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Diss Factsheets

Administrative data

Description of key information

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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
elevated humidity for 5 day of study, no adverse impact on the result
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
as above
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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)
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 between 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 Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). 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 material, 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 which 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 changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms. Pelleted 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 measurements, 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.
Route of administration:
oral: gavage
Details on route of administration:
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. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure
according to Standard Operating Procedures. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Vehicle:
polyethylene glycol
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
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (ten)
Control animals:
yes, concurrent vehicle
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 identified within the study by an ear punching system routinely used in these laboratories.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
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.

DETAILED CLINICAL OBSERVATIONS: Yes - as above
BODY WEIGHT: Yes
- Time schedule for examinations: day 1 then weekly intervals until terminal kill

FOOD CONSUMPTION: Yes
Food consumption was recorded for each cage group at weekly intervals throughout the
study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION : Yes
Water intake was observed daily, for each cage group, by visual inspection of the water
bottles for any overt changes except during Week 3 where water intake was measured
gravimetrically.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all surviving animals from each test and control group at the end of the study
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11
mol/L).

CLINICAL CHEMISTRY: Yes
-- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals:
all surviving animals from each test and control group at the end of the study
The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea, Inorganic phosphorus (P)
Glucose, Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.), Alanine aminotransferase (ALAT)
Albumin, Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat)
Sodium (Na+), Triglycerides (Trigs/Tri)
Potassium (K+), Total cholesterol (Chol)
Chloride (Cl-), Total bilirubin (Bili)
Calcium (Ca++), Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Prior to the start of treatment and on Days 6, 13, 20 and 27 all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different
stimuli. Observations were carried out from approximately two hours after dosing on each
occasion.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: behavior assessment
IMMUNOLOGY: No
Sacrifice and pathology:
Necropsy
On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -80 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
The following organs, removed from animals that were killed at the end of the dosing period were dissected free from fat and weighed before fixation:
Adrenals, Liver
Brain, Ovaries
Epididymides, Spleen
Hear,t Testes
Kidneys, Thymus
Pituitary (following partial fixation), Thyroid/Parathyroid (following partial fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids), Uterus with Cervix

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals, Ovaries
Aorta (thoracic), Pancreas
Bone & bone marrow (femur including stifle joint), Pituitary
Bone & bone marrow (sternum), Prostate
Brain (including cerebrum, cerebellum and pons), Rectum
Salivary glands (submaxillary)
Caecum, Sciatic nerve
Colon, Seminal vesicles (with coagulating glands and fluids)
Duodenum
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions, Spleen
Heart, Stomach
Ileum, Testes ♦
Jejunum, Thymus
Kidneys, Thyroid/Parathyroid
Liver, Trachea
Lungs (with bronchi)#, Urinary bladder
Lymph nodes (mandibular and mesenteric), Uterus & Cervix
Mammary gland , Vagina
Muscle (skeletal)

* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid

All tissues were dispatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues shown in bold from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the liver (both sexes), thyroids (both sexes), kidneys (males only) and mesenteric lymph nodes (females only) from animals in the ow and intermediate groups.
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. 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 analyses, 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)
Clinical signs:
effects observed, non-treatment-related
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 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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect on body weight development for treated animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no adverse effect on food consumption or food conversion efficiency for treated animals.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no adverse effect on food consumption or food conversion efficiency for treated animals.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no adverse effect on water consumption for treated animals.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined.
Clinical biochemistry findings:
effects observed, non-treatment-related
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.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
here were no toxicologically significant changes in functional performance. There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights.
An isolated, statistically significant difference noted in females (higher absolute kidney weight at 300 mg/kg) was regarded as unrelated to treatment due to the lack of a dose-related response.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations.
All of the recorded macroscopic findings at scheduled necropsy were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Findings of note were the thyroid gland abnormalities observed in females. Two control females were noted with an enlarged and discolored thyroid gland. An enlarged thyroid gland was noted for one female at 1000 mg/kg as well, and discoloration of the thyroid gland was observed in five females at 1000 mg/kg. No correlated microscopic findings were noted in the thyroid gland.
Neuropathological findings:
not examined
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Conclusions:
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:
Parental NOAEL: at least 1000 mg/kg male and females.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 full dateset available for the substance
System:
other: no toxicilogical significant effects were seen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification