Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Oct-2012 to 27-Mar-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts

EC Number:

938-654-2

Molecular formula:

not applicable

IUPAC Name:

Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts

Test material form:

other: solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 12 males and 12 females (Groups 1 to 4) and 2 males and 2 females (Group 10)
- Total Number of Animals: 50 males and 50 females
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Acclimatization): 307 - 351 g (mean 329 g) in males and 205 - 231 g (mean 218 g) in females
- Identification: Cage card and individual animal number (ear tattoo). Pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of three to five in Makrolon type-4 cages during acclimatization or individually in Makrolon type-3 cages during the treatment phase with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland, batch/lot nos. 02105120601) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands, batch 77-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Pelleted standard Harlan Teklad 2914C (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no 20/12). Analyses for contaminants were performed.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Alkenes hydroformylated, sulfosuccinates, sodium salt was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, any remaining vehicle was added if necessary.

Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE AND STABILITY OF DOSE FORMULATIONS

- Stability of Dose Formulations: For at least one week, based upon the results of stability analyses performed during the non-GLP Harlan Laboratories study D55862.
- Storage of Dose Formulations: In a refrigerator (2 ± 8 °C) in glass beakers.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (Group 1), 50 mg/kg/day (Group 2), 150 mg/kg/day (Group 3) and 450 mg/kg/day (Group 4)
- Dose Volume: 10 mL/kg
- Dose Concentrations: 0 mg/mL/day (Group 1), 5 mg/mL/day (Group 2), 15 mg/mL/day (Group 3) and 45 mg/mL/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D55862.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, this female was paired with a male of the same group which had already mated successfully. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by power regression analysis. The regression coefficients (R2) calculated were found to exceed 0.99.

The alkenes hydroformylated, sulfosuccinates, sodium salt peak was assigned in sample chromatograms by comparison to working standards. In blank sample chromatograms, no peak appeared at the retention time of alkenes hydroformylated, sulfosuccinates, sodium salt, con¬firming the absence of the test item in the vehicle control samples (bidistilled water).

The application formulations investigated during the study were found to comprise alkenes hydroformylated, sulfosuccinates, sodium salt in the range of 83.8% to 102.8%, which met the required content limit of ±20% with reference to the nominal content. The distribution of alkenes hydroformylated, sulfosuccinates, sodium salt in the preparations was found to be homogeneous; single results found did not deviate more than 6.2% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature. Recoveries met the variation limit of 10% from the time-zero (homogeneity) mean, except for stability samples of group 2 (8 days) that exceeded the acceptance criteria up to 11.1%. However, as no degradation was observed, the formulation was considered to be stable, despite exceeding the acceptance criterion (±10%).

In conclusion, the results indicate the accurate use of the test item alkenes hydroformylated, sulfosuccinates, sodium salt and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficiently stable under storage conditions.

Duration of treatment / exposure:

- Males: 57 days
- Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Details on study schedule:

MALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Blood Sampling: End of prepairing
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After treatment of at least 28 days, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Blood Sampling: End of prepairing
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Necropsy: On day 4 post partum (pups on day 4 post partum)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150 and 450 mg/kg/day
Basis:

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

Rationale for Choice of Species, Route of Administration and Dose Levels

The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.

Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (Harlan Laboratories study D55862).

Positive control:

Not required

Examinations

Parental animals: Observations and examinations:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

- Males: Pre-pairing (days 1-8, 8-13) and after pairing (days 1-5, 5-9) periods.
- Females: Pre-Pairing period days 1 - 8 and 8 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and lactation days 1 - 4.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

Animals were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were reported if noted.

FUNCTIONAL OBSERVATIONAL BATTERY

At one time during the study (females on day 3 or 4 post partum), relevant parameters from a modified Irwin screen test were performed with at least five P generation females from each group in place of the usual weekly behavioral observation. This FOB assessment was conducted following the daily dose administration.

- Grip Strength: Hind- and forelimb grip strength measurement was performed using a push-pull strain gauge (Mecmesin, AFG 25N).

- Locomotor Activity: Locomotor activity was measured quantitatively for the same animals as those scheduled for the FOB. Decreased or increased activity will be recorded. Activity was measured with an AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH. Activity of the animals was recorded for 6-minute intervals over a period of 60 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained on day 14 of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The assay was performed at Harlan Laboratories Ltd. (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.

