Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2018 to 13 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD testing guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Limit test:

no

Justification for study design:

This study was designed to use the fewest number of animals possible, consistent with the objective of the study, the scientific needs of the Sponsor, contemporary scientific standards, and in consideration of applicable regulatory requirements.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

EC Number:

946-383-6

Molecular formula:

C17H24N2O3

IUPAC Name:

Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Test material form:

liquid: viscous

Details on test material:

Name: Trixene AS
Batch no.: OP544-153/1801239973
Appearance: clear, light yellow-green, viscous liquid
Composition: Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
EINECS-No.: 946-383-6
Molecular formula: C17-H24-N2-O3
Purity: "multi-constituent substance made up of a mixture of isomers
Homogeneity: Homogeneous
Vapour pressure: not stated
Expiry date: 01. Sep. 2019
Storage Room: Temperature (20 ± 5°C)

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was selected because it is a standard species for use in toxicology studies and is also being used as a rodent species per the current OECD 422 testing guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Species and Strain: Sprague Dawley Rat
Supplier: Charles River Breeding Labs; Raleigh, NC
Method of Identification: Ear tag/cage card
Number of Animals Received: Males: 52; Females: 52
Number Used on Study: Males: 49; Females: 48
Age at First Dose: Males: 11-12 weeks; Females: 11-12 weeks
Weight Range at First Dose: Males: 381.4-467.3g; Females: 245.7-294.7g
Disposition of Extra Animals: Euthanized

Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Husbandry Information
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum, unless otherwise noted.
Water: Filtered water was provided ad libitum via an automatic watering system, supplemented with water bottles as needed
Bedding: Certified Sani Chips® hardwood bedding
Housing: Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were individually housed prior to cohabitation and after confirmation of mating. During cohabitation, one male and one female from the same group were housed together. Dams and pups were group housed.
Temperature Range: 20 to 26°C
Humidity Range: 30 to 70%
Light Cycle: 12-hour light/12-hour dark, interrupted as necessary for study-related events
Air Changes: Minimum of 10 air changes per hour

The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The water is routinely analyzed for contaminants and specific microbes. The bedding was analyzed by the manufacturer for acceptable levels of heavy metals, aflatoxins, bacteria, yeasts, molds, and organophosphates prior to certification. No contaminants were known to be present in the feed, water, or bedding at levels that might have interfered with achieving the objectives of the study.
Actual temperature and relative humidity in the animal room were monitored continuously by a computerized system. All environmental parameters were maintained within the protocol requirements.
In addition to standard husbandry procedures, environmental enrichment was provided according to company SOPs.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control substance, peanut oil, was used as received; no formulations were necessary.
Formulations were prepared four times over the course of the study and assigned a 28-day shelf life. Group 4 (500 mg/mL) formulations were prepared by adding the appropriate amount of test substance to the required amount of peanut oil, then vortexed to achieve uniformity and inverted approximately 30 times. Formulations for Groups 3 (150 mg/mL) and 2 (50 mg/mL) were prepared by serially diluting the Group 4 formulation with the required amount of peanut oil until the required final volume was reached. Formulations were vortexed to achieve uniformity and inverted approximately 30 times. All formulations were stored refrigerated (5±3°C) until used for dosing.

Details on mating procedure:

After at least 14 days of vaginal lavage and dosing, one female from each group was cohabited with one male from the corresponding group (1:1). The females were tested daily by vaginal lavage beginning on the day following cohabitation. The animals were separated when mating was confirmed or after 14 days and observed as indicated above. Day 0 of pregnancy (gestational day [GD] 0) was defined as the day on which mating evidence was confirmed (vaginal plug or presence of sperm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity analysis of the dose formulations was performed under Smithers Avanza Study No. 2386-14350 as part of the method validation.
Samples were analyzed for concentration verification by Smithers Avanza using a validated method.

Duration of treatment / exposure:

F0 males were dosed for 35 days. Animal 27455 (4m) that replaced Animal 27116 (4m) on SD 8 received 27 doses. The F0 females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. The first day of dosing was designated as SD 1 for each animal.

Frequency of treatment:

Formulations were placed at room temperature prior to dosing. The animals were dosed daily via oral gavage at a volume of 2 mL/kg. Dosing volumes were based on the animals’ most recent body weights.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Peanut oil

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 animals per dose group (12 male/12 female)

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were initially accepted into the randomization pool based upon prestudy body weights, physical examinations, and vaginal cytology. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Males and females were randomized separately. Following randomization each study animal was assigned a unique number.

