which resulted in an unrealistic high number of irregular cycle classification, the length and regularity of the estrous cycle evaluation was performed excluding this day with data of up to Week 10, Day 6 of the pre-mating period.
Most females had regular cycles of 4 to 5 days in all groups. An irregular cycle was noted for Female Nos. 113, 117 (control) and 189 (1000 mg/kg/day), all of which had normal litters. Given its incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this isolated finding in the high dose group did not indicate a relation with treatment.
The exclusion of two days in the second estrous cycle evaluation resulted in an inconclusive cycle determination for Female Nos. 118 (control), 137, 149 (250 mg/kg/day) and 158 (500 mg/kg/day). At this low incidence, the omission of estrous cycle data did not affect the overall evaluation of this study.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm motility, concentration and morphology were considered to be unaffected by treatment with the test item up to 1000 mg/kg/day.
Any statistically significant changes in sperm analysis parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and based on the direction of change (i.e. the opposite effect would be expected in case of target organ toxicity).

Reproductive performance:

no effects observed

General effects:
- Mortality: Female No. 199 (1000 mg/kg/day) was euthanized on Lactation Day 1 with a total litter loss. Its single pup was found dead with beginning autolysis at first litter check. Control female (No. 104) was euthanized as a total litter loss was suspected on Lactation Day 1. However, at necropsy this delivery appeared to be an incomplete delivery as there was still one dead fetus (with advanced autolysis) present in the uterus. Clinical signs observed were a hunched posture, a pale appearance and piloerection.

- Clinical observations: Test item-related salivation was observed in males and females in all treated groups from Weeks 2 until 12 of treatment. A dose-related trend towards slightly earlier onset and longer duration, as well as higher incidence was noted. This sign was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Other (incidental) 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. No test item-related findings were noted during the weekly arena observations in this study. For one male at 500 mg/kg/day (No. 52), lethargy was observed on one day of treatment, which was considered an isolated finding and unrelated to treatment.
- Body weight and body weight gains: For females at 500 and 1000 mg/kg/day, a trend towards slightly higher mean body weights and body weight gains was observed throughout the pre-mating period from Day 15 onwards. Changes compared with the concurrent control group were relatively slight, reaching statistical significance for body weight gain only. At the end of the premating period, mean body weight of the 500 and 1000 mg/kg/day groups was 1.02x and 1.03x of control mean, respectively. Also, during post-coitum and lactation, slightly higher mean body weights were noted at these dose levels, reaching statistical significance at 1000 mg/kg/day on all occasions, except for lactation Day 14. Body weight gain in these dams at 500 and 1000 mg/kg/day was comparable with the concurrent control, except for the means on post- coitum Day 7 and lactation Day 14 which were slightly, but statistically significantly higher or lower, respectively. Overall changes compared to the concurrent control were very minor (< 10%), with terminal body weights of 1.02x and 1.06x of control at 500 and 1000 mg/kg/day, respectively. No test item-related changes in body weights and body weight gain were observed in females at 250 mg/kg/day and in males up to 1000 mg/kg/day.
- Food consumption: Food consumption before or after correction for body weight was considered affected by treatment with the test item from 500 mg/kg/day. For males at 1000 mg/kg/day, absolute food consumption levels were normal for most weeks of treatment, with the exception of Weeks 3-6 of treatment, for which food consumption was slightly increased (1.06-1.09x of control). A trend towards higher relative food consumption levels was observed from 500 mg/kg/day. Changes were observed from Week 3 of treatment and reached statistical significance for several weeks (500 mg/kg/day) or most weeks
(1000 mg/kg/day) of treatment. For females, absolute and relative food consumption was slightly increased at 500 and
1000 mg/kg/day for most weeks of the pre-mating period reaching statistical significance for most weeks of treatment. During the post-coitum period, absolute food consumption was slightly increased from 500 mg/kg/day reaching statistical significance for most days of treatment. Relative food consumption was considered increased at 1000 mg/kg/day only. Any changes observed at 250 mg/kg/day or during the lactation period, were considered incidental and in absence of a dose related trend unrelated to treatment with the test item.
- Hematology: Hematological parameters were considered unaffected by treatment with the test item at
1000 mg/kg/day. In males, mean platelets concentrations were increased at 500 and 1000 mg/kg/day (1.11x and 1.08x of control, respectively). The following statistically significant changes were noted in treated females. Relative differences in mean values as compared to the control group are indicated between parentheses.
• A decrease in mean hemoglobin concentrations at 500 and 1000 mg/kg/day (0.94 and 0.95x, respectively)
• A decrease in mean hematocrit concentrations at 250, 500 and 1000 mg/kg/day (0.96, 0.93 and 0.96x, respectively)
• Mean corpuscular hemoglobin concentrations at 500 and 1000 mg/kg/day (0.94 and 0.95x, respectively)
All mean values remained within the historical control range and considered unrelated to treatment with the test item in absence of a clear dose response. Any statistically significant changes observed at the end of treatment in males and females at 250, 500 and/or 1000 mg/kg/day were considered unrelated to treatment with the test item as these changes occurred in the absence of a dose-related trend. In males, prothrombin time (PT) was increased at 250 and 500 mg/kg/day only. These changes were unrelated to treatment as they occurred in the absence of a dose-related trend.
- Clinical Chemistry: Clinical biochemistry parameters were considered affected by treatment with the test item at
1000 mg/kg/day. In males, mean bile acid concentration was decreased to 0.47x of control at 1000 mg/kg/day.
In females, total protein and albumin levels were increased to 1.08x of control at 1000 mg/kg/day. For both parameters, mean levels were above the upper limit of the historical control range. The decreased total bilirubin concentrations observed in females from all test item-treated groups was unrelated to treatment as no dose-related trend was evident.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Serum concentrations of TSH (thyroid stimulating hormone) and total T4 were considered not
affected by treatment with the test item.
There was a trend towards higher means of total T4 concentration with increasing doses in males starting at 250 mg/kg/day, and a lower mean concentration of total T4 in females at 1000 mg/kg/day. However, changes compared to the concurrent control group were very slight only (<10%) and did not reach statistical significance. Therefore, these changes were regarded as unrelated to treatment with the test item.
-UrinalysisIn males, a statistically significant decrease in red blood cell sedimentation score was recorded at 500 and 1000 mg/kg/day. At both dose levels, no red blood cells were found in the urine which is completely normal.
- Macroscopic Findings: All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in one control female, 6 females at 250, 7 females at 500 and one female at 1000 mg/kg/day, is related to a stage in the estrous cycle and is a normal finding.
- Organ Weights: Possible test item-related higher liver weights (absolute and relative to body weights) were noted in F0-females at 500 and 1000 mg/kg/day. Apparent significant changes were noted in the absolute weight and relative to body weight ratio of the adrenal gland of F0-males in all test item treated groups. Since there was no clear dose response or test item-related finding in of the adrenal gland, and mean values remained within the historical control range, this organ weight change was considered unrelated to treatment.
Any other differences, including those that reached statistical significance (F0-males: thyroid gland at 250 mg/kg/day, liver and kidneys at 250 and 500 mg/kg/day; F0-females: liver at 250 mg/kg/day, kidneys at 250 and 1000 mg/kg/day, adrenal gland at 1000 mg/kg/day), were considered not to be Tris(2-ethylhexyl) phosphate-related due to the lack of dose-related pattern, and/or general overlap and variability in individual values.
-Histopathology: Test item-related microscopic findings were noted in the testes of males at 250, 500 and 1000 mg/kg/day and thyroid gland of both sexes at 1000 mg/kg/day; Thyroid gland: Follicular cell hypertrophy was noted at increased incidence and/or severity at 1000 mg/kg/day in rats of both sexes (up to slight).
Testes: All test item-treated males showed intracytoplasmic eosinophilic inclusions in most of the interstitial cells of the testes. Examination of HE stained slides, revealed no dose-response in the amount of inclusions, and therefore they were not graded but recorded as present. In general, most inclusions were rectangular, and showed a crystalline appearance. There was no evidence for double breaking light when using a polarizing filter.
The Perl’s Prussian blue staining, a staining used to detect iron, did not stain the inclusions. The PAS staining coloured the inclusions very brightly, without distinction between dose levels. Based on the number and location of cells with inclusion, it was concluded that these cells represented Leydig cells. In addition, a minority of the cells within the interstitium showed PAS positive diffuse staining in the cytoplasm, comparable with the straining in the testes of control males. These PAS-positive cells were interpreted as macrophages.
Findings of note were present in the lungs of 1000 mg/kg/day group females No. 176, 179, 187 and 197 in the form of granulomatous bronchoalveolar inflammation (up to moderate degree) in one of the lung lobes of each animal and was confined to one or a few foci. This finding was considered to be related to regurgitation and/or aspiration of small amounts of test item with vehicle and was considered not to be a direct test-item related effect (Damsch et al., 2010).
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. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproduction Data - F0-Generation
- Sperm analysis: Sperm motility, concentration and morphology were considered to be unaffected by treatment with the test item up to 1000 mg/kg/day. Any statistically significant changes in sperm analysis parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and based on the direction of change (i.e. the opposite effect would be expected in case of target organ toxicity).
- Estrous Cycle: Note: For estrous cycle determination, vaginal lavages from the last two weeks of pre-mating (i.e. in Weeks 9 and 10) until Day 1 of mating were evaluated. Since several biologically non- plausible estrous stages were noted in Week 10, Day 7 of the pre-mating period, which resulted in an unrealistic high number of irregular cycle classification, the length and regularity of the estrous cycle evaluation was performed excluding this day with data of up to Week 10, Day 6 of the pre-mating period. Most females had regular cycles of 4 to 5 days in all groups. An irregular cycle was noted for Female Nos. 113, 117 (control) and 189 (1000 mg/kg/day), all of which had normal litters. Given its incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this isolated finding in the high dose group did not indicate a relation with treatment. The exclusion of two days in the second estrous cycle evaluation resulted in an inconclusive cycle determination for Female Nos. 118 (control), 137, 149 (250 mg/kg/day) and 158 (500 mg/kg/day). At this low incidence, the omission of estrous cycle data did not affect the overall evaluation of this study.
- Mating index: The mating index for males and females was 100% for all groups.
- Precoital Time: Most females were mated within the first 4 days of the mating period. Two females (one each at 250 and 500 mg/kg/day; Nos. 130 and 164) were mated after 5 days, one female after 9 days (250 mg/kg/day; No. 134), two females after 13 days (1000 mg/kg/day; Nos. 183 and 200) and two females after 14 days (one each at 250 and 1000 mg/kg/day; Nos. 143 and 178).
- Number of Implantation Sites: Number of implantation sites was unaffected by treatment with the test item up to
1000 mg/kg/day. Number of implantation sites were 12.5, 12.4, 12.9 and 12.4 for the control, 250, 500 and 1000 mg/kg/day groups, respectively.
- Fertility Index: The fertility indices for males and females were 96, 96, 100 and 96% for the control, 250, 500 and 1000 mg/kg/day groups, respectively.
A total of three females, one each in the control, 250 and 1000 mg/kg/day groups (Nos. 120, 140 and 188, respectively) were not pregnant. As these cases of non-pregnancy showed no dose-related incidence across the dose groups, it was unrelated to treatment.
- Histopathological evaluation of reproductive performance: There was 1/25 couple of the control group, 1/25 couple of the 250 mg/kg/day group and 1/25 couple of the 1000 mg/kg/day group without offspring. No histopathologically identified abnormalities were seen in the reproductive organs, that could account for their lack of offspring and these incidental lack of pregnancies in single females of the control, 250 and 1000 mg/kg/day group were regarded as unrelated to treatment. Furthermore, 1/25 control Group females and 1/25 females at 1000 mg/kg/day showed incomplete delivery or total litter loss, respectively, at Lactation Day 1. Recorded microscopic findings of the reproductive organs of these two females did fit with their total litter loss. Additional findings were present in Female No. 104 (Control Group) which were considered to be related with the intra-uterine presence of an autolytic dead fetus. These findings consisted of a reduced size of the thymus (correlating with decreased lymphoid cellularity, moderate), presence of a vascular thrombus in the lung and moderate necrosis of the liver. Based on the incidental finding in both high dose and control group, the total litter loss of the 1000 mg/kg/day female was regarded as unrelated to treatment with test item. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis of all F0-males revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Developmental Data - F0-Generation
- Gestation Index and duration: With the exception of two females (one control (No. 104) and one at 1000 mg/kg/day (199)) all pregnant females had live offspring. The gestation indices were 96, 100, 100 and 96% for the control, 250, 500 and 1000 mg/kg/day groups, respectively.
- Parturition/Maternal Care: No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Post-Implantation Survival Index: Post-implantation survival index was 94, 93, 93 and 94% for the control, 250, 500 and 1000 mg/kg/day groups, respectively.
- Litter size: Live litter sizes were 12.0, 11.5, 11.9 and 11.6 living pups/litter for the control, 250, 500 and 1000 mg/kg/day groups, respectively.
- Live Birth Index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was unaffected by treatment with the test item up to 1000 mg/kg/day. Live birth index was 99% for all groups. Four pups of the control group (Litter Nos. 105, 106, 110 and 121), three pups at 250 mg/kg/day (Litter Nos. 144 (one pup) and 148 (two pups)), four pups at 500 mg/kg/day (Litter Nos. 168 (three pups) and 175 (one pup)) and two pups at 1000 mg/kg/day (Litter Nos. 191 and 199) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. Female No. 104 (control) had an incomplete delivery (for details see section 9.2.1 and 9.4.7) and Female No. 199 (1000 mg/kg/day) had a total litter loss on lactation Day 1. At the isolated incidence, no toxicological significance was attributed to these findings.
-Viability Index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by treatment with the test item up to 1000 mg/kg/day. Viability indices were 100, 98, 100 and 99% for the control, 250, 500 and 1000 mg/kg/day groups, respectively. Five pups at 250 mg/kg/day (Litter Nos. 135, 143, 144 and 148 (two pups)), one pup at 500 mg/kg/day (Litter No. 158), and two pups at 1000 mg/kg/day (Litter Nos. 182 and 185) were found dead or missing on PND 2-4. Pups missing were most likely cannibalized. One pup at 250 mg/kg/day (Litter No. 134) was sacrificed in extremis on PND 1, due to wounds on its head and shoulders. 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.
- Weaning Index: The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item up to 1000 mg/kg/day. The weaning indices were 99, 100, 100 and 99% for the control, 250, 500 and 1000 mg/kg/day groups, respectively. One pup of the control group (Litter No. 103) was missing on PND 7 and was most likely cannibalized. One pup at 1000 mg/kg/day (Litter No. 200) was found dead on PND 19. 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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Male No. 442 of the 1000 mg/kg/day group was found dead at Day 30 and two animals of Cohort 1A and one animal of Cohort 1C were found dead after weaning on Day 6, 8 or 13 of treatment (Male No. 341 (500 mg/kg/day), Female No. 734
(1000 mg/kg/day) and Male No. 415 (1000 mg/kg/day), respectively).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In females, body weight gains were higher during Weeks 3 (250 mg/kg/day) and 4 (250, 500 and 1000 mg/kg/day) of treatment. In absence of a time and dose related trend, these changes were considered unrelated to treatment with the test item.

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 affected by treatment with the test item from 250 mg/kg/day. In the first week of treatment, a slight transient decrease in relative food consumption was noted in animals of all groups (not always dose-related and not always statistically significant); this was more pronounced in females than in males. For males at 1000 mg/kg/day, a trend towards slightly higher (relative) food consumption was observed throughout the treatment period from Week 2 onwards. Changes compared with the concurrent control group were relatively slight and not always statistically significant. Food consumption and relative food consumption were increased up to 1.10x and 1.08x, respectively, when compared with control. For females at all dose levels, a trend towards slightly higher (relative) food consumption was observed throughout the treatment period from Week 2 onwards. Changes compared with the concurrent control group were relatively slight and not always statistically significant. Food consumption and relative food consumption were increased up to 1.07-1.08x, when compared to control. Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increase in TSH in females at 250 mg/kg/day was considered unrelated to treatment in absence of a dose related trend.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Possible test item-related higher testes weights (absolute and relative to body weights) were
noted in F1-males at 1000 mg/kg/day.Apparent significant changes were noted in the absolute weight and/or relative to body weight ratio of the adrenal gland of F1-rats of Cohort 1B of both sexes at 500 and 1000 mg/kg/day within historical control data.

