Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2010 to 28 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
other: combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2010 to 28 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3550
Principles of method if other than guideline:
In addition, the procedures in this study essentially conformed to the following guidelines.

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: weight range at start of treatment was 291-323 gr (males) and 181-217 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (ad libitum): Tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 4 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.3
- Humidity (%): 37-73
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
(specific gravity 1.036)
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036) and test substance (0.98).

Storage conditions of formulations: at ambient temperature

Justification for use and choice of vehicle: based on trial formulations performed at NOTOX

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase according to a validated method (NOTOX Project 495403). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of
the Group 1 formulation prepared for use during treatment. It was not considered to derive from the formulation since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected. The maximum contribution to the other samples was 0.12% based on peak area.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Group 2 and Group 4 formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 30-44 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Three females of group 1 and one female of group 4 were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
Age at mating of the mated animals in the study: Approximately 13 weeks
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study (NOTOX 495417, See attached results).

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, at least 1-2 hours after dosing (on the peak period of anticipated effects after dosing), and if considered relevant also beyond this period. At weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. To monitor the health status, additional body weight measurements were performed for all females on Day 12 of study (data are retained in the raw data, but are not reported).

FOOD CONSUMPTION: Yes.
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : Yes
Quantitative assessment of water consumption was performed daily from Day 13 of the premating period onwards.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group).
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All males and 5 females/group were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7
Females which failed to deliver (2): Post-coitum days 25 (females with evidence of mating)
Females with total litter loss (3): within 24 hours of liter loss
Euthanized in extremis (1): when pain, distress or discomfort is considered not tranisent in nature or is likely to become more severe.
Males: following completion of the mating period (minimum of 28 days)

Samples of the following tissues and organs were collected:
- Selected 5 animals/sex/group and all animals that were killed in extremis:
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Caecum, Pituitary gland, Cervix, Preputial gland, Clitoral gland, Prostate gland, Colon, Rectum, Coagulation gland, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, (Eyes with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar-, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, (Larynx), Thyroid including parathyroid (if detectable),(Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung (infused with formalin), Urinary bladder, Lymph nodes -mandibular, mesenteric-, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions

Tissues/organs mentioned in parentheses in the above list were not examined by the pathologist, as there were no signs of toxicity or target organ involvement.

- All remaining animals, females which failed to deliver and females with total litter loss:
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes, Epididymides, Mammary gland area, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions


ORGAN WEIGHTS
- Selected 5 animals/sex/group:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY: Yes
All organs and tissues of Groups 1 and 4, all gross lesions of all animals (all dose groups), liver and kidneys of groups 2 and 3 males, liver, thymus and mammary gland of groups 2 and 3 females, organs and tissues of animal killed in extremis, additional slides of testes of group 1 and 4 males and all males suspected to be infertiel to examine staging of spermatogenesis. Reproductive organs of all surviving animals that failed to mate, conceive, sire or deliver healthy pups were checked.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on Day 5, 6 or 7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified
Description (incidence and severity):
Test substance intake: not relevant, administration via gavage
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
MORTALITY
One female at 1000 mg/kg was sacrificed in extremis on Day 14 of the post-coitum phase probably caused by a haemorrhage in the right uterine horn. Three other females at 1000 mg/kg were scheduled to be sacrificed within 24 hours after total litter loss, and hence were not directly related to treatment with the test substance. No cause of a total litter loss was found histopathologically in these females.
One of the females of this dose group with total litter loss was also noted with dark red contents in the vagina (most probably representing vaginal bleeding). In combination with the reproductive effects (described under reproduction), it was considered that this death was related to treatment with the test substance.

CLINICAL SIGNS
At 1000 mg/kg, all females were noted with hunched posture, piloerection and uncoordinated movements primarily during the premating period, and at lower incidence lean appearance, lethargy and flat posture by some females. Piloerection was also shown by several females at 300 mg/kg
towards the end of their pregnancy. Uncoordinated movements were noted by four males at 1000 mg/kg during the premating period.
Incidental findings that were noted included chromodacryorrhoea, scabs, alopecia, salivation and rales. These findings occurred 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. At the incidence
observed, these were considered to be signs of no toxicological relevance.

BODY WEIGHTS
During the last few days of the post-coitum phase, a slightly lower body weight gain was recorded for three females at 1000 mg/kg. This resulted in a statistically significant lower mean body weight gain.
The weight loss (up to 10% from Day 1 values) noted for three females at 1000 mg/kg between Days 1 and 8 of the pre-mating period, was of a temporary nature and had recovered to control values towards the end of the premating period. Hence, no toxicological relevance was ascribed to this finding.

FOOD CONSUMPTION
A slightly lower absolute and relative food consumption (statistically significant) was noted for females at 1000 mg/kg over Days 1-4 of the lactation period.
Food consumption of females at 1000 mg/kg also appeared lower during week 1 of the premating period, which corresponded to the lower body weights in that period. Since this was a temporary and slight change, this was considered to be without toxicological relevance.

WATER CONSUMPTION
Water consumption was significantly higher for females at 300 and 1000 mg/kg during the premating and post coitum period, and to a lesser extent during the lactation phase (not always statistically significant). At 1000 mg/kg, mean over mean values were 105%, 88% and 18% higher than controls
during the premating, post coitum and lactation phase respectively. At 300 mg/kg, mean over mean values were 43%, 47% and 20% higher than controls during the premating, post coitum and lactation phase, respectively. However, no histopathological kidney findings were noted in these females. Therefore, this higher water intake could not be ascribed to renal effects with certainty, and at least at 300 mg/kg this finding was considered not to represent an adverse effect. Water consumption was also higher in males at 1000 mg/kg during the premating and mating period, albeit to a lesser extent than females with mean over means being approximately 40% higher than controls in both periods.

MACROSCOPIC EXAMINATION
At 1000 mg/kg, a reduced size of the thymus was noted for three females. Enlargement of the liver was observed for two females at this dose level.
The one female at 1000 mg/kg sacrificed in extremis was noted with dark red fluid in the right uterine horn. One of the females with total litter loss was noted with one dead fetus in the left uterine horn and 2 dead fetuses in the right uterine horn, and had dark red contents in the vagina.
The black-brown discolouration of the liver noted in two females at 1000 mg/kg and one female at 300 mg/kg had no (treatment-related) histopathological correlates. Other incidental findings among control and treated animals included a yellowish soft nodule on the tail of the left epididymis, tan, reddish or a red-brown focus on or red-brown discoloration of the clitoral gland, reddish focus on the colon, alopecia on the chest and/or genital region, reddish foci on the stomach glandular mucosa or thymus, a watery-clear cyst on the left kidney, and pelvic dilation of the kidneys. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished treated animals from control animals:
- Higher absolute liver weight and liver to body weight ratio in both sexes at 300 and 1000 mg/kg (not statistically significant for absolute weights of males at 300 mg/kg). Liver to body weight ratio was 45% and 30% higher than controls at 1000 mg/kg for males and females, respectively. At
300 mg/kg, this was 12 and 14%, respectively. At 1000 mg/kg, this correlated to a minimal to slight degree of centrilobular hepatocyte hypertrophy of the liver of some males and most females. There were no microscopic changes in the liver suggestive of hepatotoxicity. Although centrilobular hepatocyte hypertrophy is commonly seen as an adaptive response associated with the metabolism of xenobiotics or their metabolites, the degree
of liver weight increase at 1000 mg/kg (i.e. notably exceeding a 10% increase from control levels) was considered to reflect an adverse effect. At 300 mg/kg, the liver weight increase was much less pronounced (i.e. just exceeding a 10% increase from control levels), and in combination with absence
of histopathological signs of hepatotoxicity considered not to be adverse. (see below)
- Higher absolute kidney weight and kidney to body weight ratio in males at 1000 mg/kg (22% higher than controls for kidney to body weight ratio).
- Lower thymus weight and thymus to body weight ratio in females at 1000 mg/kg (not statistically significant; 28% lower than controls for thymus to body weight ratio). No histopathological correlates were observed, and hence was considered to be of no toxicological relevance.
The statistically significant higher adrenal to body weight ratio of males at 300 and 1000 mg/kg and the higher ovary weight and ovary to body weight ratio of females at 300 and 1000 mg/kg occurred in the absence of a clear dose-related trend and remains within the range considered normal for rats of this age and strain. The statistically significant higher spleen to body weight ratio of males at 1000 mg/kg was also slight in nature and the mean also remained within normal ranges. The higher spleen weight of females at 100 mg/kg occurred in the absence of a dose-related trend. Moreover, no histopathological correlates were noted to support the above changes. No toxicological significance was therefore ascribed to these changes.

MICROSCOPIC EXAMINATION
There were treatment-related microscopic findings at 1000 mg/kg:
- Liver: Hypertrophy of the hepatocytes in the centrilobular area (minimal) was recorded in 2/5 males and 5/7 females which was considered to be an adaptive response.
- Kidneys (males): Tubular granular casts were present in 4/5 males (minimal-slight). An increased incidence and severity of cortical hyaline droplets was seen in 5/5 males (minimal-slight). The hyaline droplets were considered to represent alpha2µglobulin, a normal protein in male rats which
undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response which is not observed in normal female rats, and higher species of either sex, including humans, therefore not toxicologically relevant.
No abnormalities were seen in the reproductive organs of the suspected non-fertile animals of Group 4 which could account for their infertility. The remaining microscopic findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

REPRODUCTIVE DATA
Females at 1000 mg/kg had lower fertility and conception indices (both 80% as compared to 100% for controls), due to one non-pregnant female, one pregnant female with total litter loss, and one female with a post-implantation loss that was sacrificed in extremis during post-coitum due to haemorrhage from the uterine horn. One of these females showed a low number of corpora lutea and implantation sites. One additional female at 1000 mg/kg showed a low number of implantation sites. Other females of this dose group showed numbers of corpora lutea and implantation sites that were similar to control levels. The mating index and precoital time were unaffected by treatment up to 1000 mg/kg. Although the incidence of lower fertility, conception or gestation index, along with a lower number of corpora lutea and/or implantation sites may individually occur within a study, it was considered that the combined occurrence within this study and within the same group reflected a treatment related effect on reproductive performance.

GESTATION
The gestation index was low for females at 1000 mg/kg (87.5%, opposed to 100% for the control group) due to one female with post-implantation loss that was sacrificed in extremis. The duration of gestation was unaffected by treatment up to 1000 mg/kg.

PARTURITION/MATERNAL CARE
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.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) and presence of renal granular casts (m).
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effect on reproductive performance: lower female fertility, conception, and gestation index, decreased corpora lutea and/or implantation sites.
Remarks on result:
other: Generation: Reproduction (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
EARLY POSTNATAL PUP DEVELOPMENT
The number of dead pups and the number of litters with dead pups at first litter check was higher for females at 1000 mg/kg compared to controls. In total there were 11 pups out of 4 litters that were dead for the high dose females, whereas no pups were found dead at the first litter check for controls. There was also a corresponding reduction in the mean number of living pups per litter for high dose females (6.3) compared to controls (11.6). Additionally, the postnatal loss was significantly higher compared to controls. There were 6 pups from 3 litters, representing 13.6% of living pups, lost at 1000 mg/kg compared to 1 pup from 1 litter, which corresponds to 0.9% of living pups, for controls. Taken together, this resulted in a correspondingly low viability index for females at 1000 mg/kg (86.4% compared to 99.1% for controls). The ratio of male to female pups was lower at 100 mg/kg, but because this occurred in the absence of a treatment related distribution, it was not considered to be related to treatment. The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings up to 300 mg/kg, see below.

