5-bromo-1,3-dichloro-2-fluorobenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-bromo-1,3-dichloro-2-fluorobenzene

EC Number:

640-454-2

Cas Number:

17318-08-0

Molecular formula:

C6H2 BrCl2F

IUPAC Name:

5-bromo-1,3-dichloro-2-fluorobenzene

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Han Wistar rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain. Background data on the rate of spontaneous malformations have been accumulated. All animal work was conducted under authority of a Project Licence in compliance with the Animals (Scientific Procedures) Act 1986 (as amended).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty male and 48 female rats of the Crl:WI(Han) strain were supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England. The males were 8 to 9 weeks of age and the females were 11 to 12 weeks of age on arrival and on examination were found to be healthy. After at least 12 days acclimatisation, they were re-examined and confirmed to be suitable for dosing. On the first day of dosing, the males weighed 290 to 392 g and the females weighed 207 to 260 g.
Housing: groups of up to 3 males and up to 4 females, until pairing; solid floor Techniplast 1500U cages with appropriate bedding
Pairing: 1 male & 1 female were housed together in Techniplast grid-floor Type 3 cages suspended over paper-line trays
Pregnancy: females were housed individually and with their litter in North Kent Plastics RB3 solid-floor cages
Temperature: 19-23 °C
Humidity: 40-70%
Light-cycle: 12 hours light, 12 hour darkness
Diet: pelleted rodent diet VRF1 ad libitum, manufactured by SDS and supplied by Charles River (UK) Ltd, Margate, Kent, United Kingdom
Water: mains tap water ad libitum

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally, by gavage, as the oral route had been defined by the Sponsor as a possible route of human exposure.

Vehicle:

corn oil

Details on oral exposure:

Group 1: Males 1-10, females 42-51, control, dose concentration 0 mg/mL
Group 2: Males 11-20, females 53-60, 62, 63, 30 mg/kg/day, dose concentration 7.5 mg/mL
Group 3: Males 21-30, females 65-67, 69-72, 74-76, 150 mg/kg/day, dose concentration 37.5 mg/mL
Group 4: Males 31-40, females 77-86, 300 mg/kg/day, dose concentration 75.0 mg/mL
All males in Groups 1 to 4 and selected females in Groups 1 to 3, were dosed once daily, using a rubber catheter and disposable syringe, for at least 14 days before pairing and until the day before necropsy. Males were dosed before, during and after pairing; females were dosed before and during pairing, during gestation and until Day 12 of lactation. Group 4 females were dosed for 8 days only before being sent to necropsy and therefore the Group 4 males were dosed for at least 8 weeks until the day before necropsy. On Day 1 of lactation, 3
Control females and 2 Group 3 females received a marginally different dose volume than intended in error. Animals that were mid-parturition at the time of dosing were not dosed on that day. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 4 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of test item formulations prepared at concentrations of 0.25 and 75 mg/mL, spanning those used in this study (7.5 to 75 mg/mL), were examined in an earlier formulation validation study. Those formulations were found to be stable for up to 12 days when stored at room temperature or refrigerated and 35 days frozen (at approximately -20 °C).
Samples (for analysis or contingency) were taken from all formulations used on this study. Samples taken from each test item formulation prepared for the first day of dosing were analysed for the substance using a validated method (BFI078LC) to confirm homogeneity and also achieved concentrations. Having satisfactorily confirmed homogeneity on the first day of dosing, samples taken from all test item formulations prepared for use towards the end of the dosing period were analysed simply to confirm satisfactory concentration. For Controls, sets of samples (for analysis or for contingency) were taken on the first day of dosing and at the end of the dosing period from the vehicle and were analysed, using the same method, to confirm absence of test item.