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment. It should be noted that neutrophils were higher in females at 1000 mg/kg/day, but as the increase was noted in one animal only (No. 703) and was in absence of corroborating findings, this increase was considered not toxicologically relevant. Any statistically significant changes in hematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

F1-Generation General Toxicity:

DETAIL RESULTS OF F1-GENERATION:
MORTALITY: There were no test item-related premature decedents. Male No. 442 of the 1000 mg/kg/day group was found dead at Day 30. The death of this animal was likely the result of that this animal was accidentally hit by a cage on that day after which it presented with fast breathing, piloerection, flat posture and was cold. The animal remained responsive and was active, however, was found dead a few hours after being hit by the cage. Like all other test-item treated males, the presence of intracytoplasmic eosinophilic inclusions was observed in the testes of this animal. Two animals of Cohort 1A and one animal of Cohort 1C were found dead after weaning on Day 6, 8 or 13 of treatment (Male No. 341 (500 mg/kg/day), Female No. 734(1000 mg/kg/day) and Male No. 415 (1000 mg/kg/day), respectively). No clinical signs were observed for these animals in the days prior to their deaths. Cannibalism was observed for Animal Nos. 341 and 734. As the deaths of these animals occurred shortly after start of treatment and was incidental, the death of these animals was considered unrelated to treatment with the test item. The two Cohort 1A animals were therefore replaced by two other animals.
CLINICAL OBSERVATIONS: Any 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. No findings were noted during the weekly arena observations in this study. Note: “Day 1” of treatment is the day that the first animals were weaned.
BODY WEIGHTS AND BODY WEIGHT GAINS: Body weights and body weight gain were considered to have been unaffected by treatment with the test item up to 1000 mg/kg/day. In females, body weight gains were higher during Weeks 3 (250 mg/kg/day) and 4 (250, 500 and 1000 mg/kg/day) of treatment. In absence of a time and dose related trend, these changes were considered unrelated to treatment with the test item. Note: “Day 1” of treatment is the day that the first animals were weaned. Body weight of animals that were weaned during the subsequent 7 days were all entered under that day. Body weight of animals that were weaned in the subsequent week were all entered under “Day 8”. This may have resulted in the statistically significant changes as described above. Since the majority of animals were weaned within 7 days after commencement of weaning of the first animals, “Day 8” was used as the reference day for body weight gain calculations.
FOOD CONSUMPTION: Food consumption before or after correction for body weight was affected by treatment with the test item from 250 mg/kg/day. In the first week of treatment, a slight transient decrease in relative food consumption was noted in animals of all groups (not always dose-related and not always statistically significant); this was more pronounced in females than in males. For males at 1000 mg/kg/day, a trend towards slightly higher (relative) food consumption was observed throughout the treatment period from Week 2 onwards. Changes compared with the concurrent control group were relatively slight and not always statistically significant. Food consumption and relative food consumption were increased up to 1.10x and 1.08x, respectively, when compared with control. For females at all dose levels, a trend towards slightly higher (relative) food consumption was observed throughout the treatment period from Week 2 onwards. Changes compared with the concurrent control group were relatively slight and not always statistically significant. Food consumption and relative food consumption were increased up to 1.07-1.08x, when compared to control. Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.
SEXUAL MATURATION: Sexual maturation was considered unaffected by treatment with the test item up to 1000 mg/kg/day. The statistical increase in the age and body weight at attainment of balanopreputial separation at 1000 mg/kg/day was considered not relevant as the increases remained within the historical control rang. The age and body weight at attainment of vaginal opening was slightly increased at
250 mg/kg/day and was considered unrelated to treatment in absence of a dose related trend.
ESTROUS CYCLE – F1-GENERATION (COHORT 1A): 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. Most females had regular cycles of 4 to 5 days in all groups. An irregular cycle was noted for Female No. 622 (500 mg/kg/day) and for Female No. 693 (1000 mg/kg/day). Given their incidental nature and absence of a dose-related incidence these findings did not indicate a relation with treatment. For Female Nos. 494 (control), 561, 568 (250 mg/kg/day) and 627 (500 mg/kg/day) the cycle classification could not be determined.
SPERM ANALYSIS – F1-GENERATION (COHORT 1A): Sperm motility, concentration and morphology were unaffected by treatment with the test item up to 1000 mg/kg/day.
HEMATOLOGY – F1-GENERATION (COHORT 1A): Hematological parameters of treated rats were considered not to have been affected by treatment. It should be noted that neutrophils were higher in females at 1000 mg/kg/day, but as the increase was noted in one animal only (No. 703) and was in absence of corroborating findings, this increase was considered not toxicologically relevant. Any statistically significant changes in hematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
COAGULATION – F1-GENERATION (COHORT 1A): Coagulation parameters were unaffected by treatment with the test item up to 1000 mg/kg/day.
CLINICAL CHEMISTRY – F1-GENERATION (COHORT 1A): Clinical biochemistry parameters were considered unaffected by treatment with the test item at 1000 mg/kg/day. Any statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. Serum levels of TSH and T4 in F1-males and females were considered not to be affected by treatment. The statistically significant increase in TSH in females at 250 mg/kg/day was considered unrelated to treatment in absence of a dose related trend.
URINALYSIS – F1-GENERATION (COHORT 1A): Urinary parameters were unaffected by treatment with the test item up to 1000 mg/kg/day.
SPLENIC LYMPHOCYTE SUBPOPULATION – F1-GENERATION (COHORT 1A): There were no immunotoxicologically relevant effects on splenic lymphocyte subpopulations observed. Statistically lower NK-cell splenic subpopulations were observed for females at 1000 mg/kg/day. This shift was considered to represent biological variability and considered to be of no immunotoxicological relevance, because of the minimal magnitude of the shift and the absence of any findings indicative of an affected immune system.
MACROSCOPIC FINDINGS – F1-GENERATION: Cohort 1A (PND 89-101); there were no test item-related macroscopic findings. Findings of note were present in the lymph nodes (axillary, popliteal and/or iliac) consisting of enlargement and/or discoloration (dark red, greenish or tan) without dose-relationship. Microscopic correlates consisted of sinus erythrocytes for the dark red discoloration and increased number of lymphocytes and/or plasma cells for the enlargement. There was no microscopic correlate for the greenish or tan discoloration of some lymph nodes, although the presence of black pigment in the lymph nodes (most likely due to the identification procedure after birth) of some of these animals might explain the recording of a gross lesions at necropsy. The recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B (≥ PND 97); there was one finding of note in one of the kidneys of a 1000 mg/kg/day F1-female of Cohort-1B, consisting of a unilateral reduced size, yellowish, hard kidney. Since there were no test item-related macroscopic or microscopic findings in the kidneys of any other animal belonging to the parental F0-Generation or F1-Generation (Cohort 1A or 1B), this finding was regarded as unrelated to treatment with the test item. The remainder of the recorded macroscopic findings of F1-rats of Cohort 1B were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1C (males: ≥ PND 35; females: ≥ PND 25); there were no test item-related macroscopic findings. The recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort Surplus (PND 22-24; there were no test item-related macroscopic findings.
ORGAN WEIGHTS – F1-GENERATION: Cohort 1A (PND 89-101); possible test item-related organ weights differences (absolute and/or relative to body weights) were noted in the testes of 1000 mg/kg/day group males (absolute and relative to body weights). Statistically significant higher kidney to body weight ratios (+8%**) were present in F1-males of Cohort 1A in all test item-treated groups. Since there was no significant change in absolute weight, no dose response, and no test item-related microscopic finding, this apparent small increase in kidney weight, was considered to be unrelated to treatment with the test item. Apparent significant higher liver weights (absolute and relative to body weight) were present in F1-females of Cohort 1A in all test item-treated groups. There was no dose response, no test item-related morphologic correlate, and mean values remained within historical control range. Therefore, a relationship with the test item was regarded unlikely. Any other differences, including those that reached statistical significance (F1-males: relative to body weight of prostate gland at 500 mg/kg/day and absolute weight of thyroid gland at 1000 mg/kg/day; F1-females: absolute weight of mesenteric lymph node at 250 mg/kg/day) were considered not to be Tris(2-ethylhexyl) phosphate-related due to the lack of dose-related pattern, and/or general overlap and variability in individual values.
Cohort 1B (≥ PND 97); possible test item-related higher testes weights (absolute and relative to body weights) were noted in F1-males of Cohort 1B at 1000 mg/kg/day. Apparent significant changes were noted in the absolute weight and/or relative to body weight ratio of the adrenal gland of F1-rats of Cohort 1B of both sexes at 500 and 1000 mg/kg/day. This weight change was absent in Cohort 1A and mean values remained within the historical control range. Therefore, this organ weight change was considered unrelated to treatment.
Any other differences, including those that reached statistical significance (F1-males: relative to body weight of the pituitary gland at 500 mg/kg/day and of the epididymides at
1000 mg/kg/day; F1-females: absolute weight of thyroid gland at 500 and 1000 mg/kg/day and relative to body weight of the ovaries at 500 mg/kg/day) were considered not to be Tris(2-ethylhexyl) phosphate-related due to the lack of dose-related pattern, and/or general overlap and variability in individual values.
HISTOPATHOLOGY – F1-GENERATION: Test item-related microscopic findings after treatment with Tris(2-ethylhexyl) phosphate were noted in the testes at 250, 500 and 1000 mg/kg/day and thyroid gland of males at 1000 mg/kg/day. Thyroid gland: Follicular cell hypertrophy was noted at increased incidence and/or severity at 1000 mg/kg/day in males (up to slight). Testes: All test item-treated males showed similar intracytoplasmic eosinophilic inclusions in most of the interstitial cells of the testes, as in the F0-generation. Results of the additional staining’s were comparable with the F0-generation. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Ovarian Follicle and Corpora lutea Counts - Cohort 1A; there were no test item-related effects on the ovarian follicle and Corpora lutea counts in the F1-1000 mg/kg/day group females when compared to control group females. Any variation between group mean counts represented biological variability.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 14. Weekly Summary of Clinical Signs in Male and Female
		Pre mating										Repro Period					Pre mating										Repro Period
		Male													Female
Sign (Max. Grade) (location)	Week	1	2	3	4	5	6	7	8	9	10	1	2	3	Sign (Max. Grade (Location)	Week	1	2	3	4	5	6	7	8	9	10	1	2	3	4	5	6	7	8	9
Group 1 (control)
Skin/fur															Posture Hunched posture (1)
Scabs (3)	G	.	.	1	.	.	.	.	.	.	.	.	.	.		G	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.	.	.
	%	.	.	0	.	.	.	.	.	.	.	.	.	.		%	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.	.	.	.	.
Scabs (3) (Cheek left)	G	.	1	.	.	.	.	.	.	.	.	.	.	.	Skin/fur Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.	.	.
	%	.	0	.	.	.	.	.	.	.	.	.	.	.		%	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.	.	.	.	.
Scab (3) (Shoulder right)	G	.	.	1	1	1	1	1	1	1	1	.	.	.	Various Pale (3) (General)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	3	.	.	.	.	.
	%	.	.	0	0	0	0	0	0	0	0	.	.	.		%	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.	.	.	.	.
Group 2 (250 mg/kg/day)
Skin/fur
Alopecia (3) (Head)	G	.	.	.	.	.	.	.	.	.	1	1	.	.	Skin/fur Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	0	0	.	.		%	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.	.	.	.
Scabs (3)	G	.	.	1	.	.	.	.	.	.	.	.	.	.	Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.	.
	%	.	.	0	.	.	.	.	.	.	.	.	.	.		%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.	.
Scabs (3) (Head)	G	.	.	.	.	.	.	.	.	.	1	.	.	.	Scabs (3) (Head)	G	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	0	.	.	.		%	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Scabs (3) (Back)	G	1	1	1	1	1	1	1	1	1	1	1	1	.	Secretion/ excretion Salivation(3)	G	.	.	.	.	.	1	.	1	.	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	0	0	0	0	0	0	0	0	0	.		%	.	.	.	.	.	2	.	1	.	.	.	.	.	.	.	.	.	.	.
Scabs (3) (Neck)	G	.	1	1	1	1	1	1	1	1	1	1	.	.
	%	.	0	0	0	0	0	0	0	0	0	0	.	.
Scabs (3) (Flank left)	G	.	1	1	.	.	.	.	.	.	.	...
	%	.	1	0	.	.	.	.	.	.	.	.	.	.
Scabs (3) (Shoulder left)	G	.	1	.	.	.	.	.	.	.	.	.	.	.
	%	.	0	.	.	.	.	.	.	.	.	.	.	.
Scabs (3) (Shoulder right)	G	1	1	.	1	1	.	.	.	.	.	.	.	.
	%	0	1	.	0	0	.	.	.	.	.	.	.	.
Wound (3) (Head)	G	.	.	.	.	.	.	.	.	.	1	.	.	.
	%	.	.	.	.	.	.	.	.	.	0	.	.	.
Wound (3) (Neck)	G	.	.	1	1	.	.	.	.	.	.	1	1	.
	%	.	.	0	0	.	.	.	.	.	.	0	0	.
Secretion/ excretion (Salivation (3)	G	.	.	.	.	1	1	1	1	1	.	.	.	.
	%	.	.	.	.	3	3	0	0	0	.	.	.	.
Group 3 (500 mg/kg bw/day)
Skin/fur
Scabs (3) (Neck)	G	.	.	.	1	1	1	1	.	.	.	.	.	.	Skin/fur Alopecia (3)	G	.	.	.	.	.	1	1	1	1	1	1	1	1	1	.	.	.	.
	%	.	.	.	0	0	0	0	.	.	.	.	.	.		%	.	.	.	.	.	0	0	0	0	0	1	1	1	1	.	.	.	.
Secretion/excretion Salivation (3)	G	.	.	1	1	1	1	1	1	1	1	1	.	.	Alopecia (3) (Neck)	G	.	.	.	.	.	.	.	.	.	1	1	.	.	.	.	.	.	.
	%	.	.	1	0	4	6	4	4	4	5	2	.	.		%	.	.	.	.	.	.	.	.	.	0	0	.	.	.	.	.	.	.
															Scabs (3) (Cheek left)	G	.	.	.	.	.	1	1	1	.	.	.	.	.	.	.	.	.	.
																%	.	.	.	.	.	0	0	0	.	.	.	.	.	.	.	.	.	.
															Scabs (3)3 (Neck)	G	.	.	.	.	.	.	1	1	1	.	.	.	.	.	.	.	.	.
																%	.	.	.	.	.	.	0	0	0	.	.	.	.	.	.	.	.	.
															Secretion/excretion Salivation (3)	G	.	.	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.
																%	.	.	1	0	2	4	3	3	4	6	.	.	.	.	.	.	.	.
															Various Exophthalmos (1) (Eye left)	G	.	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
																%	.	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1
Group 4 (1000 mg/kg bw/day)
Skin/fur
Scabs(3) (Ear right)	G	.	.	.	1	1	.	.	.	.	.	.	.	.	Skin/fur Swelling (4) (Throat region)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	2	.	.
	%	.	.	.	0	0	.	.	.	.	.	.	.	.		%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.	.
Wound (3) (Ear right)	G	.	.	1	1	.	.	.	.	.	.	.	.	.	Alopecia (3)	G	.	.	1	1	.	.	.	1	1	1	1	2	2	1	1	1	1	1	1
	%	.	.	0	0	.	.	.	.	.	.	.	.	.		%	.	.	0	0	.	.	.	0	0	0	0	0	1	1	1	1	1	1	3
Secretion/excretion Salivation (3)	G	.	1	1	1	1	1	1	1	1	1	1	1	.	Alopecia (3) (Abdomen)	G	.	.	.	.	.	.	.	.	.	1	1	.	.	.	.	.	.	.	.
	%	.	1	2	5	6	6	8	7	4	6	2	0	.		%	.	.	.	.	.	.	.	.	.	0	0	.	.	.	.	.	.	.	.
															Secretion/excretion Salivation (3)	G	.	1	1	1	1	1	1	1	1		1	1	.	.	.	.	.	.	.
																%	.	0	3	2	3	5	4	6	3	6	1	0	.	.	.	.	.	.	.
G: Mean value of the highest individual weekly grades %: Percent of affected animals (0= less than 5%, 1 = between 5% and 15%,…, A=more than 95%) .: Observation performed, sign not present

Table 15. Summary of Clinical Signs in Male and Female

		Pre Mating											Repro Period									Pre mating										Repro Period
		Male																				Female
Sign (Max. Grade ) (Location)	Week	1	2	3	4	5	6	7	8	9	10	1	2	3	4	5	6	7	8	9	10	1	2	3	4	5	6	7	8	9	10	1	2	3	4	5	6	7	8	9	10
	Day	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	2	2	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	2	2
Group 1 (control)
No clinical signs noted
Group 2 (250 mg/kg/day)
No clinical signs noted
Group 3 (500 mg/kg/day)
Behaviour Lethargy (3)	G	.	.	.	.	.	.	1	.	.	.	.	.	.								No clinical signed noted
	%	.	.	.	.	.	.	0	.	.	.	.	.	.
Group 4 (1000 mg/kg/day)
No clinical signs noted
G: Median value of the highest individual daily grades %: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,…, A+= more than 95%) .: Observation performed, sign not present

Table 16. Mean Summary of Male Body Weights (g)  and Body Weight Gain (%)

	Body Weights (g)				Body Weight Gain (%)
	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Premating
Day 1 Week 1	180	179	177	179	0	0	0	0
Day 8 Week 2	224	222	220	223	25	24	24	25
Day 15 Week 3	262	259	259	259	45	44	46	45
Day 22 Week 4	292	290	290	290	62	62	64	63
Day 29 Week 5	317	316	316	315	76	76	78	77
Day 36 Week 6	333	333	332	332	85	86	88	87
Day 43 Week 7	351	351	347	350	95	95	96	96
Day 50 Week 8	362	362	358	260	101	102	102	102
Day 57 Week 9	375	374	370	371	108	108	109	108
Day 64 Week 10	386	387	381	383	114	116	115	114
Mating period
Day 1 Week 1	396	397	391	393	120	121	121	120
Day 8 Week 2	399	397	395	396	121	121	123	122
Day 15 Week 3	402	396	395	422	126	121	121	134
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 17. Mean Summary of  Female Body Weights (g) and Body Weight Gain (%)

	Body Weight (g)				Body Weight Gain (%)
	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Premating
Day 1 Week 1	125	122	122	122	0	0	0	0
Day 8 Week 2	145	144	145	145	16	18	19	19
Day 15 Week 3	163	162	166	167	31	33	36 **	37 **
Day 22 Week 4	178	179	182	184	43	47	48 **	51 **
Day 29 Week 5	193	193	196	198	55	58	60 *	62 **
Day 36 Week 6	204	204	209	210	63	67	71 **	72 **
Day 43 Week 7	213	211	218	220	71	73	78 **	80 **
Day 50 Week 8	215	214	223	225	73	75	82 **	84 **
Day 57 Week 9	224	220	228	231	80	81	86 *	89 **
Day 64 Week 10	228	227	233	235	83	86	91 *	93 **
Mating Period
Day 1 Week 1	232	231	239	241	87	89	95 **	98 **
Day 8 Week 2		278	---	270		116	---	113
Day 15 Week 3	---	---	---	---	---	---	---	---
Day 22 Week 4		---	---	---	---	---	---	---
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level x Explanations for excluded data are listed in the tables of the individual values

Table 18. Mean Summary of  Female Body Weights (g) and Body Weight Gains (%) -  F_0-generation

Body Weights (g)					Body Weight Gain (%)
	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
F0-Generation
Post Coitum
Day 0	228	231		235	242*	0	0	0	0
Day 4	239	242		248	256*	5	5	6	6
Day 7	244	247		254	263 **	7	7	8	9 *
Day 11	259	261		269	277 *	14	13	14	15
Day 14	265	271		277	285*	16	17	18	18
Day 17	291	294		303	310*	28	27	29	28
Day 20	329	333		341	352*	45	44	45	46
Lactation
Day 1	249	250		260	272**	0	0	0	0
Day 4	261	267		272	285 **	5	6	5	5
Day 7	269	276		281	285 **	8	10	8	8
Day 14	287	287		292	300	15	15	12	11 **
Day 21	277	282		288	294 *	12	12	11	8
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level x Explanations for excluded data are listed in the tables of the individual values

Table 19. Mean Summary of  Food Consumption (g/animal/day) and Relative Food Consumption (g/kg bw/day) in Male

Food Consumption (g/animal/day)					Relative Food consumption (g/animal/day)
	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Premating
Day 1-8 Week 1-2	20	20		20	20	91	92	92	91
Day 8-15 Week 2-3	23	23		23	23	86	88	89	88
Day 15-22 Week 3-4	23	23		23	24 *	78	79	81 *	83 **
Day 22-29 Weeks 4-5	22	22		23	24 **	69	71	71 **	75 **
Day 29-36 Week 5-6	22	22		22	23 *	65	66	67 *	69 **
Day 36-43 Week 6-7	21	21		21	22	59	59	60	62
Day 43-50 Week 7-8	20	20		20	21	55	55	56	57
Day 50-57 Week 8-9	20	20		20	20	52	52	54	55 *
Day 57-64 Week 9-10	19	20		20	21	50	52	52	54 **
Day 64-71 Weeks 10-11	19	19		19	20	48	50	51	52 *
Mean of Means over pre mating period	21	21		21	22	65	66	68	69
Mating Period
Day 1-8 Week 1-2	19	20		20	20	47	50	50	51
Day 8-15 Week 2-3	18	18		18	16	44	45	46	39
Day 15-22 Week 3-4	---	---		---	---	---	----	----	---
Mean of means over mating period	18	19		19	18	45	47	48	45
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 20. Mean Summary of  Food Consumption (g/animal/day) and Relative Food Consumption (g/kg bw/day) in Female

	Food Consumption (g/animal/day)					Relative Food consumption (g/animal/day)
	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Premating
Day 1-8 Week 1-2	14	15		14	15*	96	101	100	102 *
Day 8-15 Week 2-3	15	16		16 *	16 **	92	96	97 *	98 **
Day 15-22 Week 3-4	15	16		17 *	17 **	87	89	91	92 *
Day 22-29 Weeks 4-5	15	15		16	17 **	78	80	81	85 **
Day 29-36 Week 5-6	15	16		16 *	17 **	75	77	79 *	81 **
Day 36-43 Week 6-7	15	15		16 **	17 **	71	73	75 **	77 **
Day 43-50 Week 7-8	15	15		16 *	16 **	68	71	72 *	73 **
Day 50-57 Week 8-9	15	15		16	16	67	67	69	71
Day 57-64 Week 9-10	14	15		15 *	16 **	61	65	66 *	68 **
Day 64-71 Weeks 10-11	14	14		15 *	16 **	60	64	64*	67 **
Mean of Means over pre mating period	15	15		16	16	75	78	79	81
Mating Period
Day 1-8 Week 1-2				---				---
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 21. Mean Summary of  Food Consumption (g/animal/day) and Relative Food Consumption (g/kg bw/day) in Female -  F_0-generation

	Food Consumption (g/animal/day)					Relative Food consumption (g/animal/day)
	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Post Coitum
Days 0-4	15	16		17	19 **	64	66	67	73 **
Days 4-7	17	17		18 *	20 **	68	68	71	75 **
Days 7-11	17	18		19 *	19 *	65	68	70	70
Days 11-14	18	20 *		20 *	21 **	68	73 *	72	75 **
Days 14-17	19	19		21 *	22 **	65	66	69	72 **
Days 17-20	21	23		23	25 **	65	68	68	71 *
Mean of means	18	19		20	21	66	68	69	73
Lactation
Days 1-4	31	29		28	31	117	110	102 **	109
Days 4-7	37	38		38	41 **	138	138	135	142
Days 7-14	51	50		49	51	179	174	167	170
Days 14-21	59	58		60	61	215	207	210	206
Mean of Means	45	44		44	46	162	157	153	157
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 22. Mean Summary of Hematology Parameters in Male

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
WBC 10E9/L	7.5	6.8		6.1 *	6.7
Neutrophils 10E9/L	1.2	1.4		1.1	1.1
Lymphocytes 10E9/L	5.9	5.0		4.7	5.3
Monocytes 10E9/L	0.2	0.2		0.2	0.2
Eosinophils 10E9/L	0.1	0.1		0.1	0.1
Basophils 10E9/L	0.0	0.0		0.0	0.0
LUC 10E9/L	0.1	0.1		0.1	0.0
Red blood cells 10E12/L	8.63	8.65		8.76	8.62
Reticulocytes 10E9/L	218.9	214.2		191.9	194.5
RDW %	11.9	12.0		11.9	12.2
Haemoglobin Mmol/L	9.4	9.3		9.2 *	9.3
Haematocrit L/L	0.463	0.459		0.459	0.455
MCV fL	53.7	53.1		52.4	52.8
MCH Fmol	1.09	1.08		1.05 *	1.08
MCHC Mmol/L	20.39	20.35		19.97	20.48
Platelets 10E9/L	695	714		769 **	754 *
PT S	16.2	17.3		17.9 **	16.4
APTT S	18.9	19.1		20.1	18.6
+/++ Steel-test significant at 5% (+) or 1% (++) level */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 23. Mean Summary of Hematology Parameters in Female

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
WBC 10E9/L	5.0	4.4		5.5	4.5
Neutrophils 10E9/L	1.1	1.4		1.9	1.2
Lymphocytes 10E9/L	3.4	2.7		3.2	2.9
Monocytes 10E9/L	0.2	0.2		0.2	0.2
Eosinophils 10E9/L	0.2	0.1 +		0.1	0.1
Basophils 10E9/L	0.0	0.0		0.0	0.0
LUC 10E9/L	0.1	0.0		0.1	0.1
Red blood cells 10E12/L	8.75	8.50		8.67	8.68
Reticulocytes 10E9/L	167.0	190.6		190.7	192.1
RDW %	11.6	11.9		12.2	11.6
Haemoglobin Mmol/L	10.8	10.4		10.1 **	10.3 *
Haematocrit L/L	0.521	0.498 *		0.486 **	0.498 *
MCV fL	59.5	58.7		56.1 **	57.4
MCH Fmol	1.24	1.22		1.17 **	1.18 *
MCHC Mmol/L	20.79	20.84		20.83	20.59
Platelets 10E9/L	832	855		923	920
PT S	16.5	16.1		16.8	16.6
APTT S	20.3	19.3		21.1	21.3
+/++ Steel-test significant at 5% (+) or 1% (++) level */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 24. Mean Summary of Clinical Biochemical Parameters in Male

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
ALAT U/L	35.6	35.2		32.4	35.2
ASAT U/L	82.1	82.0		77.4	79.1
ALP U/L	121	117		119	105
Total protein g/L	61.2	63.1 *		65.0 **	63.0
Albumin g/L	32.2	32.6		33.6 **	32.4
Total bilirubin umol/L	2.7	2.6		2.6	2.7
Urea Mmol	4.5	4.5		4.2	4.6
Creatinine Umol/L	35.0	34.2		34.4	34.6
Glucose Mmol/L	9.72	8.73		8.83	9.14
Cholestrol Mmol/L	1.87	1.86		1.80	1.67
Bile Acids Umol/L	17.7	13.8		10.0	8.3 +
Sodium Mmol/L	141.1	142.3*		143.0 **	141.8
Potassium Mmol/L	3.90	4.04		3.99	4.08
Chloride Mmol/L	104	106*		106	105
Calcium Mmol/L	2.49	2.52		2.51	2.54
Inorg. Phos Mmol/L	1.97	1.83		1.74 *	1.81
TSH ulU.mL	0.103	0.212		0.246	0.262
Total T4 Ug/dL	5.57	5.81		5.92	6.02
Cohort Surplus; end of in-life
TSH ulU/mL	0.092	0.098		0.145	0.115
Total T4 ug/dL	4.93	5.00		4.98	5.44
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 25. Mean Summary of Clinical Biochemical Parameters in Female

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
ALAT U/L	38.5	36.6		42.9	35.8
ASAT U/L	95.3	93.2		96.2	87.3
ALP U/L	78	59		78	63
Total protein g/L	66.2	68.5		68.1	71.5 **
Albumin g/L	35.9	37.3		37.1	36.8 **
Total bilirubin umol/L	2.2	1.8 *		1.7 **	1.8 *
Urea Mmol	8.6	8.1		7.9	8.5
Creatinine Umol/L	39.8	42.1		40.2	41.6
Glucose Mmol/L	8.71	8.65		9.10	9.93
Cholestrol Mmol/L	2.76	2.71		2.73	2.85
Bile Acids Umol/L	40.3	13.6		23.2	16.1
Sodium Mmol/L	140.8	141.3		140.7	141.1
Potassium Mmol/L	3.46	3.29		3.35	3.33
Chloride Mmol/L	103	103		102	103
Calcium Mmol/L	2.55	2.58		2.56	2.62
Inorg. Phos Mmol/L	1.82	1.91		2.02	1.77
TSH ulU.mL	0.190	0.416		0.327	0.288
Total T4 Ug/dL	5.09	5.67		5.69	4.79
Cohort Surplus; end of in-life
TSH ulU/mL	0.085	0.076		0.086	0.102
Total T4 ug/dL	4.79	4.70		5.16	4.39
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 26. Mean Summary of Urinlysis Parameters in Male

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
Volume mL	8	7		7	6
Specific gravity	1.029	1.038		1.036	1.038
Clarity Score 1/3	1	3		3	1
Colour Score 1/5	1	2		2	2
pH	6.8	6.6		6.6	6.6
Blood score 0/3	0	0		0	0
WBC score 0/3	0	0		0	0
Bilirubin score 0/3	0	0		0	0
Urobilinogen score 0/3	0	0		0	0
Protein score 0/3	0	0		0	0
Ketones score 0/3	0	0		0	0
Glucose score 0/3	0	0		0	0
WBC-sed score 0/3	1	1		0	0
RBD-sed score 0/3	1	1		0+	0+
Casts score 0/3	0	0		0	0
Epithelial cells score 0/3	2	1		1	1
Crystals score 0/3	2	3		3	3
Bacteria score 0/3	2	2		3	3
Other score 0/3	0	0		0	0
+/++ Steel-test significant at 5% (+) or 1% (++) level */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 27. Mean Summary of Urinlysis Parameters in Female

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
Volume mL	15	13		16	11
Specific gravity	1.025	1.026		1.024	1.030
Clarity Score 1/3	1	1		1	1
Colour Score 1/5	1	1		1	1
pH	6.4	6.3		6.3	6.1
Blood score 0/3	0	0		0	0
WBC score 0/3	0	0		0	0
Bilirubin score 0/3	0	0		0	0
Urobilinogen score 0/3	0	0		0	0
Protein score 0/3	0	0		0	0
Ketones score 0/3	0	0		0	0
Glucose score 0/3	0	0		0	0
WBC-sed score 0/3	0	0		0	1
RBD-sed score 0/3	0	0		0	0
Casts score 0/3	0	0		0	0
Epithelial cells score 0/3	1	1		1	2
Crystals score 0/3	1	1		1	2
Bacteria score 0/3	0	0		2	1
Other score 0/3	3	3		3	3
+/++ Steel-test significant at 5% (+) or 1% (++) level */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 28. Summary of Macroscopic Findings in Male

	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
Animals examined	25	25		25	25
Animals without findings	18	16		17	15
Animals affected	7	9		8	10
Stomach
Focus/foci	4	2		3	4
Thickened	0	0		1	1
lleum
Focus/foci	0	0		0	1
Liver
Accentuated lobular pattern	0	0		1	1
Diaphragmatic hernia	1	0		0	0
Accessory liver	1	0		2	0
Nodules(s)	0	1		0	0
Reduced in size	0	0		1	0
Kidneys
Pelvic dilation	0	0		1	1
Cyst(s)	1	0		0	0
Irregular surface	0	2		0	1
Thyroid Gland
Reduced in size	0	0		0	1
Sleen
Ectopic splenic tissue	1	0		0	0
Thymus
Focus/foci	0	4		0	3
Discolouration	0	0		1	0
Mesenteric lymph node
Discolouration	0	0		1	0
Skin
Wound(s)	0	1		0	0
Scab formation	0	1		0	0
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 29. Summary of Macroscopic Findings in Female

	Group 1 Control		Group 2 250 mg/kg/day		Group 3 500 mg/kg/day		Group 4 1000 mg/kg/day
Intercurrent death
Animals examined	1					1
Animals affected	1					1
General observations
Incomplete delivery	1					0
Total litter loss	0					1
Discolouration	1					0
Uterus
Contents:	1					1
Thymus
Reduced in size	1					0
End of Treatment
Animals examined	24	25			25		24
Animals without findings	17	14			9		12
Animals affected	7	11			16		12
Stomach
Nodule(s)	1	0			2		0
Focus/foci	3	3			2		5
Irregular surface	0	1			0		0
Liver
Papillary process, right side constricted	0	0			0		1
Diaphragmatic hernia	1	0			1		0
Accessory liver	0	2			0		0
Reduced in size	1	0			0		1
Discolouration	0	0			0		1
Ovaries
Cyst(s)	0	1			1		0
Reduced in size	0	0			0		1
Uterus
Thickened	0	0			0		1
Contains fluid	1	6			7 #		1
Thyroid Gland
Agenesis	0	0			0		1
Thymus
Focus/foci	3	1			3		0
Reduced in size	0	0			2		3
Salivary glands
Hardened	0	0			0		1
Skin
Alopecia	0	0			0		2
Eyes
Exophthalmos	0	0			1		0
Body cavities
Nodule(s)	0	1			0		0
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 30. Mean Summary of Organ Weight (g) and Organ/Body Weight Ratio (%) in Male

	Organ Weight (g)					Organ/Body Weight Ratio (%)
	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
Body W.	385	384		379	380	-	-	-	-
Brain	2.08	2.10		2.07	2.08	0.54	0.55	0.55	0.55
Pituitary	0.009	0.009		0.009	0.009	0.002	0.002	0.002	0.002
Heart	1.038	1.052		1.044	1.030	0.270	0.274	0.276	0.271
Liver	9.22	9.59		9.46	9.18	2.39	2.49 *	2.49 *	2.41
Thyroids	0.0168	0.0192 *		0.0188	0.0185	0.0044	0.0050 *	0.0050	0.0049
Thymus	0.304	0.309		0.294	0.291	0.079	0.080	0.078	0.077
Kidney	2.41	2.55		2.52	2.47	0.63	0.67 **	0.67 **	0.65
Adrenals	0.048	0.055 **		0054 *	0.056 **	0.013	0.014 **	0.014 **	0.015 **
Spleen	0.590	0.604		0.509	0.611	0.153	0.157	0.155	0.160
Testes	3.58	3.54		3.58	3.63	0.93	0.92	0.95	0.96
Prostate	0.842	0.856		0.887	0.842	0.219	0223	0235	0.233
Epididymides	1.168	1.161		1.183	1.220	0.305	0.304	0.314	0.322
Seminal Ves	1.376	1.434		1.469	1.403	0.360	0.375	0.390	0.371
Cohort Surplus; End of in-life
Body W.	58	58		59	57	-	-	-	-
Brain	1.48	1.48		1.51	1.48	2.56	2.57	2.60	2.60
Thymus	0.227	0.233		0.230	0.220	0.394	0.401	0.395	0.386
Spleen	0.276	0.279		0.315	0.283	0.477	0.479	0.534	0.494
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 31. Mean Summary of Organ Weight (g) and Organ/Body Weight Ratio (%) in Female

	Organ Weight (g)					Organ/Body Weight Ratio (%)
	Group 1 Control		Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
End of Treatment
Body W.	236	235		240	251 *	-	-	-	-
Brain	1.89	1.90		1.91	1.91	0.80	0.81	0.80	0.77
Pituitary	0.012	0.012		0.012	0.012	0.005	0.005	0.005	0.005
Heart	0.895	0.890		0.924	0.926	0.380	0.380	0.386	0.368
Liver	9.07	9.73		10.38 **	11.02 **	3.86	4.14 *	4.32 **	4.42 **
Thyroids	0.0163	0.0163		0.0176	0.0165	0.0069	0.0070	0.0074	0.0066
Thymus	0.201	0..201		0.198	0.202	0.085	0.086	0.083	0.081
Kidney	1.91	1.99		2.01	2.08 **	0.81	0.85 *	0.84	0.83
Adrenals	0.067	0.068		0.071	0.076 *	0.029	0.029	0.30	0.030
Spleen	0.516	0.497		0.518	0.528	0.218	0.211	0.215	0.211
Ovaries	0.136	0.139		0.137	0.146	0.058	0.059	0.057	0.058
Uterus	0.664	0.744		0.745	0.716	0.282	0.319	0.314	0.287
Cohort Surplus; End of in-life
Body W.	56	54		55	55	-	-	-	-
Brain	1.42	1.46		1.47	1.47	2.60	2.72	2.69	2.71
Thymus	0.247	0.247		0.251	0.224	0.442	0.455	0.457	0.405
Spleen	0.303	0.293		0.267	0.291	0.552	0.542	0..483	0.531
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 32. Summary of Sperm Motility, Concentration and Morphology

	Group 1 Control		Group 2 250 mg/kg/day		Group 3 500 mg/kg/day		Group 4 1000 mg/kg/day
Sperm Analysis
Motile Sperm %	57	59		53		53
Progressiv. Sperm %	23	24		19		20
SPERMCOUNT EPI 10E6/gram	437.1	534.1 **		417.8		467.6
Normal morp No. of cells	177	179		177		182
Detached head No of cells	3	3		4		2
Abnormal head No of cells	2	1 +		1+		1
Coiled tail No of ells	16	16		16		13
Other tail No of cells	1	1		1		1
Abnormal neck No of cells	1	1		1		1
Combined No of cells	0	0		0		0
+/++ Steel-test significant at 5% (+) or 1% (++) level */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 33. Summary of Reproductive Data

	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Males paired	25	25	25	25
Males mated	25	25	25	25
Females paired	25	25	25	25
Females mated	25	25	25	25
Pregnant females	24	24	25	24
Females with total litter loss on Day 1	0	0	0	1
Females with living pups on Day 1	24	24	25	23
Mating Index males (5%) (Males mated/Males paired) *100	100	100	100	100
Fertility Index males (%) (Pregnant females/Males mated) *100	96	96	100	96
Mating Index females (%) (Females mated/Females paired) *100	100	100	100	100
Fertility Index females (5) (Pregnant females/Females mated) *100	96	96	100	96
Gestation Index (%) (Females with living pups on Day 1/ Pregnant females) *100	100	100	100	96

Table 34. Summary of Developmental Data

	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
F0 – Generation – Lactation
Litters
Total	23	24	25	24
Duration of Gestation	21.7	21.4	21.6	21.5
Dead pups at first litter check
Litters affected (#)	4	2	2	2
Mean (+)	0.2	0.1	0.2	0.1
Living pups at first litter check
% of living make/females (#)	47/53	48/52	48/52	51/49
Mean (+)	12.0	11.5	11.9	11.6
Postnatal Loss
% of living pups	0.0	2.2	0.3	0.7
Litters affected (#)	0	5	1	2
Mean (+)	0.0	0.3	0.0	0.1
Culled pups	97	86	103	94
Living pups day 4 P.P	7.8	76	7.7	7.6
Breeding loss days 5-21 P.P
% of living pups at day 4 P.P	0.6	0.0	0.0	0.5
Litters Affected (#)	1	0	0	1
Mean (+)	0.0	00	00	0.0
Living pups Day 21 P.P
% of Males/Females (#)	49/51	50/50	48/52	51/49
Mean (+)	7.7	7.6	7.7	7.5
Total number of offspring born	281	278	301	280
Total number of uterine implantation sites	299	298	322	297
Number of live offspring in Day 1 after littering	277	275	297	278
Number of live offspring on Day 4 (before culling)	277	269	296	276
Number of live offspring on Day 4 (after culling)	180	183	193	182
Number of live offspring on Day 21 after littering	179	183	193	181
Post-implanatation survival Index (%) (Total number of offspring borm/Total number of uterine implantation sites) * 100	94	93	93	94
Live birth Index (%) (Number of live offspring on Day 1 after littering/Total number of offspring born) * 100	99	99	99	99
Viability Index (%) (Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering) * 100	100	98	100	99
Weaning index (%) (Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling)) * 100	99	100	100	99

Table 35. Mean Summary of Body Weights of Pups F_{0-Generation Lactation} (g)

Day	Sex	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
1	M	6.4	6.3	6.6	6.7
	F	6.1	6.0	6.2	6.3
	M+F	6.3	6.2	6.4	6.5
4	M	9.8	9.7	9.9	10.0
	F	9.4	9.2	9.4	9.6
	M+F	9.6	9.5	9.6	9.8
7	M	16.2	16.2	16.1	16.5
	F	15.7	15.5	15.5	15.9
	M+F	16.0	15.8	15.8	16.2
13	M	31.5	31.1	30.8	31.2
	F	30.8	30.1	29.9	30.1
	M+F	31.4	30.7	30.3	30.7
21	M	51.5	50.8	50.8	50.7
	F	50.3	49.1	49	48.8
	M+F	51.1	50.0	49.9	49.8
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 36. Anogenital Distance and Nipple Retention Per Group F_{0-Generation Lactation} (g)

	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Anogenital dist M mm	2.87	2.82	2.80	2.84
Angogenitl dist F mm	1.1	1.06	1.08	1.02
Number of nipples	0.00	0.00	0.00	0.00
#/## fisher’s Extract test significant at 5% (#) or 1% (##) level +/++ Steel-tested significant at 5% (+) or 1% (++) level

Table 37. Summary of Corrected Anogenital Distance

	Group 1 Control	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
PND 1
Norm anog dist M Mm	1.54	1.52	1.50	1.51
Norm nog dist F Mm	0.60	0.58	0.59	0.55
+/++ Steel-tested significant at 5% (+) or 1% (++) level

Table 38. F₁ Weekly Summary of Clinical Signs in Male and Female

Sign (Max. Grade) (Location)	Male Treatment												Female Treatment
	Week	1	.	.	4	.	.	.	8	.	.	.	1	.	.	4	.	.	.	8	.	.	.	12
Group 1 (control)
Skin / fur Scabs (3)(Shoulder right)	G:	.	.	.	.	.	1	1	1	1	1	1
	%	.	.	.	.	.	0	0	0	0	0	0
Skin / fur Scabs (3)(Thigh hind left)	G:	.	.	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	.	.	.	.	0	0	.
Skin / fur Wound (3)(Shoulder right)	G:	.	.	.	.	.	1	1	1	.	.	.
	%	.	.	.	.	.	0	0	0	.	.	.
Posture Hunched posture (1)	G:												.	.	1	1	.	.	.	.	.	.	.	.
	%												.	.	0	0	.	.	.	.	.	.	.	.
Various Broken (1) (Tail apex)	G:												.	.	1	1	1	1	1	1	1	1	.	.
	%												.	.	0	0	0	1	1	1	1	0	.	.
Group 2 (250 mg/kg/day)
Skin / fur Alopecia (3)	G:												.	.	.	.	.	.	.	.	2	1	1	1
	%												.	.	.	.	.	.	.	.	0	0	1	1
Scabs (3) (Head)	G:												.	.	.	.	.	.	.	.	.	1	1	.
	%												.	.	.	.	.	.	.	.	.	0	1	.
Skin / fur Scabs (3)(Cervical region)	G:	.	.	.	.	1	1	1	1	.	.	.
	%	.	.	.	.	0	0	0	0	.	.	.
Skin / fur Scabs (3)(Shoulder left)	G:	.	.	.	1	1	1	1	1	1	1	1
	%	.	.	.	0	0	1	1	0	0	0	0
Skin / fur Scabs (3)(Shoulder right)	G:	.	.	.	.	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	1	1	1
	%	.	.	.	.	0	1	1	1	1	0	0	.	.	.	.	.	.	.	.	.	0	1	1
Wound (3)	G:	.	.	.	.	.	.	.	1	1	.	.
	%	.	.	.	.	.	.	.	0	0	.	.
Wound (3)(Cervical region)	G:	.	.	.	.	.	2	1	.	.	.	.
	%	.	.	.	.	.	0	0	.	.	.	.
Wound (3)(Shoulder left)	G:	.	.	.	.	.	1	1	.	.	.	.
	%	.	.	.	.	.	0	0	.	.	.
Wound (3)(Shoulder right)	G:	.	.	.	.	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	0	0	1	.	.	.	.	.	.	.	.	.	.	.	.	.	0	1	.
Secretion / excretionSalivation (3)	G:												.	.	.	.	.	.	.	.	.	.	1	.
	%												.	.	.	.	.	.	.	.	.	.	1	.
Various Broken (1)(Tail apex)	G:	.	.	.	.	.	.	1	1	1	1	1
	%	.	.	.	.	.	.	0	0	1	1	1
Various Broken (1)(Tail base)	G:												.	.	.	.	.	.	1	1	1	1.	.	.
	%												.	.	.	.	.	.	0	0	0	0.	.	.
Group 3 (500 mg/kg/day)
Posture Hunched posture (1)	G:	.	.	.	1	.	.	.	.	1	.	.
	%	.	.	.	0	.	.	.	.	0	.	.
Skin / fur Scabs (3) (Shoulder left)	G:	.	.	.	.	.	.	.	1	1	1	.
	%	.	.	.	.	.	.	.	0	0	0	.
Skin / fur Scabs (3)(Shoulder right)	G:	.	.	.	.	.	.	1	1	.	.	.
	%	.	.	.	.	.	.	0	0	.	.	.
Skin / fur Nodule (1) (Tail)	G:												.	.	.	1	1	1	1	1	1	1	1	1
	%												.	.	.	0	0	0	0	0	0	0	1	1
Secretion / excretionSalivation (3)	G:	.	.	.	1	1	.	.	.	.	.	.	.	.	.	.	1	.	.	.	.	1	.	1.
	%	.	.	.	0	0	.	.	.	.	.	.	.	.	.	.	0	.	.	.	.	0	.	1.
Chromodacryorrhoea (3) (Eye left)	G:	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.1	.	.	.	.	.	.
	%	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	0	.	.	.	.	.	.
Various Ptosis (3) (Eye left	G:	.	1	1	1	1	1	1	1	.	.	.
	%	.	0	0	0	0	0	0	0	.	.	.
Various Exophthalmos (1) (Eye left)	G:												.	.	.	1	1	1	1	1	1	1	.	.
	%												.	.	.	0	0	0	0	0	0	0	.	.
Broken (1) (Tail apex)	G:	.	.	.	.	.	.	.	1	1	1	1
	%	.	.	.	.	.	.	.	0	0	0	0
Group 4 (1000) mg/kg/day
Skin / fur Scabs (3)	G:	.	.	.	.	.	1	.	1	1	.	.
	%	.	.	.	.	.	0	.	0	0	.	.
Scabs (3) (Back)	G:	.	.	.	.	.	.	1	1	1	1	1
	%	.	.	.	.	.	.	0	0	0	0	0
Scabs (3) (Neck)	G:	.	.	.	.	.	1	1	1	.	.	.
	%	.	.	.	.	.	0	0	0	.	.	.
Scabs (3) (Shoulder left)	G:	.	.	.	.	.	1	1	1	1	.	.
	%	.	.	.	.	.	0	1	1	0	.	.
Scabs (3) (Shoulder right)	G:	.	.	.	.	.	1	1	1	1	1	1
	%	.	.	.	.	.	0	0	1	11	1	0
Wound (3) (Back)	G:	.	.	.	.	.	.	1	1	.	1	1
	%	.	.	.	.	.	.	0	0	.	1	1
Wound (3) (Shoulder right)	G:	.	.	.	.	.	1	1	.	.	.	.
	%	.	.	.	.	.	0	0	.	.	.	.
Secretion / excretion Salivation (3)	G:	.	.	.	.	.	.	.	.	.	1	.	.	.	1	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	.	.	.	.	.	0	.	.	.	0	.	.	.	.	.	.	2	2	.
Various Missing (1) (Toes)	G:	1	1	1	1	1	1	1	1	1	1	1	.	.	0	.	.	.	.	.	.	2	2	.
	%	0	0	0	0	0	0	0	0	0	0	0
Broken (1) (Tail apex)	G:	.	.	.	.	.	.	1	1	1	1	1
	%	.	.	.	.	.	.	1	1	1	1	1

G: Median value of the highest individual weekly grades
%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,..., A=more than 95%) .: Observation performed, sign not present, -: No signs observed

Table 39. Cohort 1A-1B-1C Mean Summary of Male Body Weights (g)  and Body Weight Gain (%)

Dose group	Female Body Weights (g)				Body Weight Gain (%)
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Day 1 Week 1	51	51	51	51	N/A	N/A	N/A	N/A
Day 8 Week 2	71	71	71	73	0	0	0	0
Day 15  Week 3	117	118	118	120	65	68	67	64
Day 22 Week 4	166	168	166	169	134	139	136	132
Day 29 Week 5	210	212	208	211	198	203	198	197
Day 36 Week 6	254	255	253	254	261	266	259	257
Day 43 Week 7	289	288	288	292	309	311	301	304
Day 50 Week 8	315	312	310	317	346	347	332	340
Day 57 Week 9	333	332	328	333	372	374	357	363
Day 64 Week 10	349	348	343	351	394	398	377	389
Day 71 Week 11	367	361	358	364	426	428	400	415
Day 78 Week 12	373	372	371	377	428	434	412	429

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level, N/A = No data collected

Table 40. Cohort 1A-1B-1C Mean Summary of  Female Body Weights (g) and Body Weight Gain (%)

Dose group	Female Body Weights (g)				Body Weight Gain (%)
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Day 1 Week 1	50	49	49	49	N/A	N/A	N/A	N/A
Day 8 Week 2	68	67	68	68	0	0	0	0
Day 15  Week 3	104	105	105	105	53	57*	54	54
Day 22 Week 4	130	135	135	135	91	103**	101*	102*
Day 29 Week 5	156	157	156	156	129	137	129	131
Day 36 Week 6	173	175	173	174	154	164	154	159
Day 43 Week 7	187	190	187	187	176	186	175	178
Day 50 Week 8	196	200	198	198	190	201	192	195
Day 57 Week 9	206	210	208	207	203	216	207	208
Day 64 Week 10	212	217	214	214	213	228	216	220
Day 71 Week 11	221	224	222	224	230	245	231	237
Day 78 Week 12	225	227	230	229	234	243	235	241
Day 85 Week 13	232	231	236	238	254	263	256	263

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level, N/A = No data collected

Table 41. F₁ Mean Summary of  Food Consumption (g/animal/day) and Relative Food Consumption (g/kg bw/day) in Male

Dose group	Food Consumption (g/animal/day)				Relative Food Consumption (g/kg bw/day)
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
DAYS 1-8 WEEKS 1-2	8	8	8	8	114	112	108*	110
DAYS 8-15 WEEKS 2-3	13	13	13	14	110	111	112	114*
DAYS 15-22 WEEKS 3-4	17	18	18	18	105	105	105	106
DAYS 22-29 WEEKS 4-5	20	21	20	21*	97	98	98	100*
DAYS 29-36 WEEKS 5-6	22	23	22	23	88	89	88	90
DAYS 36-43 WEEKS 6-7	23	23	23	24*	79	80	80	82**
DAYS 43-50 WEEKS 7-8	22	22	22	24*	70	72	71	74*
DAYS 50-57 WEEKS 8-9	22	22	21	23*	65	65	66	69**
DAYS 57-64 WEEKS 9-10	21	21	21	22**	59	60	61*	64*
DAYS 64-71 WEEKS 10-11	20	20	20	22*	55	56	57	59**
DAYS 71-78 WEEKS 11-12	19	19	19	20	52	53	53	53
DAYS 78-85 WEEKS 12-13	---	---	---	---	---	---	---	---
MEAN OF MEANS OVER TREATMENT	19	19	19	20	81	82	82	84

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 42. F₁ Mean Summary of  Food Consumption (g/animal/day) and Relative Food Consumption (g/kg bw/day) in Female

Dose group	Food Consumption (g/animal/day)				Relative Food Consumption (g/kg bw/day)
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
DAYS 1-8 WEEKS 1-2	8	8	7	7	115	113	107**	107**
DAYS 8-15 WEEKS 2-3	11	12	12	12*	108	112*	112*	114*
DAYS 15-22 WEEKS 3-4	13	15	14	14	101	109*	105	105
DAYS 22-29 WEEKS 4-5	15	16**	15	15	94	100**	96	98
DAYS 29-36 WEEKS 5-6	15	16**	15	16*	85	91**	90*	91*
DAYS 36-43 WEEKS 6-7	15	15	15	15	78	81	81*	83*
DAYS 43-50 WEEKS 7-8	14	15*	15*	15**	72	76**	76**	77**
DAYS 50-57 WEEKS 8-9	14	15*	15	15*	70	73	73	74**
DAYS 57-64 WEEKS 9-10	14	15	15	15	66	69	70*	71**
DAYS 64-71 WEEKS 10-11	14	15	15	15	64	66	66	67
DAYS 71-78 WEEKS 11-12	13	15	14	15	60	65	64	65*
DAYS 78-85 WEEKS 12-13	---	---	---	---	57	63	60	63
DAYS 85-92 WEEKS 13-14	13	14	14	14	---	---	---	---
MEAN OF MEANS OVER TREATMENT	13	14	14	14	81	85	83	85

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 43. Mean Summary of  Balanopreputial Separation, Vaginal Patency and First Estrous  

Dose group	Male				Female
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
PND (BPS) BPS PND	40.9	41.0	41.6	41.7+
BW BPS gram	172	174	175	179*
PND (VO) VO PND					29.7	31.2+	30.8	30.4
BW VO gram					86	95**	91	89
First estrus PND					33.9	34.3	34.6	35.5
Time to first E days					4.2	3.7	4.1	5.1

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level +/++ Steel-test significant at 5% (+) or 1% (++) level

Table 44. Mean Summary of Hematology Parameters in Male

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
WBC 10E9/L	6.3	6.0	6.0	6.0
Neutrophils 10E9/L	1.1	1.0	0.9	1.0
Lymphocytes 10E9/L	4.9	4.7	4.8	4.7
Monocytes 10E9/L	0.1	0.1	0.1	0.1
Eosinophils 10E9/L	0.1	0.1	0.1	0.1
Basophils 10E9/L	0.0	0.0	0.0	0.0
LUC 10E9/	0.0	0.0	0.0	0.0
Red blood cells 10E12/L	8.37	8.44	8.15	8.45
Reticulocytes 10E9/L	239.3	230.6	217.3+	226.8
RDW %	12.1	12.1	12.0	12.0
Haemoglobin mmol/L	9.2	9.1	9.1	9.2
Haematocrit L/L	0.432	0.430	0.420	0.433
MCV fL	51.6	51.0	51.6	51.3
MCH fmol	1.11	1.08	1.11	1.10
MCHC mmol/L	21.45	21.18	21.56	21.33
Platelet 10E9/L	696	715	769	792
PTs	16.6	16.7	16.6	16.4
APTTs	18.1	18.3	19.6	18.3

+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 45. Mean Summary of Hematology Parameters in Female

