(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-12-2017 to 30-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

yes elevated humidity for 5 day of study, no adverse impact on the result

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

EC Number:

272-056-6

EC Name:

(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

Cas Number:

68672-66-2

Molecular formula:

C32H33N3O5S

IUPAC Name:

(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetic acid

Test material form:

solid: particulate/powder

Details on test material:

white powder

Specific details on test material used for the study:

Identification: 1132 side chain
Appearance: White to off white powder
GSK Batch: G317115
Vendor Batch: 17TATT-S01005C
Purity/Composition: 89-100%
Test item storage: At room temperature
Stable under storage conditions until: 04 March 2021 (retest date)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Crl: WI(Han) were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 10 weeks old and weighed betw een 266 and 307 g and females were 13 weeks old and weighed between 214 and 250 g. A health inspection was performed before the initiation of dosing. Prior to start of the pretest period ( females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pretest period, reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by earmark and tattoo. Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet. The animals were allowed to acclimate to the Test Facility toxicology accommodation for 8 days prior to start of the pretest period (females) or
7 days before the commencement of dosing (males).
On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages ( Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase females were house in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding mate rial, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in whic h the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex. Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 29 to 59% (see deviation in Appendix 9). A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air chang
es per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms. Pellete d rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH,Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurement
s, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days (most females) or 60-63 days (two females of Group 3), i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before
scheduled necropsy. Females without offspring were treated for 40, 42 or 47 days. The first day of dosing was designated as Day 1.
Female nos. 55 (Group 2), 62 (Group 3) and 79 (Group 4), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmedby evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed in first instance for two control females (nos. 43 and
46). Evidence of mating was obtained by palpation (no. 46 only) and indirectly by delivery of a litter (both females). Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in these females. The mating date of these females was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In total, 16 samples were included in this study, distributed over 4 dose groups. Group 1 was the vehicle control group, Groups 2, 3 and 4 were dosed at 100, 300 and 1000 mg/kg bw/day at a dose volume of 5 mL/kg bw, respectively (test item concentrations 20, 60 and 200 mg/mL). The samples of the control Group 1 and Group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice.
Samples of treatment Groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. The mean recoveries of the procedural recovery samples fell within the criterion of 85-115%. It demonstrated that the analytical method was adequate for the determination of the test item in the test samples. In the Group 1 formulation, no test item was detected.
The determination of 1132 side chain in formulation samples was performed according to ABL analytical AWI 4305, entitled: “1132 side chain in formulations using LC-DAD by ABL.

The lower limit of quantification was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 Males/10 Females

Details on study design:

The animals were randomly allocated to treatment groups using a randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely id entified within the study by an ear punching system routinely used in these laboratories.

Examinations

Parental animals: Observations and examinations:

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F0-Generation
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – F0-Generation
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.These observations were conducted after dosing.

Body Weights – F0-Generation
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
In order to monitor the health status, animal no. 69 was also weighed on Day 1 post-coitum. A terminal weight was recorded on the day of scheduled necropsy.
Food Consumption – F0-Generation
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

Water Consumption – F0-Generation
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced for the test item groups as no effect was suspected.

Functional Tests – F0-Generation
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 9-12).These tests were performed after dosing, after completion of clinical observations.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis. From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolongedparturition or deficiencies in maternal care.

Litter observations:

Mortality/Moribundity Checks – F1-Generation
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F1-Generation
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables

Body Weights – F1-Generation
Live pups were weighed individually on PND 1, 4, 7 and 13.
Sex – F1-Generation
Sex was externally determined for all pups on PND 1 and 4.
Anogenital Distance – F1-Generation
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/Nipple Retention – F1-Generation
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Postmortem examinations (offspring):

To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.
On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female).