MORTALITY PUPS
Two pups at 100 mg/kg and seventeen pups at 1000 mg/kg were found dead or were missing during the first days of lactation. Missing pups were most likely cannibalized. One female at 1000 mg/kg that did not deliver had most likely cannibalised its pups, since this female had a normal number of implantation sites and showed body weight development that was normal for a pregnant female. Although parental effects may have affected pup development to some extent, it could not be excluded that these findings represented primary developmental toxicity.
Conversely, there were no pups in the control or 300 mg/kg groups that were found dead or went missing.

CLINICAL SIGNS PUPS
Incidental clinical symptoms of pups consisted of small size. Small, no milk, and/or dehydrated were occasionally noted for some pups on the day(s) prior to when they went missing. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT PUPS
At 1000 mg/kg, body weights of the sexes combined were significantly lower than controls on Day 1 of lactation. This difference to control values had resolved by lactation Day 4.

MACROSCOPY PUPS
Incidental macroscopic findings of pups that were found dead included small size, no milk in the stomach, and cannibalism of the thoracic and abdominal organs. Incidental macroscopic findings noted for surviving pups included small size. The nature and incidence of these findings remained
within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Conclusions:
In an OECD 422/421 oral repeated dose, reproduction/developmental screening study, a reproduction NOAEL of 300 mg/kg bw/day was determined based on effects on reproductive performance at 1000 mg/kg bw/day: lower female fertility, conception, and gestation index, decreased corpora lutea and/or implantation sites.
Executive summary:

CAT-ACID was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 30 -44 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature. Parental toxicity revealed at 1000 mg/kg bw/day, which consisted of higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) and renal granular casts (m). Indications of reproductive toxicity were also noted at 1000 mg/kg bw/day consisting of a lower fertility, conception and gestation indices, lower number of corpora lutea and/or implantation sites. The nature/severity of parental toxicity suggests that this reproductive toxicity is of a direct nature, rather than of an indirect nature. Therefore, a parental and reproductive NOAEL of 300 mg/kg bw/day were derived.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2010 to 28 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3550
Principles of method if other than guideline:
In addition, the procedures in this study essentially conformed to the following guidelines.

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: weight range at start of treatment was 291-323 gr (males) and 181-217 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (ad libitum): Tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 4 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.3
- Humidity (%): 37-73
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on oral exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036) and test substance (0.98).

Storage conditions of formulations: at ambient temperature

Justification for use and choice of vehicle: based on trial formulations performed at NOTOX

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase according to a validated method (NOTOX Project 495403). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of
the Group 1 formulation prepared for use during treatment. It was not considered to derive from the formulation since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected. The maximum contribution to the other samples was 0.12% based on peak area.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Group 2 and Group 4 formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 30-44 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Pups were only treated indirectly. Three females of group 1 and one female of group 4 were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study (NOTOX 495417, See attached results).

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, at least 1-2 hours after dosing (on the peak period of anticipated effects after dosing), and if considered relevant also beyond this period. At weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. To monitor the health status, additional body weight measurements were performed for all females on Day 12 of study (data are retained in the raw data, but are not reported).

FOOD CONSUMPTION: Yes.
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : Yes
Quantitative assessment of water consumption was performed daily from Day 13 of the premating period onwards.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation period (all before blood sampling).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test (recording period: 1-hour under normal laboratory light conditions, using a computerised monitoring system; Kinder Scientific LLC, Poway, USA).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males and 5 females/group were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7
Females which failed to deliver (2): Post-coitum days 25 (females with evidence of mating)
Females with total litter loss (3): within 24 hours of liter loss
Euthanized in extremis (1): when pain, distress or discomfort is considered not tranisent in nature or is likely to become more severe.
Males: following completion of the mating period (minimum of 28 days)

Samples of the following tissues and organs were collected:
- Selected 5 animals/sex/group and all animals that were killed in extremis:
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Caecum, Pituitary gland, Cervix, Preputial gland, Clitoral gland, Prostate gland, Colon, Rectum, Coagulation gland, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, (Eyes with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar-, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, (Larynx), Thyroid including parathyroid (if detectable),(Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung (infused with formalin), Urinary bladder, Lymph nodes -mandibular, mesenteric-, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions

Tissues/organs mentioned in parentheses in the above list were not examined by the pathologist, as there were no signs of toxicity or target organ involvement.

- All remaining animals, females which failed to deliver and females with total litter loss:
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes, Epididymides, Mammary gland area, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions


ORGAN WEIGHTS
- Selected 5 animals/sex/group:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY: Yes
All organs and tissues of Groups 1 and 4, all gross lesions of all animals (all dose groups), liver and kidneys of groups 2 and 3 males, liver, thymus and mammary gland of groups 2 and 3 females, organs and tissues of animal killed in extremis, additional slides of testes of group 1 and 4 males and all males suspected to be infertiel to examine staging of spermatogenesis. Reproductive organs of all surviving animals that failed to mate, conceive, sire or deliver healthy pups were checked.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
One female at 1000 mg/kg was sacrificed in extremis on Day 14 of the post-coitum phase probably caused by a haemorrhage in the right uterine horn. Three other females at 1000 mg/kg were scheduled to be sacrificed within 24 hours after total litter loss, and hence were not directly related to treatment with the test substance. No cause of a total litter loss was found histopathologically in these females.
One of the females of this dose group with total litter loss was also noted with dark red contents in the vagina (most probably representing vaginal bleeding). In combination with the reproductive effects (described under reproduction), it was considered that this death was related to treatment with the test substance.