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
WBC 10E9/L	4.4	3.6	3.8	4.5
Neutrophils 10E9/L	0.5	0.6	0.6	1.3
Lymphocytes 10E9/L	3.7	2.9	3.1	3.0
Monocytes 10E9/L	0.1	0.1	0.1	0.1
Eosinophils 10E9/L	0.1	0.1	0.1	0.1
Basophils 10E9/L	0.0	0.0	0.0	0.0
LUC 10E9/	0.0	0.0	0.0	0.0
Red blood cells 10E12/L	7.92	7.49**	7.75	7.94
Reticulocytes 10E9/L	209.1	220.3	201.7	222.7
RDW %	10.9	10.9	11.0	11.2
Haemoglobin mmol/L	9.0	8.7*	8.9	8.9
Haematocrit L/L	0.418	0.400*	0.407	0.411
MCV fL	52.8	53.4	52.6	51.8
MCH fmol	1.14	1.17	1.14	1.13
MCHC mmol/L	21.63	21.84	21.79	21.70
Platelet 10E9/L	696	715	769	792
PTs	16.5	16.3	16.5	16.0
APTTs	18.7	18.7	17.4	18.4

+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 46. Mean Summary of Clinical Biochemical Parameters in Male

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
ALAT U/L	38.4	34.5	35.3	34.8
ASAT U/L	86.5	86.5	82.6	81.9
ALP u/L	133	127	136	109
Total Protein g/L	62.6	62.7	62.8	64.4
Albumin g/L	33.3	33.0	33.2	33.8
Total bilirubin umol/L	2.0	1.9	2.0	2.0
Urea mmol/L	4.2	4.8	5.0	4.5
Creatinine mmol/L	36.3	36.1	36.2	35.6
Glucose mmol/L	8.93	8.81	10.10*	8.99
Cholesterol mmol/L	2.24	2.16	2.29	2.22
Bile acids umol/L	24.6	17.2	18.6	12.6
Sodium mmol/L	141.4	141.6	140.9	141.2
Potassium mmol/L	3.76	3.66	3.77	3.72
Chloride	104	102	102	103
Calcium mmol/L	2.57	2.53	2.58	2.61
Inorg.Phose mmol/L	2.01	1.96	1.85	2.01
TSH ulU/mL	0.263	0.167	0.283	0.206
Total T4 ug/dL	5.92	5.82	5.55	6.27

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 47. Mean Summary of Clinical Biochemical Parameters in Female

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
ALAT U/L	27.5	29.1	27.6	28.6
ASAT U/L	83.0	83.6	84.4	81.2
ALP u/L	57	53	48	53
Total Protein g/L	64.4	66.8	67.2	67.2
Albumin g/L	35.2	36.0	36.2	36.2
Total bilirubin umol/L	2.2	1.7**	1.7**	2.0
Urea mmol/L	5.8	5.9	6.3	5.5
Creatinine mmol/L	38.8	38.5	37.7	40.7
Glucose mmol/L	7.80	7.93	7.81	7.45
Cholesterol mmol/L	1.92	1.94	1.98	2.02
Bile acids umol/L	10.5	13.5	11.1	11.4
Sodium mmol/L	140.7	141.2	141.0	140.7
Potassium mmol/L	3.49	3.33	3.39	3.46
Chloride	104	103	103	104
Calcium mmol/L	2.59	2.62	2.68*	2.65
Inorg.Phose mmol/L	1.82	1.69	1.74	1.63
TSH ulU/mL	0.077	0.190**	0.074	0.127
Total T4 ug/dL	3.66	3.52	3.34	3.40

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 48. Mean Summary of Urinalysis Parameters in Male

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Volum mL	9	6	7	11
Specific gravity	1.028	1.032	1.032	1.027
Clarity score 1/3	1	1	1	1
Colour score 1/5	1	1	1	1
pH	6.8	6.7	6.6	6.8
Blood score 0/3	0	0	0	0
WBC score 0/3	0	0	0	0
Bilirubin score 0/3	0	0	0	0
Urobilinogen score 0/3	0	0	0	0
Protein score 0/3	0	0	0	0
Ketones score 0/3	0	0	0	0
Glucose score 0/3	0	0	0	0
WBC-sed. score 0/3	0	0	0	0
RBC-sed. score 0/3	0	0	0	0
Casts score 0/3	0	0	0	0
Epithelial cells score 0/3	1	1	1	1
Crystals score 0/3	3	3	3	3
Bacteria score 0/3	3	3	3	3
Other score 0/3	0	0	0	0

+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 49. Mean Summary of Urinlysis Parameters in Female

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Volum mL	7	8	5	8
Specific gravity	1.022	1.022	1.028	1.020
Clarity score 1/3	1	1	1	1
Colour score 1/5	1	1	1	1
pH	6.3	6.5	6.2	6.1
Blood score 0/3	0	0	0	0
WBC score 0/3	0	0	0	0
Bilirubin score 0/3	0	0	0	0
Urobilinogen score 0/3	0	0	0	0
Protein score 0/3	0	0	0	0
Ketones score 0/3	0	0	0	0
Glucose score 0/3	0	0	0	0
WBC-sed. score 0/3	1	0	0	0
RBC-sed. score 0/3	0	0	0	0
Casts score 0/3	0	0	0	0
Epithelial cells score 0/3	1	1	1	1
Crystals score 0/3	1	1	2	1
Bacteria score 0/3	2	3	3	3
Other score 0/3	0	0	0	0

+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 50. Mean Splenic Lymphocyte subpopulation analysis in Male

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
T-cells %Lymphoid cells	40.8	40.4	42.9	41.5
T-helper cells %Lymphoid cells	25.3	25.3	27.3	26.1
T-cytotoxic cell %Lymphoid cells	13.0	12.5	13.1	13.5
B-cells %Lymphoid cells	44.3	45.5	43.5	44.2
NK-cells %Lymphoid cells	4.9	4.6	4.3	4.8
Th/Tc	2.0	2.1	2.1	2.0

+/++ Steel-test significant at 5% (+) or 1% (++) level

Table 51. Mean Splenic Lymphocyte subpopulation analysis in Female

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
T-cells %Lymphoid cells	40.6	39.3	40.3	41.7
T-helper cells %Lymphoid cells	25.9	25.3	25.8	26.1
T-cytotoxic cell %Lymphoid cells	12.5	11.5	13.1	13.6
B-cells %Lymphoid cells	42.6	46.4	44.1	45.5
NK-cells %Lymphoid cells	6.0	4.7	5.1	4.3++
Th/Tc	2.1	2.3	2.0	2.0

+/++ Steel-test significant at 5% (+) or 1% (++) level

Table 52.  Summary of Macroscopic Finding in Male

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
INTERCURRENT DEATH
Animals examined				1
Animals Affected				1
General observation: Beginning autolysis				1
Lungs . Not collapsed				1
Liver . Enlarged				1
Thymus . Focus/foci				1
COHORT 1A; END OF IN-LIFE
Animals Examined	20	20	20	20
Animals without finding	13	15	17	14
Animals affected	7	5	3	6
Stomach Focus/foci	3	3	1	2
Stomach Irregular surface	1	0	0	0
Liver Accentuated lobular pattern	0	2	0	1
Liver Diaphragmatic hernia	1	0	0	1
Liver Accessory liver	1	0	0	0
Liver Grown together with:	1	0	0	0
Pancreas Nodule(s)	1	0	0	0
Kidneys Irregular surface	0	0	1	0
Thyroid gland Agenesis	0	0	1	0
Thymus Focus/foci	1	1	0	0
Thymus Enlarged	1	0	0	0
Axillary lymph node Enlarged	1	0	0	2
Axillary lymph node Discolouration	1	0	0	0
Skin . Scales	0	0	0	1
COHORT 1B; END OF IN-LIFE
Animals Examined	20	20	20	19
Animals without finding	19	18	19	14
Animals affected	1	2	1	5
Kidneys Pelvic Dilation	1	0	0	1
Adrenal Glands Enlarged	0	0	0	1
Bone Tail bent	0	1	1	3
Body Cavities Nodule(s)	0	1	0	0
COHORT 1C; END OF IN-LIFE
Animals Examined	20	20	20	20
Animals without finding	16	16	17	15
Animals affected	4	4	3	5
Liver Diaphragmatic hernia	0	1	0	2
Liver Reduced in size	1	0	0	0
Kidneys Pelvic Dilation	1	0	1	0
Kidneys Focus/foci	0	1	0	0
Preputial Glands Focus/foci	1	0	0	0
Preputial Glands Discolouration	0	0	1	2
Spleen Focus/foci	0	0	1	0
Spleen Enlarged	0	1	0	1
Thymus Focus/foci	1	1	1	2
Thymus Enlarged	1	0	0	0

 

Table 53. Summary of Macroscopic Finding in Female

Dose group	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
INTERCURRENT DEATH
Animals examined				1
Animals Affected				1
General observation Cannibalism: organ missing				1
COHORT 1A; END OF IN-LIFE
Animals Examined	20	20	20	20
Animals without finding	14	14	13	11
Animals affected	6	6	7	9
Liver Accentuated lobular pattern	0	1	0	0
Liver Diaphragmatic hernia	1	0	0	0
Accessory liver	1	0	0	0
Liver Reduced in size	0	0	0	1
Liver Discolouration	1	1	0	0
Uterus Contains fluid	0	1	3	2
Cervix Nodule(s)	0	0	1	0
Clitoral glands Focus/foci	0	0	1	2
Thyroid gland Reduced in size	1	0	0	1
Thymus Focus/foci	1	0	0	1
Axillary lymph node	1	0	1	3
Axillary Discolouration	1	0	2	0
Popliteal lymph node	0	0	0	1
Popliteal lymph Enlarged	0	0	0	1
Popliteal lymph Discolouration	0	0	0	1
Iliac lymph node Enlarged	2	3	4	0
Iliac lymph node Disculored	0	0	2	0
Skin Nodule(s)	0	1	0	0
Bone Tail bent	2	1	0	0
Eyes Exophthalmus	0	0	1	0
Eyes Enlarged	0	0	1	0
Body cavities Nodule(s)	0	0	0	1
COHORT 1B; END OF IN-LIFE
Animals Examined	20	20	20	20
Animals without finding	17	11	17	18
Animals affected	3	9	3	2
Kindney size reduced	0	0	0	1
Uterus Contains fluid	3	5	1	1
Clitorial gland focus/foci	0	2	1	0
Discolouration	0	1	0	0
Skin Alopecia	0	1	0	0
Skin Scab formation	0	1	0	0
Bone Tail bent	0	0	1	0
COHORT 1C; END OF IN-LIFE
Animals Examined	20	20	20	18
Animals without finding	20	17	18	15
Animals affected	0	3	2	3
Thymus Accessory lobe.	0	0	1	0
Thymus Focus/foci	0	2	2	3
Thymus Enlarged	0	1	0	0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 54. Mean Summary of Organ Weight (g) and Organ/Body Weight Ratio (%) in Male

Dose group	Organ Weight (g)				Organ/Body Weight Ratio (%)
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
COHORT 1A; END OF IN-LIFE
Body Weight	351	343	336	350	351	343	336	350
Brain	2.04	2.05	2.03	2.07	0.58	0.60	0.61	0.59
Pituitary	0.009	0.009	0.009	0.009	0.002	0.003	0.003	0.003
Heart	0.984	0.998	1.013	1.005	0.281	0.291	0.303	0.288
Liver	9.33	9.59	9.34	9.81	2.65	2.79	2.78	2.80
Thyroids	0.0168	0.0185	0.0187	0.0196*	0.0048	0.0055	0.0056	0.0056
Thymus	0.418	0.394	0.393	0.399	0.119	0.115	0.117	0.114
M Lymph Node	0.390	0.294	0.314	0.302	0.109	0.086	0.090	0.086
Kidney	2.28	2.39	2.38	2.43	0.65	0.70**	0.71**	0.70**
Adrenal	0.057	0.059	0.060	0.062	0.016	0.017	0.018	0.018
Spleen	0.572	0.598	0.554	0.625	0.163	0.174	0.164	0.178
Testes	3.51	3.61	3.50	3.75*	1.00	1.06	1.05	1.08*
Prostate	0.634	0.651	0.718	0.722	0.182	0.190	0.215*	0.207
AXI Lymph Node L	0.08	0.05	0.06	0.07	0.022	0.015	0.017	0.019
Epididymides	1.032	1.064	1.029	1.119*	0.295	0.311	0.308	0.321*
Seminal vesicle	1.029	0.997	1.052	1.119	0.294	0.291	0.315	0.322
COHORT 1B; END OF IN-LIFE
Body Weight	380	377	375	376	380	377	375	376
Pituitary	0.008	0.009	0.009	0.009	0.002	0.002	0.002*	0.002
Thyroids	0.0181	0.0184	0.0179	0.0183	0.0047	0.0049	0.0048	0.0049
Adrenals	0.049	0.053	0.055	0.056*	0.013	0.014	0.015*	0.015**
Testes	3.57	3.57	3.66	3.83*	0.94	0.95	0.98	1.02**
Prostate	0.681	0.773	0.752	0.720	0.179	0.205	0.201	0.195
Epididymides	1.074	1.076	1.123	1.151*	0.283	0.286	0.301	0.308**
Seminal vesicle	1.315	1.321	1.366	1.459	0.347	0.351	0.366	0.388

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 55. Mean Summary of Organ Weight (g) and Organ/Body Weight Ratio (%) in Female

Dose group	Organ Weight (g)				Organ/Body Weight Ratio (%)
	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day	Group 1 0 mg/kg/day	Group 2 250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
COHORT 1A; END OF IN-LIFE
Body Weight	203	210	202	204	203	210	202	204
Brain	1.86	1.88	1.87	1.91	0.92	0.90	0.93	0.94
Pituitary	0.011	0.010	0.010	0.011	0.005	0.005	0.005	0.005
Heart	0.673	0.694	0.688	0.700	0.332	0.330	0.341	0.344
Liver	5.77	6.57^**	6.41^*	6.40^*	2.84	3.12^**	3.18^**	3.13^**
Thyroids	0.0144	0.0151	0.0154	0.0155	0.0071	0.0072	0.0076	0.0076
Thymus	0.356	0.385	0.360	0.368	0.176	0.183	0.178	0.181
M Lymph Node	0.227	0.339^*	0.273	0.281	0.111	0.158	0.132	0.136
Kidney	1.51	1.58	1.54	1.57	0.75	0.75	0.76	0.77
Adrenal	0.070	0.069	0.069	0.073	0.035	0.033	0.034	0.036
Spleen	0.410	0.433	0.413	0.452	0.202	0.205	0.204	0.221
Ovaries	0.144	0.136	0.133	0.134	0.072	0.065	0.066	0.066
Uterus	0.507	0.606	0.548	0.573	0.251	0.290	0.273	0.281
AXI Lymph Node L	0.05	0.05	0.05	0.06	0.023	0.022	0.022	0.028
COHORT 1B; END OF IN-LIFE
Body Weight	230	235	237	234	230	235	237	234
Brain	0.011	0.012	0.011	0.011	0.005	0.005	0.005	0.005
Pituitary	0.0134	0.0148	0.0164^*	0.0163^*	0.0058	0.0063	0.0070	0.0070
Thyroids	0.0058	0.0063	0.0070	0.0070	0.024	0.027	0.028*	0.029**
Adrenal	0.024	0.027	0.028^*	0.029^**	0.003	0.003	0.005	0.005
Ovaries	0.059	0.060	0.067^*	0.060	0.059	0.060	0.067*	0.060
Uterus	0.293	0.265	0.224	0.249	0.293	0.265	0.224	0.249

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 56. Summary of Sperm Motility, Concentration and Morphology

Dose Group	Group 1 0 mg/kg/day	Group 2  250 mg/kg/day	Group 3 500 mg/kg/day	Group 4 1000 mg/kg/day
Sperm motrility (%)	66	69	65	72
Sperm Progressivity (%)	32	29	28	35
Sperm count EPI 10E6/g	497.6	454.3	455.6	488.8
Normal Morph No of cells	172	172	176	173
Detached head No of cells	2	3	3	2
Abnormal head No of cells	0	0	1	0
Coiled tail No of cells	22	23	17	21
Other tail No of cells	2	1	1	2
Abnormal neck No of cells	1	1	2	2
Combined No of cells	0	0	0	0

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level +/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 57.  Mean Summary of Follicles/Corpora Lutea

Dose groups	Group 1. 0 mg/kg/day	Group 4. 1000 mg/kg/day
Corpora lutea	82.5	83.9
Follicle Count	68.1	68.4

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Conclusions:

Based on the criteria set in Regulation (EC) No 1272/2008, Tris(2-ethylhexyl)phosphate is not classified for reproductive or developmental toxicity.

Executive summary:

OECD 443 (2020): The pre- and postnatal effects of the test item were evaluated in Wistar Han rats at dose levels of 250, 500 and 1000 mg/kg/day, based on the results of a preliminary reproductive toxicity study (simplified reproduction/developmental toxicity screening test) with oral exposure of Tris(2-ethylhexyl) phosphate in rats (Test Facility Study No. 20173303) and in an attempt to produce graded responses to the test item. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, were expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of Tris(2- ethylhexyl) phosphate on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation.

Chemical analyses of formulations were conducted at four occasions during the study to assess accuracy and homogeneity.

For the F0-generation, the following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations.

For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: estrous cycle determination, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels during Weeks 1, 13 and 22 of treatment. In Week 11, the mean accuracy of Group 3 and coefficient of variation of Group 4 were slightly outside of the acceptation criteria. As changes were slight and analysis in Weeks 1, 13 and 22 confirmed that the formulations were prepared accurately and homogeneously, the deviation from the acceptance criteria in Week 11 was considered incidental and to not have impacted the study.

F0-Generation - Parental results

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Crystalline, eosinophilic inclusions were present in the cytoplasm of Leydig cells in the testes of all males at 250, 500 and 1000 mg/kg/day. This finding is unknown as background finding in rats but has been described in a recent publication in which rats were treated with the same test item (Pelletier et al., 2020). Comparable with the findings in this study, this paper also demonstrated that the inclusions were also PAS-positive, but the composition remained unclear.