Blood was analysis for hemetolgogical paramaters as per the F0 generation.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values whereappropriate. All data were summarized in tabular form. Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ
Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA withappropriate covariates. Any
transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these a nalyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs of toxicity were noted during daily detailed clinical observations or during weekly arena observations.
Salivation was noted after dosing at all dose levels in up to all animals with a dose-related trend in frequency (particularly in males) and time of onset. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Any other clinical signs noted occurred incidentally and within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be test item-related. Female no. 69 (300 mg/kg) was euthanized for humane reasons on Day 1 of the post-coitum period (i.e. treatment Day 20; dosed for the last time on Day 19 of the treatment period) when she showed gasping, laboured respiration, rales, piloerection and ptosis. Her body weight gain during the pre-mating period was normal, but she lost some weight (2%) between post- coitum Days 0 and 1.Macroscopic findings consisted of reddish foci in the lung and thymus and a gelatinous thymus. Major microscopic findings consisted of acute erosion/ulceration in the trachea (marked) and bronchus/bronchial epithelium of the lung (slight) and the presence of necrotic luminal debris in the lung. Based on these microscopic findings and the respiratory difficulties, the moribundity of this female was most likely related to the gavage procedure and not test item-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gain of treated animals remained in the same range as
controls

Food efficiency:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food consumption before or after correction for
body weight.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in hematology parameters.
Isolated, statistically significant differences noted in females at 1000 mg/kg were regarded as unrelated to treatment due to the lack of a dose-related response and slightly low concurrent
control values (number of neutrophils) or considered to be of no toxicologically relevance due the slight magnitude of the difference from controls (number of platelets; values in treated rats remained within the historical control range

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in clinical chemistry parameters.
The statistically significantly higher mean sodium values in all male test item groups were considered to be unrelated to treatment as the differences from controls were marginal and occurred in absence of a dose-related response and all values remained within the historical control range. Other isolated statistically significant differences were regarded as unrelated to treatment due to the lack of a dose-related response (chloride in both sexes and cholesterol in females).
The slightly (about 20-25%) lower mean alanine aminotransferase (ALAT) activities noted in males at 300 and 1000 mg/kg were considered to be unrelated to treatment as the differences from controls were not statistically significant and mean values in treated males remained within the historical control range. In addition, a decrease in ALAT activities is not regarded as toxicologically relevant.
Thyroid hormone analyses:
Serum levels of T4 in F0 males were considered not to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered not to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength were regarded to be unaffected by treatment. The statistically significantly lower hind limb grip strength values noted in males at 300 and 1000 mg/kg were not attributed to treatment since the differences from control values (24 and 18%, respectively) showed no dose-related trend and grip strength values of treated males remained in the normal range

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology
encountered in rats of this age and strain. There was no test item-related alteration in the
prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment. Most females had regular cycles of four or five days during the pretest and premating period. Irregular cycles were noted during the pre-mating period in one control female (no. 49), two females at 100 mg/kg (nos. 52 and 57) andthree females at 1000 mg/kg (nos. 77, 79 and 80).
Except for female no. 80 which was not pregnant, these females had normal litters. These irregular cycles were considered not to be related to treatment because the incidences showed no dose-related trend and in 4/5 treated females the irregular cycles were followed by normal cycles (high dose female no. 79 had a 3-day cycle at the end of the premating period).
For some individual females across the groups (no. 50, 54, 56, and 76), the regularity of estrous cycles could not be determined during the premating period, as only one cycle of 4-5 days was observed in this period. This was regarded to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

One female at 100 mg/kg (no. 60, mated with male no. 20) was pregnant but did not deliver offspring (she had only one implantation site at necropsy). This failed pregnancy was considered to be related to changes in her uterus. The uterus had a pyometra-like histologic appearance in the form of a moderate granulocytic inflammation of the endometrium extending transmural to the myometrium (minimal inflammation with edema) and the presence of debris with many granulocytes in the uterus lumen. As these histopathological alterations occurred in only a single female of the lowest dose group they were regarded as
unrelated to treatment. Two females were not pregnant despite evidence of mating: 100 mg/kg female no. 53 (mated with male no. 13) and 1000 mg/kg female no. 80 (mated with male no. 40). No abnormalities were seen in the reproductive organs which could account for their lack of offspring. One female at 300 mg/kg (no. 69, mated with male no. 29) was euthanized for humane reasons on post-coitum Day 1 (her pregnancy status could not be determined at this early stage of the post-coitum period). The reproductive organs of this couple were without test item-related findings.

