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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-JUL-14 to TBD
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This data has not been finalised by LOA due to an ongoing review. The data presented in this record is from an audited draft report by the contract research organisation.
A communication from the contract research organisation stating when the finalisation of the report will occur by (31st January 2023) is included below in the "attached justification" section. LOA is committed to performing a dossier update when the full information required for ECHA to judge the read-across approach utilised in this dossier is available.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version: July 2016
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions. Please see 'Any other information on materials and methods' for details.
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
N/A
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: To be confirmed; Batch number: 10-11-2021
- Purity, including information on contaminants, isomers, etc.: Reaction mass of ethylbenzene and mxylene (ethylbenzene: 14.84 % m/m; p-xylene: 3.98 % m/m; m-xylene: 74.63 % m/m; o-xylene: 5.96 % m/m)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in corn oil vehicle for at least 24 hours at room temperature under normal laboratory light over a concentration range of 2 to 250 mg/mL (solutions); Stable in corn oil vehicle for at least 9 days in the refrigerator over a concentration range of 2 to 250 mg/mL (solutions)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS
- Specific gravity / density: To be confirmed
- Expiry date: 2026-MAR-11
- EC Number: 905-570-2
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: Males: 11-12 weeks; Females: 14-15 weeks
- Weight at study initiation: Males: 283 - 327 g; Females: 212 - 269 g
- Housing:

On arrival and during the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrol on, MIV type, height 18 cm).

During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.

During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

- Diet: pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH (Soest, Germany ) ad libitum
- Water: Municipal tap water via water bottles ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, based on the results of these analyses it was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 22°C
- Humidity (%): 46 to 68%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 2021-JUL-14 To: 2021-OCT-12
Route of administration:
oral: gavage
Remarks on MMAD:
N/A
Vehicle:
corn oil
Remarks:
Sigma-Aldrich (Steinheim, Germany)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Sigma-Aldrich, Steinheim, Germany) was used as the vehicle based on trial preparations performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of
this study and were not used for dosing.
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg bw/day dose groups, respectively.
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated.
- Any other deviations from standard protocol: Detection of mating was not confirmed in first instance for Female No. 59 (100 mg/kg/day). Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During Week 1 and 3 of treatment, dose formulation samples were collected for analysis as follows:

1. Concentration analysis: sample collected from approximately ‘Middle’ for all dose groups

2. Homogeneity analysis: sample collected from approximately ‘Top, Middle and Bottom’ for dose groups 2 and 4 only.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20275969).

For concentration: mean sample concentration results within or equal to ± 15% viscous solutions of theoretical concentration.

For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20275969) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily
Details on study schedule:
N/A
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle: Corn Oil) - Group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder study with oral gavage administration of Reaction mass of ethylbenzene and m-xylene in rats (Test Facility Study No. 20275971), and in an attempt to produce graded responses to the test material.

- Rationale for animal assignment (if not random): A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the Weeks 2 and 3 of the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

- Fasting period before blood sampling for clinical biochemistry: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No

- Dose range finding studies: 10-day Dose Range Finder study with oral gavage administration of Reaction mass of ethylbenzene and m-xylene in rats (Test Facility Study No. 20275971).
Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once daily, beginning during the first administration of the test material and lasting throughout the Dosing periods up to the day prior to necropsy. Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

All animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Except on days of receipt and necropsy where frequency was at least once daily. Arena observations were also conducted once before the first administration of the test material and weekly during the treatment Period.

BODY WEIGHT: Yes
- Time schedule for examinations:
for all animals on Day 1 of treatment (prior to dosing ) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; quantitatively measured per cage weekly, except for males and females which were housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Monitored regularly throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane); Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals:
5/sex/group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals:
5/sex/group
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid hormones)
- Time of blood sample collection:
On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals:
5/sex/group
- Parameters examined included Triiodothyronine (T3); Thyroxine (T4); and Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Functional tests were performed post dosing on 5 males per group during Week 4 of treatment and 5 females per group during the last week of lactation (i.e. PND 8-10).
- Dose groups that were examined:
All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity such as Hearing ability; Fore- and hind-limb grip strength; and Locomotor activity

IMMUNOLOGY: No

OTHER: Coagulation:
- Time schedule for collection of blood: On the day of scheduled necropsy
- How many animals: 5 animals/sex/group were selected
- Animals fasted: Males fasted overnight (maximum of 24 hours), females non-fasted
- Anaesthetic used for blood collection: Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.45 mL
- Parameters analysed: prothrombin time (PT), activated partial thromboplastin time (APTT)
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Daily vaginal lavage was performed for all females beginning 21 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis.
Sperm parameters (parental animals):
For the testes of all selected males of Group 1 (control) and Group 4 (1000 mg/kg bw/day), and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Pups were identified on postnatal Day (PND) 1, randomized per litter, and individually identified by means of subcutaneous injection of Indian ink
- All pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done.

Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable. Pups were examined for external and internal abnormalities and possible cause of death was determined for pups born or found dead].

Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination

Unscheduled Euthanasia
Recognizable fetuses of females that were euthanized in extremis (No. 72) were examined externally and sexed (both externally and internally). Live fetuses were euthanized by decapitation. Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).

Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible.

All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (see Tables 4 and 5)

Unscheduled Euthanasia
Females with total litter loss (Nos. 73 and 75)were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated within 24 hours after the last pup was found dead or missing. They underwent necropsy, and specified tissues were retained and weighed

If necessary for humane reasons, animals were deeply anesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:

Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16
Females which failed to deliver: With evidence of mating: Post-coitum Day 25-26 (Nos. 49, 52, and 77).

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy.

Necropsy:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

The organs listed in Table 4 and 5 were weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see Tables 6 and 7)

Representative samples of the tissues identified in the tables 6 and 7 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. The tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Alpha-2u-globulin staining:
Two slides per selected Group 1 and Group 4 male were shipped to CRL-Durham for HRP-polymer based chromogenic immunostaining of alpha-2u-globulin. The slides were returned to the Test Facility after the staining procedure had been completed for evaluation by the study pathologist.

Microscopic Evaluation:
All tissues as defined under Histology were examined by a board-certified toxicological pathologist. For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The slides stained for alpha-2u-globulin were evaluated by the study pathologist.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination

Unscheduled Euthanasia
Recognizable fetuses of females that were euthanized in extremis (No. 72) were examined externally and sexed (both externally and internally). Live fetuses were euthanized by decapitation. Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).

Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible.

All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Reproductive indices:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

1. Mating index (%) = (number of females mated / number of females paired) x 100

2. Precoital time = number of days between initiation of cohabitationand confirmation of mating

3. Fertility index (%): (number of pregnant females / number of females mated) x 100

4. Gestation index (%) = (number of females with living pups on Day 1/ number of pregnant females) x 100

5. Duration of gestation = number of days between confirmation of mating and the beginning of parturition

6. Post-implantation survival index (%) = (total number of offspring born / total number of uterine implantation sites) x 100

7. Live birth index (%) = (number of live offspring on Day 1 after littering / total number of offspring born) x 100

8. Percent 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

9. Percent 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

10. Lactation index (%) = (number of live offspring on Day 13 after littering / number of live offsping on Day 4 (after culling)) x 100

11. Cumulative Survival Index = ((number of live offspring on Day 13 / number of live offspring on Day 4 after culling) x (number of live offspring on Day 4 before culling / total number of offspring born)) x 100
Offspring viability indices:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

1. Viability index 1 (%) = (number of live offspring on Day 4 before culling / number of live offspring on Day 1 after litterling) x 100

2. Viability Index 2 (%) = (number of live offspring on Day 7 before culling / number of live offspring on Day 4 after litterling) x 100

3. Viability Index 3 (%) = (number of live offspring on Day 13 before culling / number of live offspring on Day 7 after litterling) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed during daily detailed clinical observations or during weekly arena observations in males and females.

Salivation observed post dosing, starting on Days 19 (males) or 36 (females) at 300 mg/kg/day and on Days 4 (males) or 11 (females) at 1000 mg/kg/day of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Incidental findings that were observed included scabs at the shoulder, alopecia and swelling of the vagina. 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 not to be signs of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One premature death (female No. 72) was observed in the study at 1000 mg/kg/day. The animal was sacrificed in extremis on post-coitum Day 21 due to acute severe clinical observations indicating a poor condition, i.e. lethargy and a pale skin (both at moderate degree), hunched posture, deep respiration, piloerection. Macroscopic observations at necropsy revealed a thymus reduced in size; the uterus of this female contained thirteen normally developed live fetuses (11 males and 2 females) and two early resorptions. Major microscopic findings were present in the glandular stomach and duodenum in the form of multiple ulcers (up to moderate) and associated inflammation (granulocytic, up to slight). Furthermore, several lymphoid organs showed lymphocyte apoptosis/necrosis (up to slight) and/or decreased cellularity (up to marked, correlating in the thymus with a reduced size). The lesions of the stomach and duodenum and the most likely secondary findings of the lymphoid organs were regarded to be related with the moribundity of this female. Since this moribundity occurred in a single 1000 mg/kg/day group female and since the combination of stomach and duodenum findings were acute to subacute in this female and absent in any other animal, these findings were regarded unrelated to test material treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes in body weight and body weight gain were observed up to dose levels of 300 mg/kg/day.

In males at 1000 mg/kg/day, reduced body weight gain was observed on Days 22 and 29 of the dosing period, which resulted in a 5% lower absolute mean body weight compared to control at the end of the dosing period (Day 29).

In females at 1000 mg/kg/day, reduced body weight gain was noted during lactation (statistically significant on Day 13 of lactation only), which resulted in a 6% lower mean body weight at the end of the dosing period (Day 13 of lactation; not statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption (before or after correction for body weight) were recorded up to 1000 mg/kg/day in male rats and up to 300 mg/kg/day in female rats.

In males at 1000 mg/kg/day, absolute and relative food consumption were slightly decreased over Days 1 8 of the dosing period (10% and 8% lower compared to control; not statistically significant), which subsequently recovered. As this finding was transient it was considered of no toxicological relevance.

In females at 1000 mg/kg/day, relative food consumption was slightly decreased over Days 1 8 of the dosing period (11% compared to control; not statistically significant), which subsequently recovered. In addition, both absolute and relative food consumption were decreased during lactation (up to 17% and 13% lower than control at the end of the dosing period, respectively, and not statistically significant).
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were unaffected by treatment with the test material.

The decreased hemoglobin concentration (HGB) observed in females at 100 and 300 mg/kg/day (0.92 and 0.94x of control, respectively) and the decrease in mean corpuscular hemoglobin concentration (MCHC) in females at 100, 300 and 1000 mg/kg/day (0.97, 0.97 and 0.98x, respectively; not always statistically significant) were considered unrelated to test material administration due to the absence of a dose response and the minimal magnitude of the change.

Other non-statistically significant changes observed in several white blood cell parameters (both sexes) were considered unrelated to test material administration due to the minimal magnitude of the change, high variation in individual values within the groups, and/or absence of a dose response.

Coagulation parameters of treated rats were considered to be unaffected by treatment. The shorter prothrombin time (PT) observed in males at 300 mg/kg/day (0.93x of control) and in females at 100 and 1000 mg/kg/day (each 0.94x of control) was considered unrelated to treatment as no dose response was observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical biochemistry parameters in female rats up to 300 mg/kg/day.

Increased alkaline phosphatase (ALP) activity was observed in male rats at 100, 300 and 1000 mg/kg/day (1.14, 1.20 and 1.27x of control, respectively; not statistically significant). Increased potassium (K) levels were observed in male rats at 1000 mg/kg/day (1.18x) and increased creatinine concentrations (CREAT) were observed in female rats at 300 and 1000 mg/kg/day (1.17 and 1.27x; not statistically significant at 300 mg/kg/day).

Increased bile acids (BILEAC) and decreased total bilirubin (TBIL) and inorganic phosphate (PHOS) levels were also observed in female rats at 100, 300 and 1000 mg/kg/day and considered related to relatively low (bile acids) or high (total bilirubin and inorganic phosphate) in the control group. Therefore, these were considered to not be toxicologically relevant.

Any other changes in clinical biochemistry parameters were considered to be unrelated to test material administration due to the minimal magnitude, variation in direction of change and/or absence of a dose-related response.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Decreased serum levels of T3, total T4 and TSH were noted in males at 1000 mg/kg/day (0.86, 0.87 and 0.70x, respectively; not statistically significant). At the individual level, 1/10 values for T3, 2/10 values for T4 and 1/10 values for TSH were below the range of the controls. These findings were regarded unrelated to treatment due to lack of a dose relationship and because individual values showed great overlap.

Any differences in mean levels of T3, total T4 and TSH in treated females compared to controls were considered to be unrelated to treatment, as no dose relation was evident.
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In male rats at 1000 mg/kg/day, grip strength of the fore legs was decreased in Week 4 of the dosing period (0.67x of control). The lower grip strength of the hind legs in male rats at 1000 mg/kg/day (0.77x of control; not statistically significant) was mainly attributed to a single male (No. 35) with a relatively low value. As individual values of other animals at the same dose level generally remained within concurrent control range, this change was considered to be unrelated to treatment.

Other functional observation parameters, including hearing ability, pupillary reflex and static righting reflex (males and females), and grip strength (females only) were considered unaffected by treatment up to 1000 mg/kg/day. The apparent increased mean grip strength of the fore legs in females at 300 mg/kg/day could mainly be attributed to a relatively low control mean compared to historical control data, high individual variability within the groups, and occurred without a dose related trend. It should be noted that for control females also the mean value for grip strength of the hind legs was relatively low compared to historical control data.

Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Possible treatment-related microscopic findings were observed in the liver and kidneys of males and thyroid gland of both sexes.

Hepatocellular hypertrophy in the liver was noted in males of the 1000 mg/kg/day group at a minimal degree. Hyaline droplet accumulation in the kidney was observed in treated rats of all dose groups up to slight degree. Immunohistochemistry for alpha 2u-globulin was performed on the kidneys of five selected males of the control group and five selected males of the 1000 mg/kg/day group. The hyaline droplets stained positive for alpha 2u-globulin in all five males at 1000 mg/kg/day.

Treatment-related increase in the incidence of follicular cell hypertrophy was observed in the thyroid gland of 1000 mg/kg/day group rats of both sexes (minimal degree).

All other microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4 days.

One female at 1000 mg/kg/day (No. 80) had an extended estrous cycle during the first week of premating. Given its incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis revealed normal progression of the spermatogenic cycle and expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One couple of the control group, one couple of the 100 mg/kg/day group and one couple of the 1000 mg/kg/day group did not produce offspring. Furthermore, total litter loss was observed in 2/10 females of the 1000 mg/kg/day group. No abnormalities were seen in the reproductive organs, which could account for their lack of healthy offspring. Additionally, one female of the 1000 mg/kg/day Group (No. 72) was euthanized in extremis at Day 21 post coitum due to acute severe clinical signs. The uterus contained 13 live pups without abnormalities and two early resorptions.

Mating index:
Not affected by treatment with the test material. All females showed evidence of mating.

Precoital time:
Not affected by treatment with the test material. All females showed evidence of mating within 5 days, except for Female No. 66 (300 mg/kg/day) for which it took 14 days before mating could be confirmed. Such longer precoital time is occasionally encountered in this type of study and in absence of any dose related occurrence, was considered not to be related to treatment with the test material.

Implantation sites:
The number of implantation sites was unaffected by treatment with the test material. Mean numbers of implantation sites were 9.4, 12.1, 12.2, and 11.2 for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

A relatively low mean number of implantation sites was observed in the control group compared to the treated groups, which was attributed to four females (Nos. 43, 46, 49 and 50) each with a low number of implantation sites (i.e. 2-5, compared to 11-16 implantation sites in the remaining control females). One of these control females (No. 49) had two implantation sites only (i.e. no offspring).

Fertility index:
Fertility index was unaffected by treatment with the test material. The fertility index was 100, 90, 100 and 90% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

One female at 100 mg/kg/day (No. 52) and one female at 1000 mg/kg/day (No. 77) were not pregnant. In the absence of a clear dose-related incidence of non-pregnancy, this was considered not to be related to treatment.
N/A
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity
Key result
Critical effects observed:
no
Clinical signs:
not examined
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
not examined
Description (incidence):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
not examined
Description (incidence and severity):
N/A
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on results:
N/A
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
N/A
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
N/A
Reproductive performance:
not examined
Description (incidence and severity):
N/A
N/A
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be treatment-related.

The nature and incidence of clinical signs, including missing toe, scabs, alopecia, dehydrated appearance, black tail apex and wound on the head remained within the range considered normal for pups of this age or occurred in absence of a dose related trend, and were therefore considered not to be treatment-related.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Gestation Index and Duration:

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test material. The gestation indices were 90, 100, 100 and 89% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

The lower gestation indices in the control and at 1000 mg/kg/day were attributed to Female No. 49 (control) which was pregnant with two implantation sites only and Female No. 72 (1000 mg/kg/day) which was pregnant with live pups but had to be sacrificed in extremis on post coitum Day 21 due to acute severe clinical signs (for details, see Section 8.2.1). All other pregnant females had live offspring.

Post-Implantation Survival Index:

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.

Post-implantation survival indices were 90, 88, 90, 78% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

The apparent lower post-implantation survival index for the 1000 mg/kg/day group was caused by Female No. 72, which was sacrificed in extremis on post coitum Day 21 (for details, see Section 8.2.1). Excluding the 15 implantation sites of this female, the post-implantation survival index was 92% and thus comparable to control.

Live birth index:

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment up to 300 mg/kg/day.

Live birth index was reduced at 1000 mg/kg/day (100, 100, 99 and 91% for the control, 100, 300 and 1000 mg/kg/day groups, respectively).

At 1000 mg/kg/day, seven pups in four different litters (one, one, three and two pups from Litter Nos. 73, 74, 78 and 79, respectively) were found dead at first litter check. No findings were noted at first litter check and/or necropsy, except for Pup No. 1 from Litter No. 73 which showed clinical signs at first litter check (i.e. cold to touch, grey discoloration of the whole body and labored respiration) and died shortly afterwards. From Litter No. 79, Pup No. 3 did not have milk in the stomach and Pup No. 4 was cannibalized.

One pup at 300 mg/kg/day (Litter No. 66) was found dead at first litter check with no milk in the stomach at necropsy. At the isolated incidence, no toxicological relevance was attached to this single dead pup in the mid-dose group.

Viability Index 1:

The viability index 1 (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not affected by treatment with the test item up to 300 mg/kg/day.

Viability index 1 was reduced at 1000 mg/kg/day (100, 100, 100 and 60% for the control, 100, 300 and 1000 mg/kg/day groups, respectively).

At 1000 mg/kg/day, a total of twenty-nine pups in four litters were found dead, sacrificed in extremis or missing on PND 2. One pup from Litter No. 71 and two pups from Litter No. 78 were missing on PND 2. In addition, Female Nos. 73 and 75 had total litter loss on PND 2. For Female No. 73, five pups were missing, eight pups were found dead, and one pup was sacrificed in extremis. For Female No. 75, three pups were missing, two pups were found dead, and seven pups were sacrificed in extremis. Pups missing were most likely cannibalized. Pups euthanized in extremis were cold to touch and had a pale appearance. No findings were noted for any of these pups at necropsy.

Viability Index 2:

The viability index 2 (number of live offspring on PND 7 after littering as percentage of number of live offspring on PND 4 (after culling) was considered not affected by treatment with the test item up to 1000 mg/kg/day.

Viability indices 2 were 100, 100, 100 and 97% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup at 1000 mg/kg/day (Litter No. 74) was found dead on PND 5. For this pup, there were no findings either during in-life or at necropsy. As all remaining seven pups in this litter survived until scheduled necropsy without any findings, no toxicological relevance was attached to this single dead pup.

Viability Index 3:

The viability index 3 (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND 7 was considered not affected by treatment with the test item up to 1000 mg/kg/day.

No pups were found dead/missing between Lactation Days 7 and 13, resulting in viability indices of 100% for all groups.

Cumulative Survival Index:

The cumulative survival index ((number of live offspring on Day 13 after litter/number of live offspring on Day 4 (after culling)) * (number of live offspring on Day 4 before culling / total number of offspring born)) * 100% was considered not to be affected by treatment with the test item up to 300 mg/kg/day.
The cumulative survival index was reduced at 1000 mg/kg/day (100, 100, 99 and 53% for the control, 100, 300 and 1000 mg/kg/day groups, respectively).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item up to 300 mg/kg/day.

At 1000 mg/kg/day, body weight of both male and female pups was decreased from PND 1 onwards (up to 17% and 24% lower than control, respectively). On PND 13, the combined (males and females) mean body weight was 19% lower than control.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T3 (up to 300 mg/kg/day) and T4 levels in PND 4 pups and T3, T4 and TSH levels in male and female PND 14 16 pups were considered not to be affected by treatment with the test item.

In PND 4 pups, serum T3 levels tended to be lower at 1000 mg/kg/day (0.75x of control). However, as only two samples were available for analysis of both T3 and T4 at 1000 mg/kg/day these data should be interpreted with caution and could not be used for toxicological evaluation.
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test material.

The lower mean for normalized anogenital distance of female pups at 100 and 300 mg/kg/day occurred without a dose-related trend. Therefore, these variations were considered not to be related to treatment.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test material up to 1000 mg/kg/day had no effect on areola/nipple retention.

For one male pup of the control and 300 mg/kg/day groups each, 1-2 nipples were observed at PND 13. As this finding occurred without a dose related trend, it was considered not to be related to treatment.
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test material. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
no effects observed
Description (incidence and severity):
Sex Ratio:

Sex ratio was considered not to be affected by treatment with the test material.

Lactation Index:

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 8 was considered not to be affected by treatment with the test item up to 1000 mg/kg/day. The lactation indices were 100% for the control, 100, 300 ,and 1000 mg/kg/day groups, respectively.

Litter Size:

Litter size was considered not affected by treatment with the test material. Mean live litter sizes were 9.4, 10.7, 10.9 and 9.0 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
other: Developmental Toxicity
Key result
Critical effects observed:
no
Clinical signs:
not examined
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
not examined
Description (incidence and severity):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
not examined
Description (incidence and severity):
N/A
Nipple retention in male pups:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Reproductive effects observed:
no

Dose Formulation Analyses

Accuracy

 

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85 115% of target concentration).

 

A small response at the retention time of the test material was observed in one of the chromatograms of the Group 1 formulation prepared for use in Week 3. It was considered not to derive from the formulation, since a similar response was obtained in the analytical blanks. The maximum contribution to Group 2 samples calculated was 0.00057%, taking the dilution factor into account and considered neglectable. In all other formulations of Group 1, no test material was detected.

 

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Table 9. Body Weights (grams) Summary (F0)

 

 

Group 1

Control

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Males

Pre-mating

 

Day 1

Week 1

Mean

302

295

303

298

St. Dev

12.1

7.0

11.1

9.0

N

10

10

10

10

 

Day 8

Week 2

Mean

320

314

321

311

St. Dev

16.1

7.8

11.8

10.8

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

335

325

334

323

St. Dev

17.7

7.3

14.5

13.0

N

10

10

10

10

 

Day 8

Week 2

Mean

344

335

340

330

St. Dev

17.7

8.0

16.4

15.5

N

10

10

10

10

 

Day 15

Week 3

Mean

357

346

352

339*

St. Dev

17.7

10.0

15.6

15.4

N

10

10

10

10

Females

Pre-mating

 

Day 1

Week 1

Mean

227

230

234

233

St. Dev

12.2

9.9

11.5

15.6

N

10

10

10

10

 

Day 8

Week 2

Mean

230

233

236

234

St. Dev

14.4

8.3

13.9

15.2

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

234

236

239

249

St. Dev

13.6

12.3

13.8

13.9

N

10

10

10

10

 

Day 8

Week 2

Mean

 

---

268

269

St. Dev

 

---

---

---

N

 

0 x

1

1

 

Day 15

Week 3

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

 

Day 22

Week 4

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

Post Coitum

 

Day 0

Mean

235

235

241

238

St. Dev

13.1

13.4

15.0

12.5

N

10

8

10

9

 

Day 4

Mean

250

250

253

250

St. Dev

13.0

12.8

16.0

12.5

N

10

8

10

9

 

Day 7

Mean

256

257

259

256

St. Dev

12.4

14.6

15.5

13.2

N

10

8

10

9

 

Day 11

Mean

270

272

275

272

St. Dev

12.4

15.0

16.5

14.5

N

10

8

10

9

 

Day 14

Mean

279

283

286

280

St. Dev

14.1

18.0

17.8

16.3

N

10

8

10

9

 

Day 17

Mean

301

307

309

301

St. Dev

16.6

17.7

21.3

18.4

N

10

8

10

9

 

Day 20

Mean

330

345

345

328

St. Dev

27.6

18.6

27.4

21.6

N

10

8

10

9

Lactation

 

Day 1

Mean

268

268

275

266

St. Dev

19.1

15.0

10.3

19.6

N

9

9

10

8

 

Day 4

Mean

277

278

279

272

St. Dev

20.1

16.6

15.8

26.0

N

9

9

10

6

 

Day 7

Mean

285

288

286

278

St. Dev

17.2

13.4

18.8

22.8

N

9

9

10

6

 

Day 13

Mean

297

296

295

279

St. Dev

17.6

18.3

23.6

23.0

N

9

9

10

6

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values in the study report

Table 10. Body Weights (%) Summary (F0)

 

 

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Males

Pre-mating

 

Day 1

Week 1

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

6

6

6

4

St. Dev

1.4

1.3

1.7

1.6

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

11

10

10

9

St. Dev

2.0

1.7

2.2

2.5

N

10

10

10

10

 

Day 8

Week 2

Mean

14

14

12

11*

St. Dev

2.3

1.6

2.8

3.0

N

10

10

10

10

 

Day 15

Week 3

Mean

18

18

16

14**

St. Dev

2.4

2.2

2.9

3.5

N

10

10

10

10

Females

Pre-mating

 

Day 1

Week 1

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

1

1

1

0

St. Dev

2.6

1.1

1.8

2.3

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

3

3

2

2

St. Dev

2.5

3.1

2.5

2.6

N

10

10

10

10

 

Day 8

Week 2

Mean

 

---

10

10

St. Dev

 

---

---

---

N

 

0 x

1

1

 

Day 15

Week 3

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

 

Day 22

Week 4

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

Post Coitum

 

Day 0

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

10

8

10

9

 

Day 4

Mean

6

7

5

5

St. Dev

1.1

2.4

1.4

2.1

N

10

8

10

9

 

Day 7

Mean

9

9

8

8

St. Dev

1.5

4.0

0.9

2.2

N

10

8

10

9

 

Day 11

Mean

15

16

14

14

St. Dev

2.3

3.9

1.9

3.6

N

10

8

10

9

 

Day 14

Mean

19

20

19

18

St. Dev

2.3

4.8

2.8

3.8

N

10

8

10

9

 

Day 17

Mean

28

31

28

27

St. Dev

3.9

3.7

5.0

5.8

N

10

8

10

9

 

Day 20

Mean

41

47

43

38

St. Dev

9.9

3.2

7.3

10.1

N

10

8

10

9

Lactation

 

Day 1

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

9

9

10

8

 

Day 4

Mean

3

4

1

1

St. Dev

2.2

2.8

3.3

2.4

N

9

9

10

6

 

Day 7

Mean

6

8

4

3

St. Dev

3.2

5.1

4.3

2.2

N

9

9

10

6

 

Day 13

Mean

11

11

7

3*

St. Dev

4.2

4.2

6.3

2.9

N

9

9

10

6

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values in the study report

 

Table 11. Summary of Select Hematology Values: Females (F0)

Day: 52 Relative to Start Date

Dose Group

 

HGB (g/L)

[G]

MCHC (g/L)

[G]

Group 1

Control

0 mg/kg/day

Mean

148.0

331.8

St. Dev

6.4

2.6

N

5

5

 

Group 2

100 mg/kg/day

Mean

136.2**

322.4

St. Dev

3.7

6.9

N

5

5

tCtrl

0.92

0.97

 

Group 3

300 mg/kg/day

Mean

139.6*

320.6*

St. Dev

3.4

4.2

N

5

5

tCtrl

0.94

0.97

 

Group 4

1000 mg/kg/day

Mean

147.4

326.8

St. Dev

5.3

9.0

N

5

5

tCtrl

1.00

0.98

[G] - Anova & Dunnett: * = p ≤ 0.05

 

Table 12. Summary of Coagulation Values: F0 Generation

Dose Group

 

PTT (sec)

[G]

APTT (sec)

[G]

Males(Day: 30 Relative to Start Date)

Group 1

Control

0 mg/kg/day

Mean

16.76

20.54

St. Dev

0.45

0.67

N

5

5

 

Group 2

100 mg/kg/day

Mean

16.12

19.56

St. Dev

0.56

0.90

N

5

5

tCtrl

0.96

0.95

 

Group 3

300 mg/kg/day

Mean

15.56**

19.16

St. Dev

0.34

1.54

N

5

5

tCtrl

0.93

0.93

 

Group 4

1000 mg/kg/day

Mean

16.50

19.08

St. Dev

0.75

2.67

N

5

5

tCtrl

0.98

0.93

Females(Day: 52 Relative to Start Date)

Group 1

Control

0 mg/kg/day

Mean

17.58

18.84

St. Dev

0.53

1.87

N

5

5

 

Group 2

100 mg/kg/day

Mean

16.48*

16.83

St. Dev

0.15

1.56

N

4

4

tCtrl

0.94

0.89

 

Group 3

300 mg/kg/day

Mean

16.90

18.66

St. Dev

0.80

1.23

N

5

5

tCtrl

0.96

0.99

 

Group 4

1000 mg/kg/day

Mean

16.60*

17.88

St. Dev

0.58

1.31

N

5

5

tCtrl

0.94

0.95

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.


Table 13. Summary of Select Clinical Chemistry Values: F0 Generation

 

 

Males(Day: 30 Relative to Start Date)

Females(Day: 52 Relative to Start Date)

Parameter

 

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

K

(mmol/L)

[G]

Mean

3.73

4.05

4.02

4.41**

4.47

5.03

4.86

4.65

St. Dev

0.11

0.27

0.20

0.23

0.31

0.44

0.56

0.33

N

5

5

5

5

5

5

5

5

tCtrl

 

1.09

1.08

1.18

 

1.13

1.09

1.04

 

CREAT

umol/L

[G1]

Mean

25.9

27.0

27.6

28.4

19.1

20.0

22.3

24.3*

St. Dev

2.4

2.5

2.1

3.1

1.7

1.8

2.1

4.6

N

5

5

5

5

5

5

5

5

tCtrl

 

1.04

1.07

1.10

 

1.05

1.17

1.27

[G] - Anova & Dunnett

[G1] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01

Table 14. Select Organ Weights (grams) Summary (F0 Males)

 

 

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Males

End of Treatment

 

Body Weight

(gram)

Mean

336

324

328

315**

St. Dev

18

9

16

14

N

10

10

10

10

 

Liver

(gram)

Mean

8.15

8.23

8.38

9.77**

St. Dev

0.85

0.30

0.67

0.86

N

5

5

5

5

 

Thymus

(gram)

Mean

0.301

0.299

0.245

0.216*

St. Dev

0.019

0.065

0.049

0.033

N

5

5

5

5

 

Spleen

(gram)

Mean

0.592

0.559

0.475**

0.514

St. Dev

0.048

0.049

0.045

0.050

N

5

5

5

5

 

Prostate

(gram)

Mean

0.858

0.858

0.811

0.720*

St. Dev

0.199

0.118

0.103

0.092

N

10

10

10

10

 

Seminal Vesicles

(gram)

Mean

1.362

1.254

1.173

1.122**

St. Dev

0.136

0.226

0.147

0.167

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 15. Select Organ / Body Weight Ratios (%) Summary (F0 Males)

 

 

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Males

End of Treatment

 

Body Weight

(gram)

Mean

336

324

328

315**

St. Dev

18

9

16

14

N

10

10

10

10

 

Liver

(%)

Mean

2.35

2.52

2.61

3.14**

St. Dev

0.27

0.08

0.11

0.23

N

5

5

5

5

 

Brain

(%)

Mean

0.59

0.62

0.62

0.64*

St. Dev

0.03

0.04

0.02

0.03

N

5

5

5

5

 

Spleen

(%)

Mean

0.170

0.171

0.149*

0.165

St. Dev

0.010

0.013

0.016

0.012

N

5

5

5

5

 

Kidney

(%)

Mean

0.63

0.62

0.65

0.75**

St. Dev

0.05

0.05

0.02

0.06

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 16. Summary of Reproduction Data

 

Group 1

Control

0 mg/kg/day

Group 2

100

mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Females paired

10

10

10

10

Females mated

10

10

10

10

Pregnant Females

10

9

10

9

Females with implantations only

1

0

0

0

Females euthanized on post-coitum Day 21

0

0

0

1

Females with living pups on Day 1

9

9

10

8

 

Mating index (%)

(Females mated / Females paired) * 100

100

100

100

100

Fertility index (%)

(Pregnant females / Females mated) * 100

100

90

100

90

Gestation index (%)

(Females with living pups on Day 1 / Pregnant females) * 100

90

100

100

89

Table 17. Pre-coital Time (F0)

Day of the Pairing Period

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000

mg/kg/day

Number of Females Mated

1

7

4

3

5

2

3

2

3

1

3

-

3

2

-

4

-

1

1

3

5

-

-

-

1

14

-

-

1

-

 

Median Pre-coital Time

1

2

2

2

Mean Pre-coital Time

1.3

2.1

3.3

2.4

N

10

10

10

10

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 18. Implantation Sites Summary (F0 – Females)

 

 

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

At Necropsy

 

Implantations

Mean

9.4

12.1

12.2

11.2

St. Dev

5.5

4.4

3.6

3.5

N

10

9

10

9

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic and reproductive toxicity No Observed Adverse Effect Levels (NOAEL) for Reaction mass of ethylbenzene and m-xylene were determined to be 1000 mg/kg/day.
Executive summary:

A key OECD Guideline 422 study was conducted to determine the potential toxic effects of the test material (Reaction mass of ethylbenzene and m-xylene) when administered orally for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

 

Parameters evaluated were mortality/moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0-males and females), gross necropsy findings, organ weights and histopathologic examinations. Additionally, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormones T3 and T4 (PND 4), and T3, T4 and TSH (PND 14-16 pups)).

 

No parental toxicity was observed up to 1000 mg/kg/day in males and females. No treatment-related changes were observed in any of the following parameters investigated in the study (i.e., mortality, clinical appearance, hematology, clotting parameters, T3, T4 and TSH thyroid hormone levels, macroscopic examination).

 

Treatment-related reduced body weight (males and females) and food consumption (females only) were observed at 1000 mg/kg/day during the lactation period. Considering the minor magnitude of the change, these effects were considered non-adverse.

 

Changes in clinical biochemistry parameters at 1000 mg/kg/day comprised of increased potassium levels in males and creatinine concentrations in females. These changes were without corroborative findings at the organ level and were therefore regarded to be non-adverse.

 

Functional tests revealed a treatment-related reduced grip strength in the fore legs of males at 1000 mg/kg/day. In absence of corroborative findings at clinical observations, motor activity and histopathology, this finding was considered non-adverse.

 

Treatment-related liver findings included hepatocellular hypertrophy (minimal degree), which correlated with higher liver weights at 1000 mg/kg/day and a minor dose-related increase of alkaline phosphatase activity (ALP) at 100, 300 and 1000 mg/kg/day. Based on the minimal severity of the microscopic finding and the absence of corroborative and degenerative findings, these effects were considered non-adverse (Palazzi 2016; Kerlin 2016).

 

Hyaline droplet accumulation was observed in the kidney of males starting at 100 mg/kg/day (up to slight degree). This finding was considered possible treatment-related at 100 and 300 mg/kg/day. As the incidence and mean severity showed a subtle increase at 1000 mg/kg/day, this finding was regarded to be treatment-related at the highest dose level. The observed hyaline droplets most likely resembled alpha 2µ-globulin, a normal protein present in the kidney of male rats, which was confirmed by immunohistochemistry. This protein is absent in female rats, as well as in humans. In absence of microscopic findings indicative of kidney damage, this finding was regarded non-adverse (Sahota 2013). Furthermore, there was a statistically significant higher kidney weight (relative to body weight) observed in male rats at 1000 mg/kg/day. Based on the absence of a microscopic correlate, these kidney changes were considered non-adverse.

 

An increased incidence of follicular cell hypertrophy in the thyroid gland of males and females at 1000 mg/kg/day (minimal degree) was considered non-adverse.

 

No reproductive toxicity and no test treatment-related changes were observed in any of the reproductive parameters investigated (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs) up to the highest dose level tested (1000 mg/kg/day).

 

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic and reproductive toxicity No Observed Adverse Effect Levels (NOAEL) for Reaction mass of ethylbenzene and m-xylene were determined to be 1000 mg/kg/day.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-Jun-22 to 2022-Mar-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This data has not been finalised by LOA due to an ongoing review. The data presented in this record is from an audited draft report by the contract research organisation. This information was requested by a National Enforcement Agency to allow a registrant to fulfill their annex VIII information requirements.
A communication from the contract research organisation stating when the finalisation of the report will occur by (31st January 2023) is included below in the "attached justification" section. LOA is committed to performing a dossier update when the full information required for ECHA to judge the read-across approach utilised in this dossier is available.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version: July 2016
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions. Please see 'Any other information on materials and methods' for details.
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
N/A
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Hanwha TOTAL (South Korea); Batch number: FB8055-15223
- Purity, including information on contaminants, isomers, etc.: Mixed Xylene (o-xylene: 20.98 WT% ; m-xylene: 49.35 WT%; and p-xylene: 23.81 WT%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in corn oil vehicle for at least 24 hours at room temperature under normal laboratory light over a concentration range of 2 to 250 mg/mL (solutions); Stable in corn oil vehicle for at least 8 days in the refrigerator over a concentration range of 2 to 250 mg/mL (solutions); 0.5 mL sample in corn oil vehicle stable for at least 3 weeks in the freezer (≤ -15°C) over a concentration range of 2 to 250 mg/mL (solutions).

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS
- Specific gravity / density: 0.8708 g/cm3 (15°C)
- Volatile: Yes, vapour pressure: 6~16 mmHg at 20°C
- Expiry date: 2023-APR-15
- CAS Number: 1330-20-7
- EC Number: 215-535-7
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: Males: 10-12 weeks; Females: 14-15 weeks
- Weight at study initiation: Males: 272 - 327 g; Females: 201 - 274 g
- Housing: On arrival and during the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).

During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.

During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

- Diet: pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH (Soest, Germany) ad libitum
- Water: Municipal tap water via water bottles ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, based on the results of these analyses it was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 39 to 67%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 2021-JUN-23 To: 2021-OCT-18
Route of administration:
oral: gavage
Remarks on MMAD:
N/A
Vehicle:
corn oil
Remarks:
Sigma-Aldrich (Steinheim, Germany)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Sigma-Aldrich, Steinheim, Germany) was used as the vehicle based on trial preparations performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and were not used for dosing.
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg bw/day dose groups, respectively.
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated.
- Any other deviations from standard protocol: Detection of mating was not confirmed in first instance for Female No. 46 (control). Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During Week 1 of treatment, dose formulation samples were collected for analysis as follows:

1. Concentration analysis: sample collected from approximately ‘Middle’ for all dose groups

2. Homogeneity analysis: sample collected from approximately ‘Top, Middle and Bottom’ for dose groups 2 and 4 only.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20275973). For concentration: mean sample concentration results within or equal to ± 15% of theoretical concentration.

For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group. Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20275973) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily
Details on study schedule:
N/A
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle: Corn Oil) - Group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a 10-Day Dose Range Finder with oral administration of Mixed Xylene (CAS 1330-20-7) in rats (Test Facility Reference No. 20275974) and in an attempt to produce graded responses to the test material.
- Rationale for animal assignment (if not random): Randomized per litter and individually identified by means of subcutaneous injection of Indian ink
- Fasting period before blood sampling for clinical biochemistry: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- Dose range finding studies: 10-Day Dose Range Finder with oral administration of Mixed Xylene (CAS 1330-20-7) in rats (Test Facility Reference No. 20275974)
Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test material and lasting throughout the Dosing periods up to the day prior to necropsy. Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

All animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Except on days of receipt and necropsy where frequency was at least once daily. Arena observations were also conducted once before the first administration of the test material and weekly during the treatment Period.

BODY WEIGHT: Yes
- Time schedule for examinations: for all animals on Day 1 of treatment (prior to dosing ) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum
and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; quantitatively measured per cage weekly, except for males and females which were housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Monitored regularly throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane); Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid hormones)
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters examined included Triiodothyronine (T3); Thyroxine (T4); and Thyroid stimulating hormone (TSH)

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed post dosing on 5 males per group during Week 4 of treatment and 5 females per group during the last week of lactation (i.e. PND 8-10).
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity such as Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; and Locomotor activity)

IMMUNOLOGY: No

OTHER: Coagulation:
- Time schedule for collection of blood: On the day of scheduled necropsy
- How many animals: 5 animals/sex/group were selected
- Animals fasted: Males fasted overnight (maximum of 24 hours), females non-fasted
- Anaesthetic used for blood collection: Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.45 mL
- Parameters analysed: prothrombin time (PT), activated partial thromboplastin time (APTT)
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
For the testes of all selected males of Group 1 (control) and Group 4 (1000 mg/kg bw/day), and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Pups were identified on postnatal Day (PND) 1, randomized per litter, and individually identified by means of subcutaneous injection of Indian ink
- All pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood
samples were collected from two of the surplus pups (from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
Pups were examined for external and internal abnormalities and possible cause of death was determined for pups born or found dead].

Unscheduled Euthanasia
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia
On PND 14-16, all remaining pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (see Tables 4 and 5)

Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:

Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16; females which failed to deliver: With evidence of mating: Post-coitum Day 25-27 (Nos. 41, 55, 64 and 76).

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy.

Necropsy:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

The organs listed in Table 4 and 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see Tables 6 and 7)

Representative samples of the tissues identified in the tables 6 and 7 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. The tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Alpha-2u-globulin staining:
Two slides per selected Group 1 and Group 4 male (e.g. lowest numbered 5 in each group) were shipped to CRL-Durham for HRP-polymer based chromogenic immunostaining of alpha-2u-globulin. The slides were returned to the Test Facility after the staining procedure has been completed for evaluation by the study pathologist.

Microscopic Evaluation:
All tissues as defined under Histology were examined by a board-certified toxicological pathologist. For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The slides stained for alpha-2u-globulin were evaluated by the study pathologist.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

Unscheduled Deaths – F1-Generation
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia – F1-Generation
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible.

All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Reproductive indices:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

1. Mating index (%) = (number of females mated / number of females paired) x 100

2. Precoital time = number of days between initiation of cohabitationand confirmation of mating

3. Fertility index (%): (number of pregnant females / number of females mated) x 100

4. Gestation index (%) = (number of females with living pups on Day 1/ number of pregnant females) x 100

5. Duration of gestation = number of days between confirmation of mating and the beginning of parturition

6. Post-implantation survival index (%) = (total number of offspring born / total number of uterine implantation sites) x 100

7. Live birth index (5) = (number of live offspring on Day 1 after littering / total number of offspring born) x 100

8. Percent 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

9. Percent 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

10. Lactation index (%) = (number of live offspring on Day 13 after littering / number of live offsping on Day 4 (after culling)) x 100

11. Cumulative Survival Index = ((number of live offspring on Day 13 / number of live offspring on Day 4 after culling) x (number of live offspring on Day 4 before culling / total number of offspring born)) x 100
Offspring viability indices:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

1. Viability index 1 (%) = (number of live offspring on Day 4 before culling / number of live offspring on Day 1 after litterling) x 100

2. Viability Index 2 (%) = (number of live offspring on Day 7 before culling / number of live offspring on Day 4 after litterling) x 100

3. Viability Index 3 (%) = (number of live offspring on Day 13 before culling / number of live offspring on Day 7 after litterling) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related toxicologically relevant clinical signs were observed through the study period.

At 1000 mg/kg/day, hunched posture was noted in one male on a single occasion in Week 2. Piloerection was observed in three females on three consecutive days during the post-coitum phase, but also in two control females on a single day. At the incidence observed, these clinical signs were cons
idered not to be treatment-related.

One male in the 100 mg/kg/day dose group had red discharge (likely blood) from the mouth on the day prior to scheduled necropsy and one female in the 1000mg/kg/day dose group was gasping on a single occasion during the post-coitum phase. As these signs were observed directly after dosing and noted at this single occasion only, they were considered gavage-related signs, rather than signs related to the test material.

Salivation seen after dosing in animals of the 300 (females) and 1000 (both sexes) mg/kg/day dose groups during most of the treatment period was considered not to be toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

No findings were noted during the weekly arena observations in this study. Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
No mortality occurred through the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes in body weights and body weight gain were observed up to 300 mg/kg/day.

In male rats treated at 1000 mg/kg/day, body weight gain was slightly lower over the whole study period compared to controls (not statistically significant, except over Days 1-8). Mean body weights were comparable to the concurrent control mean throughout treatment.

In female rats treated at 1000 mg/kg/day, slight body weight loss (up to -3%) was noted over Days 1-8, which normalized thereafter. In addition, a trend towards a lower body weight gain was noted from post-coitum Day 14 onwards until the end of the lactation period. Mean body weight at the end of the lactation period was 10% lower than the concurrent control mean. Any statistically significant changes at 100 mg/kg/day were considered unrelated to treatment with the test material, in the
absence of a dose-response relationship.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to control values up to 1000 mg/kg/day in males and up to 300 mg/kg/day in female rats.

At 1000 mg/kg/day, food consumption was lower in females during the lactation period (not statistically significant over Days 1-4) compared to controls. The mean of means for food consumption and relative food consumption over this period were 17 and 9% lower than controls, respectively.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment with the test material.

The statistically significantly higher red blood cell distribution width at 1000 mg/kg/day in females was considered to be a result of a high value for one individual animal. At the incidence observed, this was considered unrelated to treatment with the test material.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical biochemistry parameters after treatment with the test item up to 300 mg/kg/day.

At 1000 mg/kg/day, alanine aminotransferase (ALT) activity was increased in both sexes with a change of 1.33 and 1.34x (not statistically significant) of the concurrent control value, respectively.Considering the small magnitude, this was considered to be not adverse.

Aspartate aminotransferase (AST) was increased in one female at 1000 mg/kg/day. At the incidence observed, this was considered unrelated to treatment with the test material.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Total thyroxine (T4) was decreased in males at 1000 mg/kg/day (0.72x of control) but the mean value remained within the historical control range.

Thyroid hormones were considered unaffected in males up to 300 mg/kg/day and in females up to 1000 mg/kg/day.

High thyroid stimulating hormone (TSH) values at 100 and 300 mg/kg/day in both sexes occurred in the absence of a dose-response relationship and individual data generally remained within the concurrent control range. Therefore, this finding was not considered to be treatment-related.
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment with the test material up to 1000 mg/kg/day.

Any statistically significant changes in male hind grip strength were considered to derive from relatively high control values, rather than from treatment with the test material. No dose-response relationship was observed and all mean values remained within the historical range, including controls.

Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. High total movement and ambulation values for females at 1000 mg/kg/day were mainly attributed to a single female, and at this incidence observed, this was considered unrelated to treatment with the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were observed in the liver of males and females and the thyroid gland and kidney of males.

Liver:
Hepatocellular hypertrophy was present in males at 1000 mg/kg/day at a minimal to slight degree. Cytoplasmic rarefaction (likely representing glycogen) was present at increased incidence and severity in females at 1000 mg/kg/day at a minimal to slight degree.

Thyroid gland:
Follicular cell hypertrophy was present in males at 1000 mg/kg/day at a minimal to slight degree.

Kidney:
An increased incidence and severity of hyaline droplet accumulation (positive for alpha 2u-globulin) was present in males at 1000 mg/kg/day up to moderate degree. Granular casts (positive for alpha 2u-globulin) were present in males at 1000 mg/kg/day up to a slight degree.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were unaffected by treatment. All females had regular cycles of 4 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis revealed normal progression of the spermatogenic cycle and expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One couple of the control group, one couple of the 300 mg/kg/day group, and one couple of the 1000 mg/kg/day group did not produce offspring. There was one female of the 100 mg/kg/day group with implantation sites only. No abnormalities were seen in the reproductive organs, which could account for their lack of
offspring.

Mating index:
Not affected by treatment with the test material. All females showed evidence of mating.

Precoital time:
Not affected by treatment with the test material. All females showed evidence of mating within 4 days, except for one control female, which showed evidence of mating after 14 days. As this occurred in a single control animal, this was considered a background finding.

Implantation sites:
The number of implantation sites was unaffected by treatment with the test material. The mean number of implantation sites were 12.9, 12.1, 12.6, and 12.6 for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Fertility index:
Fertility index was unaffected by treatment with the test material. The fertility indices were 90, 100, 90, and 90% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
N/A
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
not examined
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
not examined
Description (incidence):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
not examined
Description (incidence and severity):
N/A
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on results:
N/A
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
N/A
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
N/A
Reproductive performance:
not examined
Description (incidence and severity):
N/A
N/A
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be treatment-related.

For one pup of a litter from the 100 mg/kg/day dose group that was missing on PND 2, paleness and dehydration were observed at first litter check. The nature and incidence of this and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Gestation Index and Duration:

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment.

Except for one female at 100 mg/kg/day (No. 55 with implantation sites only), all pregnant females had live offspring. Therefore, gestation indices were 100, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

Post-Implantation Survival Index:

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 85, 90 and 95% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. Female No. 55 (100 mg/kg/day) had 11 implantations but did not deliver a litter. As this occurred in a single female and in the low dose group only, this was considered unrelated to treatment.

Live birth index:

Live birth index was considered unaffected by treatment with the test item up to 300 mg/kg/day.

At first litter check, one pup at 100 mg/kg/day and 16 pups of the 1000 mg/kg/day group (divided over 5 litters) were found dead. Four of these 16 pups at 1000 mg/kg/day were observed to have no milk in the stomach. No macroscopic findings were noted for the other pups. At the single occurrence, the dead pup at 100 mg/kg/day was considered to be unrelated to treatment as it remained within the range considered normal for pups of this age. Live birth indices were 100, 99, 100, and 85% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Live birth index at 1000 mg/kg/day was below the historical range.

Viability Index 1:

Viability Index 1 (the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) was considered not affected by treatment with the test material. Viability indices on PND 4 were 100, 98, 99, and 98% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Two, one and two pups at 100, 300, and 1000 mg/kg/day, respectively, were found dead or missing on PND 2. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index 2:

Viability Index 2 (the number of live offspring on Day 7 compared to the number of offspring on Day 4 after culling) was unaffected by treatment. Viability indices on PND 7 were 100% for all treatment groups.

Viability Index 3:

Viability Index 3 (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND 7) was not affected by treatment with the test material. Indices were 100% for all treatment groups.

Cumulative Survival Index:

The cumulative survival index ((number of live offspring on Day 13 after litter/number of live offspring on Day 4 (after culling)) × (number of live offspring on Day 4 before culling / total number of offspring born)) × 100% was considered not to be affected by treatment up to 300 mg/kg/day. Cumulative survival indices were 100, 97, and 99% for the control, 100 and 300 mg/kg/day groups, respectively. At 1000 mg/kg/day, the cumulative survival index was decreased (i.e. 83%).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were decreased at 1000 mg/kg/day from PND 1 onwards for both sexes. Mean combined pup body weights on PND 13 were 19% lower than control. Pup body weights were considered to be unaffected up to 300 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not specified
Description (incidence and severity):
N/A
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T3 and T4 levels in male and female PND 4 and PND 14-16 pups, and TSH levels in PND 14-16 pups, were considered not to be affected by treatment. High TSH values at 100 and 300 mg/kg/day in both sexes occurred in the absence of a dose-response relationship and individual data were generally variable. Therefore, this finding was considered unrelated to treatment with the test material.
Urinalysis findings:
not specified
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
Anogenital distance (absolute and corrected for body weight) in male pups was considered not to be affected by treatment with the test item up to 1000 and in female pups up to 300 mg/kg/day. Corrected anogenital distance for female pups was increased (1.20x to control). Mean values remained within the historical control range. The apparently slightly lower corrected anogenital distance in male pups at 1000 mg/kg/day was considered to derive from overcorrection due to the low pup body weights.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test material up to 1000 mg/kg/day was considered to have had no effect on areola/nipple retention. There were one, three (in the same litter), and one male pups at the control, 100, and 1000 mg/kg/day groups, respectively, that were observed to have a single nipple on PND 13. No nipples were counted in the male pups from litters in the 300 mg/kg/day group. As this occurred in the absence of a dose-response relationship, and as one male pup in the control group was also observed to have one nipple, this finding was considered unrelated to treatment.
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test material. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
no effects observed
Description (incidence and severity):
Sex Ratio:

Sex ratio was considered not to be affected by treatment with the test material.

Lactation Index:

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 8 was unaffected by treatment with the test item. No pups were found dead/missing between lactation Days 8 and 13, resulting in a lactation index of 100% for all groups.

Litter Size:

Litter size was considered unaffected by treatment up to 300 mg/kg/day. A lower mean number of live pups was recorded at 1000 mg/kg/day (not statistically significant). Mean number of live pups were 12.3, 11.3, 11.3, and 10.1 live pups per litter for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
other: Developmental Toxicity
Key result
Critical effects observed:
no
Clinical signs:
not examined
Description (incidence and severity):
N/A
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
not examined
Description (incidence and severity):
N/A
Body weight and weight changes:
not examined
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
not examined
Description (incidence and severity):
N/A
Nipple retention in male pups:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Reproductive effects observed:
no

Dose Formulation Analyses

Accuracy

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). No test material was detected in the Group 1 formulation.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 9. Male Body Weights (g) Summary

    Control 100 mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 294 297 298 297
S.D. 17.1 9.3 12.3 10.3
N 10 10 10 10
Day 8 / Week 2 Mean 314 319 317 312
S.D. 21.7 10.8 16.9 13.6
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 329 336 331 327
S.D. 26.3 12.7 19.2 15.1
N 10 10 10 10
Day 8 /Week 2 Mean 336 348 342 332
S.D. 28.6 16.7 20.6 15.4
N 10 10 10 10
Day 16 / Week 3 Mean 345 361 352 343
S.D. 26.8 18.1 24.1 15.9
N 10 10 10 10

Table 10. Female Body Weights (g) Summary

    Control 100 mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 239 234 239 236
S.D. 20.5 15.6 14.5 12.1
N 10 10 10 10
Day 8 / Week 2 Mean 245 239 241 235
S.D. 18.7 18.6 14.8 13.4
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 251 241 246 244
S.D. 19.1 19.6 15.9 16.5
N 10 10 10 10
Day 8 /Week 2 Mean - - - -
S.D. - - - -
N 0x 0 0 0

Table 11. Female Body Weight (g) Summary

    Control

100

mg/kg/day

300

mg/kg/day

1000

mg/kg/day

Post-coitum          
Day 0 Mean 244 243 242 241
S.D. 18.8 19.1 17.1 12.8
N 8 10 9 9
Day 4 Mean 259 254 255 255
S.D. 19.7 19.5 15.6 14.3
N 8 10 9 9
Day 7 Mean 263 261 263 263
S.D. 19.7 18.3 15.8 15.6
N 8 10 9 9
Day 11 Mean 278 274 278 274
S.D. 21.6 20.1 16.6 16.7
N 8 10 9 9
Day 14 Mean 289 284 286 279
S.D. 22.6 22.7 14.6 16.3
N 8 10 9 9
Day 17 Mean 312 305 312 298
S.D. 23.3 23.3 17.5 15.8
N 8 10 9 9
Day 20 Mean 351  342  348  330
S.D. 30.5  30.5  22.0 16.3
N 8 10 9 9
Lactation          
Day 1 Mean 284  271  273  261
S.D. 25.6  21.9  17.5  16.7
N 9 9 9 9
Day 4 Mean 293  283  284  265
S.D. 32.1  24.5  20.2  14.8
N 9 9 9 9
Day 7 Mean 298  290  292  274
S.D. 24.5  22.7  18.2 16.4
N 9 9 9 9
Day 13 Mean 315  302  301  283 **
S.D. 22.6  18.9  18.7  20.2
N 9 9 9 9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 12. Male Body Weight Gain (%) Summary

    Control 100 mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 0 0 0 0
S.D. 0 0 0 0
N 10 10 10 10
Day 8 / Week 2 Mean 7 7 7 5*
S.D. 1.5 1.4 1.6 2.1
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 12 13 11 10
S.D. 3.1 2.1 2.4 2.4
N 10 10 10 10
Day 8 /Week 2 Mean 14 17 15 12
S.D. 4.2 3.1 3.3 3.2
N 10 10 10 10
Day 15 / Week 3 Mean 17 21*  18 15
S.D. 3.7 3.3 4.1 4
N 10 10 10 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 13. Female Body Weight Gain (%) Summary

    Control 100 mg/kg/day 300 mg/kg/day 1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 0 0 0 0
S.D. 0 0 0 0
N 10 10 10 10
Day 8 / Week 2 Mean 3 2 1 -1*
S.D. 2.6 2.9 1.9 2.4
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 5 3 3 3
S.D. 4.0 3.0 3.0 3.7
N 10 10 10 10
Day 8 /Week 2 Mean - - - -
S.D. - - - -
N 0x 0 0 0

Table 14. Female Body Weight Gain (%) Summary

    Control

100

mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Post-coitum          
Day 0 Mean 0 0 0 0
S.D. 0 0 0 0
N 8 10 9 9
Day 4 Mean 6 4 5 6
S.D. 1.1 2.1 2.2 1.1
N 8 10 9 9
Day 7 Mean 8 7 9 9
S.D. 1.7 2.5 3.1 1.9
N 8 10 9 9
Day 11 Mean 14  13  15  14
S.D. 3.2 . 3.7  4.2 2.2
N 8 10 9 9
Day 14 Mean 18  17  18 16
S.D. 3.9  4.4  4.0 2.9
N 8 10 9 9
Day 17 Mean 28  26  29 24
S.D. 4.4  7.0  5.0 3.8
N 8 10 9 9
Day 20 Mean 44  41  44 37
S.D. 7.3  10.9  5.9 3.1
N 8 10 9 9
Lactation          
Day 1 Mean 0 0 0 0
S.D. 0 0 0 0
N 9 9 9 9
Day 4 Mean 3 4 4 2
S.D. 3.9  2.7  2.1  3.2
N 9 9 9 9
Day 7 Mean 5 7 7 5
S.D. 3.4  3.1  1.5  3.6
N 9 9 9 9
Day 13 Mean 11 12 10 8
S.D. 7.1  2.6  2.2  4.9
N 9 9 9 9

Table 15. Female Food Consumption (g/animal/day) Summary

    Control

100

mg/kg/day

300

mg/kg/day

1000

mg/kg/day

Pre-mating           
Days 1-8 / Weeks 1-2 Mean 15 15 15 14
S.D. 0.4 0.3 0.9 0.4
N (cage) 2 2 2 2
Days 8-15 / Weeks 2-3 Mean 14 14 14 14
S.D. 0.7 0.0 0.6 0.3
N (cage) 2 2 2 2
Mean of means    15 14 14 14
Post-coitum          
Days 0-4 Mean 18 17 18 18
S.D. 2.3 1.0 1.9 1.6
N 8 10 9 9
Days 4-7 Mean 17 17 18 18
S.D. 2.8 2.1 1.9 2.6
N 8 10 9 9
Days 7-11 Mean 18 19 18 17
S.D. 3.2 2.4 2.2 2.7
N 8 10 9 9
Days 11-14 Mean 16 18 18 17
S.D. 5.1 2.6 1.2 3.7
N 8 10 9 9
Days 14-17 Mean 21 20 21 18
S.D. 2.0 2.4 2.4 3.0
N 8 10 9 9
Days 17-20 Mean 22 21 22 20
S.D. 2.5 2.6 2.5 1.6
N 8 10 9 9

Mean of means

  19 19 19 18
Lactation   19 19 19 18
Days 1-4 Mean 32 32 29 28
S.D. 8.9 5.8 3.8 6.0
N 9 9 9 9
Days 4-7 Mean 36 37 36 32*
S.D. 4.6 2.8 3.9 3.9
N 9 9 9 9
Days 7-13 Mean 52 50 48 42**
S.D. 6 3 4 5
N 9 9 9 9

Mean of means

  40 39 38 33

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 16. Summary of Haematology Values (Red Blood Cells) - Female

    RBC (10^12/L) [G1}
Control Mean 7.286
S.D. 0.444
N 5
100 mg/kg/day Mean 7.044
S.D. 0.165
N 5
tCtrl 0.97
300 mg/kg/dy Mean 7.000
S.D. 0.360
N 5
tCtrl 0.96
1000 mg/kg/day Mean 6.902
S.D. 0.455
N 5
tCtrl 0.95

[G1] - Anova & Dunnett

Table 17. Summary of Clinical Chemistry Values (ALT) - Males

  ALT (U/L) [G}
Control Mean 70.7
S.D. 25.4
N 5
100 mg/kg/day Mean 52.1
S.D. 4.2
N 5
tCtrl 0.74
300 mg/kg/dy Mean 60.5
S.D. 9.2
N 5
tCtrl 0.86
1000 mg/kg/day Mean 94.2
S.D. 33.1
N 5
tCtrl 1.33

[G] - Kruskal-Wallis & Dunn

Table 18. Summary of Clinical Chemistry Values (ALT and AST) - Females

  ALT (U/L) [G} AST (U/L) [G}
Control Mean 115.7 98.3
S.D. 16.2 12.7
N 5 5
100 mg/kg/day Mean 109.2 90.0
S.D. 9.3 9.6
N 5 5
tCtrl 0.94 0.92
300 mg/kg/dy Mean 107.2 96.7
S.D. 20.9 16.0
N 5 5
tCtrl 0.93 0.98
1000 mg/kg/day Mean 154.7 131.1
S.D. 39.7 51.1
N 5 5
tCtrl 1.34 1.33

[G] - Kruskal-Wallis & Dunn

Table 19. Summary of Endocrine Values - Male

  T3 (ng/mL) [G] T4 (ng/mL) [G] TSH (ng/mL) [G]
Control Mean 0.435 45.88 0.0943
S.D. 0.031 6.01 0.0884
N 10 10 10
100 mg/kg/day Mean 0.424 47.34 0.1172
S.D. 0.048 3.75 0.0497
N 10 10 10
tCtrl 0.97 1.03 1.24
300 mg/kg/dy Mean 0.5 51.6 0.2
S.D. 0.1230 6.9300 0.1205
N 10 10 10
tCtrl 1.13 1.13 1.75
1000 mg/kg/day Mean 0.404 33.18 ** 0.1076
S.D. 0.090 3.62 0.1038
N 10 10 10
tCtrl 0.93 0.72 1.14

[G] - Anova & Dunnett: ** = p ≤ 0.01

Table 20. Summary of Endocrine Values - Female

  T3 (ng/mL) [G] T4 (ng/mL) [G] TSH (ng/mL) [G]
Control Mean 0.279 28.24 0.196
S.D. 0.043 3.55 0.1204
N 9 9 9
100 mg/kg/day Mean 0.277 27.48 0.3558
S.D. 0.051 3.38 0.4084
N 9 9 9
tCtrl 0.99 0.97 1.82
300 mg/kg/dy Mean 0.257 24.96 0.2428
S.D. 0.0260 3.3000 0.1694
N 9 9 9
tCtrl 0.92 0.88 1.24
1000 mg/kg/day Mean 0.240 27.88 0.1379
S.D. 0.047 3.98 0.0938
N 9 9 9
tCtrl 0.86 0.99 0.7

[G] - Anova & Dunnett

Table 21. Summary of Functional Observation (Grip Strength) - Male

  Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Grip Fore Gram Mean 821 995 887 826
S.D. 120 289 208 115
N 5 5 5 5
Grip Hind Gram Mean 690  449 **  497 *  467 **
S.D. 116 74 65 105
N 5 5 5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 22. Summary fo Functional Observatiopns (Movement and Ambulations) - Female

Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Movements        
Mean 3546 3871 4873 4625
S.D. 1226 1122 483 2405
N 5 5 5 5
Ambulations        
Mean 843 981 1233 1510
S.D. 278 336 199 929
N 5 5 5 5

Table 23. Absolute Organ Weights (g) Summary - Male

  Control 100 mg/kg/day 300 mg/kg/dy 1000 mg/kg/day
Body weight Mean 327 340 331 318
S.D. 27 18 21 14
N 10 10 10 10
Liver Mean 7.38 8.51 8.81 10.14 **
S.D. 0.33 1.24 1.1 0.8
N 5 5 5 5
Kidney Mean 1.94 2.15 2.36 * 2.43 *
S.D. 0.07 0.21 0.39 0.07
N 5 5 5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 24. Relative Organ Weights (%) Summary - Male

  Control 100 mg/kg/day 300 mg/kg/dy 1000 mg/kg/day
Body weight (g) Mean 327 340 331 318
S.D. 27 18 21 14
N 10 10 10 10
Liver (%) Mean 2.37 2.52 2.64 3.17 **
S.D. 0.09 0.4 0.15 0.19
N 5 5 5 5
Kidney (%) Mean 0.62 0.64 0.71 0.76 **
S.D. 0.03 0.07 0.07 0.03
N 5 5 5 5
Brain (%) Mean 0.64 0.58 **  0.61 0.62
S.D. 0.02 0.03 0.03 0.03
N 5 5 5 5
Thyroids (%) Mean 0.0051 0.0044 *  0.005 0.005
S.D. 0.0007 0.0007 0.0004 0.0008
N 10 10 10 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 25. Absolute Organ Weights (g) Summary - Female

 

Control

100 mg/kg/day

300 mg/kg/dy

1000 mg/kg/day

Body weight

Mean

303

294

298

282

S.D.

26

21

18

15

N

10

10

10

10

Liver

Mean

12.51

12.36

13.23

13.57

S.D.

0.68

0.53

0.66

1.34

N

5

5

5

5

Kidney

Mean

2.07

1.91

2.02

1.88

S.D.

0.13

0.11

0.09

0.17

N

5

5

5

5

Spleen

Mean

0.565

0.538

0.519

0.473 *

S.D.

0.076

0.046

0.049

0.036

N

5

5

5

5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 26. Relative Organ Weights (%) Summary - Female

Female   Control 100 mg/kg/day 300 mg/kg/dy 1000 mg/kg/day
Body weight (g) Mean 303 294 298 282
S.D. 26 21 18 15
N 10 10 10 10
Liver (%) Mean 4.07 4.17 4.30 4.75 **
S.D. 0.1 0.3 0.28 0.29
N 5 5 5 5
Kidney (%) Mean 0.67 0.64 0.66 0.66
S.D. 0.02 0.03 0.02 0.03
N 5 5 5 5
Spleen (%) Mean 0.184 0.182 0.169 0.166
S.D. 0.021 0.016 0.019 0.014
N 5 5 5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 27. Summary Test Item-Related Microscopic Liver Findings

Sex Males Females
Dose level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Liver* 5 3 4 5 5 5 5 5
Hepatocellular hypertrophy              
Minimal - - - 1 - - - -
Slight - - - 4 - - - -
Cytoplasmic rarefaction                
Minimal - - - - 1 - 1 1
Slight - - - - - - - 2

*Number of tissues examined from each group

Table 28. Summary Test Item-Related Microscopic Thyroid Gland and Kidney Findings - Male

Dose level (mg/kg/day) 0 100 300 1000
Thyroid Gland* 5 3 4 5
Follicular cell hypertrophy        
Minimal - - - 3
Slight - - - 1
Kidney*: 5 3 4 5
Hyaline droplet accumulation        
Minimal 1 1 2 -
Slight 2 2 2 2
Moderate - - - 3
Granular casts        
Minimal - - - 2
Slight - - - 1

*Number of tissues examined from each group

Table 29. Developmental data - F0 Generation

    Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Dad pups at first litter check Litters affected (#) 0 1 0 5 #
Total 0 1 0 16
Mean (+) 0.0 0.1 0.0 1.8
S.D. 0.0 0.3 0.0 2.1
N 9 9 9 9
Living Pups at First Litter Check % of males/females (#) 55/45 53/47 55/45 48/52
Total 111 102 102 92
Mean (+) 12.3 11.3 11.3 10.1
S.D. 3.3 1.7 1.2 2.1
N 9 9 9 9
Postnatal loss % of living pups 0 2 1 2.2
Litters affected (#) 0 2 1 1
Total (#) 0 2 1 2
Mean (+) 0.0 0.2 0.1 0.7
S.D. 0 0.4 0.3 0.7
N 9 9 9 9
Culled Pups Total 40 28 29 21
Breeding loss Days 5-13 P.P.  % of living pups at day 4 P.P. 0 0 0 0
Litters affected (#) 0 0 0 0
Total (#) 0 0 0 0
Mean (+) 0 0 0 0
S.D. 0 0 0 0
N 9 9 9 9
Breeding loss Days 13 P.P.  % of males/females (#) 52/48 57/43 53/47 51/49
Total (#) 71 72 72 68
Mean (+) 7.9 8.0 8.0 7.6
S.D. 0.3 0.0 0.0 1.0
N 9 9 9 9
Total number of offspring born 111 103 102 107
Total number of uterine implantation sites 116 121 113 113
Number of live offspring on Day 1 after littering 111 102 102 91
Number of live offspring on Day 4 (before culling) 111 100 101 89
Number of live offspring on Day 4 (after culling) 71 72 72 686
Number of live offspring on Day 7 after littering 71 72 72 68
Number of live offspring on Day 13 after littering 71 72 72 68
Post-implantation survival index (%)   96 85 90 95
Live birth index (%) 100 99 100 85
Viability index 1 (%) 100 98 99 98
Viability index 2 (%) 100 100 100 100
Viability index 3 (%) 100 100 100 100
Lactation index (%) 100 100 100 100
Cumulative survival index (%) 100 97 99 93

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 30. Pup Body Weights (g)

Day Sex   Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
1 M Mean (+) 6.7 6.8 6.1 5.4 **
S.D. 0.7 0.7 0.6 0.7
N 9 9 9 9
F Mean (+) 6.4 6.2 5.7 5.2 ** 6.2 5.7 5.2 ** 6.2 5.7 5.2 **
S.D. 0.8 0.6 0.5 0.6
N 9 9 9 9
M+F Mean (+) 6.5 6.6 5.9 * 5.3 *
S.D. 0.8 0.6 0.6 0.6
N 9 9 9 9
4 M Mean (+) 10.1 10.4 9.4 8.4 *
S.D. 1.3 1.3 1.2 0.9
N 9 9 9 9
F Mean (+) 9.8 9.8 8.9 8.0 **
S.D. 1.5 1.2 1 0.8
N 9 9 9 9
M+F Mean (+) 10 10.1 9.2 8.2 **
S.D. 1.3 1.3 1.1 0.8
N 9 9 9 9
7 M Mean (+) 16.8 17.2 15.9 13.6 **
S.D. 1.8 1.7 1.9 1.1
N 9 9 9 9
F Mean (+) 16.2 16.1 15.2 13.1 **
S.D. 2.2 1.7 1.7 0.9
N 9 9 9 9
M+F Mean (+) 16.5 16.8 15.6 13.3 **
S.D. 2 1.7 1.8 0.9
N 9 9 9 9
13 M Mean (+) 32.3 32.6 30.8 26.3 **
S.D. 3.3 2.6 3.5 2.2
N 9 9 9 9
F Mean (+) 31.4 31.5 29.9 25.6 **
S.D. 3.5 2.4 3.3 1.9
N 9 9 9 9
M+F Mean (+) 31.9 32.2 30.4  25.9 **
S.D. 3.3 2.4 3.4 2
N 9 9 9 9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 31. Anogenital distance and nipple retention - F0 generation

    Control

100

mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Male anogenital distance (mm) Mean (+) 2.62 2.63 2.44 2.21
S.D. 0.24 0.21 0.22 0.27
N 9 9 9 9
Female anogenital distance (mm) Mean (+) 1.0 1 1.0 1.14
S.D. 0.07 0.05 0.08 0.14
N 9 9 9 9
Number of nipples Mean (+) 0.03 0.08 0.00 0.03
Median (+) 0.00 0.00 0.00 0.00
N 9 9 9 9

Table 32. Corrected Anogenital distance

PND 1   Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Male anogenital distance (mm) Mean (+) 1.39 1.39 1.34 1.26
S.D. 0.10 0.11 0.12 0.11
N 9 9 9 9
Female anogenital distance (mm) Mean (+) 0.6 0.54 0.6 0.66 +
S.D. 0.04 0.03 0.04 0.1
N 9 9 9 9

+/++ Steel-test significant at 5% (+) or 1% (++) level

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic No Observed Adverse Effect Level (NOAEL) of Mixed Xylene in rats was determined to be 300 mg/kg/day (with observed alpha-2u-globulin nephropathy taken into consideration) or at least 1000 mg/kg/day (without observed alpha 2u globulin nephropathy being considered as this is male rat specific and not relevant to humans). Based on lack of adverse reproductive toxicity observed at the highest dose tested, the NOAEL for reproductive toxicity was determined to be at least 1000 mg/kg/day.
Executive summary:

A key OECD Guideline 422 study was conducted to determine the potential toxic effects of the test material (Mixed Xylene (CAS 1330-20-7)) when administered orally for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

 

The test material was administered orally once daily via gavage to Wistar Han rats (10/sex/dose) in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg bw/day, 7 days a week for a minimum of 28 days (including at least 2 weeks of treatment prior to mating and during the mating period up to

and including the day before scheduled necropsy) for male rats and 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy for female rats.

 

Parameters evaluated were mortality/moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0-males and females), gross necropsy findings, organ weights and histopathologic

examinations. Additionally, reproduction/developmental parameters such as mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, developmental indices, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormones T3 and T4 (PND 4) and T3, T4 and TSH (PND 14-16 pups)) were evaluated.

 

No mortality and no treatment-related toxicologically relevant clinical signs were observed through the study period.At 1000 mg/kg/day, body weight gain was slightly lower during most of the treatment period for male rats and from Day 14 post-coitum onwards for female rats. In addition, slight body weight loss was observed over Days 1-8 for female rats. The lower body weight gain and body weight loss resulted in a slightly lower mean body weight at the end of treatment for females only. Correlating decreased absolute and relative food consumption was observed for females only. Considering the short period of time during which body weight loss was noted as well as the small magnitude of changes, the effects on body weight gain and food consumption were considered to be non-adverse.

 

Hematological parameters and behaviour of treated rats were considered unaffected by treatment with the test material. Non-adverse increased alanine aminotransferase (ALT) was noted in both sexes and non-adverse decreased total thyroxine (T4) was observed only in male rats. Gross necropsy did not reveal any remarkable treatment-related findings.

 

At 300 mg/kg/day, non-adverse increased absolute kidney weights were observed in males but were without histopathologic correlate. No treatment-related findings were observed in female rats at this dose level. At the 1000 mg/kg bw/day dose level, non-adverse higher liver weights in males and females that correlated with microscopic hepatocellular hypertrophy and cytoplasmic rarefaction for males and females, respectively, were observed. Non-adverse follicular cell hypertrophy was also observed in the thyroid gland in male rats dosed at 1000 mg/kg bw/day.

 

In the 1000 mg/kg bw/day dose group, adverse histopathologic findings were observed in the kidney of male rats that comprised granular casts and increased hyaline droplet accumulation. These were considered to represent a degenerative process and correlated with an increased kidney weight. The increased incidence and severity of hyaline droplet accumulation recorded in the kidney of male rats represented alpha 2u-globulin (as confirmed by immunohistochemistry), a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in mammals, including man.

 

No reproductive toxicity was observed up to the highest dose level tested 1000 mg/kg/day. No developmental toxicity was observed up to 300 mg/kg/day but at 1000 mg/kg/day, an adversely reduced live birth index (i.e. an increased incidence of pup mortality) was observed at first litter check. Consequently, litter size was adversely reduced with on average two pups per litter less than control litters, associated with a decreased cumulative survival index. Adversely reduced pup body weights (both sexes) were observed from PND 1 onwards. Non-adverse changeswere noted in (corrected) anogenital distance, which was increased in female pups.

 

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic No Observed Adverse Effect Level (NOAEL) of Mixed Xylene in rats was determined to be 300 mg/kg/day (with observed alpha-2u-globulin nephropathy taken into consideration) or at least 1000 mg/kg/day (without observed alpha 2u globulin nephropathy being considered as this is male rat specific and not relevant to humans). Based on lack of adverse reproductive toxicity observed at the highest dose tested, the NOAEL for reproductive toxicity was determined to be at least 1000 mg/kg/day.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status not known, non-guideline animal experimental study, limitations in design and/or reporting but otherwise adequate for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
One generation reproduction study in CD rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl-CD® (SC) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., St. Constance, Ontario, Canada
- Age at study initiation: 50 days
- Weight at study initiation: group mean range: 223-226 g (males), 162-165 g (females)
- Fasting period before study: none
- Housing: During the study, animals were caged individually in stainless steel wire mesh cages, with the exception of mating (nightly co-housing of males with females) and during lactation (female with the litter during non-exposure intervals)
- Diet: Purina Laboratory Chow 5002 (mash) ad libitum except during exposure periods
- Water: tap water ad libitum
- Acclimation period: 20 days (June 3-23, 1981)
- On day 21 of gestation, each female's cage was fitted with a stainless steel floor pan; these were removed on day 14 of lactation.
- Litter Kleen® hardwood shavings were added to the females cages on day 21 of gestation and fresh bedding provided as necessary. Bedding was removed on day 14 of lactation.

ENVIRONMENTAL CONDITIONS
- Temperature: 63-80°F (>90% of values between 70-75%)
- Humidity: 13-68% (>85% of values between 30-68%)
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 June 1981 To: 4 January 1982
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
The test material was measured out using a graduated cylinder into an Ehrlenmeyer flask. The test material was pumped to a JSS spraying systems atomiser using an FMI lab pump (model RPG-20) with a piston. Delivery lines used were teflon tubing. The test material was atomised with compressed air at a back pressure of 20 p.s.i and directed into the chamber inlet portal.
No further details reported.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 or 1:1
- Length of cohabitation: Mating units (1:2) did not change for the first 8 days of the mating period. After this time, males were reassigned randomly among unmated females (by group), on 1:1 ratio. The total mating period was 21 consecutive days.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

A special mating was performed to provide F0 study males at 100 days of age, with mating experience prior to initiation of the actual study mating. However, all males regardless of reproductive performance were incorporated into the mating design for the main study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Miran determinations were confirmed by gas chromatography analysis.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
For 131 days prior to mating, with exposure continued in females on gestation days 1–20 and lactation days 5–20.
Details on study schedule:
- One-half of all F0 males were sacrificed after the mating period for gross postmortem examination; the remaining half were sacrificed and examined 21 days later.
- One-half of the group I F0 females and group IV F0 females were sacrificed on GD 21 for developmental toxicity evaluation.
- The remaining F0 females were allowed to deliver litters.
Remarks:
Doses / Concentrations:
0, 60, 250 or 500 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 60±2, 250±5 or 500±13 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
0 ppm - 30 males and 60 females, 60 ppm - 10 males and 20 females, 250 ppm - 10 males and 20 females. There were three high dose groups (500 ppm): group IV contained 20 males and 40 females and both sexes were treated; group V contained 10 males and 20 females but only males were treated; group VI contained 10 males and 20 females but only females were treated.
Control animals:
yes, sham-exposed
Details on study design:
Twenty control and twelve 500 ppm females were killed on day 21 of gestation and foetuses were evaluated for external, soft tissue and/or skeletal malformations. The remaining females from all groups were allowed to deliver their litters and offspring were evaluated for growth and survival during a 21 day lactation period.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, pre- and post-exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the pre-mating period. Males and unmated females were weighed weekly throughout the mating and post-mating periods.
- Mated females weighed on days 1, 7, 13, 19 and 21 of gestation.
- Females with litters weighed on days 1, 4, 14 and 21 of lactation.

FOOD CONSUMPTION: Yes
- recorded at 2-day intervals during gestation for all mated females.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 post partum: yes. Litters were standardized by pooling all pups within each treatment group on lactation day 4 and redistributing four males and four females from this pool to each dam. However, on some days the pups could not be pooled if only one litter was available. In this case, litters were culled to four males and four females when possible.

PARAMETERS EXAMINED
The following parameters were examined offspring:
- Pups were weighed, sexed, and given a gross external examination on lactation days 1, 4, and 21.
- Randomly selected pups from each group (one/sex/litter) and all remaining F0 females with litters were sacrificed on day 21 of lactation and subjected to gross necropsy.
- The remaining pups were maintained for the postweaning interval of 28–49 days and weighed and sacrificed on day 49.
- Randomly selected pups from each group (one/sex/litter) were given a complete gross postmortem examination.

GROSS EXAMINATION OF DEAD PUPS:
- yes, gross external examination and the stomach was evaluated for the presence of milk. Visceral contents of thoracic and abdominal cavities examined.
- Pups found dead prior to day 4 of lactation were stored in 70% ethanol. pups found dead on day 5 or later were examined and discarded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: One-half of all F0 males were killed after the mating period for gross post mortem examination; the remaining half were killed and examined 21 days later.
- Maternal animals: One-half of the group I F0 females and group IV F0 females were killed on GD 21 for developmental toxicity evaluation. The remaining F0 females were killed on day 21 of lactation and subjected to gross necropsy.

POST-MORTEM EXAMINATIONS: Yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Half of the males in each group were killed after completion of the mating period. Testes, epididymides, seminal vesicles and prostate were stored in 10% neutral buffered formalin. The testes were weighed.
- The remaining males were killed 21 days later (December 14 1981). Abnormal tissue, testes, epididymides, seminal vesicles and prostate were stored in 10% neutral buffered formalin.

ORGAN WEIGHTS: The testes were weighed.
Postmortem examinations (offspring):
SACRIFICE
- Pups maintained for the postweaning interval of 28–49 days were weighed and killed on day 49. Randomly selected pups from each group (one/sex/litter) were given a complete gross post mortem examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- The following tissues were taken and stored in 10% neutral buffered formalin: adrenals, bone marrow, brain, eyes, gonads, epididymides, heart, colon, duodenum, ileum, kidneys, liver, lung, lymph node, mammary gland, pancreas, salivary gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, urinary bladder, uterus, prostate, gross lesions, tissue masses and thymus.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Testes and ovaries from all pups subjected to gross examination on lactation day 21 and day 49 post-partum were weighed.
Statistics:
Appropriate evaluations were performed between data for the control and treated groups.
Reproductive indices:
Mating, fertility and pregnancy indices.
Offspring viability indices:
Pup survival and litter survival.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- A total of 12 females (1 group II, 3 group III, 4 group IV, 1 group V and 3 group VI) did not mate during the study. Evaluation of vaginal smears indicated that with the exception of 2 group IV females, all other females were not showing normal oestrus cycling during the mating period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- The female mating index in group III and group VI was significantly lower than for controls (85 and 85%, respectively, vs. 100% for controls), however, a similar effect was not observed in group IV (500 ppm exposed males and females) and there was also an unusually high mating performance in the controls.
- The male mating index, pregnancy rate, and fertility index in exposed animals were comparable to control values.
Dose descriptor:
NOAEC
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: no systemic toxicity or effects on reproduction at the highest dose tested
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
BODY WEIGHT (OFFSPRING)
- No statistically significant decrease in mean pup body weights were observed in the exposed versus control groups at days 1 and 14. On lactation day 4, mean pup weights were statistically significantly decreased in groups II (60 ppm), III (250 ppm), and IV (500 ppm) (post-pooling) when compared with controls, but the decreases (about 8%) were not of a biologically significant magnitude. The decreased weights may have been the consequence of an elevated mean pup weight in the control group potentially caused by a smaller mean litter size (mean number of live pups per litter: 9.6, 11.8, 12.5, 12.4, 10.8, and 11.8 for groups I–VI, respectively).
- Pups from group IV had statistically significant decreased mean pup weights on lactation day 21 (90% of controls) and statistically significant decreased terminal body weights at 49 days of age (as a percentage of controls: males, 92%; females, 93%). However, despite the marginal decreases observed in mean pup weights in group IV, no decreases in body weights were observed in pups from group VI, in which dams were exposed to the same concentration of xylene (500 ppm) for the same period of time as were dams in group IV. The marginal decreases observed in mean pup weights from group IV were considered not to be an adverse effect of treatment.

ORGAN WEIGHTS (OFFSPRING)
- Female pups from the mid- and high-dose groups (groups III and IV) also had statistically significant decreased absolute (76 and 78% of controls, respectively) and relative (80% and 84% of controls, respectively) ovary weights at 21 days of age, but the decreases were not concentration related and were not observed at 49 days of age. In addition, decreases in ovary weights were also not observed in group VI pups.
Reproductive effects observed:
not specified
Conclusions:
500 ppm mixed xylene (administered for 6 hours per day for 131 days prior to mating, during mating and continuing through gestation and lactation) is a NOAEC for systemic and reproductive toxicity.
Executive summary:

Groups of male and female CD rats were exposed to 0, 60, 250, or 500 ppm mixed xylenes (groups I, II, III, and IV, respectively by inhalation. exposure was for 6 hours per day, 5 days per week, for 131 days prior to and during mating, with exposure continued in females on gestation days 1–20 and lactation days 5–20. Two additional 500 ppm groups were similarly exposed, except that only the F0 males were exposed in group V, and only the F0 females were exposed in group VI. In-life parameters evaluated in adults included body weights, observations for mortality and clinical signs, detailed weekly physical examination, maternal body weights and maternal food consumption and food efficiency. One-half of all F0 males were sacrificed after the mating period for gross post mortem examination; the remaining half were sacrificed and examined following a 21 day treatment-free period.

Litters were standardized by pooling all pups within each treatment group on lactation day 4 and redistributing (where possible) four males and four females from this pool to each dam. Pups were weighed, sexed, and given a gross external examination on lactation days 1, 4, and 21. Randomly selected pups from each group (one/sex/litter) and all remaining F0 females with litters were sacrificed on day 21 of lactation and subjected to gross necropsy. The remaining pups were maintained for the postweaning interval of 28–49 days and weighed and sacrificed on day 49. Randomly selected pups from each group (one/sex/litter) were given a complete gross postmortem examination.

The highest exposure level of 500 ppm mixed xylene administered for 6 hours per day for 131 days prior to mating, during mating and continuing through gestation and lactation is a NOAEC.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two guideline OECD 422 studies show no evidence of effects on fertility in the parental generation when exposed orally to Xylene or the Reaction Mass of Ethylbenzene and m-Xylene.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 171 mg/m³
Species:
rat
Quality of whole database:
Studies conducted in rats demonstrate no evidence that mixed xylenes or ethylbenzene adversely affect reproduction.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Non-human information


 


Data generated pre-2022


In a one-generation reproductive toxicity study (Bio/Dynamics Inc., 1983), groups of male and female CD rats were exposed to 0, 60, 250, or 500 ppm technical-grade xylene (comprising 2.4% toluene, 12.8% ethylbenzene, 20.3% p-xylene, 44.2% m-xylene, 20.4% o-xylene) by inhalation for 6 hours per day, 5 days per week, for 131 days prior to mating, with exposure continued in females on gestation days (GDs) 1–20 and lactation days 5–20. The highest exposure level of 500 ppm mixed xylene, administered for 6 hours per day for 131 days prior to mating, during mating and continuing through gestation and lactation, is a NOAEC for all endpoints measured. There was no evidence of reproductive toxicity in this study.


 


LOA acknowledges that the one generation reproduction study (Bio/Dynamics, 1983) in the dossier does not include some of the parameters measured in an OECD 443 (extended one-generation reproductive toxicity study). OECD 443 study in rats with p-xylene will be performed and read-across to o-xylene and m-xylene. The read-across is based on similarity of response of xylene isomers in sub-chronic and developmental toxicity studies. Also, Toxcast analysis on xylene isomers shows similar responses and this read-across approach helps to reduce an excessive and wasteful use of animals.


 


Data generated post-2022 as part of Integrated Assessment and Testing Approach (IATA)


In a key guideline OECD 422 study (combined repeated dose toxicity study with the reproduction/developmental toxicity screening test) (CRL 2022), 10 rats/sex/dose were exposed to 100, 300, or 1000 mg/kg/bw/day of the test material Xylene daily via oral gavage for a minimum of 28 days, including at least 2 weeks treatment prior to mating and during the mating period up to and including the day of scheduled necropsy for male rats, and at least 14 days prior to mating, duration of pregnancy, and at least 13 days after delivery including the day before scheduled necropsy for female rats.


 


Parameters evaluated included parental body weight and food consumption, oestrous cycle, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0-males and females), mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, developmental indices, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormones T3 and T4 (PND 4) and T3, T4 and TSH (PND 14-16 pups)) were evaluated.


 


No mortality and no treatment-related toxicologically relevant clinical signs were observed through the study period. At 1000 mg/kg/day, body weight gain was slightly lower during most of the treatment period for male rats and from Day 14 post-coitum onwards for female rats. In addition, slight body weight loss was observed over Days 1-8 for female rats. The lower body weight gain and body weight loss resulted in a slightly lower mean body weight at the end of treatment for females only. Correlating decreased absolute and relative food consumption was observed for females only. Considering the short period of time during which body weight loss was noted as well as the small magnitude of changes, the effects on body weight gain and food consumption were considered to be non-adverse.


 


In the 1000 mg/kg bw/day dose group, adverse histopathologic findings were observed in the kidney of male rats that comprised granular casts and increased hyaline droplet accumulation. These were considered to represent a degenerative process and correlated with an increased kidney weight. The increased incidence and severity of hyaline droplet accumulation recorded in the kidney of male rats represented alpha 2u-globulin (as confirmed by immunohistochemistry), a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in any other mammals, including humans.


 


No reproductive toxicity was observed up to the highest dose level tested 1000 mg/kg/day. No developmental toxicity was observed up to 300 mg/kg/day but at 1000 mg/kg/day, an adversely reduced live birth index (i.e., an increased incidence of pup mortality) was observed at first litter check. Consequently, litter size was adversely reduced with on average two pups per litter less than control litters, associated with a decreased cumulative survival index. Adversely reduced pup body weights (both sexes) were observed from PND 1 onwards. Non-adverse changes were noted in (corrected) anogenital distance, which was increased in female pups


 


Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic No Observed Adverse Effect Level (NOAEL) of Mixed Xylene in rats was determined to be at least 1000 mg/kg/day (without observed alpha 2u globulin nephropathy being considered as this is male rat specific and not relevant to humans). Based on lack of adverse reproductive toxicity observed at the highest dose tested, the NOAEL for reproductive toxicity was determined to be at least 1000 mg/kg/day.


 


In another key guideline OECD 422 study carried out in 10/sex/dose rats by CRL in 2022 with the Reaction Mass of Ethylbenzene and m-Xylene as the test material. The dose concentrations evaluated were 100, 300, and 1000 mg/kg/day. Parameters evaluated included parental body weight and food consumption, oestrous cycle, measurement of thyroid hormones T3, T4 and TSH (F0-males and females), mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormones T3 and T4 (PND 4), and T3, T4 and TSH (PND 14-16 pups)).


 


No parental toxicity was observed up to 1000 mg/kg/day in males and females. No treatment-related changes were observed in any of the following parameters investigated in the study (i.e., mortality, clinical appearance, haematology, clotting parameters, T3, T4 and TSH thyroid hormone levels, macroscopic examination).


 


Treatment-related reduced body weight (males and females) and food consumption (females only) were observed at 1000 mg/kg/day during the lactation period. Considering the minor magnitude of the change, these effects were considered non-adverse.


 


Hyaline droplet accumulation was observed in the kidney of males starting at 100 mg/kg/day (up to slight degree). This finding was considered possible treatment-related at 100 and 300 mg/kg/day. As the incidence and mean severity showed a subtle increase at 1000 mg/kg/day, this finding was regarded to be treatment-related at the highest dose level. The observed hyaline droplets most likely resembled alpha 2µ-globulin, a normal protein present in the kidney of male rats, which was confirmed by immunohistochemistry. This protein is absent in female rats, as well as in humans. In absence of microscopic findings indicative of kidney damage, this finding was regarded non-adverse (Sahota 2013). Furthermore, there was a statistically significant higher kidney weight (relative to body weight) observed in male rats at 1000 mg/kg/day. Based on the absence of a microscopic correlate, these kidney changes were considered non-adverse.


 


No reproductive toxicity and no test treatment-related changes were observed in any of the reproductive parameters investigated (i.e., mating and fertility indices, precoital time, number of implantations, oestrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs) up to the highest dose level tested (1000 mg/kg/day).


 


Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic and reproductive toxicity No Observed Adverse Effect Levels (NOAEL) for Reaction mass of ethylbenzene and m-xylene were determined to be at least 1000 mg/kg/day.


 


Human information


 


No human data are available.


 


Justification for selection of effect on fertility via inhalation route: 


No adverse effects on sexual function, fertility or development were apparent in a rat one generation study conducted on mixed xylenes (NOAEC exceeds 2171 mg/m^3; Bio/Dynamics, 1983).


 


Justification for selection of Effect on fertility via inhalation route: No adverse effects on fertility were observed in OECD 422 studies in rats conducted on the Reaction Mass of ethylbenzene and m-xylene or mixed xylenes.

Effects on developmental toxicity

Description of key information

Information is available on the effect of individual xylene isomers (m-, o-, and p-xylene; xylene; and the Reaction Mass of Ethylbenzene and m-Xylene via oral gavage at dose levels up to and including 1000 mg/kg bw/day. The results indicate that there was no adverse effect on pregnancy, embryonic or foetal development in rabbits exposed to xylene isomers. Results in rats indicate developmental toxicity at the highest dose tested including reduced live birth index, reduced litter size, reduced survival index and lower body weights (both sexes).

 

Information is available on the effect of individual xylene isomers (m-, o-, and p-xylene; mixed xylenes) on prenatal developmental toxicity via inhalation at concentrations up to and including 2000 ppm (8684 mg/m^3). The results indicate that maternal toxicity (reduced corrected maternal body weight gain) occurred at exposures that were lower than those causing a biologically meaningful (>10%) reduction in foetal body weight, indicating that xylene isomers are not selectively toxic towards the foetus.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-Jun-22 to 2022-Mar-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This data has not been finalised by LOA due to an ongoing review. The data presented in this record is from an audited draft report by the contract research organisation. This information was requested by a National Enforcement Agency to help a registrant fulfill their annex VIII information requirements.
A communication from the contract research organisation stating when the finalisation of the report will occur by (31st January 2023) is included below in the "attached justification" section. LOA is committed to performing a dossier update when the full information required for ECHA to judge the read-across approach utilised in this dossier is available.
Qualifier:
according to guideline
Guideline:
other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version: July 2016
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or th e interpretation of the study results and conclusions. Please see 'Any other information on materials and methods' for details.
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Hanwha TOTAL (South Korea); Batch number: FB8055-15223
- Purity, including information on contaminants, isomers, etc.: Mixed Xylene (o-xylene: 20.98 WT% ; m-xylene: 49.35 WT%; and p-xylene: 23.81 WT%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in corn oil vehicle for at least 24 hours at room temperature under normal laboratory light over a concentration range of 2 to 250 mg/mL (solutions); Stable in corn oil vehicle for at least 8 days in the refrigerator over a concentration range of 2 to 250 mg/mL (solutions); 0.5 mL sample in corn oil vehicle stable for at least 3 weeks in the freezer (≤-15°C) over a concentration range of 2 to 250 mg/mL (solutions).

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS
- Specific gravity / density: 0.8708 g/cm3 (15°C)
- Volatile: Yes, vapour pressure: 6~16 mmHg at 20°C
- Expiry date: 2023-APR-15
- CAS Number: 1330-20-7
- EC Number: 215-535-7
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: Males: 10-12 weeks; Females: 14-15 weeks
- Weight at study initiation: Males: 272 - 327 g; Females: 201 - 274 g
- Housing:

On arrival and during the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).

During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.

During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

- Diet: pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH (Soest, Germany) ad libitum
- Water: Municipal tap water via water bottles ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, based on the results of these analyses it was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 39 to 67%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 2021-JUN-23 To: 2021-OCT-18
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Sigma-Aldrich (Steinheim, Germany)
Remarks on MMAD:
N/A
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Sigma-Aldrich, Steinheim, Germany) was used as the vehicle based on trial preparations performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and were not used for dosing.
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg bw/day dose groups, respectively.
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During Week 1 of treatment, dose formulation samples were collected for analysis as follows:

1. Concentration analysis: sample collected from approximately ‘Middle’ for all dose groups

2. Homogeneity analysis: sample collected from approximately ‘Top, Middle and Bottom’ for dose groups 2 and 4 only. Analyses were performed using a validated analytical procedure (Test Facility Study No. 20275973). For concentration: mean sample concentration results within or equal to ± 15% of theoretical
concentration.

For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group. Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20275973) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until detection of mating was confirmed by evidence of sperm in the vaginallavage or by the appearance of an intravaginal copulatory plug
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated.
- Any other deviations from standard protocol: Detection of mating was not confirmed in first instance for Female No. 46 (control). Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily
Duration of test:
28 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle: Corn Oil) - Group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a 10-Day Dose Range Finder with oral administration of Mixed Xylene (CAS 1330-20-7) in rats (Test Facility Reference No. 20275974) and in an attempt to produce graded responses to the test material.

- Rationale for animal assignment (if not random): Randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

- Fasting period before blood sampling for clinical biochemistry: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No

- Dose range finding studies: 10-Day Dose Range Finder with oral administration of Mixed Xylene (CAS 1330-20-7) in rats (Test Facility Reference No. 20275974)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test material and lasting throughout the Dosing periods up to the day prior to necropsy. Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

All animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Except on days of receipt and necropsy where frequency was at least once daily. Arena observations were also conducted once before the first administration of the test material and weekly during the treatment Period.

BODY WEIGHT: Yes
- Time schedule for examinations: for all animals on Day 1 of treatment (prior to dosing ) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum
and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; quantitatively measured per cage weekly, except for males and females which were housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Monitored regularly throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane); Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid hormones)
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters examined included Triiodothyronine (T3); Thyroxine (T4); and Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed post dosing on 5 males per group during Week 4 of treatment and 5 females per group during the last week of lactation (i.e. PND 8-10).
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity such as Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; and Locomotor activity)

IMMUNOLOGY: No

OTHER: Coagulation:
- Time schedule for collection of blood: On the day of scheduled necropsy
- How many animals: 5 animals/sex/group were selected
- Animals fasted: Males fasted overnight (maximum of 24 hours), females non-fasted
- Anaesthetic used for blood collection: Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.45 mL
- Parameters analysed: prothrombin time (PT), activated partial thromboplastin time (APTT)

POST-MORTEM EXAMINATIONS
GROSS PATHOLOGY: Yes (see Tables 4 and 5)

Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:

Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16; females which failed to deliver: With evidence of mating: Postcoitum Day 25-27 (Nos. 41, 55, 64 and 76).

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy.

Necropsy:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

The organs listed in Table 4 and 5 were weighed at necropsy for all scheduled euthanasia animals.

Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see Tables 6 and 7)

Representative samples of the tissues identified in the tables 6 and 7 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solut ion, Klinipath, Duiven, The Netherlands), unless otherwise indicated. The tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Alpha-2u-globulin staining:
Two slides per selected Group 1 and Group 4 male (e.g. lowest numbered 5 in each group) were shipped to CRL-Durham for HRP-polymer based chromogenic immunostaining of alpha-2u-globulin. The slides were returned to the Test Facility after the staining procedure has been completed for evaluation by the study pathologist.

Microscopic Evaluation:
All tissues as defined under Histology were examined by a board-certified toxicological pathologist. For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The slides stained for alpha-2u-globulin were evaluated by the study pathologist.
Ovaries and uterine content:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Blood sampling:
Blood sampling:

F0-animals (selected 5/sex/group): On the day of scheduled necropsy, blood was sampled (for hematology, coagulation, clinical chemistry, and thyroid hormone analysis) from the retro-orbital sinus under anaesthesia using isoflurane between 07.00 and 10.30 a.m.

PND 4 pups at culling (2 pups/litter): On PND 4 at culling, blood was sampled (for thyroid hormone analysis) by decapitation between 7.00 and 10.30 a.m., pooled to one sample per litter (from one male and one female, if possible). If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. If no surplus pups were available, no blood sample was collected.

PND 14-16 pups (2 pups/litter): On PND 14-16, blood was sampled (for thyroid hormone analysis) by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure between 7.00 and 10.30 a.m. from two pups per litter (from one male and one female).

The parameters assessed in maternal animal blood are listed below.

Haematology:
- On the day of scheduled necropsy
- 5 animals/sex/group were selected
- Males fasted overnight (maximum of 24 hours), females non-fasted
- Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.5 mL
- Parameters analysed are listed in Table 2

Coagulation:
- On the day of scheduled necropsy
- 5 animals/sex/group were selected
- Males fasted overnight (maximum of 24 hours), females non-fasted
- Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.45 mL
- Parameters analysed: prothrombin time (PT), activated partial thromboplastin time (APTT)

Clinical chemistry:
- On the day of scheduled necropsy
- 5 animals/sex/group were selected
- Males fasted overnight (maximum of 24 hours), females non-fasted
- Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.5 mL
- Parameters analysed are listed in Table 3

Thyroid hormone:
- On the day of scheduled necropsy
- 5 animals/sex/group (F0) and 2 pups/litter (F1) were selected
- Males (F0) fasted overnight (maximum of 24 hours), females (F0) non-fasted
- Parameters analysed: thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH)
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done.

Whenever the number of male or female pups prevented having four of each sex per litter, partial a djustment (for example, five males and three females) was acceptable.
Pups were examined for external and internal abnormalities and possible cause of death was determined for pups born or found dead].

Unscheduled Euthanasia
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia
On PND 14-16, all remaining pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

POSTMORTEM EXAMINATIONS (OFFSPRING)

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

Unscheduled Deaths – F1-Generation
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia – F1-Generation
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible.

All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same
pups as selected for thyroid preservation.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Indices:
Offspring viability indices
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

1. Viability index 1 (%) = (number of live offspring on Day 4 before culling / number of live offspring on Day 1 after litterling) x 100

2. Viability Index 2 (%) = (number of live offspring on Day 7 before culling / number of live offspring on Day 4 after litterling) x 100

3. Viability Index 3 (%) = (number of live offspring on Day 13 before culling / number of live offspring on Day 7 after litterling) x 100
Historical control data:
The test facility (Charles River Den Bosch) has general and reproduction/developmental historical data in this species from the same strain and source. This data for 2017-2021 is provided in Appendix XX of the final study report.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related toxicologically relevant clinical signs were observed through the study period.

At 1000 mg/kg/day, hunched posture was noted in one male on a single occasion in Week 2. Piloerection was observed in three females on three consecutive days during the post-coitum phase, but also in two control females on a single day. At the incidence observed, these clinical signs were cons
idered not to be treatment-related.

One male in the 100 mg/kg/day dose group had red discharge (likely blood) from the mouth on the day prior to scheduled necropsy and one female in the 1000mg/kg/day dose group was gasping on a single occasion during the post-coitum phase. As these signs were observed directly after dosing and noted at this single occasion only, they were considered gavage-related signs, rather than signs related to the test material.

Salivation seen after dosing in animals of the 300 (females) and 1000 (both sexes) mg/kg/day dose groups during most of the treatment period was considered not to be toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

No findings were noted during the weekly arena observations in this study. Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
No mortality occurred through the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes in body weights and body weight gain were observed up to 300 mg/kg/ day.

In male rats treated at 1000 mg/kg/day, body weight gain was slightly lower over the whole study period compared to controls (not statistically significant, except over Days 1-8). Mean body weightswere comparable to the concurrent control mean throughout treatment.

In female rats treated at 1000 mg/kg/day, slight body weight loss (up to -3%) was noted over Days 1-8, which normalized thereafter. In addition, a trend towards a lower body weight gain was noted from post-coitum Day 14 onwards until the end of the lactation period. Mean body weight at the end of the lactation period was 10% lower than the concurrent control mean. Any statistically significant changes at 100 mg/kg/day were considered unrelated to treatment with the test material, in the absence of a dose-response relationship.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to control values up to 1000 mg/kg/day in males and up to 300 mg/kg/day in female rats.

At 1000 mg/kg/day, food consumption was lower in females during the lactation period (not statistically significant over Days 1-4) compared to controls. The mean of means for food consumption and relative food consumption over this period were 17 and 9% lower than controls, respectively.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment with the test material.

The statistically significantly higher red blood cell distribution width at 1000 mg/kg/day in females was considered to be a result of a high value for one individual animal. At the incidence observed, this was considered unrelated to treatment with the test material.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical biochemistry parameters after treatment with the test item up to 300 mg/kg/day.

At 1000 mg/kg/day, alanine aminotransferase (ALT) activity was increased in both sexes with a change of 1.33 and 1.34x (not statistically significant) of the concurrent control value, respectively.Considering the small magnitude, this was considered to be not adverse.

Aspartate aminotransferase (AST) was increased in one female at 1000 mg/kg/day. At the incidence observed, this was considered unrelated to treatment with the test material.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Total thyroxine (T4) was decreased in males at 1000 mg/kg/day (0.72x of control) but the mean value remained within the historical control range.

Thyroid hormones were considered unaffected in males up to 300 mg/kg/day and in females up to 1000 mg/kg/day.

High thyroid stimulating hormone (TSH) values at 100 and 300 mg/kg/day in both sexes occurred in the absence of a dose-response relationship and individual data generally remained within the concurrent control range. Therefore, this finding was not considered to be treatment-related.
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment with the test material up to 1000 mg/kg/day.

Any statistically significant changes in male hind grip strength were considered to derive from relatively high control values, rather than from treatment with the test material. No dose-response relationship was observed and all mean values remained within the historical range, including controls.

Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. High total movement and ambulation values for females at 1000 mg/kg/day were mainly attributed to a single female, and at this incidence observed, this was considered unrelated to treatment with the test material.
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant higher liver weights were present in males (absolute and relative to body weight) and females (relative to body weight) at 1000 mg/kg/day.

Statistically significant higher kidney weights were present in males treated at 300 (absolute) and 1000 mg/kg/day (absolute and relative to body weight).
There were no other test item-related organ weight changes.

Some organ weight differences were statistically significant (lower absolute spleen weight in females at 1000 mg/kg/day) when compared to the control group but considered to be the result of a lower final body weight. In addition, the statistically significant lower, relative to body weight, thyroid gland and brain weight in males at 100 mg/kg/day were without a dose response and therefore considered incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any remarkable findings. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were observed in the liver of males and females and the thyroid gland and kidney of males.

Liver:
Hepatocellular hypertrophy was present in males at 1000 mg/kg/day at a minimal to slight degree. Cytoplasmic rarefaction (likely representing glycogen) was present at increased incidence and severity in females at 1000 mg/kg/day at a minimal to slight degree.

Thyroid gland:
Follicular cell hypertrophy was present in males at 1000 mg/kg/day at a minimal to slight degree.

Kidney:
An increased incidence and severity of hyaline droplet accumulation (positive for alpha 2u-globulin) was present in males at 1000 mg/kg/day up to moderate degree. Granular casts (positive for alpha 2u-globulin) were present in males at 1000 mg/kg/day up to a slight degree.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle: Length and regularity of the estrous cycle were not affected by treatment with the test item. All females had regular cycles of 4 days.
Details on results:
N/A
Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 85, 90, and 95% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

Female No. 55 (100 mg/kg/day) had 11 implantations but did not deliver a litter. As this occurred in a single female and in the low dose group only, this was considered unrelated to treatment.
Total litter losses by resorption:
not specified
Description (incidence and severity):
N/A
Early or late resorptions:
not specified
Description (incidence and severity):
N/A
Dead fetuses:
no effects observed
Description (incidence and severity):
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) was considered not to be affected by treatment with the test item. Except for one female at 100 mg/kg/day, all pregnant females had live offspring. Therefore, gestation indices were 100, 90, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 85, 90 and 95% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. Female No. 55 (100 mg/kg/day) had 11 implantations but did not deliver a litter. As this occurred in a single female and in the low dose group only, this was considered unrelated to treatment with the test item.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
N/A
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of implantation sites was considered not to be affected by treatment with the test item. The mean number of implantation sites were 12.9, 12.1, 12.6, and 12.6 for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Other effects:
no effects observed
Description (incidence and severity):
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 premature birth. No deficiencies in maternal care were observed.

Fertility Index:
Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 90, 100, 90 and 90% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Details on maternal toxic effects:
N/A
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were decreased at 1000 mg/kg/day from PND 1 onwards for both sexes. Mean combined pup body weights on PND 13 were 19% lower than control. Pup body weights were considered to be unaffected up to 300 mg/kg/day.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Live birth index was considered unaffected by treatment with the test item up to 300 mg/kg/day.

At first litter check, one pup at 100 mg/kg/day and 16 pups of the 1000 mg/kg/day group (divided over 5 litters) were found dead. Four of these 16 pups at 1000 mg/kg/day were observed to have no milk in the stomach. No macroscopic findings were noted for the other pups. At the single occurrence, the dead pup at 100 mg/kg/day was considered to be unrelated to treatment as it remained within the range considered normal for pups of this age. Live birth indices were 100, 99, 100, and 85% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Live birth index at 1000 mg/kg/day was below the historical range.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment with the test material.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter Size:
Litter size was considered unaffected by treatment up to 300 mg/kg/day. A lower mean number of live pups was recorded at 1000 mg/kg/day (not statistically significant). Mean number of live pups were 12.3, 11.3, 11.3, and 10.1 live pups per litter for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

Anogenital distance of all rodent fetuses:
effects observed, treatment-related
Description (incidence and severity):
Anogenital distance (absolute and corrected for body weight) in male pups was considered not to be affected by treatment with the test item up to 1000 and in female pups up to 300 mg/kg/day. Corrected anogenital distance for female pups was increased (1.20x to control). Mean values remained within the historical control range. The apparently slightly lower corrected anogenital distance in male pups at 1000 mg/kg/day was considered to derive from overcorrection due to the low pup body weights.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
Viability Index 1:

Viability Index 1 (the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) was considered not affected by treatment with the test material. Viability indices on PND 4 were 100, 98, 99, and 98% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Two, one and two pups at 100, 300, and 1000 mg/kg/day, respectively, were found dead or missing on PND 2. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index 2:

Viability Index 2 (the number of live offspring on Day 7 compared to the number of offspring on Day 4 after culling) was unaffected by treatment. Viability indices on PND 7 were 100% for all treatment groups.

Viability Index 3:
Viability Index 3 (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND 7) was not affected by treatment with the test material. Indices were 100% for all treatment groups.

Cumulative Survival Index:
The cumulative survival index ((number of live offspring on Day 13 after litter/number of live offspring on Day 4 (after culling)) × (number of live offspring on Day 4 before culling / total number of offspring born)) × 100% was considered not to be affected by treatment up to 300 mg/kg/day. Cumulative survival indices were 100, 97, and 99% for the control, 100 and 300 mg/kg/day groups, respectively. At 1000 mg/kg/day, the cumulative survival index was decreased (i.e. 83%).
External malformations:
no effects observed
Description (incidence and severity):
No findings were noted among pups that were considered to be related to treatment with the test material. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Skeletal malformations:
not examined
Description (incidence and severity):
N/A
Visceral malformations:
not examined
Description (incidence and severity):
N/A
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be treatment-related.

Serum T3 and T4 levels in male and female PND 4 and PND 14-16 pups, and TSH levels in PND 14-16 pups, were considered not to be affected by treatment. High TSH values at 100 and 300 mg/kg/day in both sexes occurred in the absence of a dose-response relationship and individual data were generally variable. Therefore, this finding was considered unrelated to treatment with the test material.
Details on embryotoxic / teratogenic effects:
N/A
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Dose Formulation Analyses

Accuracy

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). No test material was detected in the Group 1 formulation.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 9. Male Body Weights (g) Summary

    Control 100 mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 294 297 298 297
S.D. 17.1 9.3 12.3 10.3
N 10 10 10 10
Day 8 / Week 2 Mean 314 319 317 312
S.D. 21.7 10.8 16.9 13.6
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 329 336 331 327
S.D. 26.3 12.7 19.2 15.1
N 10 10 10 10
Day 8 /Week 2 Mean 336 348 342 332
S.D. 28.6 16.7 20.6 15.4
N 10 10 10 10
Day 16 / Week 3 Mean 345 361 352 343
S.D. 26.8 18.1 24.1 15.9
N 10 10 10 10

Table 10. Female Body Weights (g) Summary

    Control 100 mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 239 234 239 236
S.D. 20.5 15.6 14.5 12.1
N 10 10 10 10
Day 8 / Week 2 Mean 245 239 241 235
S.D. 18.7 18.6 14.8 13.4
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 251 241 246 244
S.D. 19.1 19.6 15.9 16.5
N 10 10 10 10
Day 8 /Week 2 Mean - - - -
S.D. - - - -
N 0x 0 0 0

Table 11. Female Body Weight (g) Summary

    Control

100

mg/kg/day

300

mg/kg/day

1000

mg/kg/day

Post-coitum          
Day 0 Mean 244 243 242 241
S.D. 18.8 19.1 17.1 12.8
N 8 10 9 9
Day 4 Mean 259 254 255 255
S.D. 19.7 19.5 15.6 14.3
N 8 10 9 9
Day 7 Mean 263 261 263 263
S.D. 19.7 18.3 15.8 15.6
N 8 10 9 9
Day 11 Mean 278 274 278 274
S.D. 21.6 20.1 16.6 16.7
N 8 10 9 9
Day 14 Mean 289 284 286 279
S.D. 22.6 22.7 14.6 16.3
N 8 10 9 9
Day 17 Mean 312 305 312 298
S.D. 23.3 23.3 17.5 15.8
N 8 10 9 9
Day 20 Mean 351  342  348  330
S.D. 30.5  30.5  22.0 16.3
N 8 10 9 9
Lactation          
Day 1 Mean 284  271  273  261
S.D. 25.6  21.9  17.5  16.7
N 9 9 9 9
Day 4 Mean 293  283  284  265
S.D. 32.1  24.5  20.2  14.8
N 9 9 9 9
Day 7 Mean 298  290  292  274
S.D. 24.5  22.7  18.2 16.4
N 9 9 9 9
Day 13 Mean 315  302  301  283 **
S.D. 22.6  18.9  18.7  20.2
N 9 9 9 9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 12. Male Body Weight Gain (%) Summary

    Control 100 mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 0 0 0 0
S.D. 0 0 0 0
N 10 10 10 10
Day 8 / Week 2 Mean 7 7 7 5*
S.D. 1.5 1.4 1.6 2.1
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 12 13 11 10
S.D. 3.1 2.1 2.4 2.4
N 10 10 10 10
Day 8 /Week 2 Mean 14 17 15 12
S.D. 4.2 3.1 3.3 3.2
N 10 10 10 10
Day 15 / Week 3 Mean 17 21*  18 15
S.D. 3.7 3.3 4.1 4
N 10 10 10 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 13. Female Body Weight Gain (%) Summary

    Control 100 mg/kg/day 300 mg/kg/day 1000 mg/kg/day
Pre-mating           
Day 1 / Week 1 Mean 0 0 0 0
S.D. 0 0 0 0
N 10 10 10 10
Day 8 / Week 2 Mean 3 2 1 -1*
S.D. 2.6 2.9 1.9 2.4
N 10 10 10 10
Mating period          
Day 1 / Week 1 Mean 5 3 3 3
S.D. 4.0 3.0 3.0 3.7
N 10 10 10 10
Day 8 /Week 2 Mean - - - -
S.D. - - - -
N 0x 0 0 0

Table 14. Female Body Weight Gain (%) Summary

    Control

100

mg/kg/day

300

mg/kg/day

1000 mg/kg/day
Post-coitum          
Day 0 Mean 0 0 0 0
S.D. 0 0 0 0
N 8 10 9 9
Day 4 Mean 6 4 5 6
S.D. 1.1 2.1 2.2 1.1
N 8 10 9 9
Day 7 Mean 8 7 9 9
S.D. 1.7 2.5 3.1 1.9
N 8 10 9 9
Day 11 Mean 14  13  15  14
S.D. 3.2 . 3.7  4.2 2.2
N 8 10 9 9
Day 14 Mean 18  17  18 16
S.D. 3.9  4.4  4.0 2.9
N 8 10 9 9
Day 17 Mean 28  26  29 24
S.D. 4.4  7.0  5.0 3.8
N 8 10 9 9
Day 20 Mean 44  41  44 37
S.D. 7.3  10.9  5.9 3.1
N 8 10 9 9
Lactation          
Day 1 Mean 0 0 0 0
S.D. 0 0 0 0
N 9 9 9 9
Day 4 Mean 3 4 4 2
S.D. 3.9  2.7  2.1  3.2
N 9 9 9 9
Day 7 Mean 5 7 7 5
S.D. 3.4  3.1  1.5  3.6
N 9 9 9 9
Day 13 Mean 11 12 10 8
S.D. 7.1  2.6  2.2  4.9
N 9 9 9 9

Table 15. Female Food Consumption (g/animal/day) Summary

    Control

100

mg/kg/day

300

mg/kg/day

1000

mg/kg/day

Pre-mating           
Days 1-8 / Weeks 1-2 Mean 15 15 15 14
S.D. 0.4 0.3 0.9 0.4
N (cage) 2 2 2 2
Days 8-15 / Weeks 2-3 Mean 14 14 14 14
S.D. 0.7 0.0 0.6 0.3
N (cage) 2 2 2 2
Mean of means    15 14 14 14
Post-coitum          
Days 0-4 Mean 18 17 18 18
S.D. 2.3 1.0 1.9 1.6
N 8 10 9 9
Days 4-7 Mean 17 17 18 18
S.D. 2.8 2.1 1.9 2.6
N 8 10 9 9
Days 7-11 Mean 18 19 18 17
S.D. 3.2 2.4 2.2 2.7
N 8 10 9 9
Days 11-14 Mean 16 18 18 17
S.D. 5.1 2.6 1.2 3.7
N 8 10 9 9
Days 14-17 Mean 21 20 21 18
S.D. 2.0 2.4 2.4 3.0
N 8 10 9 9
Days 17-20 Mean 22 21 22 20
S.D. 2.5 2.6 2.5 1.6
N 8 10 9 9

Mean of means

  19 19 19 18
Lactation   19 19 19 18
Days 1-4 Mean 32 32 29 28
S.D. 8.9 5.8 3.8 6.0
N 9 9 9 9
Days 4-7 Mean 36 37 36 32*
S.D. 4.6 2.8 3.9 3.9
N 9 9 9 9
Days 7-13 Mean 52 50 48 42**
S.D. 6 3 4 5
N 9 9 9 9

Mean of means

  40 39 38 33

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 16. Summary of Haematology Values (Red Blood Cells) - Female

    RBC (10^12/L) [G1}
Control Mean 7.286
S.D. 0.444
N 5
100 mg/kg/day Mean 7.044
S.D. 0.165
N 5
tCtrl 0.97
300 mg/kg/dy Mean 7.000
S.D. 0.360
N 5
tCtrl 0.96
1000 mg/kg/day Mean 6.902
S.D. 0.455
N 5
tCtrl 0.95

[G1] - Anova & Dunnett

Table 17. Summary of Clinical Chemistry Values (ALT) - Males

  ALT (U/L) [G}
Control Mean 70.7
S.D. 25.4
N 5
100 mg/kg/day Mean 52.1
S.D. 4.2
N 5
tCtrl 0.74
300 mg/kg/dy Mean 60.5
S.D. 9.2
N 5
tCtrl 0.86
1000 mg/kg/day Mean 94.2
S.D. 33.1
N 5
tCtrl 1.33

[G] - Kruskal-Wallis & Dunn

Table 18. Summary of Clinical Chemistry Values (ALT and AST) - Females

  ALT (U/L) [G} AST (U/L) [G}
Control Mean 115.7 98.3
S.D. 16.2 12.7
N 5 5
100 mg/kg/day Mean 109.2 90.0
S.D. 9.3 9.6
N 5 5
tCtrl 0.94 0.92
300 mg/kg/dy Mean 107.2 96.7
S.D. 20.9 16.0
N 5 5
tCtrl 0.93 0.98
1000 mg/kg/day Mean 154.7 131.1
S.D. 39.7 51.1
N 5 5
tCtrl 1.34 1.33

[G] - Kruskal-Wallis & Dunn

Table 19. Summary of Endocrine Values - Male

  T3 (ng/mL) [G] T4 (ng/mL) [G] TSH (ng/mL) [G]
Control Mean 0.435 45.88 0.0943
S.D. 0.031 6.01 0.0884
N 10 10 10
100 mg/kg/day Mean 0.424 47.34 0.1172
S.D. 0.048 3.75 0.0497
N 10 10 10
tCtrl 0.97 1.03 1.24
300 mg/kg/dy Mean 0.5 51.6 0.2
S.D. 0.1230 6.9300 0.1205
N 10 10 10
tCtrl 1.13 1.13 1.75
1000 mg/kg/day Mean 0.404 33.18 ** 0.1076
S.D. 0.090 3.62 0.1038
N 10 10 10
tCtrl 0.93 0.72 1.14

[G] - Anova & Dunnett: ** = p ≤ 0.01

Table 20. Summary of Endocrine Values - Female

  T3 (ng/mL) [G] T4 (ng/mL) [G] TSH (ng/mL) [G]
Control Mean 0.279 28.24 0.196
S.D. 0.043 3.55 0.1204
N 9 9 9
100 mg/kg/day Mean 0.277 27.48 0.3558
S.D. 0.051 3.38 0.4084
N 9 9 9
tCtrl 0.99 0.97 1.82
300 mg/kg/dy Mean 0.257 24.96 0.2428
S.D. 0.0260 3.3000 0.1694
N 9 9 9
tCtrl 0.92 0.88 1.24
1000 mg/kg/day Mean 0.240 27.88 0.1379
S.D. 0.047 3.98 0.0938
N 9 9 9
tCtrl 0.86 0.99 0.7

[G] - Anova & Dunnett

Table 21. Summary of Functional Observation (Grip Strength) - Male

  Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Grip Fore Gram Mean 821 995 887 826
S.D. 120 289 208 115
N 5 5 5 5
Grip Hind Gram Mean 690  449 **  497 *  467 **
S.D. 116 74 65 105
N 5 5 5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 22. Summary fo Functional Observatiopns (Movement and Ambulations) - Female

Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Movements        
Mean 3546 3871 4873 4625
S.D. 1226 1122 483 2405
N 5 5 5 5
Ambulations        
Mean 843 981 1233 1510
S.D. 278 336 199 929
N 5 5 5 5

Table 23. Absolute Organ Weights (g) Summary - Male

  Control 100 mg/kg/day 300 mg/kg/dy 1000 mg/kg/day
Body weight Mean 327 340 331 318
S.D. 27 18 21 14
N 10 10 10 10
Liver Mean 7.38 8.51 8.81 10.14 **
S.D. 0.33 1.24 1.1 0.8
N 5 5 5 5
Kidney Mean 1.94 2.15 2.36 * 2.43 *
S.D. 0.07 0.21 0.39 0.07
N 5 5 5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 24. Relative Organ Weights (%) Summary - Male

  Control 100 mg/kg/day 300 mg/kg/dy 1000 mg/kg/day
Body weight (g) Mean 327 340 331 318
S.D. 27 18 21 14
N 10 10 10 10
Liver (%) Mean 2.37 2.52 2.64 3.17 **
S.D. 0.09 0.4 0.15 0.19
N 5 5 5 5
Kidney (%) Mean 0.62 0.64 0.71 0.76 **
S.D. 0.03 0.07 0.07 0.03
N 5 5 5 5
Brain (%) Mean 0.64 0.58 **  0.61 0.62
S.D. 0.02 0.03 0.03 0.03
N 5 5 5 5
Thyroids (%) Mean 0.0051 0.0044 *  0.005 0.005
S.D. 0.0007 0.0007 0.0004 0.0008
N 10 10 10 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 25. Absolute Organ Weights (g) Summary - Female

 

Control

100 mg/kg/day

300 mg/kg/dy

1000 mg/kg/day

Body weight

Mean

303

294

298

282

S.D.

26

21

18

15

N

10

10

10

10

Liver

Mean

12.51

12.36

13.23

13.57

S.D.

0.68

0.53

0.66

1.34

N

5

5

5

5

Kidney

Mean

2.07

1.91

2.02

1.88

S.D.

0.13

0.11

0.09

0.17

N

5

5

5

5

Spleen

Mean

0.565

0.538

0.519

0.473 *

S.D.

0.076

0.046

0.049

0.036

N

5

5

5

5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 26. Relative Organ Weights (%) Summary - Female

Female   Control 100 mg/kg/day 300 mg/kg/dy 1000 mg/kg/day
Body weight (g) Mean 303 294 298 282
S.D. 26 21 18 15
N 10 10 10 10
Liver (%) Mean 4.07 4.17 4.30 4.75 **
S.D. 0.1 0.3 0.28 0.29
N 5 5 5 5
Kidney (%) Mean 0.67 0.64 0.66 0.66
S.D. 0.02 0.03 0.02 0.03
N 5 5 5 5
Spleen (%) Mean 0.184 0.182 0.169 0.166
S.D. 0.021 0.016 0.019 0.014
N 5 5 5 5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 27. Summary Test Item-Related Microscopic Liver Findings

Sex Males Females
Dose level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Liver* 5 3 4 5 5 5 5 5
Hepatocellular hypertrophy              
Minimal - - - 1 - - - -
Slight - - - 4 - - - -
Cytoplasmic rarefaction                
Minimal - - - - 1 - 1 1
Slight - - - - - - - 2

*Number of tissues examined from each group

Table 28. Summary Test Item-Related Microscopic Thyroid Gland and Kidney Findings - Male

Dose level (mg/kg/day) 0 100 300 1000
Thyroid Gland* 5 3 4 5
Follicular cell hypertrophy        
Minimal - - - 3
Slight - - - 1
Kidney*: 5 3 4 5
Hyaline droplet accumulation        
Minimal 1 1 2 -
Slight 2 2 2 2
Moderate - - - 3
Granular casts        
Minimal - - - 2
Slight - - - 1

*Number of tissues examined from each group

Table 29. Developmental data - F0 Generation

    Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Dad pups at first litter check Litters affected (#) 0 1 0 5 #
Total 0 1 0 16
Mean (+) 0.0 0.1 0.0 1.8
S.D. 0.0 0.3 0.0 2.1
N 9 9 9 9
Living Pups at First Litter Check % of males/females (#) 55/45 53/47 55/45 48/52
Total 111 102 102 92
Mean (+) 12.3 11.3 11.3 10.1
S.D. 3.3 1.7 1.2 2.1
N 9 9 9 9
Postnatal loss % of living pups 0 2 1 2.2
Litters affected (#) 0 2 1 1
Total (#) 0 2 1 2
Mean (+) 0.0 0.2 0.1 0.7
S.D. 0 0.4 0.3 0.7
N 9 9 9 9
Culled Pups Total 40 28 29 21
Breeding loss Days 5-13 P.P.  % of living pups at day 4 P.P. 0 0 0 0
Litters affected (#) 0 0 0 0
Total (#) 0 0 0 0
Mean (+) 0 0 0 0
S.D. 0 0 0 0
N 9 9 9 9
Breeding loss Days 13 P.P.  % of males/females (#) 52/48 57/43 53/47 51/49
Total (#) 71 72 72 68
Mean (+) 7.9 8.0 8.0 7.6
S.D. 0.3 0.0 0.0 1.0
N 9 9 9 9
Total number of offspring born 111 103 102 107
Total number of uterine implantation sites 116 121 113 113
Number of live offspring on Day 1 after littering 111 102 102 91
Number of live offspring on Day 4 (before culling) 111 100 101 89
Number of live offspring on Day 4 (after culling) 71 72 72 686
Number of live offspring on Day 7 after littering 71 72 72 68
Number of live offspring on Day 13 after littering 71 72 72 68
Post-implantation survival index (%)   96 85 90 95
Live birth index (%) 100 99 100 85
Viability index 1 (%) 100 98 99 98
Viability index 2 (%) 100 100 100 100
Viability index 3 (%) 100 100 100 100
Lactation index (%) 100 100 100 100
Cumulative survival index (%) 100 97 99 93

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 30. Pup Body Weights (g)

Day Sex   Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
1 M Mean (+) 6.7 6.8 6.1 5.4 **
S.D. 0.7 0.7 0.6 0.7
N 9 9 9 9
F Mean (+) 6.4 6.2 5.7 5.2 ** 6.2 5.7 5.2 ** 6.2 5.7 5.2 **
S.D. 0.8 0.6 0.5 0.6
N 9 9 9 9
M+F Mean (+) 6.5 6.6 5.9 * 5.3 *
S.D. 0.8 0.6 0.6 0.6
N 9 9 9 9
4 M Mean (+) 10.1 10.4 9.4 8.4 *
S.D. 1.3 1.3 1.2 0.9
N 9 9 9 9
F Mean (+) 9.8 9.8 8.9 8.0 **
S.D. 1.5 1.2 1 0.8
N 9 9 9 9
M+F Mean (+) 10 10.1 9.2 8.2 **
S.D. 1.3 1.3 1.1 0.8
N 9 9 9 9
7 M Mean (+) 16.8 17.2 15.9 13.6 **
S.D. 1.8 1.7 1.9 1.1
N 9 9 9 9
F Mean (+) 16.2 16.1 15.2 13.1 **
S.D. 2.2 1.7 1.7 0.9
N 9 9 9 9
M+F Mean (+) 16.5 16.8 15.6 13.3 **
S.D. 2 1.7 1.8 0.9
N 9 9 9 9
13 M Mean (+) 32.3 32.6 30.8 26.3 **
S.D. 3.3 2.6 3.5 2.2
N 9 9 9 9
F Mean (+) 31.4 31.5 29.9 25.6 **
S.D. 3.5 2.4 3.3 1.9
N 9 9 9 9
M+F Mean (+) 31.9 32.2 30.4  25.9 **
S.D. 3.3 2.4 3.4 2
N 9 9 9 9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 31. Anogenital distance and nipple retention - F0 generation

    Control

100

mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Male anogenital distance (mm) Mean (+) 2.62 2.63 2.44 2.21
S.D. 0.24 0.21 0.22 0.27
N 9 9 9 9
Female anogenital distance (mm) Mean (+) 1.0 1 1.0 1.14
S.D. 0.07 0.05 0.08 0.14
N 9 9 9 9
Number of nipples Mean (+) 0.03 0.08 0.00 0.03
Median (+) 0.00 0.00 0.00 0.00
N 9 9 9 9

Table 32. Corrected Anogenital distance

PND 1   Control 100 mg/kg/day

300

mg/kg/dy

1000 mg/kg/day
Male anogenital distance (mm) Mean (+) 1.39 1.39 1.34 1.26
S.D. 0.10 0.11 0.12 0.11
N 9 9 9 9
Female anogenital distance (mm) Mean (+) 0.6 0.54 0.6 0.66 +
S.D. 0.04 0.03 0.04 0.1
N 9 9 9 9

+/++ Steel-test significant at 5% (+) or 1% (++) level

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic No Observed Adverse Effect Level (NOAEL) of Mixed Xylene in the parental male rats was determined to be 300 mg/kg/day (with observed alpha-2u-globulin nephropathy taken into consideration) or at least 1000 mg/kg/day (without observed alpha 2u globulin nephropathy being considered as this is male rat specific and not relevant to humans). The NOAEL of mixed Xylene in the parental female rats was found to be at least 1000 mg/kg/day. Based on the effects observed at the highest dose tested, the NOAEL for developmental toxicity was determined to be 300 mg/kg/day. The developmental toxicity observed in this study occurred at dose levels associated with parental toxicity. This included lower mean body weights of 10% for females, accompanied by a 17% lower absolute food consumption during the lactation period at 1000 mg/kg/day. It cannot therefore, be excluded that this effect on body weight and food intake was related to the observed developmental toxicity.
Executive summary:

A key OECD Guideline 422 study was conducted to determine the potential toxic effects of the test material (Mixed Xylene (CAS 1330-20-7)) when administered orally for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

 

The test material was administered orally once daily via gavage to Wistar Han rats (10/sex/dose) in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg bw/day, 7 days a week for a minimum of 28 days (including at least 2 weeks of treatment prior to mating and during the mating period up to

and including the day before scheduled necropsy) for male rats and 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy for female rats.

 

Parameters evaluated were mortality/moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0-males and females), gross necropsy findings, organ weights and histopathologic

examinations. Additionally, reproduction/developmental parameters such as mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, developmental indices, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormones T3 and T4 (PND 4) and T3, T4 and TSH (PND 14-16 pups)) were evaluated.

 

No mortality and no treatment-related toxicologically relevant clinical signs were observed through the study period.At 1000 mg/kg/day, body weight gain was slightly lower during most of the treatment period for male rats and from Day 14 post-coitum onwards for female rats. In addition, slight body weight loss was observed over Days 1-8 for female rats. The lower body weight gain and body weight loss resulted in a slightly lower mean body weight at the end of treatment for females only. Correlating decreased absolute and relative food consumption was observed for females only. Considering the short period of time during which body weight loss was noted as well as the small magnitude of changes, the effects on body weight gain and food consumption were considered to be non-adverse.

 

Hematological parameters and behaviour of treated rats were considered unaffected by treatment with the test material. Non-adverse increased alanine aminotransferase (ALT) was noted in both sexes and non-adverse decreased total thyroxine (T4) was observed only in male rats. Gross necropsy did not reveal any remarkable treatment-related findings.

 

At 300 mg/kg/day, non-adverse increased absolute kidney weights were observed in males but were without histopathologic correlate. No treatment-related findings were observed in female rats at this dose level. At the 1000 mg/kg bw/day dose level, non-adverse higher liver weights in males and females that correlated with microscopic hepatocellular hypertrophy and cytoplasmic rarefaction for males and females, respectively, were observed. Non-adverse follicular cell hypertrophy was also observed in the thyroid gland in male rats dosed at 1000 mg/kg bw/day.

 

In the 1000 mg/kg bw/day dose group, adverse histopathologic findings were observed in the kidney of male rats that comprised granular casts and increased hyaline droplet accumulation. These were considered to represent a degenerative process and correlated with an increased kidney weight. The increased incidence and severity of hyaline droplet accumulation recorded in the kidney of male rats represented alpha 2u-globulin (as confirmed by immunohistochemistry), a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in mammals, including man.

 

No reproductive toxicity was observed up to the highest dose level tested 1000 mg/kg/day. No developmental toxicity was observed up to 300 mg/kg/day but at 1000 mg/kg/day, an adversely reduced live birth index (i.e. an increased incidence of pup mortality) was observed at first litter check. Consequently, litter size was adversely reduced with on average two pups per litter less than control litters, associated with a decreased cumulative survival index. Adversely reduced pup body weights (both sexes) were observed from PND 1 onwards. Non-adverse changeswere noted in (corrected) anogenital distance, which was increased in female pups.

 

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic No Observed Adverse Effect Level (NOAEL) of Mixed Xylene in male parental rats was determined to be 300 mg/kg/day (with observed alpha-2u-globulin nephropathy taken into consideration) or at least 1000 mg/kg/day (without observed alpha 2u globulin nephropathy being considered as this is male rat specific and not relevant to humans). The maternal NOAEL was found to be at least 1000 mg/kg bw/day. Based on the effects observed at the highest dose tested, the NOAEL for developmental toxicity was determined to be 300 mg/kg/day. The developmental toxicity observed in this study occurred at dose levels associated with parental toxicity. This included lower mean body weights of 10% for females, accompanied by a 17% lower absolute food consumption during the lactation period at 1000 mg/kg/day. It cannot therefore, be excluded that this effect on body weight and food intake was related to the observed developmental toxicity.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-JUL-14 to TBD
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This data has not been finalised by LOA due to an ongoing review. The data presented in this record is from an audited draft report by the contract research organisation.
A communication from the contract research organisation stating when the finalisation of the report will occur by (31st January 2023) is included below in the "attached justification" section. LOA is committed to performing a dossier update when the full information required for ECHA to judge the read-across approach utilised in this dossier is available.
Qualifier:
according to guideline
Guideline:
other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version: July 2016
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions. Please see 'Any other information on materials and methods' for details.
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: To be confirmed; Batch number: 10-11-2021
- Purity, including information on contaminants, isomers, etc.: Reaction mass of ethylbenzene and mxylene (ethylbenzene: 14.84 % m/m; p-xylene: 3.98 % m/m; m-xylene: 74.63 % m/m; o-xylene: 5.96 % m/m)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in corn oil vehicle for at least 24 hours at room temperature under normal laboratory light over a concentration range of 2 to 250 mg/mL (solutions); Stable in corn oil vehicle for at least 9 days in the refrigerator over a concentration range of 2 to 250 mg/mL (solutions)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS
- Specific gravity / density: To be confirmed
- Expiry date: 2026-MAR-11
- EC Number: 905-570-2
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: Males: 11-12 weeks; Females: 14-15 weeks
- Weight at study initiation: Males: 283 - 327 g; Females: 212 - 269 g
- Housing:

On arrival and during the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrol on, MIV type, height 18 cm).

During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.

During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

- Diet: pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH (Soest, Germany ) ad libitum
- Water: Municipal tap water via water bottles ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, based on the results of these analyses it was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 22°C
- Humidity (%): 46 to 68%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 2021-JUL-14 To: 2021-OCT-12
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Sigma-Aldrich (Steinheim, Germany)
Remarks on MMAD:
N/A
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Sigma-Aldrich, Steinheim, Germany) was used as the vehicle based on trial preparations performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and were not used for dosing.
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg bw/day dose groups, respectively.
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During Week 1 and 3 of treatment, dose formulation samples were collected for analysis as follows:

1. Concentration analysis: sample collected from approximately ‘Middle’ for all dose groups

2. Homogeneity analysis: sample collected from approximately ‘Top, Middle and Bottom’ for dose groups 2 and 4 only.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20275969).

For concentration: mean sample concentration results within or equal to ± 15% viscous solutions of theoretical concentration.

For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20275969) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated.
- Any other deviations from standard protocol: Detection of mating was not confirmed in first instance for Female No. 59 (100 mg/kg/day). Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before
scheduled necropsy.

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily
Duration of test:
28 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle: Corn Oil) - Group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder study with oral gavage administration of Reaction mass of ethylbenzene and m-xylene in rats (Test Facility Study No. 20275971), and in an attempt to produce graded responses to the test material.
- Rationale for animal assignment (if not random): A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the Weeks 2 and 3 of the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. Animals were randomly assigned to groups at arrival. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- Dose range finding studies: 10-day Dose Range Finder study with oral gavage administration of Reaction mass of ethylbenzene and m-xylene in rats (Test Facility Study No. 20275971).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test material and lasting throughout the Dosing periods up to the day prior to necropsy. Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

All animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Except on days of receipt and necropsy where frequency was at least once daily. Arena observations were also conducted once before the first administration of the test material and weekly during the treatment Period.

BODY WEIGHT: Yes
- Time schedule for examinations: for all animals on Day 1 of treatment (prior to dosing ) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; quantitatively measured per cage weekly, except for males and females which were housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Monitored regularly throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane); Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid hormones)
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: 5/sex/group
- Parameters examined included Triiodothyronine (T3); Thyroxine (T4); and Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed post dosing on 5 males per group during Week 4 of treatment and 5 females per group during the last week of lactation (i.e. PND 8-10).
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity such as Hearing ability; Fore- and hind-limb grip strength; and Locomotor activity

IMMUNOLOGY: No
OTHER: Coagulation:
- Time schedule for collection of blood: On the day of scheduled necropsy
- How many animals: 5 animals/sex/group were selected
- Animals fasted: Males fasted overnight (maximum of 24 hours), females non-fasted
- Anaesthetic used for blood collection: Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.45 mL
- Parameters analysed: prothrombin time (PT), activated partial thromboplastin time (APTT)

POST-MORTEM EXAMINATIONS: Yes (see Tables 4 and 5)

Unscheduled Euthanasia
Females with total litter loss (Nos. 73 and 75)were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated within 24 hours after the last pup was found dead or missing. They underwent necropsy, and specified tissues were retained and weighed If necessary for humane reasons, animals were deeply anesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:

Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16
Females which failed to deliver: With evidence of mating: Post-coitum Day 25-26 (Nos. 49, 52, and 77).

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy.

Necropsy:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

The organs listed in Table 4 and 5 were weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see Tables 6 and 7)
Representative samples of the tissues identified in the tables 6 and 7 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. The tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Alpha-2u-globulin staining:
Two slides per selected Group 1 and Group 4 male were shipped to CRL-Durham for HRP-polymer based chromogenic immunostaining of alpha-2u-globulin. The slides were returned to the Test Facility after the staining procedure had been completed for evaluation by the study pathologist.
Ovaries and uterine content:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Blood sampling:
Blood sampling:

F0-animals (selected 5/sex/group): On the day of scheduled necropsy, blood was sampled (for hematology, coagulation, clinical chemistry, and thyroid hormone analysis) from the retro-orbital sinus under anaesthesia using isoflurane between 07.00 and 10.30 a.m.

PND 4 pups at culling (2 pups/litter): On PND 4 at culling, blood was sampled (for thyroid hormone analysis) by decapitation between 7.00 and 10.30 a.m., pooled to one sample per litter (from one male and one female, if possible). If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. If no surplus pups were available, no blood sample was collected.

PND 14-16 pups (2 pups/litter): On PND 14-16, blood was sampled (for thyroid hormone analysis) by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure between 7.00 and 10.30 a.m. from two pups per litter (from one male and one female).

The parameters assessed in maternal animal blood are listed below.

Haematology:
- On the day of scheduled necropsy
- 5 animals/sex/group were selected
- Males fasted overnight (maximum of 24 hours), females non-fasted
- Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.5 mL
- Parameters analysed are listed in Table 2

Coagulation:
- On the day of scheduled necropsy
- 5 animals/sex/group were selected
- Males fasted overnight (maximum of 24 hours), females non-fasted
- Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.45 mL
- Parameters analysed: prothrombin time (PT), activated partial thromboplastin time (APTT)

Clinical chemistry:
- On the day of scheduled necropsy
- 5 animals/sex/group were selected
- Males fasted overnight (maximum of 24 hours), females non-fasted
- Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m.
- Blood volume sampled: 0.5 mL
- Parameters analysed are listed in Table 3

Thyroid hormone:
- On the day of scheduled necropsy
- 5 animals/sex/group (F0) and 2 pups/litter (F1) were selected
- Males (F0) fasted overnight (maximum of 24 hours), females (F0) non-fasted
- Parameters analysed: thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH)
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done.

Whenever the number of male or female pups prevented having four of each sex per litter, partial a djustment (for example, five males and three females) was acceptable.
Pups were examined for external and internal abnormalities and possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

POSTMORTEM EXAMINATIONS (OFFSPRING)

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

Unscheduled Euthanasia
Recognizable fetuses of females that were euthanized in extremis (No. 72) were examined externally and sexed (both externally and internally). Live fetuses were euthanized by decapitation.

Pups that died before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible.

All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Indices:
Offspring viability indices
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

1. Viability index 1 (%) = (number of live offspring on Day 4 before culling / number of live offspring on Day 1 after litterling) x 100

2. Viability Index 2 (%) = (number of live offspring on Day 7 before culling / number of live offspring on Day 4 after litterling) x 100

3. Viability Index 3 (%) = (number of live offspring on Day 13 before culling / number of live offspring on Day 7 after litterling) x 100
Historical control data:
The test facility (Charles River Den Bosch) has general and reproduction/developmental historical data in this species from the same strain and source. This data for 2017-2021 is provided in Appendix XX of the final study report.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed during daily detailed clinical observations or during weekly arena observations in males and females.

Salivation observed post dosing, starting on Days 19 (males) or 36 (females) at 300 mg/kg/day and on Days 4 (males) or 11 (females) at 1000 mg/kg/day of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Incidental findings that were observed included scabs at the shoulder, alopecia and swelling of the vagina. 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 not to be signs of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One premature death (female No. 72) was observed in the study at 1000 mg/kg/day. The animal was sacrificed in extremis on post-coitum Day 21 due to acute severe clinical observations indicating a poor condition, i.e. lethargy and a pale skin (both at moderate degree), hunched posture, deep respiration, piloerection. Macroscopic observations at necropsy revealed a thymus reduced in size; the uterus of this female contained thirteen normally developed live fetuses (11 males and 2 females) and two early resorptions. Major microscopic findings were present in the glandular stomach and duodenum in the form of multiple ulcers (up to moderate) and associated inflammation (granulocytic, up to slight). Furthermore, several lymphoid organs showed lymphocyte apoptosis/necrosis (up to slight) and/or decreased cellularity (up to marked, correlating in the thymus with a reduced size). The lesions of the stomach and duodenum and the most likely secondary findings of the lymphoid organs were regarded to be related with the moribundity of this female. Since this moribundity occurred in a single 1000 mg/kg/day group female and since the combination of stomach and duodenum findings were acute to subacute in this female and absent in any other animal, these findings were regarded unrelated to test material treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes in body weight and body weight gain were observed up to dose levels of 300 mg/kg/day.