Positive control:

Not required

Examinations

Parental animals: Observations and examinations:

Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns.
For the FOB, animals were transported to the testing room and acclimated to white noise for at least 10 minutes prior to testing.

Clinical Pathology
Blood samples were collected from five selected animals/sex/group. Animals were fasted overnight (with water available) prior to sample collection on SD 35 (males), and PND 13 (parturient females).

See "Any other information" for further details.

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Not specified

Litter observations:

See "Any other information" for details.

Postmortem examinations (parental animals):

Termination
Moribund animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.
Nonparturient females not delivering by SD 57 were euthanized by carbon dioxide asphyxiation and exsanguination and necropsied (uterus and ovaries were examined).
On SD 36 for F0 males and F0 females, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.

Necropsy
Animals were fasted overnight (with water available) prior to necropsy. Animals were necropsied as soon as possible after the time of death or discovery. Animals were necropsied, bone marrow smears were prepared (five F0 animals/sex/group at scheduled necropsy), required organs were weighed, and protocol-specified tissues were collected and preserved. No organ weights were collected from animals found dead or euthanized prior to scheduled termination and no bone marrow smears were prepared from animals found dead.
Gross necropsy of F0 animals included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. All reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix; sex appropriate) were collected and weighed as soon as possible after dissection; paired organs were weighed together. For five selected animals/sex/group at scheduled necropsy, additional protocol-specified tissues were collected and weighed as soon as possible after dissection; paired organs were weighed together. The following organs were collected/weighed as follows: thyroid with parathyroids, seminal vesicle with coagulating gland, and uterus with cervix.
Tissues were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Tissues from animals found dead were preserved in 10% NBF only. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalization.

Histopathology
Preserved tissues from the five females and males selected in the control and high dose study groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically by a board-certified veterinary pathologist. The testis was also stained with periodic acid-Schiff stain (PAS-H) and examined microscopically by a board-certified veterinary pathologist with special emphasis on stages of spermatogenesis and interstitial testicular cell structure.
The spleen in both sexes and the testes and cecum in males were identified as potential target tissues by the pathologist, therefore these tissues from selected animals in Groups 2 and 3 were processed to slides (see above) and microscopically examined by the pathologist.

Postmortem examinations (offspring):

Termination
Moribund animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.
On PND 13 or 14 for F1 pups, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.


Necropsy
All pups (except those assigned to blood collection) were examined externally for gross abnormalities. For 2 pups/litter (1 male and 1 female, if possible), the thyroid gland was preserved in NBF and weighed after fixation.

Statistics:

Group mean, standard deviation, and sample size are reported for all quantitative measurement data.
Quantitative data (body weights, body weight changes, food consumption, clinical pathology, and organ weight data) from the treated groups were compared statistically to the data of the control group using one-way Analysis of Variance (ANOVA) techniques; sexes were analyzed separately. Prior to the ANOVA analysis, untransformed data were tested to determine if the data are normally distributed and have homogeneous variances among all groups. A Shapiro-Wilk test was used to test for normality, followed by the Levene’s test to test the hypothesis of homogeneity of variances. If either the normality or homogeneity test failed (as indicated by a Shapiro-Wilk or Levene’s test p-value ≤ 0.01), the data were transformed using log-transformed values and both the Shapiro-Wilk and Levene’s tests were repeated with the log-transformed values. If the results from either the Shapiro-Wilk or Levene’s test on the log-transformed data failed, the analysis of the data continued using rank-transformed data using Kruskal-Wallis ANOVA. If both the Shapiro-Wilk or Levene’s test were not statistically significant, the ANOVA was performed on the untransformed or log-transformed data, respectively. The Dunnett’s t-test was used to determine which groups (if any) differed from the control group. Group comparisons were evaluated at the 0.05 (two-tailed) probability level. An arcsine square root transformation was used for some proportion/percentage data which do not meet the assumptions of parametric statistical tests in the attempt to normalize the data; this data was transformed prior to the ANOVA analysis. The term “significant” is used throughout the text of the report to indicate statistical significance at p≤0.05.
Dunnett’s t-test was used to determine if differences exist between the control and treated group(s). Group comparisons were evaluated at the 5% two-tailed probability level.