The following hematology parameters were determined:

Complete Blood Cell Count

- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count

Coagulation

- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION AND NECROPSY

Males were sacrificed after treatment of 57 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 4 post partum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and 5 females of each group killed at the end of the study, the following organs were trimmed from any adherent tissue as appropriate and their wet weight taken:

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Heart including auricles
- Kidneys
- Liver
- Spleen
- Thymus

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Epididymides (fixed in Bouin's solution)
- Prostate gland and seminal vesicles incl. coagulating glands
- Testes (fixed in Bouin's solution)

The following tissue from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Ovaries

In addition, from the five male and five female animals selected from each group for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Adrenal glands
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Liver
- Lungs (preserved by inflation with fixative and then immersion)
- Lymph nodes – mesenteric and mandibular
- Rectum
- Sciatic nerve
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Trachea
- Urinary bladder (preserved by inflation with fixative and then immersion)
- Uterus (incl. oviducts, cervix and vagina)
- All gross lesions

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals which were terminated in extremis.

Qualitative assessments of the male reproductive organs were performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any females that did not give birth.

In addition, because of treatment-related findings observed in animals from the highest dosage group, the bone marrow (femur), liver, mandibular and mesenteric lymph nodes, Peyer’s patches, spleen and thymus were examined from five animals of each sex and the kidneys from five females of the lowest and intermediate dosage groups.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum by an injection of sodium pentobarbital. All pups except those excessively cannibalized sacrificed were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index.

Offspring viability indices:

From the on-line reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratio and viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day, lower mean daily food consumption and lower mean body weight in males from pre-pairing onwards and in females from gestation onwards. At 150 mg/kg bw/day lower food consumption in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day, lower mean daily food consumption and lower mean body weight in males from pre-pairing onwards and in females from gestation onwards. At 150 mg/kg bw/day lower food consumption in females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Several test item-related changes noted at all dose levels included but were not limited to minimal to severe lymphocyte depletion of the lymphoid tissues affecting all compartments of the lymph nodes and depletion of lymphocytes in the thymus of females.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day slightly lower mean litter sizes and lower gestation index.

Details on results (P0)

1. IN-LIFE DATA

VIABILITY/MORTALITY

All males survived until their respective scheduled necropsies.

After not delivering as scheduled on day 21 of gestation, one female each was killed at 50 mg/kg/day (no. 71) and 150 mg/kg/day (no. 83) on their respective days 25 of gestation. Two females at 450 mg/kg/day (nos. 91 and 92) were killed on their respective days 25 of gestation, whereas one female of this group (no. 85) was killed for ethical reasons on day 23 of gestation. Although the exact cause of death could not be ascertained from the postmortem examination, the microscopical evaluation of the tissues suggested complications from severe lymphocytic depletion.

After not delivering as scheduled on day 21 of gestation, one control female (no. 56) was killed on day 25 of gestation.

DAILY CLINICAL SIGNS OR OBSERVATIONS

- Males: There were no test item-related clinical signs during the pre-pairing, pairing and after pairing period. Transient observations such as hair loss and/or localized wounds were noted. One male treated with 450 mg/kg/day was missing the tail apex.

- Females: There were no clinical signs in any female during the pre-pairing, pairing and gestation periods. Diarrhea was noted in two females treated with 450 mg/kg/day during the lactation period. All other females were without clinical findings during the lactation period.

In the one female (no. 85) that was sacrificed for ethical reasons on day 23 of gestation, observations included general weakened condition, decreased activity, ruffled fur, chromodacryorrhea (bilateral), and reddish nasal secretion.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

- Males: There were no test item-related clinical signs during the pre-pairing, pairing and after pairing period. Transient observations without toxicological relevance were noted at all dose levels.

- Females: There were no clinical signs in any female during the pre-pairing, pairing, gestation and lactation periods.

FUNCTIONAL OBSERVATIONAL BATTERY

- Males: There were no findings evident during the functional observational battery performed during the pairing period.

The mean fore- and hind limb grip strength values of the test item-treated males compared favorably with those of the control males.

There were no test item-related differences in the mean locomotor activity of the males when compared to the control values. During the measurement interval 40 - 50 minutes, the mean locomotor activity was significantly elevated (p<0.05) in males treated with 150 mg/kg/day when compared to the control males. This transient finding was unrelated to dose and considered to be incidental.

- Females: There were no findings evident during the functional observational battery performed during the lactation period.

In females treated with 450 mg/kg/day, the mean fore limb grip strength was marginally less than that of the control females but did not attain statistical significance. This difference was considered to be related to the lower mean body weights. The mean hind limb grip strength values of these females, as well as those treated with 50 mg/kg/day and 150 mg/kg/day, were unaffected.

There were no test item-related differences in the mean locomotor activity of the females when compared to the control values.

FOOD CONSUMPTION OF MALES

The mean daily food consumption of males treated with 450 mg/kg/day was markedly lower than that of the control males. These differences were considered to be related to the treatment with the test item. The remaining males were unaffected.

- Pre-Pairing Period: Lower mean daily food consumption was noted during days 1 - 8 and 8 - 13 of the pre-pairing period in males treated with 450 mg/kg/day. The differences attained statistical significance (p<0.01) when compared to the control males.