CLINICAL SIGNS
At 1000 mg/kg, all females were noted with hunched posture, piloerection and uncoordinated movements primarily during the premating period, and at lower incidence lean appearance, lethargy and flat posture by some females. Piloerection was also shown by several females at 300 mg/kg
towards the end of their pregnancy. Uncoordinated movements were noted by four males at 1000 mg/kg during the premating period.
Incidental findings that were noted included chromodacryorrhoea, scabs, alopecia, salivation and rales. These findings occurred 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. At the incidence
observed, these were considered to be signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test
period.

BODY WEIGHTS
During the last few days of the post-coitum phase, a slightly lower body weight gain was recorded for three females at 1000 mg/kg. This resulted in a statistically significant lower mean body weight gain.
The weight loss (up to 10% from Day 1 values) noted for three females at 1000 mg/kg between Days 1 and 8 of the pre-mating period, was of a temporary nature and had recovered to control values towards the end of the premating period. Hence, no toxicological relevance was ascribed to this finding.

FOOD CONSUMPTION
A slightly lower absolute and relative food consumption (statistically significant) was noted for females at 1000 mg/kg over Days 1-4 of the lactation period.
Food consumption of females at 1000 mg/kg also appeared lower during week 1 of the premating period, which corresponded to the lower body weights in that period. Since this was a temporary and slight change, this was considered to be without toxicological relevance.

WATER CONSUMPTION
Water consumption was significantly higher for females at 300 and 1000 mg/kg during the premating and post coitum period, and to a lesser extent during the lactation phase (not always statistically significant). At 1000 mg/kg, mean over mean values were 105%, 88% and 18% higher than controls
during the premating, post coitum and lactation phase respectively. At 300 mg/kg, mean over mean values were 43%, 47% and 20% higher than controls during the premating, post coitum and lactation phase, respectively. However, no histopathological kidney findings were noted in these females. Therefore, this higher water intake could not be ascribed to renal effects with certainty, and at least at 300 mg/kg this finding was considered not to represent an adverse effect. Water consumption was also higher in males at 1000 mg/kg during the premating and mating period, albeit to a lesser extent than females with mean over means being approximately 40% higher than controls in both periods.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. The significantly higher platelet counts and significantly lower prothrombin time (PT) seen for males at 1000 mg/kg were not considered to be toxicologically relevant because they remained within the range
of data considered normal for rats of this age and strain. Also, platelet counts in the control group were considered to be slightly low.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher creatinine level in males at 300 and 1000 mg/kg, suggests that renal filtration was affected. However, in the absence of supportive morphological correlates indicative of renal dysfunction, and since the mean creatinine level only slightly exceeded the range considered normal in these type of rats, this effect was not considered to be adverse in nature.
- Higher cholesterol level in females at 1000 mg/kg (may reflect a higher functional capacity of the liver)
The higher bile acid level in females at 100, 300 and 1000 mg/kg (not statistically significant at 300 mg/kg) occurred in the absence of a dose-related trend and means remained within the range considered normal for rats of this age and strain. The statistically significant higher calcium level in
females at 1000 mg/kg occurred in conjunction with lower control values, and no dose-related trend was apparent. No toxicological significance was therefore ascribed to these changes.

MACROSCOPIC EXAMINATION
At 1000 mg/kg, a reduced size of the thymus was noted for three females. Enlargement of the liver was observed for two females at this dose level.
The one female at 1000 mg/kg sacrificed in extremis was noted with dark red fluid in the right uterine horn. One of the females with total litter loss was noted with one dead fetus in the left uterine horn and 2 dead fetuses in the right uterine horn, and had dark red contents in the vagina.
The black-brown discolouration of the liver noted in two females at 1000 mg/kg and one female at 300 mg/kg had no (treatment-related) histopathological correlates. Other incidental findings among control and treated animals included a yellowish soft nodule on the tail of the left epididymis, tan, reddish or a red-brown focus on or red-brown discoloration of the clitoral gland, reddish focus on the colon, alopecia on the chest and/or genital region, reddish foci on the stomach glandular mucosa or thymus, a watery-clear cyst on the left kidney, and pelvic dilation of the kidneys. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished treated animals from control animals:
- Higher absolute liver weight and liver to body weight ratio in both sexes at 300 and 1000 mg/kg (not statistically significant for absolute weights of males at 300 mg/kg). Liver to body weight ratio was 45% and 30% higher than controls at 1000 mg/kg for males and females, respectively. At
300 mg/kg, this was 12 and 14%, respectively. At 1000 mg/kg, this correlated to a minimal to slight degree of centrilobular hepatocyte hypertrophy of the liver of some males and most females. There were no microscopic changes in the liver suggestive of hepatotoxicity. Although centrilobular hepatocyte hypertrophy is commonly seen as an adaptive response associated with the metabolism of xenobiotics or their metabolites, the degree
of liver weight increase at 1000 mg/kg (i.e. notably exceeding a 10% increase from control levels) was considered to reflect an adverse effect. At 300 mg/kg, the liver weight increase was much less pronounced (i.e. just exceeding a 10% increase from control levels), and in combination with absence
of histopathological signs of hepatotoxicity considered not to be adverse. (see below)
- Higher absolute kidney weight and kidney to body weight ratio in males at 1000 mg/kg (22% higher than controls for kidney to body weight ratio).
- Lower thymus weight and thymus to body weight ratio in females at 1000 mg/kg (not statistically significant; 28% lower than controls for thymus to body weight ratio). No histopathological correlates were observed, and hence was considered to be of no toxicological relevance.
The statistically significant higher adrenal to body weight ratio of males at 300 and 1000 mg/kg and the higher ovary weight and ovary to body weight ratio of females at 300 and 1000 mg/kg occurred in the absence of a clear dose-related trend and remains within the range considered normal for rats of this age and strain. The statistically significant higher spleen to body weight ratio of males at 1000 mg/kg was also slight in nature and the mean also remained within normal ranges. The higher spleen weight of females at 100 mg/kg occurred in the absence of a dose-related trend. Moreover, no histopathological correlates were noted to support the above changes. No toxicological significance was therefore ascribed to these changes.