The Leydig cell inclusions were considered non-adverse. This was based on:

• the presence of normal spermatogenic profiles
• the absence of additional test-item-related microscopic findings of the testes (like atrophy or tubular degeneration)
• the absence of test-item-related alterations in sperm parameters (count and morphology)
• the successful mating and production of healthy offspring by almost all F0-males.

In both sexes at 1000 mg/kg/day, an increase in incidence and severity of follicular cell hypertrophy of the thyroid gland was observed. At the recorded severity (up to slight), and in absence of any other proliferative or degenerating change or correlating change in thyroid hormones, these changes were regarded as non-adverse.

In females at 1000 mg/kg/day, higher liver weights were observed which was considered non- adverse in absence of correlating microscopic or macroscopic findings of the liver or changes in liver enzymes.

At 1000 mg/kg/day, mean bile acid concentration was decreased in males and, total protein and albumin levels were increased in females. In absence of corroborative findings, these changes were considered non-adverse.

A trend towards slightly higher body weights and body weight gains was observed in females at 500 and 1000 mg/kg/day which may have been the result of the slightly higher food consumption levels observed at 500 and 1000 mg/kg/day. For males, the slightly higher food consumption did not result in higher body weights or body weight gains. As changes were slight, they were regarded as non-adverse.

F0-Generation - Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

F0-Generation / F1-Generation (pre-weaning) and F1-Generation - Developmental results

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

F1-Generation (post-weaning) –Developmental results

No (developmental) toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Comparable with the F0-males, crystalline, eosinophilic inclusions were present in the cytoplasm of Leydig cells in the testes of all males at 250,500 and 1000 mg/kg/day. In addition, a somewhat higher testes weight was observed in the males of the F1-generation. Since the inclusions were present in both generations in all test item-treated groups, and morphologically appeared to have equal amounts, a relationship between the higher testes weight and inclusions was regarded unlikely. The possible higher testes weight and Leydig cell inclusions were considered non-adverse for the same reasons as described for the F0- generation.

In males at 1000 mg/kg/day, an increase in incidence and severity of follicular cell hypertrophy of the thyroid gland was observed. At the recorded severity (up to slight), and in absence of any other proliferative or degenerating change or correlating change in thyroid hormone, these changes were regarded as non-adverse.

Food consumption was slightly increased from 250 mg/kg/day in females and at
1000 mg/kg/day in males. As changes were slight and body weights were normal, these changes were considered non-adverse.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect level (NOAEL) of Tris(2- ethylhexyl) phosphate were established:

General Toxicity (F0 and F1): at least 1000 mg/kg/day

Reproduction Toxicity: at least 1000 mg/kg/day

Developmental Toxicity: at least 1000 mg/kg/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The data-set for effects on fertility is consistent with data requirements under Annexes VII to X of REACH, and all studies are considered reliable and relevant for endpoint conclusions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 443 (2020): The pre- and postnatal effects of the test item was evaluated in Wistar Han rats at dose levels of 250, 500 and 1000 mg/kg/day, based on the results of a preliminary reproductive toxicity study (simplified reproduction/developmental toxicity screening test) with oral exposure of Tris(2-ethylhexyl) phosphate in rats (Test Facility Study No. 20173303) and in an attempt to produce graded responses to the test item. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of Tris(2- ethylhexyl) phosphate on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation.

F0-Generation - Parental results 

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Crystalline, eosinophilic inclusions were present in the cytoplasm of Leydig cells in the testes of all males at 250, 500 and 1000 mg/kg/day. This finding is unknown as background finding in rats but has been described in a recent publication in which rats were treated with the same test item (Pelletier et al., 2019). Comparable with the findings in this study, this paper also demonstrated that the inclusions were also PAS-positive, but the composition remained unclear.

The Leydig cell inclusions were considered non-adverse. This was based on:

• the presence of normal spermatogenic profiles


• the absence of additional test-item-related microscopic findings of the testes (like atrophy or tubular degeneration)


• the absence of test-item-related alterations in sperm parameters (count and morphology)


• the successful mating and production of healthy offspring by almost all F0-males.

In both sexes at 1000 mg/kg/day, an increase in incidence and severity of follicular cell hypertrophy of the thyroid gland was observed. At the recorded severity (up to slight), and in absence of any other proliferative or degenerating change or correlating change in thyroid hormones, these changes were regarded as non-adverse.

In females at 1000 mg/kg/day, higher liver weights were observed which was considered non- adverse in absence of correlating microscopic or macroscopic findings of the liver or changes in liver enzymes.

At 1000 mg/kg/day, mean bile acid concentration was decreased in males and, total protein and albumin levels were increased in females. In absence of corroborative findings, these changes were considered non-adverse.

A trend towards slightly higher body weights and body weight gains was observed in females at 500 and 1000 mg/kg/day which may have been the result of the slightly higher food consumption levels observed at 500 and 1000 mg/kg/day. For males, the slightly higher food consumption did not result in higher body weights or body weight gains. As changes were slight, they were regarded as non-adverse.

F0-Generation - Reproductive results 

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

F0-Generation / F1-Generation (pre-weaning) and F1-Generation - Developmental results 

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

F1-Generation (post-weaning) –Developmental results 

No (developmental) toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Comparable with the F0-males, crystalline, eosinophilic inclusions were present in the cytoplasm of Leydig cells in the testes of all males at 250,500 and 1000 mg/kg/day. In addition, a somewhat higher testes weight was observed in the males of the F1-generation. Since the inclusions were present in both generations in all test item-treated groups, and morphologically appeared to have equal amounts, a relationship between the higher testes weight and inclusions was regarded unlikely. The possible higher testes weight and Leydig cell inclusions were considered non-adverse for the same reasons as described for the F0- generation.

In males at 1000 mg/kg/day, an increase in incidence and severity of follicular cell hypertrophy of the thyroid gland was observed. At the recorded severity (up to slight), and in absence of any other proliferative or degenerating change or correlating change in thyroid hormone, these changes were regarded as non-adverse.

Food consumption was slightly increased from 250 mg/kg/day in females and at
1000 mg/kg/day in males. As changes were slight and body weights were normal, these changes were considered non-adverse.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect level (NOAEL) of Tris(2- ethylhexyl) phosphate were established:

General Toxicity (F0 and F1): at least 1000 mg/kg/day

Reproduction Toxicity: at least 1000 mg/kg/day

Developmental Toxicity: at least 1000 mg/kg/day

Effects on developmental toxicity

Description of key information

OECD 414  in rats (2008):  NOEL (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg body weight/day.

OECD 414 in rabbits (2020): NOAEL ( (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg body weight/day.)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-May-2019 - 01-Jul-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidlines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

yes

Remarks:

Female#55 at 300 mg/kg/day was dosed slightly longer i.e. on day of necropsy. Fetuses of litter A035 at 100 mg/kg/day had an early delivery on post- coitum Day 27 were examined externally.The uterus weight was not determined for females#70 at 1000 mg/kg.

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

yes

Remarks:

Female#55 at 300 mg/kg/day was dosed slightly longer i.e. on day of necropsy. Fetuses of litter A035 at 100 mg/kg/day had an early delivery on post- coitum Day 27 were examined externally.The uterus weight was not determined for females#70 at 1000 mg/kg.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

yes

Remarks:

Female#55 at 300 mg/kg/day was dosed slightly longer i.e. on day of necropsy. Fetuses of litter A035 at 100 mg/kg/day had an early delivery on post- coitum Day 27 were examined externally.The uterus weight was not determined for females#70 at 1000 mg/kg.

Principles of method if other than guideline:

Yes

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: On 19 Jul 2019, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France).
- Females (if applicable) nulliparous and non-pregnant: Time mated female were used, Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
- Age at study initiation: 17-20 weeks old
- Weight at study initiation: Females: 3.052 - 4.062 kg.
- Fasting period before study: No
- Housing: shelters: Individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: 2 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20ºC
- Humidity (%): 61 to 97 %
- Air changes (per hr): =>10 air changes/hour with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 19th Jul 2019 To Day 29 post-coitum

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% Aqueous carboxymethyl cellulose (CMC) with 0.1% Tween 80

Details on exposure:

 PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at least every 2 weeks and formulated in daily portions used within 6 hours after after removal from the refrigerator. 

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. 1% Aqueous carboxymethyl cellulose (CMC) with 0.1% Tween 80 was considered suitable.
- Concentration in vehicle: concentration range of 0 to 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg (c.f. 5 ml/kg).
- Lot/batch no. (if required): No specified.
- Purity: Not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20173310).

Details on mating procedure:

Mating procedure was not disclosed but the females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

Day 6 to Day 28 post-coitum

Frequency of treatment:

Once daily

Duration of test:

23 days

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with the test item by oral gavage at a dose volume of 5 mL/kg body weight at 0, 125, 250 and 500 mg/kg/day were administered. At 500 mg/kg/day, 1 out of 6 animals showed notable weight loss of 5% over Days 6-9 post-coitum, followed by (on most occasions) weight gain on subsequent days. This same animal was found to be non-gravid. All other animals of this dose group showed a similar level of weight gain as recorded among control animals. Food intake at 250 and 500 mg/kg/day was temporarily lower over Days 6-9 post-coitum, but recovered to levels similar to control means as treatment progressed. No necropsy findings were recorded that were considered to be related to treatment. No effects on examined developmental parameters were recorded in this study. For comparison, in the Tolerability Study (non-pregnant rabbits; Test Facility Study No. 20173147), 3 out of 3 animals showed weight loss up to 5% during the first 3-4 days of treatment at 500 mg/kg/day. These data suggest that –in contrast to what would normally be expected – pregnant animals show a lower degree of toxicity than non- pregnant animals. At subsequent dose levels tested in this Tolerability Study (750 and 1000 mg/kg/day), a similar degree of weight loss was recorded for most or all animals, I.e. no clear dose related-response was observed over the dose levels tested. Overall, these data indicate that the dose level of 500 mg/kg/day used as high dose in the Dose Range Finding Study may not result in a sufficient degree of toxicity in the Main Study. Therefore, based on the results of the Tolerability Study in combination with the observed lower degree of toxicity in pregnant animals vs non-pregnant animals, the limit dose of 1000 mg/kg/day was selected as high dose for the Main Study.
- Rationale for animal assignment (if not random): NA
 - Other: The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe.
The dosing formulations were stirred continuously during dose administration.
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
 - Time schedule: during clinical observations
 - Cage side observations checked in table [No.?]: Yes

DETAILED CLINICAL OBSERVATIONS: Yes 
 - Time schedule: From Day 6 post-coitum onwards up to the day prior to necropsy, animals will be observed at least once daily.
Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed and graded for severity with the maximum grade predefined at 1, 3 or 4. Grades coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1)were scored.

 BODY WEIGHT: Yes 
 - Time schedule for examinations: Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

 FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes 
 - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: On days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum. .

 WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: monitored on regular basis throughout the study by visual inspection of the water bottles/containers.
 
 POST-MORTEM EXAMINATIONS: Yes
 - Sacrifice on gestation day # GD 29
- Organs examined: Yes, external and visceral organs

 OTHER: The following data were recorded i.e.
* The number of corpora lutea.
* The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
* The number of implantation sites.
* The number and distribution of live and dead fetuses.
* The number and distribution of embryo-fetal deaths.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:

- Gravid uterus weight: Yes 

- Number of corpora lutea: Yes 

- Number of implantations: Yes 

- Number of early resorptions: Yes 

- Number of late resorptions: Yes

 - Other: Yes, gross evaluation of placenta, pregnancy status

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:
- Other: The following litter data were recorded:
* Total number of fetuses
* Number of live fetuses
* Number of dead fetuses
* Individual fetal body weight
* Fetus sex

Statistics:

STATISTICAL ANALYSIS
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. This included Dunnett-test (many-to-one-t-test), Steel-test (many-to-one rank test), Kruskal-Wallis nonparametric ANOVA test and Mann Whitney test.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Indices including; pre and post-implantation loss (%) and fetuses viability index (%).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental occurrence of clinical findings without any dose-related response were observed but considered not to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Variation in body weight were observed but all within historical control range and as such not considered toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 (0.33x of control for relative food consumption; outside historical control data range) and 27 to 29 post-coitum (0.72x of control for relative food consumption; not statistically significant for relative food consumption and within historical control data range).
At 300 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 post-coitum (0.53x of control for relative food consumption; outside historical control data range).
At 100 mg/kg/day, absolute and relative food consumption was similar to control levels.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Morality:
- One control group animal (No. 4) was sacrificed for humane reasons on post-coitum Day 15. This animal showed weight loss (up to 5%) between post-coitum Days 6 and 15 and did (almost) not consume food during this period. Other than notably reduced faeces production, this animal showed no relevant clinical signs. Necropsy showed a black-brown focus (1x3 mm) on the glandular mucosa of the stomach. This female was pregnant.
- One animal at 100 mg/kg/day (No. 35) had an early delivery on post-coitum Day 27. This animal showed piloerection and lean appearance prior to early delivery, showed weight loss (up to 3%) between post-coitum Days 21 and 24 and did (almost) not consume food between post-coitum Days 21 and 27. Except for reduced faeces production, none of the clinical signs recorded for this animal were observed among other animals of this dose group. Necropsy showed no lesions.
- One animal at 300 mg/kg/day (No. 52) was found dead on post-coitum Day 29. This animal showed piloerection, reduced faeces production, and diarrhoea prior to being found dead, and did (almost) not consume food between post-coitum Days 21 and 29. Necropsy showed a beginning stage of autolysis. This female was pregnant.
- One animal at 1000 mg/kg/day (No. 82) was euthanized for humane reasons on Day 12 post-coitum. She was noted with faeces containing mucus (starting on Day 10 post-coitum) and red fluid on the manure tray mixed with faeces (starting on Day 11 post-coitum). In addition, she had severely reduced /no food intake from Days 6 to 12 post-coitum, along with a 12% body weight loss over days 6 to 12 and severely reduced faeces production from Day 9 post-coitum onwards. This female was examined by the veterinarian and found to be cold by touch. At necropsy, reddish and gelatinous content of the rectum was noted. This female was not pregnant.

Clinical Observations:
Reduced faeces production was recorded at increased incidence over the dose groups, when compared to the control group, and occurred from post-coitum Day 9 onwards.
One female at 300 mg/kg/day (No. 66) showed diarrhoea on post-coitum Days 22 and 23 and one other female at this dose (No. 65) showed pale faeces and lean appearance on post-coitum Day 28. Given the incidental occurrence of these findings, and since these did not show a dose-related response, these were considered not to be related to treatment with the test item.
Other findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Body Weights and Body Weight Gains:

At 1000 mg/kg/day, slight mean weight loss was recorded over post-coitum Days 6 to 9 (-2%). Also, statistically significantly lower mean body weights were recorded on post- coitum Days 27 and 29 (5% lower than control means on both occasions). Although mean body weight gain at this dose was statistically significantly lower throughout most of the treatment period, means did not show a clear dose-related response over the dose groups. Overall mean weight gain over the treatment period was 6% at 1000 mg/kg/day vs. 11% in the control group. Mean body weights remained well within the historical control data range.
Corrected mean weight gain for gravid uterus at 1000 mg/kg/day was slightly outside the historical range for a total of 6 out of 20 animals. The mean corrected weight remained within the historical control range.
At 300 mg/kg/day, slight mean weight loss was recorded over post-coitum Days 6 to 9 (-1%). On subsequent days, body weight gain remained essentially similar to control means and means recorded at 100 and 1000 mg/kg/day (i.e. a clear dose-related trend was absent). Mean body weights remained well within the historical control data range. Therefore, the statistically significantly lower mean body weight gain on post-coitum Days 21 and 24 was considered not to represent a test item-related effect.
At 100 mg/kg/day, body weights and body weight gain remained similar to the control group. Corrected mean weight gain for gravid uterus at 100 and 300 mg/kg/day remained within the historical control range (in the control group, 100 and at 300 mg/kg/day, a total of 1/19, 2/20 and 2/20 values were slightly outside the historical control range, respectively).

Food Consumption:
At 1000 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 (0.33x of control for relative food consumption; outside historical control data range) and 27 to 29 post-coitum (0.72x of control for relative food consumption; not statistically significant for relative food consumption and within historical control data range).
At 300 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 post-coitum (0.53x of control for relative food consumption; outside historical control data range).
At 100 mg/kg/day, absolute and relative food consumption was similar to control levels.

Gross Pathology:
Macroscopic observations at necropsy of animals that survived until the scheduled treatment duration did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

A total of 2 control animals (Nos. 7 and 20), one animal at 100 mg/kg/day (No. 26), one animal at 300 mg/kg/day (No. 64) and two animals at 1000 mg/kg/day (Nos. 82 (euthanized on Day 12) and 88) were not pregnant. The incidence of non-pregnancy did not show a relationship to treatment with the test item and was within the historical control range. These incidences of non-pregnancy still allowed a meaningful assessment of the data.
The numbers of pregnant females, corpora lutea and implantation sites, pre- and postimplantation loss and early and late resorptions in the control and test groups were similar and in the range of normal biological variation.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects was observed up to the highest tested group

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were considered not affected by treatment with the test item.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment with the test item up to 1000 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects on litter size of any group.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded in one control fetus and two fetuses each at 100, 300 and 1000 mg/kg/day. Some of these malformations were not previously encountered in historical control fetuses, or the incidence slightly exceeded this historical data range. However, the single occurrence, group distribution and/or occurrence in a control fetus of these malformations did not indicate a relationship to treatment with the test item. Therefore, these malformations were considered to be spontaneous in origin.
At 1000 mg/kg/day, these malformations consisted of a hind limb with syndactyly, brachydactyly and tarsal flexure in one fetus (A070-07). The latter malformation also occurred in this group in fetus A080-02 that had gastroschisis as well, and a flexure of both carpals occurred in one fetus at 300 mg/kg/day (A053-06). There was no underlying skeletal anomaly for both affected fetuses at 1000 mg/kg/day. The other externally affected fetus at 300 mg/kg/day (A059-01) had omphalocele that also occurred in one fetus at 100 mg/kg/day (A039-01) and in a late resorption at 1000 mg/kg/day (A080-04). Remaining malformations in this study were facial cleft and cleft palate (one fetus at 100 mg/kg/day (A027-02)) and short tail (control fetus A003-03; confirmed at skeletal examination).