Duration of treatment / exposure:

All males were dosed for at least 14 days before pairing, during pairing and after pairing. Males in the group receiving 300 mg/kg bw/day were dosed for at least 8 weeks.
Females were dosed for at least 14 days before pairing, during pairing and gestation. Females with litters were dosed until day 12 of lactation. Females without litters were dosed until day 25 post-mating. Females where all pups had died in the litter were dosed until the litter death. Females receiving 300 mg/kg bw/day were dosed for 8 days only before being sent to necropsy.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control groups were receiving corn oil at same volume as animals receiving test material.

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Dose concentration 7.5 mg/mL

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Dose concentration 37.5 mg/mL

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dose concentration 75.0 mg/mL

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

P GENERATION CLINICAL OBSERVATIONS AND MEASUREMENTS
CLINICAL OBSERVATIONS
Animals were examined twice daily for mortality and morbidity. A detailed clinical examination was done weekly. Animals were observed before and shortly after dosing. On weekdays, animals were also observed 1 hour after dosing, and a final check was made around 4 hours after dosing or at the end of the working day. On weekends, animals were observed at the end of the working day. In the pre-pairing period and for males in the post-pairing period, observations were done based on group housed animals. For males and females during pairing and for females after pairing, observations were based on individual animals.

BODY WEIGHT
Male bodyweights were recorded on the first day of dosing and then at weekly intervals throughout the study. Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Body weight was determined on days 0, 7, 14 and 20 of gestation and on days 1, 4, 7, 10 and 13 of lacation, or on the day of necropsy for non-pregnant females.

FOOD INTAKE
The amount of food consumed was recorded at weekly intervals for males and females during the pre-pairing period. Individual food intake of females was recorded over days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over days 1 to 4, 4 to 7, 7 to 10 and 10 to 13 of lactation. Two weeks after the start of pairing, food intake for males was again recorded.

BEHAVIOURAL OBSERVATIONS
All animals were observed once weekly, starting from the final week of the acclimatisation period, for their behaviour within the cage and then after placement in an open arena. Observations were made about at the same time of day during the afternoon. Observations were not conducted for any female that was mid-parturition.

FUNCTIONAL OBSERVATION BATTERY
Sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for the first five surviving males and females in each group during week 7.

OESTROUS CYCLING MONITORING
From 14 days before the start of dosing and until the day of pairing, vaginal smears were taken daily by lavage. The smears were examined under light microscopy and the stage of the oestrous cycle was determined.

PAIRING AND DETECTION OF MATING
After the pre-pairing dosing period of 14 days, each female in the control and dose groups (except females in the group receiving 300 mg/kg bw/day) was paired with a male from the same dose group for up to 14 days. On confirmation of mating, the males were returned to the group cages and the females were housed individually. During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed by sperm being found in the smears. The smears were examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was recorded to give an assessment of the mating activity of the animals. The day on which sperm were detected was designated Day 0 of gestation.

PARTURITION OBSERVATIONS
Females were observed from day 21 of gestation until all animals had littered or until the start of the working day when the last pregnant female was on day 26 after mating. Following completion of parturition, pups were designated as day 0 of age with the next day classified as day 1 of age. Dead offspring were not removed from the litter until parturition had been completed.

OBSERVATION OF LITTERING FEMALES
The females were allowed to rear their offspring to day 13 of lactation. Abnormalities of nesting or nursing behaviour were recorded.

F1a GENERATION
LITTER SIZE, SEXES AND CULLING
The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from day 1 of age. On day 4 of age, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomised basis. Non-selected pups were killed and discarded after blood sample collection.

IDENTIFICATION
Pups wer not individually identified. Identification numbers were assigned for the purposes of recording total T4 concentrations and organ weights.

CLINICAL OBSERVATIONS
Animals were examined twice daily for mortality and morbidity and were examined daily for clinical signs of toxicity or changes in behaviour and appearance.

BODY WEIGHTS
Pups were weighed individually on days 1, 4, 7, 10 and 13 of age.

ANOGENITAL DISTANCE
The anogenitcal distance of the F1a pups was measured on day 1 of age.

NUMBER OF NIPPLES/AREOLAE
For each male pup, the number of nipples/aerolae was counted on day 12 of age.