Reproductive indices:

Statistical analysis on reproductivel parameters (as deemed appropriate) was conducted using SigmaStat™ Statistical Software, Version 1 (Jandel Scientific, San Rafael, CA). Data were analyzed using the Kolmogorov-Smirnov test for normality, the Levene Median test for equal variance, and by one-way Analysis of Variance (ANOVA). If either the normality or equal variance test failed, then the analysis continued using the non-parametric Kruskal-Wallis ANOVA on rank-transformed data. For parametric data, if the ANOVA indicates statistical significance among experimental groups then the Dunnett’s t-test was used to delineate which groups (if any) differs from the control. For non-parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunn’s test was used to delineate which groups (if any) differs from the control. The probability value of less than 0.05 (two tailed) was used as the critical level of significance for all tests.

Offspring viability indices:

Statistical analysis on developmental parameters (as deemed appropriate) was conducted using SigmaStat™ Statistical Software, Version 1 (Jandel Scientific, San Rafael, CA). Data were analyzed using the Kolmogorov-Smirnov test for normality, the Levene Median test for equal variance, and by one-way Analysis of Variance (ANOVA). If either the normality or equal variance test failed, then the analysis continued using the non-parametric Kruskal-Wallis ANOVA on rank-transformed data. For parametric data, if the ANOVA indicates statistical significance among experimental groups then the Dunnett’s t-test was used to delineate which groups (if any) differs from the control. For non-parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunn’s test was used to delineate which groups (if any) differs from the control. The probability value of less than 0.05 (two tailed) was used as the critical level of significance for all tests.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS did not affect physical examinations or cageside observations. Observations noted during physical examinations were considered incidental and not related to test substance due to lack of an associated dose response. In addition to the observations listed above, the following abnormalities were observed in the males at various study intervals: alopecia of the forepaws (1m 27053) or hindlimbs/forepaw digits (4m-27117), hunched posture (4m-27117), and abrasion of the neck (3m 27097; 300 mg/kg/day).
The following observations were observed in females at various study intervals: alopecia on the forepaw digits (2f-27090; 3f-27107) or forelimbs (2f-27083) abrasion/alopecia of the abdomen (1f-27061), and alopecia of the chest (3f-27114). No abnormalities were observed during cageside observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment with Trixene AS did not affect mortality. There was one male and three females that died during the study, but all deaths were unrelated to the test substance. Animal 27116 (4m; 1000 mg/kg/day) had a rough haircoat and was vocalizing continuously on SD 8. This animal also had body weight loss and low food consumption. The attending veterinarian examined the animal and treated with a diet gel recovery pack. The animal was euthanized as moribund on SD 8 and replaced with Animal 27455. The death was not considered test substance related due to the low incidence morbidity with observations (low body weight and food consumption) that were not observed in other animals. Additionally, all other males survived until the scheduled termination.
Females 27063 (1f; 0 mg/kg/day) and 27064 (1f, 0 mg/kg/day) were pale with a rough haircoat on GD 27, and Female 27064 was also severely languid on GD 23. Female 27063 (1f) had suspected delayed parturition and after examination by the attending veterinarian, both animals were euthanized as moribund on the respective gestational days. Animal 27131 (4f; 1000 mg/kg/day) was found dead on GD 23. Red discharge from the vagina was noted at necropsy in all three animals, however the cause of death was undetermined based on microscopic evaluation of limited tissues by the pathologist. All of the unscheduled deaths were considered unrelated to the test substance and they were suspected to be a complication with pregnancy, as they occurred during late gestation and occurred in the control group and at low incidence in Group 4 females. All other females survived until the scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment with Trixene AS impacted body weights and body weight changes of males. Mean body weights of Groups 2 (100 mg/kg/day) and 4 (1000 mg/kg/day) were significantly lower than the control from SD 15 to 35. Mean body weights of Group 3 (300 mg/kg/day) were significantly lower on SD 29 and 35. Mean body weight changes of treated groups were significantly lower than the control at various study intervals, and all groups had significantly lower absolute body weight change from SD 1 to 35. Although, males in all groups had a significantly lower body weight change, all groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.
Treatment with Trixene AS impacted body weights of females. Mean body weight of females were significantly lower than the control at the following intervals: SD 15 2f, GD 0–2f, 3f; GD 7–2f, 3f; GD 14–all; GD 20 – 3f; PND 0–all; PND 4–all, PND 7–4f. Treated groups often had significantly lower mean body weight change compared to the controls. Mean absolute body weight change from SD 1 to 15 was significantly lower in all treated groups compared to the control. Mean body weight change from PND 4 to 7 was significantly higher in all treated groups. Although, females in all groups had variable body weight changes compared to the controls at varying intervals, all groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment with Trixene AS impacted food consumption. Mean total food consumption (SD 1-15) of treated groups was lower compared to the control, with significant differences for Groups 2 (100 mg/kg/day) and males in Group 4 (1000 mg/kg/day). Females in Groups 3 and 4 from SD 1-8 and females in Group 2 from SD 8- 15 had significantly lower mean food consumption compared to the control. Mean food consumption of females in Group 4 was also significantly lower than the control from PND 7-12. Although, there were variable effects on food consumption and body weight changes throughout the study, all animals gained weight. Thus, the effects on food consumption were not considered adverse.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males (Study Day 36)
Test substance-related decreases in measures of erythroid mass (red blood cell count, hemoglobin concentration and hematocrit percent) with concurrent increases in reticulocyte counts occurred in males at all doses. In males administered 1000 mg/kg/day, decreases in erythroid parameters ranged from 12 14% and reticulocyte counts were 47% increased. In males administered 300 mg/kg/day, decreases in erythroid parameters ranged from 9-11% and reticulocyte counts were 14% increased. In males administered 100 mg/kg/day, decreases in erythroid parameters ranged from 6-7% and reticulocyte counts were 12% increased. No correlating changes in bone marrow morphology were observed microscopically; however, minimally increased erythropoiesis was observed in the spleen of 4 of 5 males administered 1000 mg/kg/day. Decreases in measures of erythroid mass with concurrent increases in reticulocyte count were suggestive of blood loss and/or erythrocyte injury with a normal regenerative response.
Test substance-related changes in white blood cell counts consisted of a mild (66%) increase in lymphocyte counts at a dose level of 1000 mg/kg/day. Although increases in lymphocyte counts were observed at dose levels of 100 or 300 mg/kg/day, the magnitudes of the increases were within normal variation and could not be attributed definitively to the test substance. Decreases in eosinophil counts at all doses were considered equivocal due to the lack of a dose-response; however, would be consistent with stress in a rodent.
No test substance-related changes in coagulation parameters were observed.