MICROSCOPIC EXAMINATION
There were treatment-related microscopic findings at 1000 mg/kg:
- Liver: Hypertrophy of the hepatocytes in the centrilobular area (minimal) was recorded in 2/5 males and 5/7 females which was considered to be an adaptive response.
- Kidneys (males): Tubular granular casts were present in 4/5 males (minimal-slight). An increased incidence and severity of cortical hyaline droplets was seen in 5/5 males (minimal-slight). The hyaline droplets were considered to represent alpha2µglobulin, a normal protein in male rats which
undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response which is not observed in normal female rats, and higher species of either sex, including humans, therefore not toxicologically relevant.
No abnormalities were seen in the reproductive organs of the suspected non-fertile animals of Group 4 which could account for their infertility. The remaining microscopic findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) presence of renal granular casts (m), and hypertrophy of hepatocytes (m,f).
Critical effects observed:
not specified
Conclusions:
In an OECD 422/421 oral repeated dose/reproduction screening study, a NOAEL of 300 mg/kg bw/day was determined based on effects at 1000 mg/kg bw/day: higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) presence of renal granular casts (m), and hypertrophy of hepatocytes (m,f).
Executive summary:

CAT-ACID was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 30-44 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature. Parental toxicity revealed at 1000 mg/kg bw/day, which consisted of higher kidney (m) and liver (m,f) weight, higher cholesterol level (f), higher water intake (m,f) and renal granular casts (f). No clear and consistent pattern of occurrence of clinical signs was noted during the inlife phase. Also slight changes in body weight and food intake levels were noted, each during different phases of the treatment period. Functional observation tests did not reveal treatment-related changes. Therefore, a parental NOAEL of 300 mg/kg bw/d was derived.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Health Effects Test Guidelines, OPPTS 870.3550
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures in this study essentially conformed to the following guidelines.

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
EC Number:
619-508-4
Cas Number:
381209-09-2
Molecular formula:
C13 H24 O2
IUPAC Name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
Details on test material:
- Name of test material (as cited in study report): CAT-Acid
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH: 5.4-5.2 (1% in water)
- Density: 0.98 g/ml

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: weight range at start of treatment was 291-323 gr (males) and 181-217 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (ad libitum): Tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 4 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.3
- Humidity (%): 37-73
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
(specific gravity 1.036)
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036) and test substance (0.98).

Storage conditions of formulations: at ambient temperature

Justification for use and choice of vehicle: based on trial formulations performed at NOTOX

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase according to a validated method (NOTOX Project 495403). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of
the Group 1 formulation prepared for use during treatment. It was not considered to derive from the formulation since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected. The maximum contribution to the other samples was 0.12% based on peak area.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Group 2 and Group 4 formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or 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.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 30-44 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Three females of group 1 and one female of group 4 were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Duration of test:
Males: 28 days
Females: 30-44 days
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study (NOTOX 495417, See attached results).

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, at least 1-2 hours after dosing (on the peak period of anticipated effects after dosing), and if considered relevant also beyond this period. At weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. To monitor the health status, additional body weight measurements were performed for all females on Day 12 of study (data are retained in the raw data, but are not reported).