The males treated with 150 mg/kg/day had significantly reduced mean daily food consumption (p<0.05) from days 8 - 13 of the pre-pairing period when compared with the control males.

Males treated with 50 mg/kg/day consumed similar amounts of feed as the control males.

- After Pairing Period: The statistically significant difference in mean daily food consumption seen in males at 450 mg/ kg/day during the preparing period continued during the after pairing period. The mean daily food consumption remained lower during days 1 - 5 (p<0.01) and 5 - 9 (p<0.01) of the after pairing period.

Significantly lower mean daily food consumption was also noted during days 5 - 9 in males at 50 mg/kg/day (p<0.05) and at 150 mg/kg/day (p<0.05) when compared with the control males.

FOOD CONSUMPTION OF FEMALES

Females treated with 450 mg/kg/day had test item-related reductions of mean daily food consumption during all intervals. This difference was also noted in females treated with 150 mg/kg/day with slightly less (i.e. dose-related) severity. Females at 50 mg/kg/day were unaffected.

- Pre-Pairing, Gestation and Lactation Periods: Females treated with 450 mg/kg/day consumed significantly less feed than the control females during days 1 - 8 of the pre-pairing period (p<0.01), the gestation period (days 0 - 7 and days 7 - 14, both p<0.01, and days 14 - 21, p<0.05) and the lactation period (days 1 - 4, p<0.01).

At 150 mg/kg/day, significantly lower mean daily food consumption was noted only during days 0 - 7 of the gestation period (p<0.05).

No differences in mean daily food consumption were noted in females treated with 50 mg/kg/day when compared with the control females.

BODY WEIGHTS OF MALES

- Pre-Pairing, Pairing and After Pairing Periods: Males treated with 450 mg/kg/day had significantly lower mean body weight beginning on day 3 (p<0.05) and continuing from days 4 - 14 (p<0.01) of the pre-pairing period. This difference continued throughout days 1 - 23 of the pairing period (p<0.01), and days 1 - 9 of the after pairing period (p<0.01). These differences were considered to be test item-related.

Although the differences did not attain statistical significance, marginally to slightly lower body weights were also noted in males treated with 50 mg/kg/day and 150 mg/kg/day when compared with the controls.

BODY WEIGHTS OF FEMALES

- Pre-Pairing, Pairing, Gestation and Lactation Periods: No differences in mean body weights were noted in test item-treated females during the pre-pairing period or pairing period (all females were mated within 3 days). At 450 mg/kg/day, lower body weights were noted from day 4 of gestation onwards, with the difference attaining statistical significance from days 4 - 17 (p<0.01 or p<0.05) and again on day 21 (p<0.05). Significantly lower mean body weights were also recorded during days 1 - 4 of the lactation period (p<0.01).

The mean body weight of the females treated with 50 mg/kg/day or 150 mg/kg/day were considered to be unaffected by the treatment with the test item.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

- Males: At 450 mg/kg/day, test item-related changes included significantly elevated white blood cell count, due largely to significant elevations in mean absolute neutrophils (p<0.01), basophils (p<0.01), lymphocytes (p<0.01) and monocytes (p<0.01) and large unstained cells (p<0.01) that mostly exceeded the upper ranges of the historical control data. These changes were considered to be test item related. The mean relative eosinophil count was significantly lower (p<0.05) when compared with the controls.

At 150 mg/kg/day, slightly elevated white blood cell count was noted in males and was largely due to an increased lymphocyte count. Although these values did not exceed the upper ranges of the historical control data, they were dose-related and considered to be related to the treatment with the test item.

At 50 mg/kg/day, no test item-related effects on hematology parameters were noted in males.

- Females: At 450 mg/kg/day, test item-related changes included significantly elevated white blood cell count, due largely to significant elevations in mean absolute neutrophils (p<0.01), lymphocytes (p<0.05) and monocytes (p<0.01) and large unstained cells (p<0.01) that mostly exceeded the upper ranges of the historical control data. These changes were considered to be test item related.

At 150 mg/kg/day, a mildly elevated white blood cell count was noted in females but was considered to be within the range of typical biological variation. The mean relative eosinophil count was significantly lower (p<0.05) when compared with the controls but considered to be within the range of typical biological variation.

At 50 mg/kg/day, no test item-related effects on hematology parameters were noted in females.

CLINICAL BIOCHEMISTRY

- Males: At 450 mg/kg/day, test item-related changes included significantly increased activities of aspartate aminotransferase (p<0.05), alanine aminotransferase (p<0.01) and alkaline phosphatase (p<0.01), as well as increased sodium (p<0.05) and reduced total protein and globulin (both p<0.01, but considered to be due to high control values). The changes seen in the hepatic enzymes were considered to be the results of metabolic adaptation rather than indications of systemic toxicity. The mean cholesterol level was significantly reduced (p<0.01) when compared with the controls. This finding, combined with the reduction in triglycerides, was considered to be a non-specific test item-related effect upon lipid metabolism.

At 150 mg/kg/day, the creatine level was significantly increased (p<0.05) but was unrelated to dose and therefore considered to be incidental. The mean protein level was significantly lower than that of the controls (p<0.05) but was considered to be the result of a high control value.

At 50 mg/kg/day, significantly elevated potassium levels (p<0.05) were unrelated to dose and considered to be of no toxicological relevance. Slight but statistically significant reduced total protein level noted in these males was considered incidental and an artifact of a slightly higher control value.

- Females: At 450 mg/kg/day, elevated activities of liver enzymes were noted (alanine aminotransferase, p<0.05 and alkaline phosphatase, p<0.01). However, only alkaline phosphatase activity exceeded the upper range of the historical control data. Reduction of phosphorus was also statistically significant (p<0.05), but remained within the range of the historical control values.

At 150 mg/kg/day, globulin levels were increased (p<0.05) when compared with the control but the differences was unrelated to dose and considered to be incidental.

At 50 mg/kg/day, reduced phosphorus was statistically significant (p<0.05) but within the range of the historical control data. Dose-unrelated increased globulin levels (p<0.01) were considered to be incidental.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

- Males: At 450 mg/kg/day, the mean liver-to-body weight ratio was significantly higher in males (p<0.01) when compared with the controls. The mean liver-to-brain weight was also increased in these males but did not attain statistical significance. Mean absolute thymus weights, mean thymus-to-body weight and mean thymus-to-brain weight ratios were significantly reduced in males (all p<0.01).

The mean absolute heart weight and heart-to-brain weight ratio of males treated with 450 mg/kg/day were significantly lower (p<0.01), and the mean brain-to-body weight ratio of these males was significantly elevated (p<0.01); these differences were considered to be body weight effects.

At 50 and 150 mg/kg/day, there were no statistically significant differences to the control males, although a trend for lower thymus weights were noted. In the absence of a clear dose-response relationship, these differences were considered to be unrelated to the treatment with the test item. All other organ weight and ratios were generally similar to the control males.

- Females: At 450 mg/kg/day, significantly higher mean liver-to-body weight ratios (p<0.01) and significantly higher mean liver-to-brain weight ratios (p<0.05) were noted when compared with the controls. The mean absolute liver weight was also elevated in these females, but did not attain statistical significance. The mean absolute thymus weight, the mean thymus-to-body weight ratio and the mean thymus-to-brain weight ratio were significantly reduced (p<0.01) when compared to the controls. The mean brain-to-body weight and the mean kidney-to-body weight ratios were significantly elevated (both p<0.05) but these were considered to be body weight effects.

At 150 mg/kg/day, the mean absolute thymus weight, the mean thymus-to-body weight ratio and the mean thymus-to-brain weight ratio were significantly reduced (p<0.05) when compared to the controls.

At 50 mg/kg/day, there were no statistically significant differences to the control females.

MACROSCOPICAL FINDINGS

There were no test item-related macroscopical findings at any dose level in males or females.

HISTOPATHOLOGY FINDINGS

Findings in Decedents:

Animal no. 85, a female given 450 mg/kg/day, was killed in a moribund condition on study day 37. The animal’s moribund condition was most probably associated with severe lymphocyte depletion but the precise cause was not established.

Findings in Surviving Rats:

Changes considered to be associated with the administration of the test item were present in the bone marrow, liver, lymphoid tissues (mandibular and mesenteric lymph nodes, Peyer’s patches, spleen and thymus) of both sexes and the kidneys of females.

- Bone Marrow (Femur): The myeloid/erythroid ratio of the femoral bone marrow was greater in both sexes given 450 mg/kg/day than in controls. In addition, the amount of fat in the bone marrow of all the treated male groups and females given 50 or 450 mg/kg/day was greater than in controls. This finding was considered to indicate an overall decrease in hemopoietic cells in the marrow. Since there was no effect on bone marrow observed in females given 150 mg/kg/day it is likely that the greater incidence in bone marrow fat seen in the lowest female dosage group occurred fortuitously.

- Kidneys: Minimal or moderate diffuse cortical tubular fat vacuolation was seen in 4/6 females given 450 mg/kg/day. This finding was not observed in any of the other animals on study. No other evidence of degenerative change was seen in the kidneys of the affected animals.

- Liver: Mild periportal fat vacuolation was observed in 2/5 females given 150 mg/kg/day, minimal or mild centrilobular fat vacuolation in 4/5 males given 450 mg/kg/day and moderate or marked centrilobular or diffuse fat vacuolation in all the females given 450 mg/kg/day. Fat vacuolation was responsible for the clay-colored livers seen at necropsy in two of the highest dosage females and probably for the increase in mean liver weights recorded in both sexes given the highest dosage.