Sacrifice and pathology:

NECROPSY
Females with litters were killed and subjected to necropsy on day 13 of lactation. Females which did not litter were killed on day 26 post mating and females where all pups had died in the litter were subjected to necropsy following the litter death. Surviving females in the group treated at 300 mg/kg bw/day were killed on day 9 of the study.
Males were killed and subjected to necropsy after the females in the same treatment group had littered.
Dead body weight was recorded and thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs and uterus were examined. The number of implantation scars/sites for each female killed on day 13 of lactation was recorded. Organs or tissues showing any macroscopic abnormalities were recorded and retained.

ORGAN WEIGHTS
The following organs were weighed after trimming of fat and other contiguous tissue. Contralateral organs were weighed together:
adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate and seminal vesicles (including coagulating gland), spleen, testes, thymus, thyroids (including parathyroids), uterus including cervix weighed whole with cervix and oviducts)

MACROSCOPIC AND MICROSCOPIC PATHOLOGY
PARENT GENERATION
For all animals, with the exception of the eyes, bone marrow smear, optic nerves and testes, either whole organs or samples of the tissues listed below were preserved in neutral buffered formaldehyde. The eyes and optic nerves were fixed in Davidson’s solution and the testes were fixed in Modified Davidson’s. In the absence of any haematological reasons for doing so, the bone marrow smears were not examined.
adrenal glands, aorta (thoracic), bone marrow smear, brain (3 levels examined), caecum, colon, duodenum, epididymides, eyes (including optic nerves), femur & femorotibial joint (including marrow), gut associated lymphoid tissue (GALT)1, head (including teeth, 1 LS section2) spinal cord (3 levels examined LS and TS), heart, ileum, jejunum, kidneys (1 LS and 1 TS), lacrimal glands, larynx, liver, lungs (including mainstem bronchi), mammary gland (collected with inguinal skin), mesenteric lymph nodes, nasal turbinates (1 section), nasopharynx, oesophagus, ovaries, pancreas, pharynx, pituitary gland, prostate & seminal vesicles (incl. coagulating gland), rectum, salivary gland (submandibular), sciatic nerve, skeletal muscle, skin (collected with mammary gland), spleen, sternum (including marrow), stomach (fore stomach and glandular), submandibular lymph nodes, testes, thymus, thyroids (including parathyroids), trachea, urinary bladder, uterus (including uterine cervix and oviducts3), vagina, all gross lesions
For all animals, the tissues specified were wax embedded, cut at a nominal thickness of 4 μm to 5 μm and stained with haematoxylin and eosin. For all Control and high dose animals and any premature decedents, the tissues were examined microscopically. Due to test item related findings the liver, thymus, mesenteric lymph nodes, oesophagus, forestomach and head from males and females, thyroids and adrenals from males in the low and intermediate groups (Groups 2 and 3) were examined microscopically. An internal peer review was performed in accordance with current standard operating procedures. An external peer review was also conducted by the Sponsor’s pathologist.

F1a NECROPSY
Pups culled on day 4 of age: Culled pups were killed by an intraperitoneal injection of sodium pentobarbitone solution or by decapitation for those subjected to blood sampling and discarded without necropsy.
Pups culled on day 13 of age: Pups were killed by an intraperitoneal injection of sodium pentobarbitone solution or by decapitation for those subjected to blood sampling. Any pups killed or found dead during lactation and all pups killed on Day 13 of lactation, were examined externally for gross abnormalities only, with particular attention paid to the external reproductive genitals. On Day 13 of age, the thyroid from 1 male and 1 female pup/litter (from pups that had not been sampled for thyroid hormone assessments or killed by decapitation) were preserved in neutral buffered formaldehyde and weighed (after fixation). A dead body weight was recorded for these pups. Due to the age and size of pups, and to prevent damage, the thyroid was left in situ on the larynx and retained in neutral buffered formaldehyde. Following fixation of the carcass, the thyroid was removed and weighed. All remaining pups, including those sampled for thyroid hormone assessment, were discarded without further examination.

Other examinations:

CLINICAL LABORATORY STUDIES
BLOOD SAMPLE COLLECTION
Blood samples of 1.5 mL were taken under isoflurane anaesthesia from the sublingual vein of the 5 males and 5 females with the highest identification numbers on day 14 of dosing. Blood samples were taken into anticoagulent as follows: 0.5 mL into EDTA for haematology, 0.5 mL into 3.2% w/v aqueous trisodium citrate for coagulation, 0.5 mL into lithium heparin for blood chemistry.

HAEMATOLOGY
The following parameters were measured: haemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, red blood cell distribution width, platelet count, total leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, reticulocytes

COAGULATION
Parameters: prothrombin time, fibrinogen, activate partial thromboplastin time

BLOOD CHEMISTRY
Parameters: urea, creatinine, glucose, alkaline phosphatase, alkaline aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, cholesterol, triglyceride, sodium, chloride, potassium

THYROID HORMONE ASSESSMENTS
BLOOD SAMPLING
Samples were taken from all animals in the morning into tubes with no anticoagulant and allowed to clot for 30 minutes at room temperature. Once clotted, samples were centrifuged at 3000 g for 10 minutes at 4 °C. For P generation animals, the resultant serum was divided equally into two aliquots, before being stored frozen (<= -70 °C) along with the remaining serum samples.

P GENERATION ANIMALS
A blood sample of 0.5 mL was taken on the day of necropsy (day 13 of lactation for females) from the sublingual vein under isoflurane anaesthesia.

F1a PUPS - DAY 4 OF AGE
Where possible, a culled pup/sex/litter was bled on day 4 of age following decapitation; 1 pooled sample, of as much blood as possible, was collected per litter.

F1a PUPS - DAY 13 OF AGE
One pup/sex/litter (not from pups with gross external abnormalities) was sampled on day 13 of age, following decapitation; as much blood as possible was taken.

SAMPLE ANALYSIS
P generation male and female samples (set 1) and the day 4 and day 13 pup samples were analysed; all other serum samples were retained for possible future analysis. Samples were analysed for total thyroxine (T4) using a previously validated method and a Calbiotech Mouse/Rat Total T4 ELISA assay kit.

Statistics:

General Approach: two-sided statistical tests with minimum significance levels of 5% and 1%. Non-parametric statistics not routinely conducted. Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Data were examined for unusually high or low values (possible outliers). After examining for any outliers, if the variances were clearly heterogeneous, transformations (e.g. log, double arcsine or square root) used to stabilise the variances. If the transformations failed, the data set was examined and a decision taken on further action.
For Quantitative Data: Body weight, food intake, cumulative body weight gain from the start of dosing (throughout gestation and lactation), pup body weights, cumulative pup body weight gain, litter size, oestrous cycle number and length, gestation length, pre-coital interval and total T4 concentrations analysed using ANOVA.
Group mean anogenital distance analysed by ANCOVA using body weight as covariate.
Organ weights analysed using ANOVA for the absolute weights and ANCOVA using terminal kill body weight as covariate. Group summaries (mean, standard deviation and number of observations) and individual values presented for organ weights as a percentage of body weight.
For Percentages: Fertility, mating, implantation, pup survival, pup sex ratios and litter based mean percentages and fertility and copulation indices were analysed using a parametric ANOVA, following a double arcsine transformation.
Outliers: Exclusion of individual outlier values was considered unnecessary, as it was decided that the data reflected variation expected.
Dunnett’s test: for parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test used to compare the control and treated groups, based on the error mean square. Performed for all continuous data parameters, and statistical flags were presented in the tables of results in the final report.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Female 62 given 30 mg/kg/day and Females 65 and 76 given 150 mg/kg/day were killed on Day 3 of lactation as all the litter had died. There were no adverse effects on the body weight gain or food intake of these dams before loss of the litter and no macroscopic or microscopic findings.
Abnormally white teeth were noted in males given 150 or 300 mg/kg/day from Day 43 of dosing and in females given 150 mg/kg/day from Day 3 of lactation (approximately Day 30 of dosing). Male 32 given 300 mg/kg/day also had piloerection, partially closed eyes and a cold body surface on Day 52 of dosing.