FOOD CONSUMPTION: Yes.
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : Yes
Quantitative assessment of water consumption was performed daily from Day 13 of the premating period onwards.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation period (all before blood sampling).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test (recording period: 1-hour under normal laboratory light conditions, using a computerised monitoring system; Kinder Scientific LLC, Poway, USA).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
SACRIFICE
Pups surviving to planned termination were killed by decapitation on Day 5, 6 or 7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Indices:
Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, all females were noted with hunched posture, piloerection and uncoordinated movements primarily during the premating period, and at lower incidence lean appearance, lethargy and flat posture by some females. Piloerection was also shown by several females at 300 mg/kg
towards the end of their pregnancy. Uncoordinated movements were noted by four males at 1000 mg/kg during the premating period.
Incidental findings that were noted included chromodacryorrhoea, scabs, alopecia, salivation and rales. These findings occurred 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. At the incidence
observed, these were considered to be signs of no toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female at 1000 mg/kg was sacrificed in extremis on Day 14 of the post-coitum phase probably caused by a haemorrhage in the right uterine horn. Three other females at 1000 mg/kg were scheduled to be sacrificed within 24 hours after total litter loss, and hence were not directly related to treatment with the test substance. No cause of a total litter loss was found histopathologically in these females.
One of the females of this dose group with total litter loss was also noted with dark red contents in the vagina (most probably representing vaginal bleeding). In combination with the reproductive effects (described under reproduction), it was considered that this death was related to treatment with the test substance.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During the last few days of the post-coitum phase, a slightly lower body weight gain was recorded for three females at 1000 mg/kg. This resulted in a statistically significant lower mean body weight gain.
The weight loss (up to 10% from Day 1 values) noted for three females at 1000 mg/kg between Days 1 and 8 of the pre-mating period, was of a temporary nature and had recovered to control values towards the end of the premating period. Hence, no toxicological relevance was ascribed to this finding.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slightly lower absolute and relative food consumption (statistically significant) was noted for females at 1000 mg/kg over Days 1-4 of the lactation period.
Food consumption of females at 1000 mg/kg also appeared lower during week 1 of the premating period, which corresponded to the lower body weights in that period. Since this was a temporary and slight change, this was considered to be without toxicological relevance.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was significantly higher for females at 300 and 1000 mg/kg during the premating and post coitum period, and to a lesser extent during the lactation phase (not always statistically significant). At 1000 mg/kg, mean over mean values were 105%, 88% and 18% higher than controls
during the premating, post coitum and lactation phase respectively. At 300 mg/kg, mean over mean values were 43%, 47% and 20% higher than controls during the premating, post coitum and lactation phase, respectively. However, no histopathological kidney findings were noted in these females. Therefore, this higher water intake could not be ascribed to renal effects with certainty, and at least at 300 mg/kg this finding was considered not to represent an adverse effect. Water consumption was also higher in males at 1000 mg/kg during the premating and mating period, albeit to a lesser extent than females with mean over means being approximately 40% higher than controls in both periods.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The significantly higher platelet counts and significantly lower prothrombin time (PT) seen for males at 1000 mg/kg were not considered to be toxicologically relevant because they remained within the range of data considered normal for rats of this age and strain. Also, platelet counts in the control group were
considered to be slightly low.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher creatinine level in males at 300 and 1000 mg/kg,
- Higher cholesterol level in females at 1000 mg/kg.
The higher bile acid level in females at 100, 300 and 1000 mg/kg (not statistically significant at 300 mg/kg) occurred in the absence of a dose-related trend and means remained within the range considered normal for rats of this age and strain. The statistically significant higher calcium level in females at 1000 mg/kg occurred in conjunction with lower control values, and no dose-related trend was apparent. No toxicological significance was therefore ascribed to these changes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished treated animals from control animals:
- Higher absolute liver weight and liver to body weight ratio in both sexes at 300 and 1000 mg/kg (not statistically significant for absolute weights of males at 300 mg/kg). Liver to body weight ratio was 45% and 30% higher than controls at 1000 mg/kg for males and females, respectively. At
300 mg/kg, this was 12 and 14%, respectively. At 1000 mg/kg, this correlated to a minimal to slight degree of centrilobular hepatocyte hypertrophy of the liver of some males and most females. There were no microscopic changes in the liver suggestive of hepatotoxicity. Although centrilobular hepatocyte hypertrophy is commonly seen as an adaptive response associated with the metabolism of xenobiotics or their metabolites, the degree
of liver weight increase at 1000 mg/kg (i.e. notably exceeding a 10% increase from control levels) was considered to reflect an adverse effect. At 300 mg/kg, the liver weight increase was much less pronounced (i.e. just exceeding a 10% increase from control levels), and in combination with absence
of histopathological signs of hepatotoxicity considered not to be adverse. (see below)
- Higher absolute kidney weight and kidney to body weight ratio in males at 1000 mg/kg (22% higher than controls for kidney to body weight ratio).
- Lower thymus weight and thymus to body weight ratio in females at 1000 mg/kg (not statistically significant; 28% lower than controls for thymus to body weight ratio). No histopathological correlates were observed, and hence was considered to be of no toxicological relevance.
The statistically significant higher adrenal to body weight ratio of males at 300 and 1000 mg/kg and the higher ovary weight and ovary to body weight ratio of females at 300 and 1000 mg/kg occurred in the absence of a clear dose-related trend and remains within the range considered normal for rats of this age and strain. The statistically significant higher spleen to body weight ratio of males at 1000 mg/kg was also slight in nature and the mean also remained within normal ranges. The higher spleen weight of females at 100 mg/kg occurred in the absence of a dose-related trend. Moreover, no histopathological correlates were noted to support the above changes. No toxicological significance was therefore ascribed to these changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, a reduced size of the thymus was noted for three females. Enlargement of the liver was observed for two females at this dose level.
The one female at 1000 mg/kg sacrificed in extremis was noted with dark red fluid in the right uterine horn. One of the females with total litter loss was noted with one dead fetus in the left uterine horn and 2 dead fetuses in the right uterine horn, and had dark red contents in the vagina.
The black-brown discolouration of the liver noted in two females at 1000 mg/kg and one female at 300 mg/kg had no (treatment-related) histopathological correlates. Other incidental findings among control and treated animals included a yellowish soft nodule on the tail of the left epididymis, tan, reddish or a red-brown focus on or red-brown discoloration of the clitoral gland, reddish focus on the colon, alopecia on the chest and/or genital region, reddish foci on the stomach glandular mucosa or thymus, a watery-clear cyst on the left kidney, and pelvic dilation of the kidneys. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These findings were therefore considered to be of no toxicological relevance.
Description (incidence and severity):
There were treatment-related microscopic findings at 1000 mg/kg:
- Liver: Hypertrophy of the hepatocytes in the centrilobular area (minimal) was recorded in 2/5 males and 5/7 females which was considered to be an adaptive response.
- Kidneys (males): Tubular granular casts were present in 4/5 males (minimal-slight). An increased incidence and severity of cortical hyaline droplets was seen in 5/5 males (minimal-slight). The hyaline droplets were considered to represent alpha2µglobulin, a normal protein in male rats which
undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response which is not observed in normal female rats, and higher species of either sex, including humans, therefore not toxicologically relevant.
No abnormalities were seen in the reproductive organs of the suspected non-fertile animals of Group 4 which could account for their infertility. The remaining microscopic findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Maternal developmental toxicity

Other effects:
effects observed, treatment-related
Description (incidence and severity):
REPRODUCTIVE DATA
Females at 1000 mg/kg had lower fertility and conception indices (both 80% as compared to 100% for controls), due to one non-pregnant female, one pregnant female with total litter loss, and one female with a post-implantation loss that was sacrificed in extremis during post-coitum due to haemorrhage from the uterine horn. One of these females showed a low number of corpora lutea and implantation sites. One additional female at 1000 mg/kg showed a low number of implantation sites. Other females of this dose group showed numbers of corpora lutea and implantation sites that were similar to control levels. The mating index and precoital time were unaffected by treatment up to 1000 mg/kg. Although the incidence of lower fertility, conception or gestation index, along with a lower number of corpora lutea and/or implantation sites may individually occur within a study, it was considered that the combined occurrence within this study and within the same group reflected a treatment related effect on reproductive performance.