In addition, minimal centrilobular hypertrophy of hepatocytes was observed in 3/5 males and 1/6 females given 450 mg/kg/day. This finding was also probably partly responsible for the increase in mean liver weights. In those animals with moderate or marked centrilobular or diffuse fat vacuolation it is possible that the large fat vacuoles masked any underlying hypertrophy.

Hepatocellular brown pigment and periportal pigment-laden macrophages were seen in a few of the treated females. This is a common background finding in rats of this strain and the pigment was different in character and appearance from that seen in the lymph nodes. It was, therefore, considered a spontaneous change with no relationship to treatment.

Lymphoid Tissues:

Lymphocyte depletion was the main finding seen in the lymphoid tissues. It ranged in severity from minimal to severe and affected all compartments of the lymph nodes. Animals with severe depletion had virtually no lymphocytes in the node. Females were more severely affected than males and in the lymphoid tissues draining the gut (mesenteric lymph nodes and Peyer’s patches) the affect was dosage-related and occurred at all dosage levels. In the more distant lymphoid tissue of the mandibular lymph node and spleen the affect was seen at the highest dosage level only. Depletion of lymphocytes in the thymus of females was also seen at all dosage levels and in two control animals. It is probable that, in this tissue, the test-item related change was compounded by stress. In the mandibular and mesenteric lymph nodes intrasinusal pigmented macrophages were observed which might have resulted from ingestion of test item or metabolite. In the mesenteric lymph node the depletion of lymphocytes was accompanied by minimal or mild fibrosis.

- Mesenteric Lymph Node: In animals of both sexes from all the treated groups there was minimal to severe depletion of lymphocytes from the mesenteric lymph nodes. This finding was dosage-related in severity and affected all compartments of the node. The finding was accompanied by minimal or mild fibrosis and the presence of minimal to moderate levels of intrasinusal pigmented (yellow-brown) macrophages. The pigment may represent ingested test item or metabolite.

- Peyer’s Patches: Dosage-related minimal to marked lymphocyte depletion was also present in Peyer’s patches. Probably as a result of the reduction in size brought about by depletion, the number of patches presented for microscopy from females given 150 or 450 mg/kg/day was low; 3/5 and 1/6, respectively.

- Mandibular Lymph Node: In this node minimal to marked lymphocyte depletion was confined to 4/5 males and all the females given the highest dosage. Minimal levels of intrasinusal pigmented macrophages were observed in 1/5 males and 5/6 females given this dosage. There was no fibrosis.

- Spleen: Minimal or moderate lymphocyte depletion was confined to 2/5 males and 2/6 females given 450 mg/kg/day.

- Thymus: Minimal or mild lymphocyte depletion was observed in 1/5 males given 150 mg/kg/day and 5/5 males given 450 mg/kg/day but was not seen in controls or males given 50 mg/kg/day. In females there was a dosage-related greater incidence and severity of lymphocyte depletion in all the treated groups when compared to controls. Two control females had minimal or mild lymphocyte depletion and it is probable that, in this tissue, a part of the depletion could be attributed to stress. This finding correlated with the reduction in size of the tissue seen at necropsy in a few animals given 450 mg/kg/day and the statistically significant decrease in mean organ weights seen in females given 150 mg/kg/day and both sexes given 450 mg/kg/day.

There were no other treatment-related findings. All of the other histopathological findings were considered to have occurred spontaneously or post mortem.

Testes - Detailed Examination Including Sperm Staging

In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.

4. REPRODUCTION AND BREEDING

MATING PERFORMANCE AND FERTILITY

The mean pre-coital time of the test item-treated females was similar to that of the control females.

The percentage mating, fertility index and conception rate of the test item-treated females compared favorably with those of the control females.

As a result of the lower number of females with living pups, the gestation index of the females treated with 450 mg/kg/day was lower than that of the control females and other test item-treated females. This was considered to be a test item-related effect.

DURATION OF GESTATION

The duration of gestation was unaffected at all dose levels.

CORPORA LUTEA COUNT

The mean number of corpora lutea was marginally but not significantly lower in females treated with 450 mg/kg/day, and was considered to be within the range of typical biological variation. All other groups compared favorably.

IMPLANTATION RATE AND POST IMPLANTATION LOSS

The mean implantation rates of females treated with 150 and 450 mg/kg/day were slightly but not significantly lower when compared to the control females. Minor differences in the mean post implantation loss of the test item-treated females were unrelated to dose and therefore considered to be incidental.