Mortality:

mortality observed, treatment-related

Description (incidence):

Females 77 and 81 given 300 mg/kg/day were euthanised due to a decline in clinical condition and excessive body weight loss on Day 6. The remainder of the females given 300 mg/kg/day were euthanised on Day 9. Most females at this dose level lost weight during the dosing period, losing up to 17% of their body weight; observations of piloerection and cold body surface were also noted. Male 37 given 300 mg/kg/day was killed prematurely on Day 6 following excessive body weight loss. These deaths were considered to be test item related.
Female 68 given 150 mg/kg/day, died following the first dose administration as a result of the dosing procedure; this female was replaced by Female 72 and any data for Female 68 has not been included in this report.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration of the substance was associated with dose-related reductions in body weight gain, or body weight loss, in both males and females.
Males gained 17%, 31% or 58% less weight than Controls during the dosing period at 30, 150 and 300 mg/kg/day, respectively.
Females given 150 mg/kg/day gained less weight than Controls during the pre-pairing period and gestation (70% and 19% respectively), and body weight loss was also observed during the first week of dosing. Females given 30 mg/kg/day gained less weight than Controls during gestation only (12%). During lactation, body weight gain was similar to Controls in the groups given 30 or 150 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Both males and females given 150 or 300 mg/kg/day consumed significantly less diet than Control animals during the first week of dosing (p≤0.01 Week 0-1). Food intake improved thereafter so that food intake for the remainder of the dosing period in males, and throughout gestation for females was comparable with Control animals.
During lactation, females given 30 or 150 mg/kg/day consumed 11% or 28%, respectively, less than Controls.

Food efficiency:

not specified

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):