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment with 1000
mg/kg/day.

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Litter Size:
Mean litter sizes were 10.2, 8.9, 8.7 and 8.9 fetuses/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Sex ratio:
Mean sex ratios (males:females) were 60:40, 50:50, 49:51 and 56:44 for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Fetal Body Weights:
Mean combined (male and female) fetal body weights were 37.7, 38.7, 40.5 and 38.4 gram for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Fetal Morphological Examinations:
The numbers of fetuses (litters) available for fetal morphological examination were 193 (19), 178 (20), 174 (20) and 178 (20) in the control, 100, 300 and 1000 mg/kg/day Groups, respectively. External and visceral examinations were done for all fetuses, soft tissue cephalic examination was done for approximately half of the fetuses of the same litter for all groups, and skeletal examination was done for all fetuses of Groups 1 and 4.
Dams A004, A052 and A082 (control, 300 and 1000 mg/kg/day group, respectively) did not survive until the planned day of necropsy. In addition, one dam at 100 mg/kg/day (A035) had an early delivery
External Malformations and Variations:
External malformations were recorded in one control fetus and two fetuses each at 100, 300 and 1000 mg/kg/day. Some of these malformations were not previously encountered in historical control fetuses, or the incidence slightly exceeded this historical data range. However, the single occurrence, group distribution and/or occurrence in a control fetus of these malformations did not indicate a relationship to treatment with the test item. Therefore, these malformations were considered to be spontaneous in origin.

At 1000 mg/kg/day, these malformations consisted of a hind limb with syndactyly, brachydactyly and tarsal flexure in one fetus (A070-07). The latter malformation also occurred in this group in fetus A080-02 that had gastroschisis as well, and a flexure of both carpals occurred in one fetus at 300 mg/kg/day (A053-06). There was no underlying skeletal anomaly for both affected fetuses at 1000 mg/kg/day. The other externally affected fetus at 300 mg/kg/day (A059-01) had omphalocele that also occurred in one fetus at 100 mg/kg/day (A039-01) and in a late resorption at 1000 mg/kg/day (A080-04). Remaining malformations in this study were facial cleft and cleft palate (one fetus at 100 mg/kg/day (A027-02)) and short tail (control fetus A003-03; confirmed at skeletal examination).

Visceral Malformations and Variations:
Visceral malformations were recorded in one control fetus and two fetuses (two litters) at 100 mg/kg/day, 2 fetuses (one litter) at 300 mg/kg/day and five fetuses (four litters) at 1000 mg/kg/day. Although some of these malformations were not previously encountered in historical control fetuses, or the incidence slightly exceeded this historical data range, the single occurrence and/or group distribution of these malformations did not indicate a relationship to treatment with the test item. Therefore, these malformations were considered to be spontaneous in origin. At 1000 mg/kg/day, the fetus with gastroschisis as external malformation (A080-02) also had abnormal liver lobation and a malpositioned kidney. The other affected fetuses in this group had tetralogy of Fallot (A067-06), absent kidney and ureter (A080-1), a large heart and fluid-filled body cavities (A081-07) and internal hydrocephaly (A086-09). At 300 mg/kg/day, one of the affected fetuses (A060-03) also had a malpositioned kidney, while a littermate (A060-05) had a cyst associated with the diaphragm in the thoracic cavity. At 100 mg/kg/day, visceral malformations observed were abnormal lung lobation (A038-05) and a multiple cardiovascular abnormality (A040-05). One control fetus (A005-02) had a malpositioned kidney and testis.
All visceral variations noted were considered unrelated to treatment with the test item as they occurred in the absence of a dose-related trend, occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Skeletal Malformations and Variations:

Skeletal malformations occurred in one control and five fetuses (four litters) at 1000 mg/kg/day. Although the incidence of some of these malformations slightly exceeded the historical data range, the single occurrence and/or group distribution of these malformations did not indicate a relationship to treatment with the test item. At 1000 mg/kg/day, these malformations consisted of a vertebral anomaly with or without associated rib anomaly in three fetuses (A075-05, A080-02 and -06), and the other two fetuses had either a costal cartilage anomaly (A067-06) or rib anomaly (A070-04). The affected control fetus (A008- 09) had a sternal anomaly. Although vertebral anomalies occurred in three fetuses at 1000 mg/kg/day only, this was considered not test-item related as a vertebral anomaly is the most common skeletal malformation among historical control fetuses.
Moreover, two cases were observed in litter A080 (-02 and -06) and because in that litter other malformations occurred in fetuses A080-01 and -02 and late resorption A080-04 (see above), it could be considered that the malformations had a maternal or hereditary origin. The other malformations at 1000 mg/kg/day (costal cartilage and rib anomaly) also occurred incidentally and incidences remained within the historical control data range. Therefore, these malformations were considered chance findings.
Noteworthy were the two related variations 13th full ribs and caudal shift of pelvic girdle that occurred at significantly higher incidences at 1000 mg/kg/day. Mean litter incidences were 71.7% and 38.2% per litter, respectively, compared to respective control values 50.5% and 13.4% per litter. Nevertheless, because the high-dose values were near the historical control maximum values of 71.4% and 38.5% for 13th full ribs and caudal shift of pelvic girdle, respectively, they were considered not test item-related.
All other variations noted were also considered not to be related to treatment with the test item as they occurred infrequently, occurred in control fetuses only and/or at frequencies that were within the range of available historical control data.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed up to limit dose of 1000 mg/kg bw/day

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 4. Summary of Clinical Signs in Females

Sign (Max.Grade Location)	Treatment
	Week	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	4	.	.
	Day	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3
Group 1(Control)
Skin/fur Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	0
Skin/fur Scabs (3) (Mouth)	G	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.
Skin/fur Scabs (3) (Nose)	G	.	.	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.
Skin/furScabs (3) (Flews)	G	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	1	1	1
	%	0	1	1	1	1	1	1	0	0	0	.	.	.	.	.	.	.	.	.	.	.	0	0	0
Skin/fur Wound (3) (Nose)	G	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Skin/fur Wound (3) (Flews)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.	.	.
Red Staining (1) (Vagina)	G	.	.	.	.	.	.	.	.	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Secretion / excretion Watery discharge from eye (3) (Eye left)	G	.	.	.	1	1	1	1	1	3	3	2	1	2	2	2	2	2	2	1	1	1	1	1	1
	%	.	.	.	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Secretion / excretion Faeces production reduced (3)	G	.	.	.	2	2	2	2	2	2	1	1	1	1	1	1	1	1	1	1	2	2	2	2	2
	%	.	.	.	1	1	1	1	1	1	5	5	5	4	4	4	2	2	2	2	2	2	2	5	5
Group 2 (100 mg/kg/day)
Skin / fur Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	1	1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	0
Skin/fur Scabs (3) (Nose)	G	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.
	%	0	0		1	1	1	1	1	1	1	1	1	1	0	.	.	.	.	.	.	.	.	.	.
Skin/furScabs (3) (Flews)	G	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	1	1	1	1	1	1	1	0	0	0	0	.	.	.	.	.	.	.	.	.	.
Skin/furScabs (3) (Snout)	G	.	.	.	.	.	.	.	.	.	.1	1	1	1	1	1	1	1	1	1	1	1	1	.	.
	%	.	.	.	.	.	.	.	.	.	0	0	0	0	0	0	0	0	0	0	0	0	0	.	.
Secretion / excretion Faeces production reduced (3)	G	.	.	.	1	1	1	1	1	1	2	2	2	2	2	2	2	2	2	2	1	1	1	2	1
	%	.	.	.	5	5	5	2	2	2	6	6	5	5	5	5	3	3	3	5	5	5	5	66
Various Lean (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.
Broken (1) (Upper incisors)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	0	0	.	.	.	.	.	.
Group 3 (300 mg/kg/day)
Skin / fur Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.
Skin / fur Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.
Skin / fur Scars (3) (Flews)	G	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.
	%	.	.	.	.	.	.	.	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	.
Skin / fur Scabs (3) (Mouth)	G	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.
Skin / fur Scabs (3) (Nose)	G	.	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.	.
Skin / fur Scabs (3) (Flews)	G	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.
	%	0	1	1	0	1	1	1	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.
Secretion / excretion Diarrhoea (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.	.	.	.	.	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.	.	.	.	.	A
Secretion / excretion Faeces pale (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	0	.
Secretion / excretion Faeces production reduced(1)	G	.	.	.	2	2	2	1	1	1	1	1	1	1	1	1	2	2	2	2	2	2	1	1	.
	%	.	.	.	7	7	7	1	1	1	6	6	5	4	4	4	2	2	2	3	3	3	6	6	.
Various Lean (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.
Group 4 (1000 mg/kg/day)
Skin / fur Scabs (3) (Mouth)	G	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	0	0	0	1	1	0	0	0	0	.	.	.	.		.	.	.	.	.	.	.
Skin / fur Scabs (3) (Nose)	G	.	.	.	.	.	.	1	1	1	1	1	1	1	1	.		.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.
Skin / fur Scabs (3) (Flews)	G	1	1	1	1	1	1	1	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1
	%	0	0	0	1	1	1	1	.	.	.	.	.	.	0	0	0	0	0	0	0	0	0	0	0
Secretion / excretion Faeces containing mucus (1)	G	.	.	.	.	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	0	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Secretion / excretion Faeces production reduced (3)	G	.	.	.	2	2	2	2	1	1	2	2	2	2	2	2	2	1	1	1	2	2	2	1	1
	%	.	.	.	A	A	A	4	3	3	5	5	5	5	5	5	5	3	3	3	4	4	4	6	6
Secretion / excretionRed fluid on manure tray (3)	G	.	.	.	.	.	.	2	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.

G: median value of the highest individual dailyy grades; %: percentage of afftected animals (0=less than 5%, 1= bewteen 5% and 15%,..., A=more than 95%); .: Observation performed, sign not presnt.

 

Table 5. Summary of Mean Body Weights  and body weight gains of F0 generations.

Post Coitum day	Body Weights (g)				Body weight gains (%)
	Group 1 (Control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)	Group 1 (Control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
0	3606	3637	3579	3574	-	-	-	-
6	3575	3554	3562	3550	0	0	0	0
9	3640	3587	3530	3474	2	1	-1**	-2**
12	3687	3656	3629	3573	3	3	2	1**
15	3742	3721	3677	3648	5	5	3	3
18	3809	3743	3715	3672	7	5	4	3*
21	3855	3766	3742	3720	8	6	5*	5*
24	3890	3809	3778	3753	9	7	6*	6*
27	3927	3807	3807	3740*	10	7	7	5**
29	3957	3845	3820	3754*	11	9	7	6**

*/** Dunnett-test based on pooled variance sigificant at 5%(*) or 1% (**) level; - Not applicable.

 

Table 6. Summary of Mean food consumption  and relative food consuption of F0 generations.

Post Coitum Day	Food consumption (g/animal/day)				Relative food consumption (g/kgbw/day)
	Group 1 (control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)	Group 1(Control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
6-9	132	111	66**	40**	36	31	19**	12**
9-12	120	128	125	101	33	35	34	28
12-15	87	79	85	81	23	21	23	22
15-18	106	91	101	93	28	24	27	25
18-21	125	117	117	121	32	31	31	33
21-24	111	96	98	97	28	25	26	26
24-27	90	77	87	72	23	20	23	19
27-29	99	84	88	69*	25	22	23	18
Mean of means	109	98	96	84	29	26	24	23

*/** Dunnett-test based on pooled variance sigificant at 5%(*) or 1% (**) level

 

Table 7. Summary Macroscopic Findings in F0 generations.

Post Coitum	Group 1 (control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Animals examined	22	22	22	22
Animals without findings	18	21	20	18
Animalw affected	4	1	2	4
General observations: Beginning autolysis	4	1	2	4
General Observations: Early delivery	0	0	1	0
Lung: focus/Foci	0	1	0	0
Stomach: focus/foci	0	0	0	1
Rectum	1	0	0	0
Contents: Discolouration	0	0	0	1
Oviduts Cyst(s)	0	0	0	1
Skin	2	0	0	2
Scab formation	1	0	0	0
scar (s)	0	0	1	0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

Table 8. Summary of Maternal Survival & Pregnacy Status (%).

Dose Group	1 (0 mg/kg)		2 (100 mg/kg)		3 (300 mg/kg)		4 (1000 mg/kg)
Females on study	No.	%	No.	%	No.	%	No.	%
Female that aborted or delivered	0	0.0	1	4.5	0	0.0	0	0.0
Female that died	0	0.0	0	0.0	1	4.5	0	0.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Female that were euthanized	1	4.5	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	100.0	0	0.0	0	0.0	0	0.0
Female examined at sechduled necropsy	21	95.5	21	95.5	21	95.5	21	95.5
Nongravid	2	9.5	1	4.8	1	4.8	1	4.8
Gravid	19	90.5	20	95.2	20	95.2	20	95.2
With resorption only	1	100.0	0	0.0	0	0.0	0	0.0
with viable fetuses	19	100.0	20	100.0	20	100.0	20	100.0
Total female gravid	20	90.9	21	95.5	21	95.5	20	95.5

 

Table 9. Mean Summary of Fetal Data at Scheduled Necropsy.

Group	Sex		Viable fetuses	Dead Fetuses	Resorption		Post Implantation Loss	Implantation	Corpora Lutea	Pre Implatation Loss	Fetal Weight (g)	No. of Gravid Female
	Male	Female			Early	Late
Group 1 (0 mg/kg)	6.0	4.2	10.2	0.0	0.3	0.2	0.5	10.6	10.9	0.3	37.7	19
2 (100 mg/kg)	4.6	4.6	8.9	0.0	0.4	0.4	0.8	9.7	10.7	1.0	38.7	20
3 (300 mg/kg)	4.4	4.3	8.7	0.0	0.2	0.1	0.3	9.0	9.4	0.4	40.5	20
4 (1000 mg/kg)	4.9	4.1	8.9	0.0	0.5	0.2	0.7	9.6	10.5	0.9	38.4	20

 

Table 10. Mean Summary of Fetal Data at Scheduled Necropsy (% per litter).

Group	Copora lutea	Implantation site	viable fetuses	Dead fetuses	Early resorption	Late resorption	Total resorption	Pre-implantation	Post-implantation	Male	Female	Male fetal weight	Female fetal weight	Combined fetal weight
1 (0 mg/kg)	10.9	10.6	95.7	0.0	2.4	1.9	4.3	2.6	4.3	59.9	40.1	38.4	36.4	37.7
2 (100 mg/kg)	10.7	9.7	93.0	0.0	3.7	3.4	7.0	9.7	7.0	49.6	50.4	39.1	37.8	38.7
3 (300 mg/kg	9.4	9.0	97.6	0.0	1.5	0.9	2.4	5.2	2.4	49.3	50.7	40.6	40.4	40.5
4 (1000 mg/kg)	10.5	9.6	93.0	0.0	5.4	1.6	7.0	8.6	7.0	56.2	43.8	38.6	37.9	38.4

Proportional (%) data compared using the mann-whitney test fetal weights compared using dunnett's test
none significantly different from control group

Table 10. Mean Summary Of Fetuses And Litters With Malformations [Absolute No.]

Dose group	Fetuses				Litters
	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
Number examined externally	193	178	174	178	19	20	20	20
Tail- absent, short or filamentous	1	0	0	0	1	0	0	0
Carpal and/or tarsal flexure	0	0	1	2	0	0	1	2
Facial cleft	0	1	0	0	0	1	0	0
Cleft palate	0	1	0	0	0	1	0	0
Trunk- omphalocele	0	1	1	0	0	1	1	0
Trunk- gastroschisis	0	0	0	1	0	0	0	1
Syndactyly	0	0	0	1	0	0	0	1
Brachydactyly	0	0	0	1	0	0	0	1
Number examined viscerally	193	178	174	178	19	20	20	20
Tetralogy of fallot	0	0	0	1	0	0	0	1
Kidney(s)- malpositioned	1	0	1	1	1	0	1	1
Testis- malpositioned	1	0	0	0	1	0	0	0
Kidney(s) and/or ureter(s)- absent	0	0	0	1	0	0	0	1
Liver- abnormal lobation	0	0	0	1	0	0	0	1
Heart- large	0	0	0	1	0	0	0	1
Body cavity fluid-filled0	0	0	0	1	0	0	0	1
Lung- abnormal lobation	0	0	1	0	0	0	0	1
Diaphram - cyst	0	0	1	0	0	0	1	0
Aortic arch- dilated	0	1	0	0	0	1	0	0
Pulmonary trunk- narrow	0	1	0	0	0	1	0	0
Ventricular septum defect	0	1	0	0	0	1	0	0
Hydrocephaly- internal	0	0	0	1	0	0	0	1
Number examined skeletally	193	-	-	178	19	-	-	20
Costal cartilage anomaly	0	-	-	1	0	-	-	1
Rib anomaly	0	-	-	1	0	-	-	1
Vertebral anomaly with or without associated rib anomaly	0	-	-	3	0	-	-	2
Sternal anomaly	1	-	-	0	1	-	-	0
Total number with malformations external	1	2	2	2	1	2	2	2
Soft tissue	1	2	2	5	1	2	1	2
Skeletal	1	-	-	5	1	-	-	4
Combined	3	4	4	9	3	4	3	6

 

Table 11. Mean Summary Of Litter Proportions of  Malformations [% per Litter]

Dose group	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
Number examined externally	19	20	20	20
Tail- absent, short or filamentous	0.4	0.0	0.0	0.0
Carpal and/or tarsal flexure	0.0	0.0	0.5	1.5
Facial cleft	0.0	0.6	0.0	0.0
Cleft palate	0.0	0.6	0.0	0.0
Trunk- omphalocele	0.0	0.5	1.7	0.0
Trunk- gastroschisis	0.0	0.0	0.0	0.8
Syndactyly	0.0	0.0	0.0	0.6
Brachydactyly	0.0	0.0	0.0	0.6
Tetralogy of fallot	0.0	0.0	0.0	0.6
Kidney(s)- malpositioned	0.5	0.0	0.5	0.8
Testis- malpositioned	0.5	0.0	0.0	0.0
Kidney(s) and/or ureter(s)- absent	0.0	0.0	0.0	0.8
Liver- abnormal lobation	0.0	0.0	0.0	0.8
Heart- large	0.0	0.0	0.0	0.4
Body cavity fluid-filled	0.0	0.0	0.0	0.4
Lung- abnormal lobation	0.0	0.6	0.0	0.0
Diaphram - cyst	0.0	0.0	0.5	0.0
Aortic arch- dilated	0.0	0.6	0.0	0.0
Pulmonary trunk- narrow	0.0	0.6	0.0	0.0
Ventricular septum defect	0.0	0.6	0.0	0.0
Hydrocephaly- internal	0.0	0.0	0.0	0.5
Costal cartilage anomaly	0.0	-	-	0.6
Rib anomaly	0.0	-	-	0.6
Vertebral anomaly with or without associated rib anomaly	0.0	-	-	2.1
Sternal anomaly	0.0	-	-	0.0
Percent per litter with external malformation	0.4	1.1	2.2	1.5
Percent per litter with soft tissue malformation	0.5	1.2	0.9	3.2
Percent per litter with skeletal malformation	0.6	-	-	3.3
Total percent per litter with malformation	1.5	2.2	3.1	5.7

 

Table 12. Mean Summary Of Fetuses Litter with variations [Absolute No.]