Females (Post Natal Day 13)
Test substance-related decreases in erythroid parameters (red blood cell count, hemoglobin concentration and hematocrit percent) with concurrent increases in reticulocyte counts occurred in females at ≥300 mg/kg/day. In females administered 1000 mg/kg/day, decreases in erythroid parameters ranged from 7-9% and reticulocyte counts were 45% increased. In females administered 300 mg/kg/day, decreases in erythroid parameters ranged from 5-6% and reticulocyte counts were 29% increased. No correlating changes in bone marrow morphology were observed microscopically; however, mildly increased erythropoiesis was observed in the spleen of one female administered 1000 mg/kg/day. Decreases in measures of erythroid mass with concurrent increases in reticulocyte count were suggestive of blood loss and/or erythrocyte injury with a normal regenerative response.
No test substance-related changes in coagulation parameters were observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males (Study Day 36)
Test substance-related changes in clinical chemistry consisted of nearly 2-fold increases in alanine transaminase activities in males administered 1000 mg/kg/day that were consistent with minimal hepatocellular injury; however, no correlating microscopic observations occurred in the liver, therefore this was not considered an adverse effect.
Females (Post Natal Day 13)
Test substance-related changes in clinical chemistry consisted of 2-fold increases in alanine transaminase in females administered 1000 mg/kg/day that were consistent with minimal hepatocellular injury and approximately 2-fold increases in alkaline phosphatase activities in females administered ≥300 mg/kg/day. No correlating microscopic observations occurred in the liver.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on the functional observation battery. A score of zero was considered normal. During handheld observations, Animal 27068 (2m) had no reactivity to handling (score of 2) and Animal 27137 (4f) exhibited salivation (score of 1). Animal 27121 (4m) exhibited low activity during the open field observations (score of 1). These observations were not considered test substance-related due to the low incidence. Means of quantitative measures (grip strength, hindlimb splay, grooms, fecal boli, urine pools, and rears) were comparable across groups.