GESTATION
The gestation index was low for females at 1000 mg/kg (87.5%, opposed to 100% for the control group) due to one female with post-implantation loss that was sacrificed in extremis. The duration of gestation was unaffected by treatment up to 1000 mg/kg.

PARTURITION/MATERNAL CARE
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.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
One female at 1000 mg/kg was sacrificed in extremis on Day 14 of the post-coitum phase probably caused by a haemorrhage in the right uterine horn. Three other females at 1000 mg/kg were scheduled to be sacrificed within 24 hours after total litter loss, and hence were not directly related to treatment with the test substance. No cause of a total litter loss was found histopathologically in these females.
One of the females of this dose group with total litter loss was also noted with dark red contents in the vagina (most probably representing vaginal bleeding). In combination with the reproductive effects (described under reproduction), it was considered that this death was related to treatment with the test substance.

CLINICAL SIGNS
At 1000 mg/kg, all females were noted with hunched posture, piloerection and uncoordinated movements primarily during the premating period, and at lower incidence lean appearance, lethargy and flat posture by some females. Piloerection was also shown by several females at 300 mg/kg
towards the end of their pregnancy. Uncoordinated movements were noted by four males at 1000 mg/kg during the premating period.
Incidental findings that were noted included chromodacryorrhoea, scabs, alopecia, salivation and rales. These findings occurred 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. At the incidence
observed, these were considered to be signs of no toxicological relevance.

BODY WEIGHTS
During the last few days of the post-coitum phase, a slightly lower body weight gain was recorded for three females at 1000 mg/kg. This resulted in a statistically significant lower mean body weight gain.
The weight loss (up to 10% from Day 1 values) noted for three females at 1000 mg/kg between Days 1 and 8 of the pre-mating period, was of a temporary nature and had recovered to control values towards the end of the premating period. Hence, no toxicological relevance was ascribed to this finding.

FOOD CONSUMPTION
A slightly lower absolute and relative food consumption (statistically significant) was noted for females at 1000 mg/kg over Days 1-4 of the lactation period.
Food consumption of females at 1000 mg/kg also appeared lower during week 1 of the premating period, which corresponded to the lower body weights in that period. Since this was a temporary and slight change, this was considered to be without toxicological relevance.

WATER CONSUMPTION
Water consumption was significantly higher for females at 300 and 1000 mg/kg during the premating and post coitum period, and to a lesser extent during the lactation phase (not always statistically significant). At 1000 mg/kg, mean over mean values were 105%, 88% and 18% higher than controls
during the premating, post coitum and lactation phase respectively. At 300 mg/kg, mean over mean values were 43%, 47% and 20% higher than controls during the premating, post coitum and lactation phase, respectively. However, no histopathological kidney findings were noted in these females. Therefore, this higher water intake could not be ascribed to renal effects with certainty, and at least at 300 mg/kg this finding was considered not to represent an adverse effect. Water consumption was also higher in males at 1000 mg/kg during the premating and mating period, albeit to a lesser extent than females with mean over means being approximately 40% higher than controls in both periods.

MACROSCOPIC EXAMINATION
At 1000 mg/kg, a reduced size of the thymus was noted for three females. Enlargement of the liver was observed for two females at this dose level.
The one female at 1000 mg/kg sacrificed in extremis was noted with dark red fluid in the right uterine horn. One of the females with total litter loss was noted with one dead fetus in the left uterine horn and 2 dead fetuses in the right uterine horn, and had dark red contents in the vagina.
The black-brown discolouration of the liver noted in two females at 1000 mg/kg and one female at 300 mg/kg had no (treatment-related) histopathological correlates. Other incidental findings among control and treated animals included a yellowish soft nodule on the tail of the left epididymis, tan, reddish or a red-brown focus on or red-brown discoloration of the clitoral gland, reddish focus on the colon, alopecia on the chest and/or genital region, reddish foci on the stomach glandular mucosa or thymus, a watery-clear cyst on the left kidney, and pelvic dilation of the kidneys. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished treated animals from control animals:
- Higher absolute liver weight and liver to body weight ratio in both sexes at 300 and 1000 mg/kg (not statistically significant for absolute weights of males at 300 mg/kg). Liver to body weight ratio was 45% and 30% higher than controls at 1000 mg/kg for males and females, respectively. At
300 mg/kg, this was 12 and 14%, respectively. At 1000 mg/kg, this correlated to a minimal to slight degree of centrilobular hepatocyte hypertrophy of the liver of some males and most females. There were no microscopic changes in the liver suggestive of hepatotoxicity. Although centrilobular hepatocyte hypertrophy is commonly seen as an adaptive response associated with the metabolism of xenobiotics or their metabolites, the degree
of liver weight increase at 1000 mg/kg (i.e. notably exceeding a 10% increase from control levels) was considered to reflect an adverse effect. At 300 mg/kg, the liver weight increase was much less pronounced (i.e. just exceeding a 10% increase from control levels), and in combination with absence
of histopathological signs of hepatotoxicity considered not to be adverse. (see below)
- Higher absolute kidney weight and kidney to body weight ratio in males at 1000 mg/kg (22% higher than controls for kidney to body weight ratio).
- Lower thymus weight and thymus to body weight ratio in females at 1000 mg/kg (not statistically significant; 28% lower than controls for thymus to body weight ratio). No histopathological correlates were observed, and hence was considered to be of no toxicological relevance.
The statistically significant higher adrenal to body weight ratio of males at 300 and 1000 mg/kg and the higher ovary weight and ovary to body weight ratio of females at 300 and 1000 mg/kg occurred in the absence of a clear dose-related trend and remains within the range considered normal for rats of this age and strain. The statistically significant higher spleen to body weight ratio of males at 1000 mg/kg was also slight in nature and the mean also remained within normal ranges. The higher spleen weight of females at 100 mg/kg occurred in the absence of a dose-related trend. Moreover, no histopathological correlates were noted to support the above changes. No toxicological significance was therefore ascribed to these changes.