There was a dose-related, statistically significant, increase (p≤0.01) in group mean platelet counts for males given 150 or 300 mg/kg/day compared with Controls. As these group mean values were above the upper limit of the historical Control range of means, this effect was considered to be potentially adverse.
There was a statistically significant approximate 2-fold increase (p≤0.01) in group mean absolute monocyte count in males given 300 mg/kg/day compared with the Control mean. However, the mean was within the historical Control range of means, and as such this change was considered to be of no toxicological significance.
Group mean prothrombin time for males given the substance was between 1.6 and 3.3 seconds longer than the Control mean. Although these inter-group differences were dose-related and achieved statistical significance (p≤0.0.5 at 30 and 150 mg/kg/day and p≤0.01 at 300 mg/kg/day), they were within the historical Control range of means. As similar findings were not seen in females these differences were considered to be of no toxicological significance.
Mean red cell distribution width (p≤0.01) and percent and absolute reticulocyte count (p≤0.05) were lower in females given 150 mg/kg/day than the Control means. In these same females, total white cell count was statistically significantly higher (p≤0.01) than the Control mean, primarily due to a slightly increased absolute lymphocyte count and a 2.8-fold increase in large unstained cell count. The group mean values for all the above changes in females were within the historical Control ranges of means.
There were no effects on haematology or coagulation parameters for females given 30 mg/kg/day.
Other statistically significant inter-group differences in males were not strictly dose-related and therefore were considered unlikely to be related to administration of of the substance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males given 300 mg/kg/day, there were marked statistically significant increases (p≤0.01) in mean plasma alanine aminotransferase (2-fold) and gamma glutamyl transpeptidase activities (19-fold) together with an increased cholesterol concentration compared with the Control means. As these values were above the upper limit of the historical Control ranges of means, these changes were considered to be potentially adverse. Although it did not achieve statistical significance, there was also an increase in plasma bilirubin concentration at 300 mg/kg/day and this was considered likely to be associated with the increased cholesterol concentration. In addition, mean alkaline phosphatase activity was higher than the Control mean (p≤0.05) and mean plasma glucose concentration was lower than the Control mean (p≤0.01). However, the mean values for the parameters were within the historical Control range of means.
In females given 150 mg/kg/day, mean plasma glucose concentration was also lower than the Control mean (p≤0.05), while plasma total protein and triglyceride concentrations in these females were higher than the Control mean (p≤0.05). These mean values all fell within the historical Control ranges of means.
Other statistically significant, apparent intergroup differences, were either not strictly doserelated (total protein and albumin concentrations in males) or were in the opposite direction to that normally associated with toxicity (creatinine concentration in females) and were therefore considered to be due to normal biological variation rather than any effect of the test item.
There were no effects on any blood chemistry parameters for either sex given 30 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males given 150 or 300 mg/kg/day, there were marked, statistically significant, dose related increases, in group mean absolute and adjusted liver weight (p≤0.01) compared with Control ranging from 95% to 178%. In addition, there was a smaller increase in group mean absolute (11% higher than Controls) and adjusted (15% higher than Controls) liver weights in males given 30 mg/kg/day with an adjusted weight achieving statistical significance (p≤0.01) when compared with Controls. Smaller, 17% to 24%, increases (p≤0.01) were seen in females given 150 mg/kg/day. The mean values for both sexes given 150 mg/kg/day and males given 300 mg/kg/day were above the upper limit of the historical Control range of means and these weight changes correlated with microscopic findings of hepatic hypertrophy.
In males given 300 mg/kg/day, there was a 40% reduction (p≤0.01) in absolute thymus weight compared with the Control mean that correlated with thymic atrophy seen microscopically. The group mean value however, was within the historical Control range of means.
In treated males, group mean adjusted kidney weight was significantly higher than Controls at all dose levels (p ≤0.05 to p ≤0.01). However, these differences were not strictly dose-related and, in the absence of corroborative microscopic findings in the kidney, they were considered not to be of toxicological significance.
In females given 150 mg/kg/day, group mean absolute spleen weight and spleen weight adjusted for body weight were 15% and 12% lower respectively, than the Control means (p<0.01). As there were no microscopic correlates for these findings, they were considered not to be of toxicological significance.
Other statistically significant inter-group differences were considered to be a consequence of reduced body weight gain rather than a direct effect of the test item.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings recorded at necropsy considered to be related to the treatment were seen in the liver, teeth and thymus (females only).
Abnormally coloured teeth (white) were seen in the majority of males (8/10) in the group given 300 mg/kg/day, and in occasional males and females given 150 mg/kg/day.
Typically, this appearance of the teeth is due to increased opacity of the enamel, which masks the darker coloured dentine beneath, giving a whiter appearance. As the enamel is removed during the decalcification process, changes to the enamel cannot be examined microscopically; however because of these changes, a detailed histological examination of the enamel organ (ameloblasts) was undertaken.
Large livers were noted in the majority of males (7/10 and 8/10 respectively) given 150 or 300 mg/kg/day. In the animals given 150 mg/kg/day accentuated liver pattern was recorded in 1/10 males and 2/10 females given 150 mg/kg/day.
The macroscopic liver changes were associated with higher liver weights in males given 30, 150 and 300 mg/kg/day. In females the liver weights were higher at 150 mg/kg/day, with smaller increases in animals given 30 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related to treatment were recorded in the liver (hepatocyte hypertrophy and necrosis of occasional hepatocytes), teeth (atrophy of the ameloblasts), mesenteric lymph nodes (decreased cellularity, paracortex), thymus (atrophy), oesophagus (hyperkeratosis), forestomach (epithelial hyperplasia with associated hyperkeratosis in a few animals) and thyroid glands (epithelial hypertrophy and increased basophilia of colloid).
In the liver, the hypertrophy ranged from minimal to slight centrilobular hypertrophy in the animals given 30 mg/kg/day to a marked diffuse hypertrophy in the males given 300 mg/kg/day. The females given 300 mg/kg/day were only dosed for a short period, and the hypertrophy was less marked than in the males, but the incidence of necrotic hepatocytes was the same and there was also some vacuolation in this group. The dose related increase in hypertrophy closely correlated with a similar dose related higher liver weight, and the macroscopic findings of large liver and accentuated liver pattern.
The observed liver changes were consistent with altered ALT, γGT, cholesterol, bilirubin and TG values. Higher ALT and γGT levels were seen in the high dose males, together with higher TG and cholesterol values in the mid and high dose, and bilirubin in the high dose only. In females the TG, ALT (very questionable in terms of magnitude of change) and cholesterol levels were higher at 150 mg/kg/day, with TG also higher at 30 mg/kg/day.
Ameloblast atrophy was seen predominantly in males and females in the groups given 150 and 300 mg/kg/day, reflecting the gross findings. One female in the group given 30 mg/kg/day showed atrophy accompanied by an area of degeneration in the enamel matrix. There was no clear dose relationship for this finding.
Decreased cellularity of the mesenteric lymph nodes was seen in both males and females given 300 mg/kg/day and in females given 150 mg/kg/day. Other elements of the lymphoreticular-endothelial system appeared unaffected. Given the status of the animals (lower body weights and significant liver hypertrophy) it is considered that these changes may be non-specific and not directly related to the test item.
Hyperkeratosis of the oesophagus (slight/moderate) was seen in all treated animals, with increasing severity as the dose increased. In the forestomach treated males (2/10, 2/10, 1/10, respectively) and females (0/10, 4/10, 5/10, respectively) showed epithelial hyperplasia, often accompanied by hyperkeratosis. These findings are considered to reflect local irritancy, possibly as a consequence of synergy between the test article and the vehicle (corn oil).
Epithelial hypertrophy accompanied by increased basophilia of the colloid was seen in the thyroid glands of males given 30, 150 or 300 mg/kg/day. The change was also seen in females given 150 mg/kg/day. The basophilia is likely to be associated with increased colloid turnover.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Small thymuses were seen in 2 males given 300 mg/kg/day and in 2, 1 and 3 females in the groups given 30, 150 or 300 mg/kg/day, respectively. This finding was associated with the thymic atrophy seen microscopically. There was a small increase in the incidence of thymic atrophy in males given 300 mg/kg/day. In the females, corpora lutea of pregnancy were seen in all animals except those given 300 mg/kg/day, which were not pregnant.

