[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Feb - 07 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

limited set of parameters investigated

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 Feb - 07 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP-Guideline study, tested with the source substance glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277 - 353 g (males) and 183 - 225 g (females)
- Housing: 5 animals of the same sex per cage in Macrolon cages (MIV type). Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenised to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

DOSE VOLUME:
5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 h.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 5 females (main study)
5 males and 5 females (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 10-day dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males: Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.

Sacrifice and pathology:

DAY OF NERCROPSY:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanised in extremis.

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
From the first 5 Main animals/sex/group, all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):
Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

From all remaining animals and females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse)

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg bw/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg bw/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg bw/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg bw/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time in males at 1000 mg/kg bw/day and lower relative lymphocyte counts in females at 300 mg/kg bw/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg bw/day, and higher chloride levels in males at 100 mg/kg bw/day. Changes at 1000 mg/kg bw/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg bw/day had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg bw/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg bw/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg bw/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg bw/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg bw/day and the end of treatment, lower heart weight in females at 1000 mg/kg bw/day, higher heart to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg bw/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg bw/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discolouration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discolouration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg bw/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to and including the highest dose level.

Key result

Critical effects observed:

no

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Feb - 07 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only external abnormalities and mainly macroscopic examination of soft tissues performed, no skeletal examination of pups

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-Quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: approximately 12 weeks.
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).

Duration of treatment / exposure:

41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

daily, 7 days/week

Duration of test:

41-49 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 10-day dose range finding study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes

For further details see Section 7.5.1

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: No

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes, partly (see details below)
- Skeletal examinations: No
- Head examinations: No

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Pups surviving to planned termination were killed by decapitation on lactation Day 5.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Indices:

For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse); for details please refer to section 7.5.1 oral repeated dose toxicity

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects observed (for details please refer to section 7.5.1 oral repeated dose toxicity).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects observed (for details please refer to section 7.5.1 oral repeated dose toxicity).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects observed (for details please refer to section 7.5.1 oral repeated dose toxicity).

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse and treatment-related effects observed up to and including the highest tested dose level.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of live pups: 432
Mortality: 1/119, 1/116. 1/126 and 1/71 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. This is not considered to be treatment-related, as this falls within the expected range for this strain and age.
A statistically significant lower mean number of living pups at first litter check observed at 1000 mg/kg/day was due to a low number of pups (5 in total) for one female. Since the number of pups of other females of this dose group was within the range considered normal, no toxicological relevance was ascribed to this change.
Viability index, body weights of pups, external examination for abnormalities and clinical signs of pups did not indicate any treatment-related abnormalities

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects observed up to and including the highest tested dose level.

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

anogenital distance and nipple retention in males not determined

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates

EC Number:

293-170-2

EC Name:

Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates

Cas Number:

91052-13-0

Molecular formula:

C15H26O6, C19H34O6, C21H38O6, C25H46O6

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-Quality)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).

Following a minimum of 14 days of exposure for the males and females, one Repro female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was not confirmed for animal no. 98 which did deliver. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating. After 14 days of mating, females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.

Duration of treatment / exposure:

41-49 days,
i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

Offspring were euthanized at the age of 4 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 10-day dose range finding study

Examinations

Parental animals: Observations and examinations:

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1

Oestrous cyclicity (parental animals):

Uterus epithelium was analysed histologically for estrus, proestrus and cystic endometrial glands.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: testis weight, epididymis weight, histology of testes
Of the first 5 Main males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Litter observations:

On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals which delivered: on lactation Day 5
- Maternal animals which failed to deliver (4 animals): Post-coitum Day 26 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1

Postmortem examinations (offspring):

SACRIFICE
- Pups surviving to planned termination were killed by decapitation on lactation Day 5.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Reproductive indices:

For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Offspring viability indices:

For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse), for details please refer to section 7.5.1 oral repeated dose toxicity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects observed, for details please refer to section 7.5.1 oral repeated dose toxicity.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

as found by histological examination of uterus

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

as found by histological examination of testes

Reproductive performance:

no effects observed

Details on results (P0)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment related influences on estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment related effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

parental fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Number of pups: 432
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed up to and including the highest tested dose level

Overall reproductive toxicity

Key result

Reproductive effects observed:

no