LITTER SIZE AT FIRST LITTER CHECK

At 450 mg/kg/day, the mean litter size was slightly lower than that of the control group; all other test item-treated groups were considered to be unaffected.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

< 50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

< 50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day, slighly lower litter sizes and increased total and mean postnatal loss. Lower number of living pups on day 4 post partum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day reduced mean body weights in pups of both genders.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

At 450 mg/kg/day, the total postnatal loss was significantly elevated (p<0.01), as was the number of litters affected (p<0.05) when compared with the controls. The mean postnatal loss was clearly, but not significantly, elevated when compared to the controls. The mean number of living pups on day 4 post partum was accordingly lower and was considered to be a test item-related effect.

At 150 mg/kg/day, marginally higher postnatal loss was noted when compared with the controls, but these values were considered to be within the range of typical biological variation.

The mean postnatal loss at 50 mg/kg/day was identical to that of the controls.

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

There were no abnormal findings noted during the first litter check in any pup at any dose level.

During lactation, findings were noted in three litters of dams treated with 450 mg/kg/day. These findings included absence of milk in the stomach and reduced body temperature (13 pups and 10 pups, respectively) in litter no. 88, and absence of milk in the stomach (three pups) in litter no. 95.

PUP SEX RATIOS

There were no effects upon the sex ratios at any dose level.

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

At 450 mg/kg/day, the mean body weights of male pups and female pups were significantly reduced (p<0.01) on day 1 post partum when compared with the controls. On day 4 post partum, the values remained clearly lower than those of the control pups, although without statistical significance.

The mean pup weights at 50 mg/kg/day and 150 mg/kg/day were unaffected.

MACROSCOPICAL FINDINGS IN PUPS

There were no test item-related macroscopical findings in either male or female pups.

Effect levels (F1)

1

Dose descriptor:

NOEL

Remarks:

for reproduction / developmental toxicity

Generation:

F1

Effect level:

150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Test item-related effects at 450 mg/kg/day (reduced mean body weights of male and female pups, slightly lower mean litter sizes, lower gestation index, increased total and mean postnatal loss, lower mean number of living pups on day 4 post partum)

2

Dose descriptor:

NOAEL

Remarks:

for reproduction / developmental toxicity

Generation:

F1

Effect level:

150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Test item-related effects at 450 mg/kg/day (reduced mean body weights of male and female pups, slightly lower mean litter sizes, lower gestation index, increased total and mean postnatal loss, lower mean number of living pups on day 4 post partum)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Allocation and Dose Levels mg/kg bw/day	Group 1 Control 0	Group 2 50	Group 3 150	Group 4 450
Female numbers	49-60	61-72	73-84	85-96
Number of females paired	12	12	12	12
Number of females mated	12	12	12	12
Number of females not pregnant	1	1	1	1
Number of premature decedent females	1	1	1	3
Number of females which reared their pups until day 4 post partum	11	11	11	9

 

 

Applicant's summary and conclusion

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Alkenes hydroformylated, sulfosuccinates, sodium salt to rats over approximately 28 days. Alkenes hydroformylated, sulfosuccinates, sodium salt was administered in bidistilled water as vehicle at dosages of 50, 150, and 450 mg/kg body weight/day, and controls received the vehicle only. Alkenes hydroformylated, sulfosuccinates, sodium salt was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

EVALUATION OF THE SYSTEMIC TOXICITY PARAMETERS

No test item-related deaths at any dose level (although lymphocyte depletion contributed to the death of one female treated with 450 mg/kg/day), no test item-related daily or weekly clinical signs in males during the pre-pairing, pairing and after pairing periods, no clinical signs in females during the pre-pairing, pairing and gestation periods, no findings evident during the functional observational battery in females during the lactation period, no effects on grip strength or locomotor activity in males or females.

TEST ITEM-RELATED EFFECTS UPON SYSTEMIC TOXICITY PARAMETERS

At 450 mg/kg/day, lower mean daily food consumption values were noted in males and females, lower mean body weight were noted in males during the pre-pairing, pairing and after pairing periods and in females during the gestation and lactation periods, increased white cell counts at in males and females, increased activities of liver enzymes in males and females (considered to be the results of metabolic adaptation rather than indications of systemic toxicity), slight reductions in hepatic lipid metabolism, higher mean liver-to-body weight and liver-to-brain weight ratios were noted higher in males and females when compared with the controls, and reduced mean absolute thymus weights, mean thymus-to-body weight and mean thymus-to-body weight ratios in males and females. Test item-related microscopical changes included lymphocyte depletion in the mandibular lymph node and spleen, a greater myeloid/erythroid ratio of the femoral bone marrow in both sexes and a greater amount of fat in the bone marrow of male and females; minimal or moderate diffuse cortical tubular fat vacuolation in the kidneys of most females. The livers showed minimal or mild centrilobular fat vacuolation in most males and moderate or marked centrilobular or diffuse fat vacuolation in all females. Minimal centrilobular hypertrophy of hepatocytes was noted in most males and one female.