Details on results:

EFFECTS ON REPRODUCTIVE ENDPOINTS
- There was no effect on oestrous cycles or mean cycle length during the pre-pairing period.
- There was no effect on the fertility or mating performance of animals given the substance, all animals mated within four days. One female in each of the groups given 0 or 30 mg/kg/day was not pregnant. All animals given 150 mg/kg/day were pregnant.
- There was no effect on the mean duration of gestation or parturition.
- At 30 or 150 mg/kg/day, there was a slight, dose-related decrease in the mean number of pups born, however there was no increase in post-implantation losses as there were lower mean numbers of implantation scars in these groups compared with the Control; the decrease was considered to be not related to the test item.
- In P generation males, there was a dose-related reduction in serum thyroxine (T4) concentration (p≤0.01); T4 concentrations were 5%, 58% or 68% lower than Controls in males given 30, 150 or 300 mg/kg/day, respectively. Females given 150 mg/kg/day had a statistically significant (p≤0.01) 43% reduction in T4 concentrations when compared with Controls. Group mean values for females given 30 mg/kg/day were lower than the Control, but did not achieve statistical significance. The reductions in T4 concentrations were considered to be secondary consequence of the microscopic changes in the liver and thyroids.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity in the P generation could not be established based on histopathological changes at 30 mg/kg/day in the teeth (atrophy of ameloblasts) in females and lower body weight gain and histopathological changes in the liver, thyroid gland and forestomach in males.