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):

Males (Study Day 36)
Test substance-related changes occurred in the spleen and the testes. In the spleen, minimally increased erythropoiesis was observed in the red pulp of 4 of 5 males administered 1000 mg/kg/day and correlated with increased spleen weight parameters. In the testes of 3 of 5 males administered 1000 mg/kg/day, spermatid retention was characterized by spermatozoa at the luminal surface of some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules. In affected tubules there were also a few spermatozoa basally located within Sertoli cell cytoplasm oriented parallel to the basement membrane.
A finding of uncertain relationship to test substance administration consisted of minimal to mild mucosal regeneration with minimal to mild lymphocytic infiltration of the lamina propria in the cecum of males at all doses. Two of 5 males administered 100 mg/kg/day, 4 of 5 males administered 300 mg/kg/day and 3 of 5 males administered 1000 mg/kg/day were affected. These changes were characterized by the expansion of the lamina propria by a cell population of primarily lymphocytes and macrophages with regeneration of the overlying mucosa characterized by increased basophilia of the epithelium. Given the lack of similar changes in females, the lack of correlating clinical observations, and the lack of a clear dose response, the relationship of this change to the test substance was uncertain.

Females (Post Natal Day 13)
Test substance-related changes occurred in the spleen at doses ≥300 mg/kg/day. In the spleen, mildly increased erythropoiesis was observed in the red pulp of 1 of 5 females administered 1000 mg/kg/day and correlated with increased spleen weight parameters in that individual (27135). Additionally, 3 of 5 females administered 300 mg/kg/day had minimally increased erythropoiesis in the spleen.
Notably, one female administered 300 mg/kg/day had mildly increased hematopoiesis in the spleen. This was not considered test substance-related, as this individual also had a cutaneous ulcer that was considered the likely cause for the increased hematopoiesis in the spleen.
All other microscopic alterations seen in the current study were typical of sporadic, naturally occurring background changes commonly observed in rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hormone Levels
Treatment with Trixene AS affected thyroxine (T4) levels of males. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. Although there was a test-substance related decrease in T4 levels, there was no associated effects on thyroid organ weight and histopathological effects, therefore this was not considered an adverse effect.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on vaginal cytology. All females were cycling prior to dosing. 91.7% of females in Groups 1 (0 mg/kg/day) and 3 (300 mg/kg/day), and 100% of females in Groups 2 (100 mg/kg/day) and 4 (1000 mg/kg/day) were cycling regularly. The following percent of females were cycling regularly during the first 14 days of dosing: Group 1 – 81.8%; Group 2 – 100%, Group 3 – 75.0%; Group 4 – 81.8%. Any differences in estrus cycling were not considered test substance related due lack of an associated dose response. The mean number of cycles during the 14-day cytology period ranged from 2.45 (Groups 1 and 4) to 2.58 (Group 2). Mean cycle length ranged from 4.10 days (Group 2) to 4.75 days (Group 1).

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.

Reproductive performance:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS did not impact pregnancy rate or affect mating performance. Mating success was 91.7% (Groups 2; 100 mg/kg/day and 3; 300 mg/kg/day) and 100% (Groups 1; 0 mg/kg/day and 4; 1000 mg/kg/day). The percent of dams that littered were 83.3% (Group 1), 100% (Group 2), 91.7% (Group 3), and 83.3% (Group 4). Mean gestation periods were comparable, ranging from 22.1 days to 22.6 days.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

cauda epididymis

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment of the dam with Trixene AS did not affect clinical observations of the pups.
Incidental findings included alopecia, cold to touch, and pale which were all observed in control pups. One dam in Group 1 and three dams in Group 4 had pups with no visible milk in the stomach on PND 0 and/or 4.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Treatment with Trixene AS had no effect on litter size but impacted offspring survival. On PND 0, no statistically significant difference in litter size was noted, although there was a reduction in mean live litter size (13.2 pups/dam Group 1 (0 mg/kg/day) versus 9.7 pups/dam in Group 4 (1000 mg/kg/day) in a dose-dependent manner. For Group 4 (1000 mg/kg/day), the post-natal survival rate of the live offspring between birth and PND 0 (82.5%) and birth and PND 4 (51.8%) was decreased compared to the control (98.5% and 97.7%, respectively). There were three litters with total litter loss that resulted from premature deaths or cannibalization of the offspring by PND 4. The percentage of offspring surviving to PND 4 in Group 4 was 61.4% compared to the control offspring 99.3%. This was considered test substance-related due to the magnitude of the response. All offspring survived from PND 7 to 13.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS did not affect body weights of pups. Mean pup body weight increased over the course of the study. No statistically significant differences were observed between Group 1 and the other treatment groups on any post-natal day. No trend was noted within any post-natal day period in males, females or combined sexes.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on AGD. Mean AGD and AGD corrected for body weights were comparable across groups; no significant differences were observed.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on nipple retention; no males retained nipples when examined on PND 13