Mating index was not affected by treatment. All females showed evidence of mating (for some females indirectly by delivery of a litter). Mating index was 100%for all groups.
Precoital time was considered not to be affected by treatment. Most females were mated within four days. Longer precoital intervals were noted for two females at 300 mg/kg (11 or 13 days) and one female at 1000 mg/kg (6 days). These longer intervals were considered not to reflect an effect of the test item due to the lack of a dose-related trend.
Number of implantation sites was considered not to be affected by treatment. One female of the 100 mg/kg group had only one implantation site (no. 60; she had no offspring). This incidental finding in a low-dose animal was regarded as unrelated to treatment.

Fertility index was considered not to be affected by treatment. The fertility indices were 100, 90, 90 and 90% for the control, 100, 300 and 1000 mg/kg groups, respectively. Two females with evidence of mating were not pregnant: nos. 53 (100 mg/kg) and 80 (1000 mg/kg). Pregnancy status could not be determined for female no. 69 (300 mg/kg) which was euthanized for humane reasons on post-coitum Day 1. These cases of non-pregnancy, without related histopathology changes in reproductive organs, were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other: no effects seen

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99% for the 300 mg/kg group and 100% for the other groups.
The incidental death of one pup (litter no. 67) of the mid-dose group which went missing on PND 2 was unrelated to treatment.
The mean number of living pups at first litter check (live litter size) was considered not to be affected by treatment. Mean litter size at 100 mg/kg was lower than the control group mean (9.9 versus 11.9; not statistically significant). In absence of a dose-related trend this finding was not attributed to treatment

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not affected by treatment

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were not affected by treatment

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were not affected by treatment.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment
Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment. Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment. The survival indices were 95, 82, 93 and 90% for the control, 100, 300 and 1000 mg/kg groups, respectively. The slightly low post-implantation survival index for the 100 mg/kg group could largely be explained by a high number of unaccounted for sites in female no. 52 (8/13 implantation sites were unaccounted for). This finding in a single low-dose female was regarded as unrelated to treatment. For females no. 67 (300 mg/kg), 71 (1000 mg/kg) and 78 (1000 mg/kg) the number of pups
born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

The mean number of living pups at first litter check (live litter size) was considered not to be affected by treatment. Mean litter size at 100 mg/kg was lower than the control group mean (9.9 versus 11.9; not statistically significant). In absence of a dose-related trend this finding was not attributed to treatment.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100% for the control and 100 mg/kg groups and 98% for the 300 and 1000 mg/kg groups. At first litter check, two pups at 300 mg/kg (litter nos. 63 and 70) and two pups at 1000 mg/kg (litter no. 77) were found dead. This incidental pup mortality was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within normal limits.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NO treatment related effects were seen in the F1 generation

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F2 generation

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Wistar Han rats were treated with 1132 Side Chain by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg. The rats of the control group received the vehicle, propylene glycol, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 14-16 days of lactation (for 50-63 days). Females that failed to deliver pups were treated for 40, 42 or 47 days.
Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted the reproductive parameters examined (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results
No treatment-related changes were noted in the developmental parameters examined (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, live litter size, maternal care, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum T4 thyroid hormone levels (PND 14-16) and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of 1132 Side Chain were established:
Reproduction NOAEL: at least 1000 mg/kg.
Developmental NOAEL: at least 1000 mg/kg.

Executive summary:

The objectives of this study were to determine the potential toxic effects of 1132 Side Chain when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parental parameters were evaluated in this study: mortality/moribundity, clinical signs, functional tests, body weight, food consumption, estrous cycle length and regularity, clinical pathology, serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examination. Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously.

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). The only treatment-related finding in this study consisted of slight salivation after dosing, noted at all dose levels in up to all animals with a dose-related trend in frequency and time of onset.  This was regarded as a physiological response rather than a sign of systemic toxicity. No reproduction or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).