Dose group	Fetuses				Litters
	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
									Number examined viscerally	193	178	174	178	19	20	20	20
Number with findings	0	0	0	0	0	0	0	0
Number examined viscerally	193	178	174	178	19	20	20	20
Left carotid- originating from brachiocephalic trunk	5	2	3	9	3	1	2	4
Gallbladder- absent or small	2	4	0	1	2	4	0	1
Retrocaval ureter	7	5	1	2	6	4	1	2
Lung- absent accessory lobe	3	6	4	4	2	2	2	2
Aortic arch- supernumerary artery	0	1	0	5	0	1	0	3
Gallbladder- large	0	0	1	0	0	0	1	0
Adrenal gland- displaced	1	0	0	0	1	0	0	0
Renal papilla(e)- absent and/or small	1	1	0	0	1	1	0	0
Right subclavian- retroesophageal	0	1	0	0	0	1	0	0
Number examined skeletally	193	-	-	178	19	-	-	20
13th full rib(s)	96	-	-	125	19	-	-	20
Sternebra(e) malaligned	35	-	-	20	15	-	-	12
Sternebrae fused	2	-	-	0	2	-	-	0
Sternebra(e) #5 and/or #6 unossified	64	-	-	35	13	-	-	13
Pelvic girdle- caudal shift	24	-	-	65	7	-	-	14
13th rudimentary rib(s)	14	-	-	6	9	-	-	5
7th cervical ossification site(s)	2	-	-	2	1	-	-	2
Vertebral centra- reduced ossification	1	-	-	4	1	-	-	4
Metacarpal (s) and /or metatarsal (s) unossified	8	-	-	10	4	-	-	4
Tarsal (s) unossified	1	-	-	1	1	-	-	1
Caudal vertebral anomaly	1	-	-	1	1	-	-	1
Rib(s)- nodulated	0	-	-	1	0	-	-	1
Skull- supernumerary site	1	-	-	1	1	-	-	1
Number examined skeletally	193	-	-	178	19	-	-	20
Sternum- supernumerary ossification site	2	-	-	0	1	-	-	0
Hyoid body and/or arches unossified	0	-	-	1	0	-	-	1
Reduced ossification of the skull	0	-	-	1	0	-	-	1
hyoid arch(es) bent	1	-	-	0	1	-	-	0

 

Table 13. Mean Summary Of Fetuses Litter with variations [% per litter]

Dose Group	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
Number examined	19	20	20	20
Numbers of litters with findings	0	0	0	0
Left carotid- originating from brachiocephalic trunk	2.7	1.0	1.3	5.2
Gallbladder- absent or small	0.9	2.2	0.0	0.6
Retrocaval ureter	3.8	2.6	0.4	1.0
Lung- absent accessory lobe	1.5	3.3	1.7	2.8
Aortic arch- supernumerary artery	0.0	0.5	0.0	2.7
Gallbladder- large	0.0	0.0	0.5	0.0
Adrenal gland- displaced	0.5	0.0	0.0	0.0
Renal papilla(e)- absent and/or small	0.0	0.6	0.0	0.0
Right subclavian- retroesophageal	0.0	0.6	0.0	0.0
Number examined skeletally	19	-	-	20
13th full rib(s)	50.5	-	-	71.1*
Sternebra(e) malaligned	18.9	-	-	11.5
Sternebrae fused	1.1	-	-	0.0
Sternebra(e) #5 and/or #6 unossified	34.6	-	-	19.2
Pelvic girdle- caudal shift	13.4	-	-	38.2*
13th rudimentary rib(s)	6.7	-	-	3.0
7th cervical ossification site(s)	1.0	-	-	1.0
Vertebral centra- reduced ossification	0.4	-	-	2.6
Metacarpal (s) and /or metatarsal (s) unossified	4.0	-	-	4.4
Tarsal (s) unossified	0.5	-	-	0.6
Caudal vertebral anomaly	0.5	-	-	0.7
Rib(s)- nodulated	0.5	-	-	0.7
Skull- supernumerary site	0.5	-	-	0.5
Number examined skeletally	19	-	-	20
Sternum- supernumerary ossification site	1.0	-	-	0.0
Hyoid body and/or arches unossified	0.0	-	-	0.8
Reduced ossification of the skull	0.0	-	-	0.8
hyoid arch(es) bent	0.4	-	-	0.0
Number of litter examined	19	20	20	20
Total variations Percetage per litter with external variation	0.0	0.0	0.0	0.0
Total variations percentage per litter with soft tissue varations	8.3	9.7	3.5	12.3
Total variations - percentage per litter with sekeletal variations	78.0	-	-	86.2

Conclusions:

Based on the results in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Tris (2- ethylhexyl)phosphate was established as being at least 1000 mg/kg/day.

Executive summary:

The potential of Tris (2-ethylhexyl)phosphate to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post- coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels the study were selected to be 0, 100, 300 and 1000 mg/kg/day, based on the results of the dose range finder (Test Facility Study No. 20173163).

The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

Maternal findings
No adverse maternal findings were recorded up to a dose of 1000 mg/kg/day.

At 300 and 1000 mg/kg/day, a non-adverse slight mean weight loss (-1% or -2%) and lower mean absolute and relative food consumption (0.53x and 0.33x of control, respectively, for relative food intake) was recorded over post-coitum Days 6 to 9. From post-coitum Day 9 onwards, reduced faeces production was recorded at increased incidence over the dose groups. Food intake however remained similar to control levels during this period, except for post-coitum Days 27 to 29, when a lower mean absolute and relative food consumption (0.72x of control for relative food consumption) and lower mean body weights were recorded (0.95x of control) at 1000 mg/kg/day. In the period between PND 9 and 27, there were no clear differences in body weight and food intake between treated groups and the control group that could be ascribed to the test-item (statistically significant changes in that period occurred in the absence of a clear dose-related trend). Overall, mean weight gain at 1000 mg/kg/day over the treatment period was 6% vs. 11% in the control group, but mean body weights remained well within the historical control data range.

Also, corrected weight gain for gravid uterus at 1000 mg/kg/day was slightly outside the historical range for a total of 6 out of 20 animals (the mean corrected weight remained within the historical control range). Overall, it was considered that these changes in body weight and food intake did not represent adverse maternal effects, since these variations were not consistently seen with continuing treatment and generally remained within the historical control data range. Also, there were no indications among fetuses that pointed towards a secondary effect of these maternal variations in body weight and food intake.

At 100 mg/kg/day, no test item related maternal effects were recorded. No test item-related macroscopic lesions were recorded at any dose level. Fetal Examinations

No test item-related changes were noted up to 1000 mg/kg/day in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Rat, HanRcc: WIST(SPF Quality)

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
a) A copulation plug was observed, and / or
b) The daily vaginal smear was sperm positive.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used only for mating. These male rats are in the possession of RCC and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.

Duration of treatment / exposure:

Day 6 - 20 post coitum

Frequency of treatment:

Daily.

Duration of test:

At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.

Remarks:

Doses / Concentrations:
Group 1: 0 mg/kg body weight/day (control group); Group 2: 500 mg/kg body weight/day; Group 3: 1000 mg/kg body weight/day.
Basis:

No. of animals per sex per dose:

Each group consisted of 24 mated female rats.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no data

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Analysis of dose formulations:

The concentration of the test substance in all samples ranged from 97.7 to 106.6% of the nominal concentrations. In addition, the homogenous distribution of the test item in corn oil was demonstrated. The dose formulations were demonstrated to be stable for at least 7 days when kept refrigerated, which was the maximum time of storage and use of dosing preparations in this study.

Maternal data:

Mortality and clinical signs or observations:

All females survived until the scheduled necropsy. No clinical signs or signs of discomfort were noted for any female in any group during the study.

Food consumption (g/animal/day) of dams post coitum:

Food consumption was similar in all dose groups during the treatment period.

Body weights (gram) of dams post coitum, body weight gain (gram) of dams post coitum and corrected body weight gain of dams: There were no effects on mean body weight, mean body weight gain or mean body weight corrected for uterus weight in any dose group.

Reproduction data: Reproduction processes, distribution within uterus and contents of uterus (plan view):

No treatment-related effects were noted on reproduction parameters in any dose group. Post-implantation loss and the number of embryonic resorptions per litter were statistically significantly lower at 1000 mg/kg/day compared to the control group. However, these values were within the range of the historical control data and treatment-related effects are typically associated with increases in post-implantation loss and embryonic resorptions. Therefore these values reflect normal variability and were not related to treatment.Macroscopical findings:

No macroscopical findings were noted for any female in any group.

Fetal data:

External examination of fetuses:

No test item-related findings were noted during external examination of the fetuses. One fetus at 1000 mg/kg/day had a shortened lower jaw (brachygnathia). This was within the range of historical control external findings and was considered to be incidental.

Sex ratios:

The sex ratio of the fetuses was close to 50% in all dose groups.

Body weights:

The mean body weight of the fetuses was similar in all groups and there was no effect of treatment at 500 or 1000 mg/kg/day.

Visceral examination of fetuses (microdissection technique):

A total of 4 fetuses with visceral abnormalities were noted. At 1000 mg/kg/day, two fetuses from a single litter were noted with multiple changes. One fetus exhibited multiple abnormalities of the craniofacial region, which correlated with the external finding of shortened lower jaw. The second fetus from this litter exhibited multiple cardiovascular changes and situs inversus. Both of these fetuses had the accompanying abnormality of pituitary small/misshapen. At 500 mg/kg/day, two fetuses from separate litters exhibited abnormalities; one fetus exhibited thoracic and abdominal situs inversus, and the other exhibited a heart interventricular septal defect. The low incidences of these abnormalities were within the historical control range and considered unrelated to treatment. The total incidence of visceral abnormalities and variations combined in each group was: 71 out of 151 examined fetuses (in 22 of 24 litters) of group 1 ( 0 mg/kg) 63 out of 144 examined fetuses (in 22 of 23 litters) of group 2 ( 500 mg/kg) 67 out of 143 examined fetuses (in 20 of 23 litters) of group 3 (1000 mg/kg) There was no statistically significant difference from the control group in the total incidence of abnormalities or of abnormalities and variations combined at 500 or 1000 mg/kg/day. For the individual variation of long thymus (cranial), the fetal incidence at 1000 mg/kg/day (22) was statistically significantly higher compared to the control group (10), but the litter incidence (11) was no different from the control (7). Conversely, the variation of subcutaneous haemorrhage was statistically significantly lower than the control at 1000 mg/kg/day on both a fetal and litter basis. Both of these variations are common in the historic control data, with the latter finding described as “localized haemorrhage”.

Considering the high background incidence of these variations, and the presence of both higher and lower incidences in the 1000 mg/kg/day group, these variations are considered incidental to treatment.

Overall, the visceral findings noted were common findings for this species and strain of rats, and therefore, they were considered unrelated to treatment.

Skeletal examinations - bone and cartilage abnormalities and variations:

A single fetus at 1000 mg/kg/day was noted with a cartilage finding (severe dumbbell-shaped cervical vertebral body 1), without any abnormalities in bone structures. This single incidence is not considered treatment-related. The total incidence of bone and cartilage abnormalities and variations in each group was: 37 out of 138 examined fetuses (in 19 of 23 litters) of group 1 ( 0 mg/kg) 30 out of 134 examined fetuses (in 17 of 23 litters) of group 2 ( 500 mg/kg) 29 out of 126 examined fetuses (in 15 of 23 litters) of group 3 (1000 mg/kg) There were no statistically significant differences from the control group in the incidence of total skeletal findings or any individual skeletal findings. The bone and cartilage variations that were noted were generally observed at similar incidence in control and treated groups, and in the historic control data, and were considered not to be test item-related.

Bone examinations - ossification stage / supernumerary ribs:

Bone examination for ossification stage and/or supernumerary ribs did not reveal any test item related findings. On a fetal basis, certain isolated structures had statistically significantly lower incidences of incomplete ossification or non-ossified in 500 and/or 1000 mg/kg/day treatment groups. However, these were not statistically significant on a litter basis, and the direction of change and their sporadic occurrence did not indicate a treatment-related effect.

Cartilage examinations - additional variations:

Cartilage additional variations were noted at similar incidence in control and treated groups, and there was no effect of treatment. Branched xiphoid cartilage was observed at lower incidence at 1000 mg/kg/day than in the control group on both a fetal and litter basis, but a lower incidence of this common finding is considered normal variability and not an effect of treatment.

Conclusion:

The test item was administered orally by gavage once daily from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) to pregnant rats at dose levels of 0, 500 and 1000 mg/kg/day. No maternal toxicity was noted in any of the treatment groups. The reproduction data of the test item-treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any test item-related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was considered to be 1000 mg/kg body weight/day. Report Number:

Executive summary:

The purpose of this study according to OECD Guideline 414 was to evaluate any effects of the test material on the pregnant female rat and development of the embryo and fetus consequent to exposure of the female to the test item. Each group consisted of 24 mated female rats. The test substance was administered orally, by gavage, once daily from day 6 through to day 20 post coitum (last treatment) at dose levels of:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 500 mg/kg body weight/day

Group 3: 1000 mg/kg body weight/day.

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females in the main groups were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examination of dams and fetuses was performed in accordance with international recommendations. Maternal data:

General tolerability:

All females survived until the scheduled necropsy. No clinical signs or signs of discomfort were noted for any female in any group during the study.

Food consumption:

Food consumption was similar in all dose groups during the treatment period.

Body weights:

Absolute body weights, mean body weight gain and corrected body weight gain were not affected by treatment with the test item. Reproduction data:

No effects were noted due to treatment with the test item in the reproduction data.

Macroscopical data:

No macroscopical findings were noted for any female in any group.

Fetal data:

External examination of fetuses:

At scheduled Caesarean section, no test item-related findings were observed.

Sex ratios:

The sex ratio of the fetuses was close to 50% in all dose groups.

Body weights:

The mean body weight of the fetuses was similar in all groups.

Visceral examination of fetuses (microdissection technique):

No test item-related findings were noted.

Skeletal examinations - bone and cartilage abnormalities and variations:

There were no treatment-related effects on the incidence of skeletal abnormalities and variations.

Bone examinations - ossification stage / supernumerary ribs:

Skeletal examination (ossification stage), which was tabulated separately to evaluate changes in degree of ossification and thus the relative stage of development of fetuses, did not reveal any test item-related findings. There was no effect of treatment on the incidence of supernumerary ribs.

Cartilage examinations - additional variations:

Cartilage additional variations were noted at similar incidence in control and treated groups, and there was no effect of treatment.

Conclusion:

The test substance was administered orally by gavage once daily from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) to pregnant rats at dose levels of 0, 500 and 1000 mg/kg/day. No maternal toxicity was noted in any of the treatment groups. The reproduction data of the test item-treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any test item-related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The data-set for effects on development is consistent with data requirements under Annexes VII to X of REACH, and both key studies are considered reliable and relevant for endpoint conclusions.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a study according to OECD 414 the test substance was administered orally by gavage once daily from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) to pregnant rats at dose levels of 0, 500 and 1000 mg/kg/day. No maternal toxicity was noted in any of the treatment groups. The reproduction data of the test item-treated groups and the control group were similar. Fetal pathology (external, visceral, and skeletal examinations) did not reveal any test item-related findings. Based on the results of this study, the NOEL (No Observed Effect Level) for maternal and developmental findings was 1000 mg/kg body weight/day (Whitlow, 2008).

In an OECD 414 (2020), the test item and vehicle were administered to pregnant rabbits via oral gavage once daily, 7 days a week from Day 6 to Day 28 post-coitum, inclusive at dose levels of 0, 100, 300 and 1000 mg/kg/day. No adverse maternal findings were recorded up to a dose of 1000 mg/kg/day. No test item-related changes were noted up to 1000 mg/kg/day in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

The assumed main metabolite, 2-ethylhexanol, also showed no developmental effects in inhalation and dermal developmental studies (Tyl, 1992; Nelson, 1989).

Classification: Classification is not required.

Mode of Action Analysis / Human Relevance Framework

There was no evidence of reproductive or developmental toxicity that requires analysis of the mode of action of the substance, nor any relevance to humans.

Justification for classification or non-classification

The study does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.