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on thyroid weights; no significant differences were noted.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Offspring toxicity consisted of a reduction in the number of live births and a decreased postnatal survival from PND 0 to 4. Observations on the morphology of the pups were performed from birth to PND 13 and there was no indication that the test substance induced specific malformations. Therefore, it is unknown what resulted in the low offspring survival rate but potentially could be due to maternal or paternal toxicity rather than an assumption of impaired development in utero. However, there is still a potential for impaired development that could not be determined in this study

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

This study determined the potential toxicity of Trixene AS when administered daily for at least 28 days via oral gavage to male and female Sprague Dawley rats and the potential reproductive and developmental toxicity.

Mortality was not effected by administration of the test substance. Two control females and one male and female administered 1000 mg/kg/day were found dead or euthanized in a moribund condition. The cause of death was undetermined based on gross observations and limited microscopic tissue evaluation. However, the deaths were not considered test substance-related because the deaths in the females were considered to be complications of parturition and because of the low incidence of deaths in males.

Test substance-related changes in body weight and food consumption were observed but were not considered adverse. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore the effect on body weight was not considered adverse.

Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered adverse. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed.
Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.

The only other test substance-related microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.

The test substance, Trixene AS, did induce developmental toxicity at doses of 1000 mg/kg/day. Offspring toxicity consisted of a reduction in the number of live births and a decreased postnatal survival from PND 0 to 4. Observations on the morphology of the pups were performed from birth to PND 13 and there was no indication that the test substance induced specific malformations. Therefore, it is unknown what resulted in the low offspring survival rate but potentially could be due to maternal or paternal toxicity rather than an assumption of impaired development in utero. However, there is still a potential for impaired development that could not be determined in this study.

In summary, Trixene AS induced repeat dose toxicity and reproductive and developmental toxicity in male and female rats. The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested. The developmental NOAEL was determined to be 300 mg/kg/day dose due to the decreased survival of the offspring at the high dose.

Executive summary:

Trixene AS: Combined Oral Gavage Repeated Dose Toxicity Study Including Reproduction/Developmental Toxicity Screening in Male and Female Sprague Dawley Rats

 

 

The purpose of this study was to determine the potential toxicity of Trixene AS when administered once daily for at least 28 days via oral gavage to male and female Sprague Dawley rats and to determine the potential reproductive and developmental toxicity.

 

Ninety-six (48/sex) Sprague Dawley rats were randomly assigned to four groups (12 animals/sex). Animals were administered control substance (Peanut Oil) or Trixene AS at 100, 300, or 1000 mg/kg once daily via oral gavage for at least 28 days. Females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. Animals were subjected to a full gross necropsy on Study Day (SD) 36 (F0 males), or PND 13 or 14 (parturient F0 females and pups). Females that did not litter were subjected to a full gross necropsy on SD 57.

 

Parameters evaluated during the study for the F0 generation included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, functional observation battery, vaginal cytology, clinical pathology (clinical chemistry, hematology, and coagulation), thyroid hormone (T4) analysis (males only), gross pathology findings, absolute and relative organ weights, and histopathology findings. Parameters evaluated during the study for the F1 generation included mortality (litter sex and size), clinical observations, body weights, ano-genital distance, nipple retention (males pups only), T4 analysis, and thyroid weights.

 

Treatment with Trixene AS at doses ≥300 mg/kg/day had no effect on the parameters listed above. Test substance-related changes in body weight and food consumption were observed but were not considered adverse at doses ≥100 mg/kg/day. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore, the effect on body weight and food consumption were not considered adverse.

 

Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered an adverse response. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed.

 

Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.

 

The only other test substance-related microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.

 

Trixene AS induced developmental toxicity at doses of 1000 mg/kg/day. Pup toxicity consisted of reduction in the number of live births and a decreased postnatal survival from PND 0 to 4. Observations on the morphology of the pups were performed from birth to PND 13 and there was no indication that the test substance induced specific malformations. Therefore, the cause of the low pup survival rate is unknown but potentially could be due to maternal or paternal toxicity rather than an assumption of impaired development in utero. However, there is still a potential for impaired in utero development that could not be determined in this study. 

 

In summary, Trixene AS induced repeat dose toxicity and reproductive and developmental toxicity in male and female rats at >300 mg/kg/day. The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested. The developmental NOAEL was determined to be 300 mg/kg/day dose due to the decreased survival of the offspring at the high dose.