MICROSCOPIC EXAMINATION
There were treatment-related microscopic findings at 1000 mg/kg:
- Liver: Hypertrophy of the hepatocytes in the centrilobular area (minimal) was recorded in 2/5 males and 5/7 females which was considered to be an adaptive response.
- Kidneys (males): Tubular granular casts were present in 4/5 males (minimal-slight). An increased incidence and severity of cortical hyaline droplets was seen in 5/5 males (minimal-slight). The hyaline droplets were considered to represent alpha2µglobulin, a normal protein in male rats which
undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response which is not observed in normal female rats, and higher species of either sex, including humans, therefore not toxicologically relevant.
No abnormalities were seen in the reproductive organs of the suspected non-fertile animals of Group 4 which could account for their infertility. The remaining microscopic findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of dead pups and the number of litters with dead pups at first litter check was higher for females at 1000 mg/kg compared to controls. In total there were 11 pups out of 4 litters that were dead for the high dose females, whereas no pups were found dead at the first litter check for controls.
There was also a corresponding reduction in the mean number of living pups per litter for high dose females (6.3) compared to controls (11.6). Additionally, the Taken together, this resulted in a correspondingly low viability index for females at 1000 mg/kg (86.4% compared to 99.1% for controls).
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The ratio of male to female pups was lower at 100 mg/kg, but because this occurred in the absence of a treatment related distribution, it was not considered to be related to treatment.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, body weights of the sexes combined were significantly lower than controls on Day 1 of lactation. This difference to control values had resolved by lactation Day 4.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
postnatal loss was significantly higher compared to controls. There were 6 pups from 3 litters, representing 13.6% of living pups, lost at 1000 mg/kg compared to 1 pup from 1 litter, which corresponds to 0.9% of living pups, for controls.
External malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: lower viability index

Details on embryotoxic / teratogenic effects:
EARLY POSTNATAL PUP DEVELOPMENT
The number of dead pups and the number of litters with dead pups at first litter check was higher for females at 1000 mg/kg compared to controls. In total there were 11 pups out of 4 litters that were dead for the high dose females, whereas no pups were found dead at the first litter check for controls. There was also a corresponding reduction in the mean number of living pups per litter for high dose females (6.3) compared to controls (11.6). Additionally, the postnatal loss was significantly higher compared to controls. There were 6 pups from 3 litters, representing 13.6% of living pups, lost at 1000 mg/kg compared to 1 pup from 1 litter, which corresponds to 0.9% of living pups, for controls. Taken together, this resulted in a correspondingly low viability index for females at 1000 mg/kg (86.4% compared to 99.1% for controls). The ratio of male to female pups was lower at 100 mg/kg, but because this occurred in the absence of a treatment related distribution, it was not considered to be related to treatment. The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings up to 300 mg/kg, see below.

MORTALITY PUPS
Two pups at 100 mg/kg and seventeen pups at 1000 mg/kg were found dead or were missing during the first days of lactation. Missing pups were most likely cannibalized. One female at 1000 mg/kg that did not deliver had most likely cannibalised its pups, since this female had a normal number of implantation sites and showed body weight development that was normal for a pregnant female. Although parental effects may have affected pup development to some extent, it could not be excluded that these findings represented primary developmental toxicity.
Conversely, there were no pups in the control or 300 mg/kg groups that were found dead or went missing.

CLINICAL SIGNS PUPS
Incidental clinical symptoms of pups consisted of small size. Small, no milk, and/or dehydrated were occasionally noted for some pups on the day(s) prior to when they went missing. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT PUPS
At 1000 mg/kg, body weights of the sexes combined were significantly lower than controls on Day 1 of lactation. This difference to control values had resolved by lactation Day 4.

MACROSCOPY PUPS
Incidental macroscopic findings of pups that were found dead included small size, no milk in the stomach, and cannibalism of the thoracic and abdominal organs. Incidental macroscopic findings noted for surviving pups included small size. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
In an OECD 422/421 oral repeated dose, reproduction/developmental screening study, a NOAEL of 300 mg/kg bw/day was determined based on an increased post natal loss and an increased number of dead pups at 1000 mg/kg bw/day.
Executive summary:

CAT-ACID was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 30 -44 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature. Parental toxicity revealed at 1000 mg/kg bw/day, which consisted of higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) and renal granular casts (m). Indications of developmental toxicity were also noted at 1000 mg/kg bw/day consisting of an increased number of dead pups and number of litters with dead pups, reduction in mean number of living pups per litter and a significantly higher postnatal loss, resulting in a low viability index. The nature/severity of parental toxicity suggests that this developmental toxicity is of a direct nature, rather than of an indirect nature. Therefore, a parental and developmental NOAEL of 300 mg/kg bw/day were derived.