In males at 1000 mg/kg/day, reduced body weight gain was observed on Days 22 and 29 of the dosing period, which resulted in a 5% lower absolute mean body weight compared to control at the end of the dosing period (Day 29).

In females at 1000 mg/kg/day, reduced body weight gain was noted during lactation (statistically significant on Day 13 of lactation only), which resulted in a 6% lower mean body weight at the end of the dosing period (Day 13 of lactation; not statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption (before or after correction for body weight) were recorded up to 1000 mg/kg/day in male rats and up to 300 mg/kg/day in female rats.

In males at 1000 mg/kg/day, absolute and relative food consumption were slightly decreased over Days 1 8 of the dosing period (10% and 8% lower compared to control; not statistically significant), which subsequently recovered. As this finding was transient it was considered of no toxicological relevance.

In females at 1000 mg/kg/day, relative food consumption was slightly decreased over Days 1 8 of the dosing period (11% compared to control; not statistically significant), which subsequently recovered. In addition, both absolute and relative food consumption were decreased during lactation (up to 17% and 13% lower than control at the end of the dosing period, respectively, and not statistically significant).
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were unaffected by treatment with the test material.

The decreased hemoglobin concentration (HGB) observed in females at 100 and 300 mg/kg/day (0.92 and 0.94x of control, respectively) and the decrease in mean corpuscular hemoglobin concentration (MCHC) in females at 100, 300 and 1000 mg/kg/day (0.97, 0.97 and 0.98x, respectively; not always statistically significant) were considered unrelated to test material administration due to the absence of a dose response and the minimal magnitude of the change.

Other non-statistically significant changes observed in several white blood cell parameters (both sexes) were considered unrelated to test material administration due to the minimal magnitude of the change, high variation in individual values within the groups, and/or absence of a dose response.

Coagulation parameters of treated rats were considered to be unaffected by treatment. The shorter prothrombin time (PT) observed in males at 300 mg/kg/day (0.93x of control) and in females at 100 and 1000 mg/kg/day (each 0.94x of control) was considered unrelated to treatment as no dose response was observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical biochemistry parameters in female rats up to 300 mg/kg/day.

Increased alkaline phosphatase (ALP) activity was observed in male rats at 100, 300 and 1000 mg/kg/day (1.14, 1.20 and 1.27x of control, respectively; not statistically significant). Increased potassium (K) levels were observed in male rats at 1000 mg/kg/day (1.18x) and increased creatinine concentrations (CREAT) were observed in female rats at 300 and 1000 mg/kg/day (1.17 and 1.27x; not statistically significant at 300 mg/kg/day).

Increased bile acids (BILEAC) and decreased total bilirubin (TBIL) and inorganic phosphate (PHOS) levels were also observed in female rats at 100, 300 and 1000 mg/kg/day and considered related to relatively low (bile acids) or high (total bilirubin and inorganic phosphate) in the control group. Therefore, these were considered to not be toxicologically relevant.

Any other changes in clinical biochemistry parameters were considered to be unrelated to test material administration due to the minimal magnitude, variation in direction of change and/or absence of a dose-related response.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Decreased serum levels of T3, total T4 and TSH were noted in males at 1000 mg/kg/day (0.86, 0.87 and 0.70x, respectively; not statistically significant). At the individual level, 1/10 values for T3, 2/10 values for T4 and 1/10 values for TSH were below the range of the controls. These findings were regarded unrelated to treatment due to lack of a dose relationship and because individual values showed great overlap.

Any differences in mean levels of T3, total T4 and TSH in treated females compared to controls were considered to be unrelated to treatment, as no dose relation was evident.
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In male rats at 1000 mg/kg/day, grip strength of the fore legs was decreased in Week 4 of the dosing period (0.67x of control). The lower grip strength of the hind legs in male rats at 1000 mg/kg/day (0.77x of control; not statistically significant) was mainly attributed to a single male (No. 35) with a relatively low value. As individual values of other animals at the same dose level generally remained within concurrent control range, this change was considered to be unrelated to treatment.

Other functional observation parameters, including hearing ability, pupillary reflex and static righting reflex (males and females), and grip strength (females only) were considered unaffected by treatment up to 1000 mg/kg/day. The apparent increased mean grip strength of the fore legs in females at 300 mg/kg/day could mainly be attributed to a relatively low control mean compared to historical control data, high individual variability within the groups, and occurred without a dose related trend. It should be noted that for control females also the mean value for grip strength of the hind legs was relatively low compared to historical control data.

Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related higher liver and kidney weights were observed in male rats at 1000 mg/kg/day. Statistically significant higher liver weights (absolute and relative to body weight) were observed in male rats at 1000 mg/kg/day. Statistically significant higher kidney weights (relative to body weight) were noted in male rats at 1000 mg/kg/day.

Thymus weights in treated rats of both sexes at 300 and 1000 mg/kg/day tended to be lower compared to the control group/s. In males at 1000 mg/kg/day, statistical significance was only reached for the absolute thymus weight. In females at 1000 mg/kg/day, there was no dose-response and at 1000 mg/kg/day there were large differences between the individual thymus weights. Furthermore, there were no treatment-related microscopic findings observed in the thymus of scheduled sacrificed rats. Therefore, a relationship with the test material seemed unlikely.

The absolute mean weights of the prostate gland and seminal vesicle of 1000 mg/kg/day dose group males were statistically significantly lower when compared to the control group but were considered to be the result of lower final body weight and/or variability in individual values.

Any other differences (including statistically significantly higher relative brain weight of 1000 mg/kg/day group males and lower absolute and relative spleen weight of 300 mg/kg/day group males) were not considered to be treatment-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross observations in male rats. Reduced size of the thymus, present in Female No. 72 (sacrificed in extremis) and in Female No. 73 (with total litter loss of the 1000 mg/kg/day group), was regarded to be secondary to these conditions. The microscopic correlate was decreased lymphoid cellularity. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Possible treatment-related microscopic findings were observed in the liver and kidneys of males and thyroid gland of both sexes.

Hepatocellular hypertrophy in the liver was noted in males of the 1000 mg/kg/day group at a minimal degree. Hyaline droplet accumulation in the kidney was observed in treated rats of all dose groups up to slight degree. Immunohistochemistry for alpha 2u-globulin was performed on the kidneys of five selected males of the control group and five selected males of the 1000 mg/kg/day group. The hyaline droplets stained positive for alpha 2u-globulin in all five males at 1000 mg/kg/day.

Treatment-related increase in the incidence of follicular cell hypertrophy was observed in the thyroid gland of 1000 mg/kg/day group rats of both sexes (minimal degree).

All other microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4 days.

One female at 1000 mg/kg/day (No. 80) had an extended estrous cycle during the first week of premating. Given its incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Details on results:
N/A
Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites): Unaffected by treatment

Post-implantation survival indices were 90, 88, 90, 78% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

The apparent lower post-implantation survival index for the 1000 mg/kg/day group was caused by Female No. 72, which was sacrificed in extremis on post coitum Day 2. Excluding the 15 implantation sites of this female, the post-implantation survival index was 92% and thus comparable to control
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One premature death was observed in the study: Female No. 72 at 1000 mg/kg/day was sacrificed in extremis on post-coitum Day 21 and macroscopic observations at necropsy revealed that the uterus of this female contained thirteen normally developed live fetuses (11 males and 2 females) and two early resorptions. These findings were regarded unrelated to treatment.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
One premature death was observed in the study: Female No. 72 at 1000 mg/kg/day was sacrificed in extremis on post-coitum Day 21 and macroscopic observations at necropsy revealed that the uterus of this female contained thirteen normally developed live fetuses (11 males and 2 females) and two early resorptions. These findings were regarded unrelated to treatment.
Dead fetuses:
no effects observed
Description (incidence and severity):
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test material. The gestation indices were 90, 100, 100, and 89% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

The lower gestation indices in the control and at 1000 mg/kg/day were attributed to Female No. 49 (control) which was pregnant with two implantation sites only and Female No. 72 (1000 mg/kg/day) which was pregnant with live pups but had to be sacrificed in extremis on post coitum Day 21 due to acute severe clinical signs. All other pregnant females had live offspring.

Post-Implantation Survival Index (total number of offspring born compared to the total number of uterine implantations) was considered not to be affected by treatment with the test material. Post-implantation survival indices were 90, 88, 90, 78% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

The apparent lower post-implantation survival index for the 1000 mg/kg/day group was caused by Female No. 72, which was sacrificed in extremis on post coitum Day 21. Excluding the 15 implantation sites of this female, the post-implantation survival index was 92% and thus comparable to control.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
N/A
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Fertility index was considered not to be affected by treatment with the test material. The fertility index was 100, 90, 100 and 90% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

One female at 100 mg/kg/day (No. 52) and one female at 1000 mg/kg/day (No. 77) were not pregnant. In the absence of a clear dose-related incidence of non-pregnancy, this was considered not to be related to treatment with the test material.
Other effects:
no effects observed
Description (incidence and severity):
Parturition/maternal care:
No signs of difficult or prolonged parturition were noted among the pregnant females.

Female No 72 at 1000 mg/kg/day was sacrificed in extremis on Day 21 post coitum due to acute severe clinical observations (i.e. lethargy, hunched posture, deep respiration, piloerection and pale skin) and suspected problems with parturition. However, no evidence was not observed at necropsy and therefore this sacrifice was not attributed to a difficult parturition.

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:
N/A
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Abnormalities:
effects observed, non-treatment-related
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item up to 300 mg/kg/day.

At 1000 mg/kg/day, body weight of both male and female pups was decreased from PND 1 onwards (up to 17% and 24% lower than control, respectively). On PND 13, the combined (males and females) mean body weight was 19% lower than control.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item up to 300 mg/kg/day.

Live birth index was reduced at 1000 mg/kg/day (100, 100, 99 and 91% for the control, 100, 300 and 1000 mg/kg/day groups, respectively).

At 1000 mg/kg/day, seven pups in four different litters (one, one, three and two pups from Litter Nos. 73, 74, 78 and 79, respectively) were found dead at first litter check. No findings were noted at first litter check and/or necropsy, except for Pup No. 1 from Litter No. 73 which showed clinical signs at first litter check (i.e. cold to touch, grey discoloration of the whole body and labored respiration) and died shortly afterwards. From Litter No. 79, Pup No. 3 did not have milk in the stomach and Pup No. 4 was cannibalized.

One pup at 300 mg/kg/day (Litter No. 66) was found dead at first litter check with no milk in the stomach at necropsy. At the isolated incidence, no toxicological relevance was attached to this single dead pup in the mid-dose group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment with the test material.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was considered not affected by treatment with the test material.

Mean live litter sizes were 9.4, 10.7, 10.9, and 9.0 living pups/litter for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test material.

The lower mean for normalized anogenital distance of female pups at 100 and 300 mg/kg/day occurred without a dose-related trend. Therefore, these variations were considered not to be related to treatment.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
Viability Index 1:

The viability index 1 (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not affected by treatment with the test item up to 300 mg/kg/day.

Viability index 1 was reduced at 1000 mg/kg/day (100, 100, 100 and 60% for the control, 100, 300 and 1000 mg/kg/day groups, respectively).

At 1000 mg/kg/day, a total of twenty-nine pups in four litters were found dead, sacrificed in extremis or missing on PND 2. One pup from Litter No. 71 and two pups from Litter No. 78 were missing on PND 2. In addition, Female Nos. 73 and 75 had total litter loss on PND 2. For Female No. 73, five pups were missing, eight pups were found dead, and one pup was sacrificed in extremis. For Female No. 75, three pups were missing, two pups were found dead, and seven pups were sacrificed in extremis. Pups missing were most likely cannibalized. Pups euthanized in extremis were cold to touch and had a pale appearance. No findings were noted for any of these pups at necropsy.

Viability Index 2:

The viability index 2 (number of live offspring on PND 7 after littering as percentage of number of live offspring on PND 4 (after culling) was considered not affected by treatment with the test item up to 1000 mg/kg/day.

Viability indices 2 were 100, 100, 100 and 97% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup at 1000 mg/kg/day (Litter No. 74) was found dead on PND 5. For this pup, there were no findings either during in-life or at necropsy. As all remaining seven pups in this litter survived until scheduled necropsy without any findings, no toxicological relevance was attached to this single dead pup.

Viability Index 3:

The viability index 3 (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND 7 was considered not affected by treatment with the test item up to 1000 mg/kg/day.
No pus were found dead/missing between Lactation Days 7 and 13, resulting in viability indices of 100% for all groups.

Cumulative Survival Index:

The cumulative survival index ((number of live offspring on Day 13 after litter/number of live offspring on Day 4 (after culling)) * (number of live offspring on Day 4 before culling / total number of offspring born)) * 100% was considered not to be affected by treatment with the test item up to 300 mg/kg/day.
The cumulative survival index was reduced at 1000 mg/kg/day (100, 100, 99 and 53% for the control, 100, 300 and 1000 mg/kg/day groups, respectively).
External malformations:
no effects observed
Description (incidence and severity):
No findings were noted among pups that were considered to be related to treatment with the test material. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Skeletal malformations:
not examined
Description (incidence and severity):
N/A
Visceral malformations:
not examined
Description (incidence and severity):
N/A
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Areola/Nipple Retention:
Treatment with the test material up to 1000 mg/kg/day had no effect on areola/nipple retention.

For one male pup of the control and 300 mg/kg/day groups each, 1-2 nipples were observed at PND 13. As this finding occurred without a dose related trend, it was considered not to be related to treatment.

Clinical Biochemistry (Thyroid Hormone Levels):
Serum T3 (up to 300 mg/kg/day) and T4 levels in PND 4 pups and T3, T4 and TSH levels in male and female PND 14 16 pups were considered not to be affected by treatment with the test material.

In PND 4 pups, serum T3 levels tended to be lower at 1000 mg/kg/day (0.75x of control). However, as only two samples were available for analysis of both T3 and T4 at 1000 mg/kg/day these data should be interpreted with caution and could not be used for toxicological evaluation
Details on embryotoxic / teratogenic effects:
N/A
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Dose Formulation Analyses

Accuracy

 

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85 115% of target concentration).

 

A small response at the retention time of the test material was observed in one of the chromatograms of the Group 1 formulation prepared for use in Week 3. It was considered not to derive from the formulation, since a similar response was obtained in the analytical blanks. The maximum contribution to Group 2 samples calculated was 0.00057%, taking the dilution factor into account and considered neglectable. In all other formulations of Group 1, no test material was detected.

 

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Table 9. Body Weights (grams) Summary (F0)

 

 

Group 1

Control

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Males

Pre-mating

 

Day 1

Week 1

Mean

302

295

303

298

St. Dev

12.1

7.0

11.1

9.0

N

10

10

10

10

 

Day 8

Week 2

Mean

320

314

321

311

St. Dev

16.1

7.8

11.8

10.8

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

335

325

334

323

St. Dev

17.7

7.3

14.5

13.0

N

10

10

10

10

 

Day 8

Week 2

Mean

344

335

340

330

St. Dev

17.7

8.0

16.4

15.5

N

10

10

10

10

 

Day 15

Week 3

Mean

357

346

352

339*

St. Dev

17.7

10.0

15.6

15.4

N

10

10

10

10

Females

Pre-mating

 

Day 1

Week 1

Mean

227

230

234

233

St. Dev

12.2

9.9

11.5

15.6

N

10

10

10

10

 

Day 8

Week 2

Mean

230

233

236

234

St. Dev

14.4

8.3

13.9

15.2

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

234

236

239

249

St. Dev

13.6

12.3

13.8

13.9

N

10

10

10

10

 

Day 8

Week 2

Mean

 

---

268

269

St. Dev

 

---

---

---

N

 

0 x

1

1

 

Day 15

Week 3

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

 

Day 22

Week 4

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

Post Coitum

 

Day 0

Mean

235

235

241

238

St. Dev

13.1

13.4

15.0

12.5

N

10

8

10

9

 

Day 4

Mean

250

250

253

250

St. Dev

13.0

12.8

16.0

12.5

N

10

8

10

9

 

Day 7

Mean

256

257

259

256

St. Dev

12.4

14.6

15.5

13.2

N

10

8

10

9

 

Day 11

Mean

270

272

275

272

St. Dev

12.4

15.0

16.5

14.5

N

10

8

10

9

 

Day 14

Mean

279

283

286

280

St. Dev

14.1

18.0

17.8

16.3

N

10

8

10

9

 

Day 17

Mean

301

307

309

301

St. Dev

16.6

17.7

21.3

18.4

N

10

8

10

9

 

Day 20

Mean

330

345

345

328

St. Dev

27.6

18.6

27.4

21.6

N

10

8

10

9

Lactation

 

Day 1

Mean

268

268

275

266

St. Dev

19.1

15.0

10.3

19.6

N

9

9

10

8

 

Day 4

Mean

277

278

279

272

St. Dev

20.1

16.6

15.8

26.0

N

9

9

10

6

 

Day 7

Mean

285

288

286

278

St. Dev

17.2

13.4

18.8

22.8

N

9

9

10

6

 

Day 13

Mean

297

296

295

279

St. Dev

17.6

18.3

23.6

23.0

N

9

9

10

6

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values in the study report

Table 10. Body Weights (%) Summary (F0)

 

 

Group 1

Control

0 mg/kg/day

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

1000 mg/kg/day

Males

Pre-mating

 

Day 1

Week 1

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

6

6

6

4

St. Dev

1.4

1.3

1.7

1.6

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

11

10

10

9

St. Dev

2.0

1.7

2.2

2.5

N

10

10

10

10

 

Day 8

Week 2

Mean

14

14

12

11*

St. Dev

2.3

1.6

2.8

3.0

N

10

10

10

10

 

Day 15

Week 3

Mean

18

18

16

14**

St. Dev

2.4

2.2

2.9

3.5

N

10

10

10

10

Females

Pre-mating

 

Day 1

Week 1

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

1

1

1

0

St. Dev

2.6

1.1

1.8

2.3

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

3

3

2

2

St. Dev

2.5

3.1

2.5

2.6

N

10

10

10

10

 

Day 8

Week 2

Mean

 

---

10

10

St. Dev

 

---

---

---

N

 

0 x

1

1

 

Day 15

Week 3

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

 

Day 22

Week 4

Mean

 

---

 

 

St. Dev

 

---

 

 

N

 

0 x

 

 

Post Coitum

 

Day 0

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

10

8

10

9

 

Day 4

Mean

6

7

5

5

St. Dev

1.1

2.4

1.4

2.1

N

10

8

10

9

 

Day 7

Mean

9

9

8

8

St. Dev

1.5

4.0

0.9

2.2

N

10

8

10

9

 

Day 11

Mean

15

16

14

14

St. Dev

2.3

3.9

1.9

3.6

N

10

8

10

9

 

Day 14

Mean

19

20

19

18

St. Dev

2.3

4.8

2.8

3.8

N

10

8

10

9

 

Day 17

Mean

28

31

28

27

St. Dev

3.9

3.7

5.0

5.8

N

10

8

10

9

 

Day 20

Mean

41

47

43

38

St. Dev

9.9

3.2

7.3

10.1

N

10

8

10

9

Lactation

 

Day 1

Mean

0

0

0

0

St. Dev

0.0

0.0

0.0

0.0

N

9

9

10

8

 

Day 4

Mean

3

4

1

1

St. Dev

2.2

2.8

3.3

2.4

N

9

9

10

6

 

Day 7

Mean

6

8

4

3

St. Dev

3.2

5.1

4.3

2.2

N

9

9

10

6

 

Day 13

Mean

11

11

7

3*

St. Dev

4.2

4.2

6.3

2.9

N

9

9

10

6

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values in the study report

 

Table 11. Summary of Select Hematology Values: Females (F0)

Day: 52 Relative to Start Date

Dose Group

 

HGB (g/L)

[G]

MCHC (g/L)

[G]

Group 1

Control

0 mg/kg/day

Mean

148.0

331.8

St. Dev

6.4

2.6

N

5

5

 

Group 2

100 mg/kg/day

Mean

136.2**

322.4

St. Dev

3.7

6.9

N

5

5

tCtrl

0.92

0.97

 

Group 3

300 mg/kg/day

Mean

139.6*

320.6*

St. Dev

3.4

4.2

N

5

5

tCtrl

0.94

0.97

 

Group 4

1000 mg/kg/day

Mean

147.4

326.8

St. Dev

5.3

9.0

N

5

5

tCtrl

1.00

0.98