Executive summary:

The repeated dose toxicity of the substance to the rat was studied under GLP in a combined repeated dose/reproductive and developmental toxicity screening study performed to OECD TG 422. This study summary covers only the effects on the parent generation.

Four groups of ten male and ten female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (corn oil), 30, 150 and 300 mg/kg bw/day. Males were dosed for 14 days before and during pairing, and until the day before necropsy. Females given 300 mg/kg bw/day were dosed for 8 days before being sent to necropsy. Remaining females were dosed for 14 days before pairing, during pairing and gestation until day 12 of lactation.

Before the start of the dosing period, the oestrous cycles of 12 females per group were monitored and the first 10 in each group, which were cycling normally, were selected to be dosed on the study. Oestrous cycle monitoring continued through the pre-pairing phase of the study. All selected animals were examined for effects on general condition, body weight and food intake. Functional observations were monitored throughout the study and on Day 14 of dosing (before pairing), blood samples were taken for assessment of clinical pathology parameters. During the pairing period, vaginal smears were taken daily until sperm were detected. The females were allowed to litter and rear their offspring to Day 13 of age. All parental males and females, where possible, had blood samples taken for thyroid hormone analysis and analysed.

A necropsy was performed on all P generation animals and a selection of organs were weighed, fixed and examined microscopically.

There were a number of premature decedents during the study; 2 females given 300 mg/kg/day were euthanised on Day 6 due to excessive body weight loss, and the remainder of the females at this dose were killed on Day 9 due to a decline in clinical condition and body weight loss. One male given 300 mg/kg/day was also killed on Day 6 as a result of excessive body weight loss.

Abnormally white teeth were noted in males given 150 or 300 mg/kg/day from Day 43 of dosing and in females given 150 mg/kg/day from Day 3 of lactation (approximately Day 30 of dosing). One male given 300 mg/kg/day also had piloerection, partially closed eyes and cold body surface on Day 52 of dosing.

Administration of CA5528 was associated with dose-related reductions in body weight gain, or body weight loss, in both males and females, however female body weight gain during lactation was similar to Controls.

At 150 or 300 mg/kg/day, there were initial reductions in food intake during the first week of dosing, thereafter, improvement was noted in both males and females. During lactation, females given 30 or 150 mg/kg/day ate a reduced amount relative to Controls.

There were no changes in behaviour or observations in the home cage or open field arena, and no effects on sensorimotor responses, grip strength or motor activity.

Administration of 150 or 300 mg/kg/day to males was associated with increased plasma cholesterol and bilirubin concentrations and increased alanine aminotransferase and gamma glutamyl transpeptidase activities.

There was no effect on oestrous cycling, fertility and mating performance, or on gestation and parturition.

There was a dose-related reduction in serum thyroxine (T4) concentration in males given 30, 150 or 300 mg/kg/day (5%, 58% or 68% lower than Controls, respectively) and females given 150 mg/kg/day (43% lower than Controls).

In males given 30, 150 or 300 mg/kg/day and in females given 150 mg/kg/day, there was an increase in liver weight, associated with hepatic hypertrophy. There was also a reduction in thymus weight in males given 300 mg/kg/day which correlated with microscopic findings of thymic atrophy.

Macroscopic findings recorded at necropsy considered to be related to treatment were seen in the liver, teeth and thymus (females only). Abnormally coloured teeth (white) were seen in the majority of males (8/10) given 300 mg/kg/day, and in occasional males and females given 150 mg/kg/day.

Microscopic findings considered to be related to treatment were recorded in the liver, teeth, mesenteric lymph nodes, thymus, oesophagus, forestomach, thyroid glands. The changes in the liver and teeth were considered likely to be primary response to the test item. The liver changes were associated with a substantial increase in liver weights in animals given 150 or 300 mg/kg/day, and smaller increases in animals given 30 mg/kg/day.