At 150 mg/kg/day, a dose-related reduction of mean daily food consumption was noted in females, and increased white cell counts in males. Test item-related microscopical changes included mild periportal fat vacuolation in the liver of some females.

At 50 mg/kg/day, there were no test item-related findings of in-life parameters, but test item-related microscopical changes included greater amounts of fat in the bone marrow of males and females.

Test item-related changes noted at all dose levels included minimal to severe lymphocyte depletion of the lymphoid tissues affecting all compartments of the lymph nodes and females more severely than males. In the lymphoid tissues draining the gut (mesenteric lymph nodes and Peyer’s patches) the effect was dose-related and occurred at all dosage levels. Depletion of lymphocytes in the thymus of females was also seen at all dosage levels as well as in two control animals. Lymphocyte depletion was considered to be a test-item related change compounded by stress. In the mandibular and mesenteric lymph nodes, intrasinusal pigmented macrophages were observed which might have resulted from ingestion of test item or metabolite. In the mesenteric lymph node, lymphocyte depletion was accompanied by minimal or mild fibrosis.

EVALUATION OF THE REPRODUCTION PARAMETERS AND OFFSPRING

There were no effects upon pre-coital time, percentage mating, fertility index and conception rate, mean number of corpora lutea, post implantation loss, duration of gestation, sex ratios of pups or macroscopical findings of parent animals. There were no findings at first litter check and no macroscopical findings in any pup.

TEST ITEM-RELATED EFFECTS UPON REPRODUCTION PARAMETERS AND OFFSPRING

At 450 mg/kg/day, there were reduced mean body weights of male and female pups, slightly lower mean litter sizes, lower gestation index, increased total and mean postnatal loss, lower mean number of living pups on day 4 post partum.

All other reproduction parameters and offspring data were considered to be unaffected by treatment with the test item.


The NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females was considered to be below 50 mg/kg/day.

The NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered to be 150 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Alkenes hydroformylated, sulfosuccinates, sodium salt on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Alkenes hydroformylated, sulfosuccinates, sodium salt was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

 

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

Group 2: 50 mg/kg body weight/day

Group 3: 150 mg/kg body weight/day

Group 4: 450 mg/kg body weight/day

 

Control animals were dosed with the vehicle (bidistilled water) alone.

 

The following results were obtained:

 

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All males survived until their respective scheduled necropsies.

 

After not delivering as scheduled on day 21 of gestation, one female each was killed at 50 mg/kg/day and 150 mg/kg/day on their respective day 25 of gestation. Two females at 450 mg/kg/day were killed on their respective day 25 of gestation. One control female was killed on day 25 of gestation.

 

One female at 450 mg/kg/day was killed for ethical reasons on day 23 of gestation. Although the exact cause of death could not be ascertained, the microscopical evaluation of the tissues suggested complications from severe lymphoid depletion.

 

FUNCTIONAL OBSERVATIONAL BATTERY AND LOCOMOTOR ACTIVITY IN PARENTAL ANIMALS

 

No test item-related effects were noted during functional observational battery or locomotor activity in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

The mean daily food consumption of males treated with 450 mg/kg/day was markedly lower than that of the control males. These differences were considered to be related to the treatment with the test item.

Females treated with 450 mg/kg/day had test item-related reductions of mean daily food consumption during all intervals. This difference was also noted in females treated with 150 mg/kg/day with slightly less (i.e. dose-related) severity. Females at 50 mg/kg/day were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

Males treated with 450 mg/kg/day had lower mean body weight from days 3 - 14 of the pre-pairing period. This difference continued throughout days 1 - 23 of the pairing period and days 1 - 9 of the after pairing period. These differences were considered to be test item-related. Marginally to slightly lower body weights were also noted in males treated with 50 mg/kg/day and 150 mg/kg/day when compared with the controls.

 

No differences in mean body weights were noted in test item-treated females during the pre-pairing period or pairing period. At 450 mg/kg/day, lower body weights were noted from day 4 of gestation onwards. Lower mean body weights were also recorded during days 1 - 4 of the lactation period. The mean body weight of the females treated with 50 mg/kg/day or 150 mg/kg/day were considered to be unaffected by the treatment with the test item.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 

Test item-related changes in hematology parameters included increased white cell counts at 150 mg/kg/day in males and 450 mg/kg/day in males and females.

 

Changes in clinical biochemistry parameters considered to be test item-related included increased activities liver enzymes (aspartate aminotransferase in males (p<0.05), alanine aminotransferase in males (p<0.01) and females (p<0.05) and alkaline phosphatase (p<0.01) in males and females). However, these changes were considered to be the results of metabolic adaptation rather than indications of systemic toxicity.

 

REPRODUCTION AND BREEDING DATA OF PARENTAL ANIMALS

 

No effect on mating performance or fertility was observed at any dose level. The duration of gestation was similar at all dose levels.

 

The number of corpora lutea and mean implantation rate at 450 mg/kg/day were marginally lower than that of the controls. The mean implantation rate of the females treated with 150 mg/ kg/day was also slightly reduced. No conclusive differences in the post implantation losses were noted.

 

At 450 mg/kg/day, the mean litter size was slightly lower than that of the control group; all other test item-treated groups were considered to be unaffected.

 

There were no test item-related differences in the sex ratios at any dose level.

 

At 450 mg/kg/day, the total postnatal loss was elevated, as was the number of litters affected when compared with the controls. The mean postnatal loss was elevated when compared to the controls. The mean number of living pups on day 4 post partum was accordingly lower and was considered to be a test item-related effect. At 150 mg/kg/day, a marginally higher postnatal loss was noted was considered to be within the range of typical biological variation. The mean postnatal loss at 50 mg/kg/day was identical to that of the controls.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

At 450 mg/kg/day, the mean liver-to-body weight ratio and the mean liver-to-brain weight were higher in males and considered to be test item-related but likely to be due to metabolic activation. The reduction of mean absolute and relative thymus weights in males and females were also considered to be related to the test item and considered to the consequence of chemical stress. All other organ weight and ratios were generally similar to the control males.

 

Similarly, higher mean liver-to-body weight ratios and higher mean liver-to-body weight ratios were noted in the females at this dose level when compared with the controls. The mean absolute liver weight was also elevated in these females and, as in the males, considered to be test item-related but likely due to metabolic activation.

 

At 150 mg/kg/day, the mean absolute thymus weight, the mean thymus-to-body weight ratio and the mean thymus-to-brain weight ratio were significantly reduced (p<0.05) in females when compared to the controls. These differences were also considered to be related to the test item and the result of chemical stress.

 

All other organ weight and ratios were generally similar to the control females.

 

MACROSCOPICAL FINDINGS IN PARENTAL ANIMALS

 

There were no test item-related macroscopical findings at any dose level in males or females.

 

HISTOPATHOLOGICAL EXAMINATION OF PARENTAL ANIMALS

 

The following treatment-related microscopic changes were noted:

 

- In comparison to controls, a greater myeloid/erythroid ratio of the femoral bone marrow in both sexes given 450 mg/kg/day and a greater amount of fat in the bone marrow of all the treated male groups and females given 50 or 450 mg/kg/day.

- Minimal or moderate diffuse cortical tubular fat vacuolation in the kidneys of 4/6 females given 450 mg/kg/day.

- In the liver mild periportal fat vacuolation in 2/5 females given 150 mg/kg/day, minimal or mild centrilobular fat vacuolation in 4/5 males given 450 mg/kg/day and moderate or marked centrilobular or diffuse fat vacuolation in all the females given 450 mg/kg/day. Minimal centrilobular hypertrophy of hepatocytes in 3/5 males and 1/6 females given 450 mg/kg/day.

- Lymphocyte depletion of the lymphoid tissues, ranging in severity from minimal to severe, affecting all compartments of the lymph nodes and females more severely than males. In the lymphoid tissues draining the gut (mesenteric lymph nodes and Peyer’s patches) the affect was dosage-related and occurred at all dosage levels. In the more distant lymphoid tissue of the mandibular lymph node and spleen the affect was seen at the highest dosage level only. Depletion of lymphocytes in the thymus of females was also seen at all dosage levels and in two control animals. It is probable that, in this tissue, the test-item related change was compounded by stress. In the mandibular and mesenteric lymph nodes intrasinusal pigmented macrophages were observed which might have resulted from ingestion of test item or metabolite. In the mesenteric lymph node the depletion of lymphocytes was accompanied by minimal or mild fibrosis.

- Severe lymphocyte depletion was considered a contributory cause of the moribund condition of the only premature decedent, Animal No 85, a female given 450 mg/kg/day.

 

All of the other histopathological findings encountered were considered to have arisen spontaneously or at post mortem.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

There were no abnormal findings noted during the first litter check in any pup at any dose level.

 

During lactation, findings were noted in three litters of dams treated with 450 mg/kg/day. In one litter, these findings were considered likely to be the result of maternal neglect.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

At 450 mg/kg/day, the mean body weights of male pups and female pups were reduced on days 1 - 4 post partum when compared with the controls. The mean pup weights at 50 mg/kg/day and 150 mg/kg/day were unaffected.

 

MACROSCOPICAL FINDINGS IN PUPS

 

There were no test item-related macroscopical findings in either male or female pups.

 

CONCLUSION

 

The NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level)for general toxicity in males and females was considered to be below 50 mg/kg/day.

 

The NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered to be 150 mg/kg/day.